advertisement
Browse Health
Internist, Infectious Disease Specialist (virus, bacteria, parasites)
28 years of experience
Accepting new patients

Credentials

Education ?

Medical School Score Rankings
Harvard University (1984)
  •  
Top 25%

Awards & Distinctions ?

Appointments
Massachusetts General Hospital
Associations
American Board of Internal Medicine

Affiliations ?

Dr. Johnson is affiliated with 3 hospitals.

Hospital Affiliations

Score

Rankings

  • Massachusetts General Hospital
    55 Fruit St, Boston, MA 02114
    •  
    Top 25%
  • Mass General Hospital
  • Massachusetts Eye And Ear Infirmary
    243 Charles St, Boston, MA 02114
  • Publications & Research

    Dr. Johnson has contributed to 258 publications.
    Title Concerted Vs Stepwise Mechanisms in Dehydro-diels-alder Reactions.
    Date February 2012
    Journal The Journal of Organic Chemistry
    Excerpt

    The Diels-Alder reaction is not limited to 1,3-dienes. Many cycloadditions of enynes and a smaller number of examples with 1,3-diynes have been reported. These "dehydro"-Diels-Alder cycloadditions are one class of dehydropericyclic reactions which have long been used to generate strained cyclic allenes and other novel structures. CCSD(T)//M05-2X computational results are reported for the cycloadditions of vinylacetylene and butadiyne with ethylene and acetylene. Both concerted and stepwise diradical routes have been explored for each reaction, with location of relevant stationary points. Relative to 1,3-dienes, replacement of one double bond by a triple bond adds 6-6.5 kcal/mol to the activation barrier; a second triple bond adds 4.3-4.5 kcal/mol to the barrier. Product strain decreases the predicted exothermicity. In every case, a concerted reaction is favored energetically. The difference between concerted and stepwise reactions is 5.2-6.6 kcal/mol for enynes but diminishes to 0.5-2 kcal/mol for diynes. Experimental studies on intramolecular diyne + ene cycloadditions show two distinct reaction pathways, providing evidence for competing concerted and stepwise mechanisms. Diyne + yne cycloadditions connect with arynes and ethynyl-1,3-cyclobutadiene. This potential energy surface appears to be flat, with only a minute advantage for a concerted process; many diyne cycloadditions or aryne cycloreversions will proceed by a stepwise mechanism.

    Title Sers from Two-tier Sphere Segment Void Substrates.
    Date January 2012
    Journal Physical Chemistry Chemical Physics : Pccp
    Excerpt

    Sphere segment void, or inverse opal, films prepared from a variety of coinage metals have shown promise as reliable and reproducible substrates for surface enhanced Raman spectroscopy (SERS). Sphere segment void substrates are prepared from colloidal templates consisting of one or more layers of polystyrene spheres. In this paper, we investigate the reflection spectra and SERS for a gold film consisting of two-tiers of spherical cavities, and show that the best SERS enhancements are obtained from substrates consisting of just a single layer of sphere segment voids.

    Title Pruritic Rash on Trunk.
    Date October 2011
    Journal The Journal of Family Practice
    Excerpt

    The patient had been treated with topical antifungals and steroids without relief, but a more detailed history suggested a serious infectious etiology.

    Title Characterization of Killer Immunoglobulin-like Receptor Genetics and Comprehensive Genotyping by Pyrosequencing in Rhesus Macaques.
    Date September 2011
    Journal Bmc Genomics
    Excerpt

    Human killer immunoglobulin-like receptors (KIRs) play a critical role in governing the immune response to neoplastic and infectious disease. Rhesus macaques serve as important animal models for many human diseases in which KIRs are implicated; however, the study of KIR activity in this model is hindered by incomplete characterization of KIR genetics.

    Title Sers from Molecules Bridging the Gap of Particle-in-cavity Structures.
    Date September 2011
    Journal Chemical Communications (cambridge, England)
    Excerpt

    We demonstrate that by combining silver nanoparticles and structured gold SSV surfaces the SERS for those molecules that bridge the nanoparticle-cavity gap is preferentially enhanced using 4-mercaptoaniline and 4-mercaptobenzoic acid as examples.

    Title Flow Cytometry Based Identification of Simian Immunodeficiency Virus Env-specific B Lymphocytes.
    Date September 2011
    Journal Journal of Immunological Methods
    Excerpt

    SIV infection of macaques is the most widely employed model for preclinical AIDS vaccine and pathogenesis research. In macaques, high-titer virus-specific antibodies are induced by infection, and antibody responses can drive evolution of viral escape variants. However, neutralizing antibodies (Nabs) induced in response to SIVmac239 and SIVmac251 infection or immunization are generally undetectable or of low titer, and the identification and cloning of potent Nabs from SIVmac-infected macaques remains elusive. Based on recent advances in labeling HIV-specific B lymphocytes [1-3], we have generated recombinant, secreted, soluble SIVmac envelope (Env) proteins (gp120 and gp140) for detection and quantification of SIVmac Env-specific B lymphocytes. In contrast to HIV-1, we found that soluble SIVmac239 gp140 retains the ability to form stable oligomers without the necessity for introducing additional, stabilizing modifications. Soluble oligomeric gp140 reacted with rhesus anti-SIV Env-specific monoclonal antibodies (MAbs), and was used to deplete Env-specific antibodies with SIV neutralization capability from plasma taken from a rhesus macaque immunized with live attenuated SIVmac239∆nef. Soluble gp120 and gp140 bound to SIV-specific immortalized B cells, and to SIV Env-specific B lymphocytes in peripheral blood of immunized animals. These reagents will be useful for analyzing development of Env-specific B cell responses in preclinical studies using SIV-infected or vaccinated rhesus macaques.

    Title Reduction of Co2 on a Tricarbonyl Rhenium(i) Complex: Modeling a Catalytic Cycle.
    Date July 2011
    Journal The Journal of Physical Chemistry. A
    Excerpt

    Tricarbonyl rhenium(I) complexes, such as Re(bpy)(CO)(3)Cl where bpy = 2,2'-bipyridyl, have demonstrated superior activity in catalyzing CO(2) reduction in the presence of sacrificial electron donors. We have utilized density functional theory (DFT) to investigate a potential pathway for formate production via a rhenium-hydride insertion mechanism in the presence of triethylamine (TEA). On the basis of prior studies, we re-examined the role of TEA and studied a catalytic cycle for CO(2) reduction in which TEA functions as both the hydrogen atom and the electron donor for reducing CO(2) into formate. The catalytic cycle is found to be exothermic with inclusion of solvation and may be viewed as a two-electron reduction of CO(2) because the net result is a transfer of hydride from TEA to CO(2). In addition, we have identified structures of key intermediates in the CO(2)-reduction process and found that the insertion step has a very modest barrier in acetonitrile. These findings provide a molecular-level understanding to formate production via CO(2) reduction mediated by transition-metal complexes. A theoretical investigation is underway to elucidate the formation of carbon monoxide, another common product in Re-catalyzed CO(2) reduction.

    Title Kir Polymorphisms Modulate Peptide-dependent Binding to an Mhc Class I Ligand with a Bw6 Motif.
    Date July 2011
    Journal Plos Pathogens
    Excerpt

    Molecular interactions between killer immunoglobulin-like receptors (KIRs) and their MHC class I ligands play a central role in the regulation of natural killer (NK) cell responses to viral pathogens and tumors. Here we identify Mamu-A1*00201 (Mamu-A*02), a common MHC class I molecule in the rhesus macaque with a canonical Bw6 motif, as a ligand for Mamu-KIR3DL05. Mamu-A1*00201 tetramers folded with certain SIV peptides, but not others, directly stained primary NK cells and Jurkat cells expressing multiple allotypes of Mamu-KIR3DL05. Differences in binding avidity were associated with polymorphisms in the D0 and D1 domains of Mamu-KIR3DL05, whereas differences in peptide-selectivity mapped to the D1 domain. The reciprocal exchange of the third predicted MHC class I-contact loop of the D1 domain switched the specificity of two Mamu-KIR3DL05 allotypes for different Mamu-A1*00201-peptide complexes. Consistent with the function of an inhibitory KIR, incubation of lymphocytes from Mamu-KIR3DL05(+) macaques with target cells expressing Mamu-A1*00201 suppressed the degranulation of tetramer-positive NK cells. These observations reveal a previously unappreciated role for D1 polymorphisms in determining the selectivity of KIRs for MHC class I-bound peptides, and identify the first functional KIR-MHC class I interaction in the rhesus macaque. The modulation of KIR-MHC class I interactions by viral peptides has important implications to pathogenesis, since it suggests that the immunodeficiency viruses, and potentially other types of viruses and tumors, may acquire changes in epitopes that increase the affinity of certain MHC class I ligands for inhibitory KIRs to prevent the activation of specific NK cell subsets.

    Title Gene Cluster Conferring Streptomycin, Sulfonamide, and Tetracycline Resistance in Escherichia Coli O157:h7 Phage Types 23, 45, and 67.
    Date May 2011
    Journal Applied and Environmental Microbiology
    Excerpt

    Multidrug resistance to streptomycin, sulfonamide, and tetracycline (AMR-SSuT) was identified in 156 of 171 isolates of Escherichia coli O157:H7 of phage types 23, 45, and 67. In 154 AMR-SSuT isolates, resistance was encoded by strA, strB, sul2, and tet(B), which in 59 of 63 tested isolates were found clustered together on the chromosome within the cdiA locus.

    Title Cloning and Characterization of Rhesus Il-18 Binding Protein, a Natural Antagonist to Il-18.
    Date November 2010
    Journal Cytokine
    Excerpt

    IL-18 is a proinflammatory cytokine that is important for host defense, but is also involved in the pathogenesis of a number of disease processes, ranging from autoimmune disorders to atherosclerosis. IL-18 binding protein (IL-18BP) is a constitutively expressed glycoprotein that specifically neutralizes the effects of IL-18, resulting in decreased production of IFN-gamma and reduction in Th1 immune responses. In this study we cloned and sequenced a full-length cDNA of the rhesus IL-18BP (RhIL-18BP) from the spleen of rhesus macaques (Macaca mulatta) and compared its nucleotide and amino acid sequences to the functional murine and human IL-18BP orthologues. In addition, we fused RhIL-18BP to the Fc portion of human IgG1 to make recombinant RhIL-18BP x Fcgamma1 in order to facilitate its detection by Western blot analysis and determined the approximate molecular weight of RhIL-18BP x Fcgamma1 to be 66 kD. With this fusion protein, we showed that RhIL-18BP was functional and could significantly reduce murine IL-18 and LPS-induced IFN-gamma production by murine splenocytes. Furthermore, we demonstrated the expression of IL-18BP in atherosclerotic lesions in a rhesus model of atherosclerosis, underscoring the need to fully understand the role of this protein as a primary negative regulator of IL-18 in multiple disease processes.

    Title Survival of the Fittest: Positive Selection of Cd4+ T Cells Expressing a Membrane-bound Fusion Inhibitor Following Hiv-1 Infection.
    Date November 2010
    Journal Plos One
    Excerpt

    Although a variety of genetic strategies have been developed to inhibit HIV replication, few direct comparisons of the efficacy of these inhibitors have been carried out. Moreover, most studies have not examined whether genetic inhibitors are able to induce a survival advantage that results in an expansion of genetically-modified cells following HIV infection. We evaluated the efficacy of three leading genetic strategies to inhibit HIV replication: 1) an HIV-1 tat/rev-specific small hairpin (sh) RNA; 2) an RNA antisense gene specific for the HIV-1 envelope; and 3) a viral entry inhibitor, maC46. In stably transduced cell lines selected such that >95% of cells expressed the genetic inhibitor, the RNA antisense envelope and viral entry inhibitor maC46 provided the strongest inhibition of HIV-1 replication. However, when mixed populations of transduced and untransduced cells were challenged with HIV-1, the maC46 fusion inhibitor resulted in highly efficient positive selection of transduced cells, an effect that was evident even in mixed populations containing as few as 1% maC46-expressing cells. The selective advantage of the maC46 fusion inhibitor was also observed in HIV-1-infected cultures of primary T lymphocytes as well as in HIV-1-infected humanized mice. These results demonstrate robust inhibition of HIV replication with the fusion inhibitor maC46 and the antisense Env inhibitor, and importantly, a survival advantage of cells expressing the maC46 fusion inhibitor both in vitro and in vivo. Evaluation of the ability of genetic inhibitors of HIV-1 replication to confer a survival advantage on genetically-modified cells provides unique information not provided by standard techniques that may be important in the in vivo efficacy of these genes.

    Title Simian Immunodeficiency Virus Infection Induces Expansion of Alpha4beta7+ and Cytotoxic Cd56+ Nk Cells.
    Date September 2010
    Journal Journal of Virology
    Excerpt

    Herein we demonstrate that chronic simian immunodeficiency virus (SIV) infection induces significant upregulation of the gut-homing marker alpha4beta7 on macaque NK cells, coupled with downregulation of the lymph node-trafficking marker, CCR7. Interestingly, in naïve animals, alpha4beta7 expression was associated with increased NK cell activation and, on CD16(+) NK cells, delineated a unique dual-function cytotoxic-CD107a(+)/gamma interferon (IFN-gamma)-secreting population. However, while SIV infection increased CD107a expression on stimulated CD56(+) NK cells, alpha4beta7(+) and alpha4beta7(-) NK cells were affected similarly. These findings suggest that SIV infection redirects NK cells away from the lymph nodes to the gut mucosae but alters NK cell function independent of trafficking repertoires.

    Title Oral Delivery Systems for Encapsulated Bacteriophages Targeted at Escherichia Coli O157:h7 in Feedlot Cattle.
    Date September 2010
    Journal Journal of Food Protection
    Excerpt

    Bacteriophages are natural predators of bacteria and may mitigate Escherichia coli O157:H7 in cattle and their environment. As bacteriophages targeted to E. coli O157:H7 (phages) lose activity at low pH, protection from gastric acidity may enhance efficacy of orally administered phages. Polymer encapsulation of four phages, wV8, rV5, wV7, and wV11, and exposure to pH 3.0 for 20 min resulted in an average 13.6% recovery of phages after release from encapsulation at pH 7.2. In contrast, untreated phages under similar conditions had a complete loss of activity. Steers (n = 24) received 10(11) CFU of naladixic acid-resistant E. coli O157:H7 on day 0 and were housed in six pens of four steers. Two pens were control (naladixic acid-resistant E. coli O157:H7 only), and the remaining pens received polymer-encapsulated phages (Ephage) on days -1, 1, 3, 6, and 8. Two pens received Ephage orally in gelatin capsules (bolus; 10(10) PFU per steer per day), and the remaining two pens received Ephage top-dressed on their feed (feed; estimated 10(11) PFU per steer per day). Shedding of E. coli O157:H7 was monitored for 10 weeks by collecting fecal grab and hide swab samples. Acceptable activity of mixed phages at delivery to steers was found for bolus and feed, averaging 1.82 and 1.13 x 10(9) PFU/g, respectively. However, Ephage did not reduce shedding of naladixic acid-resistant E. coli O157:H7, although duration of shedding was reduced by 14 days (P < 0.1) in bolus-fed steers as compared with control steers. Two successful systems for delivery of Ephage were developed, but a better understanding of phage-E. coli O157:H7 ecology is required to make phage therapy a viable strategy for mitigation of this organism in feedlot cattle.

    Title Competing Mechanistic Channels in the Oxidation of Aldehydes by Ozone.
    Date August 2010
    Journal The Journal of Organic Chemistry
    Excerpt

    The reaction of ozone with aldehydes has been studied intermittently for over 100 years, but its mechanism remains uncertain. Experimental results support two reaction channels: radical abstraction of the acyl hydrogen and addition to form a five-membered ring tetroxolane. We have studied the aldehyde-ozone reaction by DFT and CCSD(T) calculations. CCSD(T)/6-311+G(d,p)//M05-2X)/6-311+G(d,p) calculations predict two competitive pathways for the oxidation of formaldehyde by ozone. Abstraction of the acyl hydrogen by ozone has a barrier of 16.2 kcal/mol, leading to a radical pair, which can combine to form a hydrotrioxide; this species may subsequently decompose to a carboxylic acid and singlet oxygen. In the second reaction channel, addition of ozone to the carbonyl is stepwise, with barriers of 19.1 and 23.0 kcal/mol, leading to a five-membered ring tetroxolane intermediate. This process may be reversible, consistent with earlier observations of isotopic exchange. The two channels connect by an intramolecular hydrogen abstraction. Ring opening of the tetroxolane by an alternate O-O bond cleavage, followed by spin inversion in the resulting diradical intermediate, can give a carbonyl oxide plus (3)O(2). It is also possible that reaction of triplet oxygen with carbonyl oxides can produce ozone by the reverse route. These two stepwise reaction channels, hydrogen abstraction and addition to the C=O bond, explain much of what has been observed in the long history of ozone-aldehyde chemistry. Known reaction rates and the substantial barriers to both channels support an earlier conclusion that aldehyde oxidation by ozone is too slow to be of importance in atmospheric chemistry.

    Title Gamma-ray Emission Concurrent with the Nova in the Symbiotic Binary V407 Cygni.
    Date August 2010
    Journal Science (new York, N.y.)
    Excerpt

    Novae are thermonuclear explosions on a white dwarf surface fueled by mass accreted from a companion star. Current physical models posit that shocked expanding gas from the nova shell can produce x-ray emission, but emission at higher energies has not been widely expected. Here, we report the Fermi Large Area Telescope detection of variable gamma-ray emission (0.1 to 10 billion electron volts) from the recently detected optical nova of the symbiotic star V407 Cygni. We propose that the material of the nova shell interacts with the dense ambient medium of the red giant primary and that particles can be accelerated effectively to produce pi(0) decay gamma-rays from proton-proton interactions. Emission involving inverse Compton scattering of the red giant radiation is also considered and is not ruled out.

    Title Pressure-dependent Contribution of Rho Kinase-mediated Calcium Sensitization in Serotonin-evoked Vasoconstriction of Rat Cerebral Arteries.
    Date August 2010
    Journal The Journal of Physiology
    Excerpt

    Our understanding of the cellular signalling mechanisms contributing to agonist-induced constriction is almost exclusively based on the study of conduit arteries. Resistance arteries/arterioles have received less attention as standard biochemical approaches lack the necessary sensitivity to permit quantification of phosphoprotein levels in these small vessels. Here, we have employed a novel, highly sensitive Western blotting method to assess: (1) the contribution of Ca(2+) sensitization mediated by phosphorylation of myosin light chain phosphatase targeting subunit 1 (MYPT1) and the 17 kDa PKC-potentiated protein phosphatase 1 inhibitor protein (CPI-17) to serotonin (5-HT)-induced constriction of rat middle cerebral arteries, and (2) whether there is any interplay between pressure-induced myogenic and agonist-induced mechanisms of vasoconstriction. Arterial diameter and levels of MYPT1 (T697 and T855), CPI-17 and 20 kDa myosin light chain subunit (LC(20)) phosphorylation were determined following treatment with 5-HT (1 micromol l(1)) at 10 or 60 mmHg in the absence and presence of H1152 or GF109203X to suppress the activity of Rho-associated kinase (ROK) and protein kinase C (PKC), respectively. Although H1152 and GF109203X suppressed 5-HT-induced constriction and reduced phospho-LC(20) content at 10 mmHg, we failed to detect any increase in MYPT1 or CPI-17 phosphorylation. In contrast, an increase in MYPT1-T697 and MYPT1-T855 phosphorylation, but not phospho-CPI-17 content, was apparent at 60 mmHg following exposure to 5-HT, and the phosphorylation of both MYPT1 sites was sensitive to H1152 inhibition of ROK. The involvement of MYPT1 phosphorylation in the response to 5-HT at 60 mmHg was not dependent on force generation per se, as inhibition of cross-bridge cycling with blebbistatin (10 micromol l(1)) did not affect phosphoprotein content. Taken together, the data indicate that Ca(2+) sensitization owing to ROK-mediated phosphorylation of MYPT1 contributes to 5-HT-evoked vasoconstriction only in the presence of pressure-induced myogenic activation. These findings provide novel evidence of an interplay between myogenic- and agonist-induced vasoconstriction in cerebral resistance arteries.

    Title Spectrum of the Isotropic Diffuse Gamma-ray Emission Derived from First-year Fermi Large Area Telescope Data.
    Date June 2010
    Journal Physical Review Letters
    Excerpt

    We report on the first Fermi Large Area Telescope (LAT) measurements of the so-called "extragalactic" diffuse gamma-ray emission (EGB). This component of the diffuse gamma-ray emission is generally considered to have an isotropic or nearly isotropic distribution on the sky with diverse contributions discussed in the literature. The derivation of the EGB is based on detailed modeling of the bright foreground diffuse Galactic gamma-ray emission, the detected LAT sources, and the solar gamma-ray emission. We find the spectrum of the EGB is consistent with a power law with a differential spectral index gamma = 2.41 +/- 0.05 and intensity I(>100 MeV) = (1.03 +/- 0.17) x 10(-5) cm(-2) s(-1) sr(-1), where the error is systematics dominated. Our EGB spectrum is featureless, less intense, and softer than that derived from EGRET data.

    Title Fermi Large Area Telescope Measurements of the Diffuse Gamma-ray Emission at Intermediate Galactic Latitudes.
    Date June 2010
    Journal Physical Review Letters
    Excerpt

    The diffuse galactic gamma-ray emission is produced by cosmic rays (CRs) interacting with the interstellar gas and radiation field. Measurements by the Energetic Gamma-Ray Experiment Telescope (EGRET) instrument on the Compton Gamma-Ray Observatory indicated excess gamma-ray emission greater, > or approximately equal to 1 GeV relative to diffuse galactic gamma-ray emission models consistent with directly measured CR spectra (the so-called "EGRET GeV excess"). The Large Area Telescope (LAT) instrument on the Fermi Gamma-Ray Space Telescope has measured the diffuse gamma-ray emission with improved sensitivity and resolution compared to EGRET. We report on LAT measurements for energies 100 MeV to 10 GeV and galactic latitudes 10 degrees < or = |b| < or = 20 degrees. The LAT spectrum for this region of the sky is well reproduced by a diffuse galactic gamma-ray emission model that is consistent with local CR spectra and inconsistent with the EGRET GeV excess.

    Title Cd16- Natural Killer Cells: Enrichment in Mucosal and Secondary Lymphoid Tissues and Altered Function During Chronic Siv Infection.
    Date June 2010
    Journal Blood
    Excerpt

    Natural killer (NK) cells contribute to control of HIV/SIV infection. We defined macaque NK-cell subsets based on expression of CD56 and CD16 and found their distribution to be highly disparate. CD16(+) NK cells predominated in peripheral blood, whereas most mucosal NK cells were CD56(+), and lymph nodes contained both CD56(+) and CD16(-)CD56(-) (double-negative [DN]) subsets. Functional profiles were also distinct among subsets--CD16(+) NK cells expressed high levels of cytolytic molecules, and CD56(+) NK cells were predominantly cytokine-secreting cells, whereas DN NK possessed both functions. In macaques chronically infected with SIV, circulating CD16(+) and DN NK cells were expanded in number and, although markers of cytoxicity increased, cytokine secretion decreased. Notably, CD56(+) NK cells in SIV-infected animals up-regulated perforin, granzyme B, and CD107a. In contrast, the lymph node-homing molecules CD62 ligand (CD62L) and C-C chemokine receptor type 7 (CCR7), which are expressed primarily on CD56(+) and DN NK cells, were significantly down-regulated on NK cells from infected animals. These data demonstrate that SIV infection drives a shift in NK-cell function characterized by decreased cytokine production, expanded cytotoxicity, and trafficking away from secondary lymphoid organs, suggesting that the NK-cell repertoire is not only heterogeneous but also plastic.

    Title Fermi Large Area Telescope Search for Photon Lines from 30 to 200 Gev and Dark Matter Implications.
    Date June 2010
    Journal Physical Review Letters
    Excerpt

    Dark matter (DM) particle annihilation or decay can produce monochromatic gamma rays readily distinguishable from astrophysical sources. gamma-ray line limits from 30 to 200 GeV obtained from 11 months of Fermi Large Area Space Telescope data from 20-300 GeV are presented using a selection based on requirements for a gamma-ray line analysis, and integrated over most of the sky. We obtain gamma-ray line flux upper limits in the range 0.6-4.5x10{-9} cm{-2} s{-1}, and give corresponding DM annihilation cross-section and decay lifetime limits. Theoretical implications are briefly discussed.

    Title Spallation Neutron Source Saddle Antenna H- Ion Source Project.
    Date May 2010
    Journal The Review of Scientific Instruments
    Excerpt

    In this project we are developing an H(-) source which will synthesize the most important developments in the field of negative ion sources to provide high current, high brightness, good lifetime, high reliability, and high power efficiency. We describe two planned modifications to the present spallation neutron source external antenna source in order to increase the plasma density near the output aperture: (1) replacing the present 2 MHz plasma-forming solenoid antenna with a 13 MHz saddle-type antenna and (2) replacing the permanent multicusp magnetic system with a weaker electromagnet.

    Title Electrode Activation in Cesium-free Negative Ion Sources.
    Date May 2010
    Journal The Review of Scientific Instruments
    Excerpt

    Features of emission electrode activation leading to enhancement of negative ion emission in cesium-free discharges are discussed. In some ion sources with cesium-free discharges, the emission of negative ions has been increased significantly by emission electrode activation using strong heating of the negative biased electrode by discharge plasma. A simple explanation of this enhancement is that it is due to an accumulation on the emission surface of the plasma electrode of impurities with low ionization potential that decreases in surface work function and increases the secondary emission of negative ions similar to "Cesiation." The negative biasing of emission surface is important for accumulation and trapping the impurities on the emission surface. To effectively control the activation process it is important to directly detect the evolution of the work function and the impurity concentration during electrode activation with enhancement of negative ion emission.

    Title Host Range and Lytic Capability of Four Bacteriophages Against Bovine and Clinical Human Isolates of Shiga Toxin-producing Escherichia Coli O157:h7.
    Date May 2010
    Journal Journal of Applied Microbiology
    Excerpt

    To evaluate host range and lytic capability of four bacteriophages (rV5, wV7, wV8 and wV11) against Escherichia coli O157:H7 (STEC O157:H7) from cattle and humans.

    Title Definition of Epitopes and Antigens Recognized by Vaccinia Specific Immune Responses: Their Conservation in Variola Virus Sequences, and Use As a Model System to Study Complex Pathogens.
    Date March 2010
    Journal Vaccine
    Excerpt

    In the last few years, a wealth of information has become available relating to the targets of vaccinia virus (VACV)-specific CD4(+) T cell, CD8(+) T cell and antibody responses. Due to the large size of its genome, encoding more than 200 different proteins, VACV represents a useful model system to study immunity to complex pathogens. Our data demonstrate that both cellular and humoral responses target a large number of antigens and epitopes. This broad spectrum of targets is detected in both mice and humans. CD4(+) T cell responses target late and structural antigens, while CD8(+) T cells preferentially recognize early antigens. While both CD4(+) and CD8(+) T cell responses target different types of antigens, the antigens recognized by T(H) cells are highly correlated with those recognized by antibody responses. We further show that protein abundance and antibody recognition can be used to predict antigens recognized by CD4(+) T cell responses, while early expression at the mRNA level predicts antigens targeted by CD8(+) T cells. Finally, we find that the vast majority of VACV epitopes are conserved in variola virus (VARV), thus suggesting that the epitopes defined herein also have relevance for the efficacy of VACV as a smallpox vaccine.

    Title Lineage and Host Source Are Both Correlated with Levels of Shiga Toxin 2 Production by Escherichia Coli O157:h7 Strains.
    Date March 2010
    Journal Applied and Environmental Microbiology
    Excerpt

    Escherichia coli O157:H7 strains fall into three major genetic lineages that differ in their distribution among humans and cattle. Several recent studies have reported differences in the expression of virulence factors between E. coli O157:H7 strains from these two host species. In this study, we wished to determine if important virulence-associated "mobile genetic elements" such as Shiga toxin 2 (Stx2)-encoding prophage are lineage restricted or are host source related and acquired independently of the pathogen genotype. DNA sequencing of the stx(2) flanking region from a lineage II (LII) strain, EC970520, revealed that the transcriptional activator gene Q in LI strain EDL933 (upstream of stx(2)) is replaced by a pphA (serine/threonine phosphatase) homologue and an altered Q gene in this and all other LII strains tested. In addition, nearly all LI strains carried stx(2), whereas all LII strains carried variant stx(2c) and 4 of 14 LI/II strains had copies of both stx(2) and variant stx(2c). Real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that LI and LI/II strains produce significantly more stx(2) mRNA and Stx2 than LII strains. However, among LI strains significantly more Stx2 is also produced by strains from humans than from cattle. Therefore, lineage-associated differences among E. coli O157:H7 strains such as prophage content, toxin type, and toxin expression may contribute to host isolation bias. However, the level of Stx2 production alone may also play an important role in the within-lineage association of E. coli O157:H7 strains with human clinical disease.

    Title Vaccination with Sivmac239deltanef Activates Cd4+ T Cells in the Absence of Cd4 T-cell Loss.
    Date January 2010
    Journal Journal of Medical Primatology
    Excerpt

    Pathogenic HIV and SIV infections characteristically deplete central memory CD4(+) T cells and induce chronic immune activation, but it is controversial whether this also occurs after vaccination with attenuated SIVs and whether depletion or activation of CD4(+) T-cell play roles in protection against wild-type virus challenge.

    Title Effective Simian Immunodeficiency Virus-specific Cd8+ T Cells Lack an Easily Detectable, Shared Characteristic.
    Date January 2010
    Journal Journal of Virology
    Excerpt

    The immune correlates of human/simian immunodeficiency virus control remain elusive. While CD8(+) T lymphocytes likely play a major role in reducing peak viremia and maintaining viral control in the chronic phase, the relative antiviral efficacy of individual virus-specific effector populations is unknown. Conventional assays measure cytokine secretion of virus-specific CD8(+) T cells after cognate peptide recognition. Cytokine secretion, however, does not always directly translate into antiviral efficacy. Recently developed suppression assays assess the efficiency of virus-specific CD8(+) T cells to control viral replication, but these assays often use cell lines or clones. We therefore designed a novel virus production assay to test the ability of freshly ex vivo-sorted simian immunodeficiency virus (SIV)-specific CD8(+) T cells to suppress viral replication from SIVmac239-infected CD4(+) T cells. Using this assay, we established an antiviral hierarchy when we compared CD8(+) T cells specific for 12 different epitopes. Antiviral efficacy was unrelated to the disease status of each animal, the protein from which the tested epitopes were derived, or the major histocompatibility complex (MHC) class I restriction of the tested epitopes. Additionally, there was no correlation with the ability to suppress viral replication and epitope avidity, epitope affinity, CD8(+) T-cell cytokine multifunctionality, the percentage of central and effector memory cell populations, or the expression of PD-1. The ability of virus-specific CD8(+) T cells to suppress viral replication therefore cannot be determined using conventional assays. Our results suggest that a single definitive correlate of immune control may not exist; rather, a successful CD8(+) T-cell response may be comprised of several factors.

    Title Inhibitory Tcr Coreceptor Pd-1 is a Sensitive Indicator of Low-level Replication of Siv and Hiv-1.
    Date January 2010
    Journal Journal of Immunology (baltimore, Md. : 1950)
    Excerpt

    Ongoing antigenic stimulation appears to be an important prerequisite for the persistent expression of programmed death 1 (PD-1), an inhibitory TCR coreceptor of the CD28 family. Although recent publications have emphasized the utility of PD-1 as a marker for dysfunctional T cells in chronic viral infections, its dependence on antigenic stimulation potentially renders it a sensitive indicator of low-level viral replication. To explore the antigenic threshold for the maintenance of PD-1 expression on virus-specific T cells, we compared PD-1 expression on virus-specific and memory T cell populations in controlled and uncontrolled SIV and HIV-1 infection. In both controlled live attenuated SIV infection in rhesus macaques and HIV-1 infection in elite controllers, elevated levels of PD-1 expression were observed on SIV- and HIV-1-specific CD8(+) T cells. However, in contrast to chronic wild-type SIV infection and uncontrolled HIV-1 infection, controlled SIV/HIV-1 infection did not result in increased expression of PD-1 on total memory T cells. PD-1 expression on SIV-specific CD8(+) T cells rapidly decreased after the emergence of CTL escape in cognate epitopes, but was maintained in the setting of low or undetectable levels of plasma viremia in live attenuated SIV-infected macaques. After inoculation of naive macaques with a single-cycle SIV, PD-1 expression on SIV-specific CD8(+) T cells initially increased, but was rapidly downregulated. These results demonstrate that PD-1 can serve as a sensitive indicator of persistent, low-level virus replication and that generalized PD-1 expression on T lymphocytes is a distinguishing characteristic of uncontrolled lentiviral infections.

    Title Risk Factors Associated with Escherichia Coli O157:h7 in Ontario Beef Cow-calf Operations.
    Date December 2009
    Journal Preventive Veterinary Medicine
    Excerpt

    The aim of this study was to identify farm management factors associated with the prevalence of Escherichia coli O157:H7 among cattle in Ontario beef cow-calf operations. A total of 119 cow-calf operations with more than 50 cows in southern Ontario were visited between June and December 2002. From each farm, 65 fresh fecal samples were collected and cultured for E. coli O157:H7. Colonies of E. coli O157:H7 were isolated using immunomagnetic separation and standard microbiological techniques. Final confirmation of suspected colonies was based on identifying E. coli O157:H7-specific genes by PCR and serotyping of representative isolates. A questionnaire was administered to collect information on farm size, cattle demographics, farm management practices, the presence of other livestock and wildlife, and other aspects of the farm environment. Associations between the prevalence of E. coli O157:H7 in cattle feces and management factors were determined using a multivariable logistic regression model that included random effects for farm and county. The presence of pigs on farm, use of corn silage supplementation in winter, number of times cattle were taken to a show in the previous 12 months and the percentage of cows on farm were significant risk factors for the presence of E. coli O157:H7 in fecal pat samples, after controlling for region and the age group of the sampled animals. These findings highlight the potential roles of biosecurity and avoiding mixed animal agriculture in controlling the prevalence of E. coli O157:H7 in beef cow-calf operations.

    Title Adp Signaling in Vascular Endothelial Cells: Adp-dependent Activation of the Endothelial Isoform of Nitric-oxide Synthase Requires the Expression but Not the Kinase Activity of Amp-activated Protein Kinase.
    Date December 2009
    Journal The Journal of Biological Chemistry
    Excerpt

    ADP responses underlie therapeutic approaches to many cardiovascular diseases, and ADP receptor antagonists are in widespread clinical use. The role of ADP in platelet biology has been extensively studied, yet ADP signaling pathways in endothelial cells remain incompletely understood. We found that ADP promoted phosphorylation of the endothelial isoform of nitric-oxide synthase (eNOS) at Ser(1179) and Ser(635) and dephosphorylation at Ser(116) in cultured endothelial cells. Although eNOS activity was stimulated by both ADP and ATP, only ADP signaling was significantly inhibited by the P2Y(1) receptor antagonist MRS 2179 or by knockdown of P2Y(1) using small interfering RNA (siRNA). ADP activated the small GTPase Rac1 and promoted endothelial cell migration. siRNA-mediated knockdown of Rac1 blocked ADP-dependent eNOS Ser(1179) and Ser(635) phosphorylation, as well as eNOS activation. We analyzed pathways known to regulate eNOS, including phosphoinositide 3-kinase/Akt, ERK1/2, Src, and calcium/calmodulin-dependent kinase kinase-beta (CaMKKbeta) using the inhibitors wortmannin, PD98059, PP2, and STO-609, respectively. None of these inhibitors altered ADP-modulated eNOS phosphorylation. In contrast, siRNA-mediated knockdown of AMP-activated protein kinase (AMPK) inhibited ADP-dependent eNOS Ser(635) phosphorylation and eNOS activity but did not affect eNOS Ser(1179) phosphorylation. Importantly, the AMPK enzyme inhibitor compound C had no effect on ADP-stimulated eNOS activity, despite completely blocking AMPK activity. CaMKKbeta knockdown suppressed ADP-stimulated eNOS activity, yet inhibition of CaMKKbeta kinase activity using STO-609 failed to affect eNOS activation by ADP. These data suggest that the expression, but not the kinase activity, of AMPK and CaMKKbeta is necessary for ADP signaling to eNOS.

    Title A Limit on the Variation of the Speed of Light Arising from Quantum Gravity Effects.
    Date November 2009
    Journal Nature
    Excerpt

    A cornerstone of Einstein's special relativity is Lorentz invariance-the postulate that all observers measure exactly the same speed of light in vacuum, independent of photon-energy. While special relativity assumes that there is no fundamental length-scale associated with such invariance, there is a fundamental scale (the Planck scale, l(Planck) approximately 1.62 x 10(-33) cm or E(Planck) = M(Planck)c(2) approximately 1.22 x 10(19) GeV), at which quantum effects are expected to strongly affect the nature of space-time. There is great interest in the (not yet validated) idea that Lorentz invariance might break near the Planck scale. A key test of such violation of Lorentz invariance is a possible variation of photon speed with energy. Even a tiny variation in photon speed, when accumulated over cosmological light-travel times, may be revealed by observing sharp features in gamma-ray burst (GRB) light-curves. Here we report the detection of emission up to approximately 31 GeV from the distant and short GRB 090510. We find no evidence for the violation of Lorentz invariance, and place a lower limit of 1.2E(Planck) on the scale of a linear energy dependence (or an inverse wavelength dependence), subject to reasonable assumptions about the emission (equivalently we have an upper limit of l(Planck)/1.2 on the length scale of the effect). Our results disfavour quantum-gravity theories in which the quantum nature of space-time on a very small scale linearly alters the speed of light.

    Title High-temporal Contrast Using Low-gain Optical Parametric Amplification.
    Date October 2009
    Journal Optics Letters
    Excerpt

    We demonstrate the use of low-gain optical parametric amplification (OPA) as a means of improving temporal contrast to a detection-limited level 10(-10). 250 microJ, 500 fs pulses of 1053 nm are frequency doubled and subsequently restored to the original wavelength by OPA with >10% efficiency.

    Title Subcutaneous Nodules with Sporotrichoid Spread.
    Date September 2009
    Journal Dermatology Online Journal
    Excerpt

    Mycobacterium marinum is an atypical mycobacterium found worldwide and associated with swimming pools and aquariums. Infections typically present with subcutaneous nodules and lymphangitis.

    Title Adherence of Escherichia Coli O157:h7 Mutants in Vitro and in Ligated Pig Intestines.
    Date September 2009
    Journal Applied and Environmental Microbiology
    Excerpt

    There are contradictory literature reports on the role of verotoxin (VT) in adherence of enterohemorrhagic Escherichia coli O157:H7 (O157 EHEC) to intestinal epithelium. There are reports that putative virulence genes of O island 7 (OI-7), OI-15, and OI-48 of this pathogen may also affect adherence in vitro. Therefore, mutants of vt2 and segments of OI-7 and genes aidA(15) (gene from OI-15) and aidA(48) (gene from OI-48) were generated and evaluated for adherence in vitro to cultured human HEp-2 and porcine jejunal epithelial (IPEC-J2) cells and in vivo to enterocytes in pig ileal loops. VT2-negative mutants showed significant decreases in adherence to both HEp-2 and IPEC-J2 cells and to enterocytes in pig ileal loops; complementation only partially restored VT2 production but fully restored the adherence to the wild-type level on cultured cells. Deletion of OI-7 and aidA(48) had no effect on adherence, whereas deletion of aidA(15) resulted in a significant decrease in adherence in pig ileal loops but not to the cultured cells. This investigation supports the findings that VT2 plays a role in adherence, shows that results obtained in adherence of E. coli O157:H7 in vivo may differ from those obtained in vitro, and identified AIDA-15 as having a role in adherence of E. coli O157:H7.

    Title Enhanced Laser-driven Ion Acceleration in the Relativistic Transparency Regime.
    Date September 2009
    Journal Physical Review Letters
    Excerpt

    We report on the acceleration of ion beams from ultrathin diamondlike carbon foils of thickness 50, 30, and 10 nm irradiated by ultrahigh contrast laser pulses at intensities of approximately 7 x 10;{19} W/cm;{2}. An unprecedented maximum energy of 185 MeV (15 MeV/u) for fully ionized carbon atoms is observed at the optimum thickness of 30 nm. The enhanced acceleration is attributed to self-induced transparency, leading to strong volumetric heating of the classically overdense electron population in the bulk of the target. Our experimental results are supported by both particle-in-cell (PIC) simulations and an analytical model.

    Title Diverse Recognition of Conserved Orthopoxvirus Cd8+ T Cell Epitopes in Vaccinated Rhesus Macaques.
    Date September 2009
    Journal Vaccine
    Excerpt

    Vaccinia virus (VACV) induces a vigorous virus-specific CD8+ T cell response that plays an important role in control of poxvirus infection. To identify immunodominant poxvirus proteins and to facilitate future testing of smallpox vaccines in non-human primates, we used an algorithm for the prediction of VACV peptides able to bind to the common macaque MHC class I molecule Mamu-A*01. We synthesized 294 peptides derived from 97 VACV ORFs; 100 of these peptides did not contain the canonical proline at position three of the Mamu-A*01 binding motif. Cellular immune responses in PBMC from two vaccinia-vaccinated Mamu-A*01+ macaques were assessed by IFNgamma ELISPOT assays. Vaccinated macaques recognized 17 peptides from 16 different ORFs with 6 peptides recognized by both macaques. Comparison with other orthopoxvirus sequences revealed that 12 of these epitopes are strictly conserved between VACV, variola, and monkeypoxvirus. ELISPOT responses were also observed to eight epitopes that did not contain the canonical P3 proline. These results suggest that the virus-specific CD8+ T cell response is broadly directed against multiple VACV proteins and that a subset of these T cell epitopes is highly conserved among orthopoxviruses.

    Title Detection of 16 Gamma-ray Pulsars Through Blind Frequency Searches Using the Fermi Lat.
    Date August 2009
    Journal Science (new York, N.y.)
    Excerpt

    Pulsars are rapidly rotating, highly magnetized neutron stars emitting radiation across the electromagnetic spectrum. Although there are more than 1800 known radio pulsars, until recently only seven were observed to pulse in gamma rays, and these were all discovered at other wavelengths. The Fermi Large Area Telescope (LAT) makes it possible to pinpoint neutron stars through their gamma-ray pulsations. We report the detection of 16 gamma-ray pulsars in blind frequency searches using the LAT. Most of these pulsars are coincident with previously unidentified gamma-ray sources, and many are associated with supernova remnants. Direct detection of gamma-ray pulsars enables studies of emission mechanisms, population statistics, and the energetics of pulsar wind nebulae and supernova remnants.

    Title A Population of Gamma-ray Millisecond Pulsars Seen with the Fermi Large Area Telescope.
    Date August 2009
    Journal Science (new York, N.y.)
    Excerpt

    Pulsars are born with subsecond spin periods and slow by electromagnetic braking for several tens of millions of years, when detectable radiation ceases. A second life can occur for neutron stars in binary systems. They can acquire mass and angular momentum from their companions, to be spun up to millisecond periods and begin radiating again. We searched Fermi Large Area Telescope data for pulsations from all known millisecond pulsars (MSPs) outside of globular clusters, using rotation parameters from radio telescopes. Strong gamma-ray pulsations were detected for eight MSPs. The gamma-ray pulse profiles and spectral properties resemble those of young gamma-ray pulsars. The basic emission mechanism seems to be the same for MSPs and young pulsars, with the emission originating in regions far from the neutron star surface.

    Title Detection of High-energy Gamma-ray Emission from the Globular Cluster 47 Tucanae with Fermi.
    Date August 2009
    Journal Science (new York, N.y.)
    Excerpt

    We report the detection of gamma-ray emissions above 200 megaelectron volts at a significance level of 17sigma from the globular cluster 47 Tucanae, using data obtained with the Large Area Telescope onboard the Fermi Gamma-ray Space Telescope. Globular clusters are expected to emit gamma rays because of the large populations of millisecond pulsars that they contain. The spectral shape of 47 Tucanae is consistent with gamma-ray emission from a population of millisecond pulsars. The observed gamma-ray luminosity implies an upper limit of 60 millisecond pulsars present in 47 Tucanae.

    Title Identification and Functional Characterization of Protein Kinase A-catalyzed Phosphorylation of Potassium Channel Kv1.2 at Serine 449.
    Date August 2009
    Journal The Journal of Biological Chemistry
    Excerpt

    Vascular smooth muscle Kv1 delayed rectifier K+ channels (KDR) containing Kv1.2 control membrane potential and thereby regulate contractility. Vasodilatory agonists acting via protein kinase A (PKA) enhance vascule smooth muscle Kv1 activity, but the molecular basis of this regulation is uncertain. We characterized the role of a C-terminal phosphorylation site, Ser-449, in Kv1.2 expressed in HEK 293 cells by biochemical and electrophysiological methods. We found that 1) in vitro phosphorylation of Kv1.2 occurred exclusively at serine residues, 2) one major phosphopeptide that co-migrated with 449pSASTISK was generated by proteolysis of in vitro phosphorylated Kv1.2, 3) the peptide 445KKSRSASTISK exhibited stoichiometric phosphorylation by PKA in vitro, 4) matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy (MS) and MS/MS confirmed in vitro Ser-449 phosphorylation by PKA, 5) in situ phosphorylation at Ser-449 was detected in HEK 293 cells by MALDI-TOF MS followed by MS/MS. MIDAS (multiple reaction monitoring-initiated detection and sequencing) analysis revealed additional phosphorylated residues, Ser-440 and Ser-441, 6) in vitro 32P incorporation was significantly reduced in Kv1.2-S449A, Kv1.2-S449D, and Kv1.2-S440A/S441A/S449A mutant channels, but Kv1.2-S440A/S441A was identical to wild-type Kv1.2 (Kv1.2-WT), and 7) bath applied 8-Br-cAMP or dialysis with PKA catalytic subunit (cPKA) increased Kv1.2-WT but not Kv1.2-S449A current amplitude. cPKA increased Kv1.2-WT current in inside-out patches. Rp-CPT-cAMPS reduced Kv1.2-WT current, blocked the increase due to 8-Br-cAMP, but had no effect on Kv1.2-S449A. cPKA increased current due to double mutant Kv1.2-S440A/S441A but had no effect on Kv1.2-S449D or Kv1.2-S440A/S441A/S449A. We conclude that Ser-449 in Kv1.2 is a site of PKA phosphorylation and a potential molecular mechanism for Kv1-containing KDR channel modulation by agonists via PKA activation.

    Title Microwave Flash Pyrolysis.
    Date August 2009
    Journal The Journal of Organic Chemistry
    Excerpt

    In a microwave reactor, graphite heats rapidly to high surface temperatures; applications of graphite thermal "sensitization" have been described previously. We report here that microwave thermal sensitization with graphite, carbon nanotubes, or silicon carbide can be used to carry out reactions more typically accomplished by flash vacuum pyrolysis (FVP) and which usually require temperatures much higher than the nominal limit of a microwave reactor. The graphite-sensitized microwave reaction of azulene in the solid phase at temperatures of 100 to 300 degrees C affords rapid rearrangement to naphthalene, a reaction typically observed by FVP at 700-900 degrees C. Multiwall carbon nanotubes give similar results when used as a thermal sensitizer. Other graphite-sensitized reactions that we have observed include the following: conversion of 2-ethynylbiphenyl to phenanthrene, fragmentation of phthalic anhydride to benzyne, cleavage of iodobenzene to phenyl radical, aryl-aryl bond cleavage, and a variety of cycloaromatizations. An advantage is seen for less volatile substrates. Rearrangement of azulene and generation of benzyne from phthalic anhydride have also been observed on powdered silicon carbide. Because of the high temperature, rapid heating, and frequent ejection of material from the irradiation zone, we refer to this general method as microwave flash pyrolysis (MFP).

    Title Ca2+ Sensitization Via Phosphorylation of Myosin Phosphatase Targeting Subunit at Threonine-855 by Rho Kinase Contributes to the Arterial Myogenic Response.
    Date August 2009
    Journal The Journal of Physiology
    Excerpt

    Ca(2+) sensitization has been postulated to contribute to the myogenic contraction of resistance arteries evoked by elevation of transmural pressure. However, the biochemical evidence of pressure-induced increases in phosphorylated myosin light chain phosphatase (MLCP) targeting subunit 1 (MYPT1) and/or 17 kDa protein kinase C (PKC)-potentiated protein phosphatase 1 inhibitor protein (CPI-17) required to sustain this view is not currently available. Here, we determined whether Ca(2+) sensitization pathways involving Rho kinase (ROK)- and PKC-dependent phosphorylation of MYPT1 and CPI-17, respectively, contribute to the myogenic response of rat middle cerebral arteries. ROK inhibitors (Y27632, 0.03-10 micromol l(-1); H1152, 0.001-0.3 micromol l(-1)) and PKC inhibitors (GF109203X, 3 micromol l(-1); Gö6976; 10 micromol l(-1)) suppressed myogenic vasoconstriction between 40 and 120 mmHg. An improved, highly sensitive 3-step Western blot method was developed for detection and quantification of MYPT1 and CPI-17 phosphorylation. Increasing pressure from 10 to 60 or 100 mmHg significantly increased phosphorylation of MYPT1 at threonine-855 (T855) and myosin light chain (LC(20)). Phosphorylation of MYPT1 at threonine-697 (T697) and CPI-17 were not affected by pressure. Pressure-evoked elevations in MYPT1-T855 and LC(20) phosphorylation were reduced by H1152, but MYPT1-T697 phosphorylation was unaffected. Inhibition of PKC with GF109203X did not affect MYPT1 or LC(20) phosphorylation at 100 mmHg. Our findings provide the first direct, biochemical evidence that a Ca(2+) sensitization pathway involving ROK-dependent phosphorylation of MYPT1 at T855 (but not T697) and subsequent augmentation of LC(20) phosphorylation contributes to myogenic control of arterial diameter in the cerebral vasculature. In contrast, suppression of the myogenic response by PKC inhibitors cannot be attributed to block of Ca(2+) sensitization mediated by CPI-17 or MYPT1 phosphorylation.

    Title Measurement of the Cosmic Ray E+ +e- Spectrum from 20 Gev to 1 Tev with the Fermi Large Area Telescope.
    Date July 2009
    Journal Physical Review Letters
    Excerpt

    Designed as a high-sensitivity gamma-ray observatory, the Fermi Large Area Telescope is also an electron detector with a large acceptance exceeding 2 m;{2} sr at 300 GeV. Building on the gamma-ray analysis, we have developed an efficient electron detection strategy which provides sufficient background rejection for measurement of the steeply falling electron spectrum up to 1 TeV. Our high precision data show that the electron spectrum falls with energy as E-3.0 and does not exhibit prominent spectral features. Interpretations in terms of a conventional diffusive model as well as a potential local extra component are briefly discussed.

    Title Evaluation of Bacteriophages for Prevention and Treatment of Diarrhea Due to Experimental Enterotoxigenic Escherichia Coli O149 Infection of Pigs.
    Date July 2009
    Journal Veterinary Microbiology
    Excerpt

    The objective of this study was to determine the efficacy of selected phages individually and in combination in prevention and treatment of diarrhea due to experimental O149:H10:F4 enterotoxigenic Escherichia coli (ETEC) in weaned pigs. For prophylaxis, the phages were administered orally shortly after challenge, and for therapeutic use, were given 24h after challenge, following the onset of diarrhea. The parameters used to assess outcomes were weight change, duration of diarrhea, severity of diarrhea, composite diarrhea score, and extent of shedding of the challenge ETEC over 6 days. Six phages that were tested individually in a prophylactic mode were effective as determined by a significant change in each of the parameters, although the phages were not present at titres greater than 10(3)PFU/g of feces. A modified protocol involving pre-treatment of the pigs with florfenicol and oral administration of sodium bicarbonate prior to the ETEC challenge and phage administration resulted in high levels of phages in the feces. Using this protocol, a combination of three phages that was tested in the prophylactic mode significantly reduced the severity of diarrhea and the composite diarrhea score. A mixture of two phages given therapeutically significantly improved each of the outcome parameters, without perturbation of the total fecal E. coli flora. Enumeration of phages in feces after treatment indicated that the phages were replicating to high titres in the intestinal tract of ETEC infected pigs within 1-2 days before declining progressively. These findings indicate that the selected phages were effective in moderating the course of experimental O149:H10:F4 ETEC diarrhea in weaned pigs when given prophylactically or therapeutically.

    Title The Genome and Proteome of a Virulent Escherichia Coli O157:h7 Bacteriophage Closely Resembling Salmonella Phage Felix O1.
    Date June 2009
    Journal Virology Journal
    Excerpt

    Based upon whole genome and proteome analysis, Escherichia coli O157:H7-specific bacteriophage (phage) wV8 belongs to the new myoviral genus, "the Felix O1-like viruses" along with Salmonella phage Felix O1 and Erwinia amylovora phage phiEa21-4. The genome characteristics of phage wV8 (size 88.49 kb, mol%G+C 38.9, 138 ORFs, 23 tRNAs) are very similar to those of phage Felix O1 (86.16 kb, 39.0 mol%G+C, 131 ORFs and 22 tRNAs) and, indeed most of the proteins have their closest homologs within Felix O1. Approximately one-half of the Escherichia coli O157:H7 mutants resistant to phage wV8 still serotype as O157:H7 indicating that this phage may recognize, like coliphage T4, two different surface receptors: lipopolysaccharide and, perhaps, an outer membrane protein.

    Title Oral and Rectal Administration of Bacteriophages for Control of Escherichia Coli O157:h7 in Feedlot Cattle.
    Date April 2009
    Journal Journal of Food Protection
    Excerpt

    This study compared oral and rectal administration of O157-specific bacteriophages for mitigating the fecal shedding of Escherichia coli O157 by experimentally inoculated steers. Fecal shedding of nalidixic acid-resistant (Nal(R)) E. coli O157:H7 was monitored over 83 days after oral (ORL; 3.3 x 10(11) PFU), rectal (REC; 1.5 x 10(11) PFU), both oral and rectal (O+R; 4.8 x 10(11) PFU), or no (CON; control) treatment with a four-strain O157-specific bacteriophage cocktail in multiple doses. Bacteriophages were enumerated by plaque assay, and NalR E. coli O157:H7 by direct plating on sorbitol MacConkey agar supplemented with cefixime, potassium tellurite, and nalidixic acid. Orally treated steers produced the fewest Nal(R) E. coli O157:H7 culture-positive samples (P < 0.06) compared with REC and O+R steers, but this number was only nominally lower (P = 0.26) than that for the CON steers. The overall mean shedding level (log CFU per gram of feces) was higher for REC steers (P < 0.10) than for steers of the other treatment groups. Despite the shedding of higher mean bacteriophage levels (log PFU per gram of feces) by ORL and O+R than by CON and REC steers, there was no difference (P > 0.05) in the number of E. coli O157-positive samples among treatments. Bacteriophage was isolated from CON steers, indicating that these steers acquired the bacteriophage from the environment and shed the phage at a level similar to that of REC steers (P = 0.39). Continuous bacteriophage therapy may be an efficacious method for mitigating shedding of E. coli O157:H7 in cattle, providing that the host bacterium does not develop resistance. This therapy may be especially advantageous if nontreated cattle can acquire this biocontrol agent from the feedlot environment.

    Title Fermi Observations of High-energy Gamma-ray Emission from Grb 080916c.
    Date April 2009
    Journal Science (new York, N.y.)
    Excerpt

    Gamma-ray bursts (GRBs) are highly energetic explosions signaling the death of massive stars in distant galaxies. The Gamma-ray Burst Monitor and Large Area Telescope onboard the Fermi Observatory together record GRBs over a broad energy range spanning about 7 decades of gammaray energy. In September 2008, Fermi observed the exceptionally luminous GRB 080916C, with the largest apparent energy release yet measured. The high-energy gamma rays are observed to start later and persist longer than the lower energy photons. A simple spectral form fits the entire GRB spectrum, providing strong constraints on emission models. The known distance of the burst enables placing lower limits on the bulk Lorentz factor of the outflow and on the quantum gravity mass.

    Title Shock Formation and the Ideal Shape of Ramp Compression Waves.
    Date April 2009
    Journal Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics
    Excerpt

    We derive expressions for shock formation based on the local curvature of the flow characteristics during dynamic compression. Given a specific ramp adiabat, calculated for instance from the equation of state for a substance, the ideal nonlinear shape for an applied ramp loading history can be determined. We discuss the region affected by lateral release, which can be presented in compact form for the ideal loading history. Example calculations are given for representative metals and plastic ablators. Continuum dynamics (hydrocode) simulations were in good agreement with the algebraic forms. Example applications are presented for several classes of laser-loading experiment, identifying conditions where shocks are desired but not formed, and where long-duration ramps are desired.

    Title Bacteriophages for Prophylaxis and Therapy in Cattle, Poultry and Pigs.
    Date March 2009
    Journal Animal Health Research Reviews / Conference of Research Workers in Animal Diseases
    Excerpt

    The successful use of virulent (lytic) bacteriophages (phages) in preventing and treating neonatal enterotoxigenic Escherichia coli infections in calves, lambs and pigs has prompted investigation of other applications of phage therapy in food animals. While results have been very variable, some indicate that phage therapy is potentially useful in virulent Salmonella and E. coli infections in chickens, calves and pigs, and in control of the food-borne pathogens Salmonella and Campylobacter jejuni in chickens and E. coli O157:H7 in cattle. However, more rigorous and comprehensive research is required to determine the true potential of phage therapy. Particular challenges include the selection and characterization of phages, practical modes of administration, and development of formulations that maintain the viability of phages for administration. Also, meaningful evaluation of phage therapy will require animal studies that closely represent the intended use, and will include thorough investigation of the emergence and characteristics of phage resistant bacteria. As well, effective use will require understanding the ecology and dynamics of the endemic and therapeutic phages and their interactions with target bacteria in the farm environment. In the event that the potential of phage therapy is realized, adoption will depend on its efficacy and complementarity relative to other interventions. Another potential challenge will be regulatory approval.

    Title Enumeration of Bacteriophages by Double Agar Overlay Plaque Assay.
    Date March 2009
    Journal Methods in Molecular Biology (clifton, N.j.)
    Excerpt

    The determination of the concentration of infectious phage particles is fundamental to many protocols in phage biology, genetics, and molecular biology. In this chapter the classical overlay protocol is described.

    Title Enumeration of Bacteriophages by the Direct Plating Plaque Assay.
    Date March 2009
    Journal Methods in Molecular Biology (clifton, N.j.)
    Excerpt

    A method is described for determination of the concentration of infectious phage particles by the direct plating plaque assay, which is simpler and faster than the double agar overlay plaque procedure outlined in the previous chapter.

    Title Enumeration of Bacteriophages Using the Small Drop Plaque Assay System.
    Date March 2009
    Journal Methods in Molecular Biology (clifton, N.j.)
    Excerpt

    The determination of the concentration of infectious phage particles is fundamental to many protocols in phage biology, genetics, and molecular biology. Described here is a drop plaque assay, which, being simpler, faster and more efficient than either the classical overlay or direct plating methods, enhances efficiency in processing large numbers of samples.

    Title Preparation and Characterization of Anti-phage Serum.
    Date March 2009
    Journal Methods in Molecular Biology (clifton, N.j.)
    Excerpt

    This chapter describes a method for the generation of polyclonal antibodies against bacteriophages and how these may be assayed immunochemically and biologically.

    Title Generalized Transduction by Lytic Bacteriophages.
    Date March 2009
    Journal Methods in Molecular Biology (clifton, N.j.)
    Excerpt

    As interest in lytic phages as antimicrobial therapies or as treatments to reduce environmental contamination with pathogenic bacteria has increased, so has the need to determine if the use of lytic phages may lead to dissemination of virulence factors through generalized transduction, as occurs with temperate phages. Here we describe simple methods we have developed to determine if a lytic phage, rV5, can mediate generalized transduction in Escherichia coli O157:H7. These sensitive methods can be easily adapted to study generalized transduction between virulent and avirulent strains of bacteria.

    Title Determination of Bacteriophage Genome Size by Pulsed-field Gel Electrophoresis.
    Date March 2009
    Journal Methods in Molecular Biology (clifton, N.j.)
    Excerpt

    Standard agarose gel electrophoresis is extensively used to resolve DNA fragments from 0.2 to 40-50 kb. Larger fragments of genomic DNA or whole viral genomes can only effectively be resolved by pulsed-field gel electrophoresis (PFGE), which extends the range of molecular separation from 200 bp to 12 Mb.

    Title Prevalence and Impact of Bacteriophages on the Presence of Escherichia Coli O157:h7 in Feedlot Cattle and Their Environment.
    Date March 2009
    Journal Applied and Environmental Microbiology
    Excerpt

    The relationship between endemic bacteriophages infecting E. coli O157:H7 (referred to as "phage") and levels of shedding of E. coli O157:H7 by cattle was investigated in two commercial feedlots in southern Alberta, Canada. Between May and November 2007, 10 pens of cattle were monitored by collection of pooled fecal pats, water with sediment from troughs, manure slurry from the pen floor, and rectal fecal samples from individual animals (20 per pen) at two separate times. Bacteriophages infecting E. coli O157:H7 were detected more frequently (P<0.001) after 18 to 20 h enrichment than by initial screening and were recovered in 239 of 855 samples (26.5% of 411 pooled fecal pats, 23.8% of 320 fecal grab samples, 21.8% of 87 water trough samples, and 94.6% of 37 pen floor slurry samples). Overall, prevalence of phage was highest (P<0.001) in slurry. Recovery of phage from pooled fecal pats was highest (P<0.05) in May. Overall recovery did not differ (P>0.10) between fecal grab samples and pooled fecal pats. A higher prevalence of phage in fecal pats or water trough samples was associated (P<0.01) with reduced prevalence of E. coli O157:H7 in rectal fecal samples. There was a weak but significant negative correlation between isolation of phage and E. coli O157:H7 in fecal grab samples (r= -0.11; P<0.05). These data demonstrate that the prevalence of phage fluctuates in a manner similar to that described for E. coli O157:H7. Phage were more prevalent in manure slurry than other environmental sources. The likelihood of fecal shedding of E. coli O157:H7 was reduced if cattle in the pen harbored phage.

    Title Thermal Rearrangements of 2-ethynylbiphenyl: a Dft Study of Competing Reaction Mechanisms.
    Date February 2009
    Journal The Journal of Organic Chemistry
    Excerpt

    Mechanistic pathways for high-temperature rearrangements of 2-ethynylbiphenyl have been investigated by calculations at the B3LYP/6-31G(d) level of theory, with free energy estimates at 625 degrees C. Two different routes for high temperature thermal rearrangement can lead to phenanthrene, which was the major product observed by Brown and co-workers (J. Chem. Soc. Chem. Commun. 1974, 123). 1,2-Hydrogen shift (Hopf type B mechanism) affords a vinylidene which proceeds to the major product by sequential electrocyclic closure and a 1,2-shift, rather than the expected aryl C-H insertion. Alternatively, insertion of the vinylidene into a ring double bond would lead directly to the observed minor product, benzazulene. Along a competitive pathway, electrocyclic closure to an isophenanthrene is predicted to be nearly isoenergetic. This intermediate should have a planar allene structure, with substantial diradical character. Sequential hydrogen shifts lead to phenanthrene but with higher cumulative barriers than for the vinylidene route. Calculation of 625 degrees C free energies shows that the carbene mechanism is of lower energy, primarily because of the lower entropic cost. Predictions are made for the unusually facile hydrogen atom dissociation from isoaromatics at high temperature, a consequence of aryl radical formation. Isophenanthrene, isobenzene (1,2,4-cyclohexatriene) and several isonaphthalenes are also predicted to have unusually low C-H bond dissociation energies. Potential significance as a source of aryl radicals in high temperature and combustion chemistry is discussed.

    Title Scaling Laws for Energetic Ions from the Commissioning of the New Los Alamos National Laboratory 200 Tw Trident Laser.
    Date February 2009
    Journal The Review of Scientific Instruments
    Excerpt

    The recent Los Alamos National Laboratory Trident laser enhanced from 30 to 200 TW in power allows more than 100 J to be delivered on target in 500 fs with a spot size smaller than 12 microm at full width at half maximum. 15 microm flat-foil targets have been observed to produce proton beams in excess of 50 MeV at an intensity of only approximately 4x10(19) W/cm(2) with efficiencies approaching 5%. The Trident laser beam characteristics are presented along with the data compared to published scaling laws for proton acceleration.

    Title Trident High-energy-density Facility Experimental Capabilities and Diagnostics.
    Date February 2009
    Journal The Review of Scientific Instruments
    Excerpt

    The newly upgraded TRIDENT high-energy-density (HED) facility provides high-energy short-pulse laser-matter interactions with powers in excess of 200 TW and energies greater than 120 J. In addition, TRIDENT retains two long-pulse (nanoseconds to microseconds) beams that are available for simultaneous use in either the same experiment or a separate one. The facility's flexibility is enhanced by the presence of two separate target chambers with a third undergoing commissioning. This capability allows the experimental configuration to be optimized by choosing the chamber with the most advantageous geometry and features. The TRIDENT facility also provides a wide range of standard instruments including optical, x-ray, and particle diagnostics. In addition, one chamber has a 10 in. manipulator allowing OMEGA and National Ignition Facility (NIF) diagnostics to be prototyped and calibrated.

    Title A Novel Backscatter Focus Diagnostic for the Trident 200 Tw Laser.
    Date February 2009
    Journal The Review of Scientific Instruments
    Excerpt

    Here we present the first direct focal spot images and analysis of an ultrahigh intensity short-pulse laser focus (>5x10(19) W/cm(2)) on target. Such a focal spot characterization is typically done previous to the shot with a low-power alignment beam using equivalent plane imaging techniques. The resulting intensity of the shot is then inferred from these results. We report on the development of a backscatter focus diagnostic, which enables imaging of the on-target full-power focal spot.

    Title Using a Short-pulse Diffraction-limited Laser Beam to Probe Filamentation of a Random Phase Plate Smoothed Beam.
    Date February 2009
    Journal The Review of Scientific Instruments
    Excerpt

    A short pulse (few picoseconds) laser probe provides high temporal resolution measurements to elucidate details of fast dynamic phenomena not observable with typical longer laser pulse probes and gated diagnostics. Such a short pulse laser probe (SPLP) has been used to measure filamentation of a random phase plate (RPP) smoothed laser beam in a gas-jet plasma. The plasma index of refraction due to driven density and temperature fluctuations by the RPP beam perturbs the phase front of a SPLP propagating at a 90 degree angle with respect to the RPP interaction beam. The density and temperature fluctuations are quasistatic on the time scale of the SPLP (approximately 2 ps). The transmitted near-field intensity distribution from the SPLP provides a measure of the phase front perturbation. At low plasma densities, the transmitted intensity pattern is asymmetric with striations across the entire probe beam in the direction of the RPP smoothed beam. As the plasma density increases, the striations break up into smaller sizes along the direction of the RPP beam propagation. The breakup of the intensity pattern is consistent with self-focusing of the RPP smoothed interaction beam. Simulations of the experiment using the wave propagation code, PF3D, are in qualitative agreement demonstrating that the asymmetric striations can be attributed to the RPP driven density fluctuations. Quantification of the beam breakup measured by the transmitted SPLP could lead to a new method for measuring self-focusing of lasers in underdense plasmas.

    Title Production of Verotoxin and Distribution of O Islands 122 and 43/48 Among Verotoxin-producing Escherichia Coli O103:h2 Isolates from Cattle and Humans.
    Date January 2009
    Journal Applied and Environmental Microbiology
    Excerpt

    This study investigated variations in the occurrence of markers of O islands 122 and 43/48 and in verotoxin 1 production in 91 verotoxin-producing Escherichia coli (VTEC) O103:H2 strains of bovine and human origins. None of the genes that were investigated appear to be virulence indicators for human O103:H2 VTEC.

    Title Phenotypic and Genotypic Characterization of Verotoxin-producing Escherichia Coli O103:h2 Isolates from Cattle and Humans.
    Date December 2008
    Journal Journal of Clinical Microbiology
    Excerpt

    Characterization of important non-O157 verotoxin-producing Escherichia coli (VTEC) has lagged considerably behind that of O157:H7 strains. This study characterized 91 VTEC O103:H2 strains from bovine and human sources and of North American and European origins by virulence or putative virulence genes, pulsed-field gel electrophoresis (PFGE) patterns, plasmid profiles, antimicrobial resistance, and colicin production. All strains were positive for vt1 and eae-epsilon; 97% were positive for ehxA; and all were negative for hlyA. Two strains carried vt2. There were 66 PFGE patterns grouped in six clusters, and there were 25 different plasmid profiles. Plasmid-encoded katP and etp genes were significantly more frequent in European than in North American human strains. The distribution of selected phenotypes was as follows: enterohemorrhagic E. coli (EHEC) hemolysin, 95%; colicin production, 38%; antimicrobial resistance, 58%. All the strains were negative for the alpha-hemolytic phenotype. In conclusion, the VTEC O103:H2 strains were diverse, as shown by PFGE, plasmid profiles, virulence markers, and antimicrobial resistance patterns, and all strains showed an EHEC hemolytic phenotype instead of the alpha-hemolytic phenotype that has been shown previously.

    Title The Fermi Gamma-ray Space Telescope Discovers the Pulsar in the Young Galactic Supernova Remnant Cta 1.
    Date December 2008
    Journal Science (new York, N.y.)
    Excerpt

    Energetic young pulsars and expanding blast waves [supernova remnants (SNRs)] are the most visible remains after massive stars, ending their lives, explode in core-collapse supernovae. The Fermi Gamma-Ray Space Telescope has unveiled a radio quiet pulsar located near the center of the compact synchrotron nebula inside the supernova remnant CTA 1. The pulsar, discovered through its gamma-ray pulsations, has a period of 316.86 milliseconds and a period derivative of 3.614 x 10(-13) seconds per second. Its characteristic age of 10(4) years is comparable to that estimated for the SNR. We speculate that most unidentified Galactic gamma-ray sources associated with star-forming regions and SNRs are such young pulsars.

    Title Prevalence and Characterization of Verotoxin-producing Escherichia Coli (vtec) in Cattle from an Ontario Abattoir.
    Date October 2008
    Journal Canadian Journal of Veterinary Research = Revue Canadienne De Recherche Vétérinaire
    Excerpt

    This study determined the prevalence of verotoxin (VT)-producing Escherichia coli (VTEC) in Ontario beef cattle at slaughter and characterized the isolates by serotype, virulence factors, virulence markers, and antimicrobial resistance. Cultures of rectal feces from 500 animals were screened for VT by an enzyme-linked immunosorbent assay (ELISA) and by polymerase chain reaction (PCR) for genes vt1, vt2, and eae. The VT-ELISA-positive samples were tested by a VT-immunoblot to isolate VTEC colonies. The prevalence rates of VTEC by VT-ELISA and PCR were 10.2% [95% confidence interval (CI), 7.8% to 13.2%] and 6.2% (95% CI, 4.4% to 8.7%), respectively. Colonies of VTEC were isolated from 27 (53%) of the 51 VT-ELISA-positive samples and belonged to 24 serotypes, which did not include O157:H7. Twelve of the serotypes have been implicated in disease in humans. Virulence profiling of the isolates by PCR revealed that 2 (8%) were eae-positive, 5 (21%) had vt1 only, and 19 (79%) had vt2, of which 3 had vt2 only, 7 had vt1 + vt2, 4 had vt2 + vt2c, 2 had vt2 + vt2c + vt2d, 2 had vt1 + vt2 + vt2c, and 1 had vt1 + vt2 + vt2c + vt2d. The distribution of selected plasmid-encoded putative virulence genes was as follows: ehxA, 63%; espP, 46%; saa, 67%; and subA, 54%. Nine of the 24 isolates were resistant to 1 or more antimicrobials. Major conclusions are that the VTEC prevalence of 10.2% was among the lower rates reported for beef cattle, a high proportion of the isolates had vt2 genes, the subA gene was reported for the 1st time in Canadian VTEC, and the absence of O157 VTEC likely reflects the use of a technique that detected all VTEC.

    Title What is Your Diagnosis? Cutaneous Mastocytosis (urticaria Pigmentosa).
    Date September 2008
    Journal Cutis; Cutaneous Medicine for the Practitioner
    Title Efficient Entry Inhibition of Human and Nonhuman Primate Immunodeficiency Virus by Cell Surface-expressed Gp41-derived Peptides.
    Date September 2008
    Journal Gene Therapy
    Excerpt

    Membrane-anchored C-peptides (for example, maC46) derived from human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp41 effectively inhibit HIV-1 entry in cell lines and primary human CD4+ cells in vitro. Here we evaluated this gene therapy approach in animal models of AIDS. We adapted the HIV gp41-derived maC46 vector construct for use in rhesus monkeys. Simian immunodeficiency virus (SIV and SHIV) sequence-adapted maC46 peptides, and the original HIV-1-derived maC46 expressed on the surface of established cell lines blocked entry of HIV-1, SIVmac251 and SHIV89.6P. Furthermore, primary rhesus monkey CD4+ T cells expressing HIV sequence-based maC46 peptides were also protected from SIV entry. Depletion of CD8+ T cells from PBMCs enhanced the yield of maC46-transduced CD4+ T cells. Supplementation with interleukin-2 (IL-2) increased transduction efficiency, whereas IL-7 and/or IL-15 provided no additional benefit. Phenotypic analysis showed that maC46-transduced and expanded cells were predominantly central memory CD4+ T cells that expressed low levels of CCR5 and slightly elevated levels of CD62L, beta7-integrin and CXCR4. These findings show that maC46-based cell surface-expressed peptides can efficiently inhibit primate immunodeficiency virus infection, and therefore serve as the basis for evaluation of this gene therapy approach in an animal model for AIDS.

    Title Instability of Retroviral Vectors with Hiv-1-specific Rt Aptamers Due to Cryptic Splice Sites in the U6 Promoter.
    Date August 2008
    Journal Aids Research and Therapy
    Excerpt

    BACKGROUND: Internal polymerase III promoters in retroviral vectors have been used extensively to express short RNA sequences, such as ribozymes, RNA aptamers or short interfering RNA inhibitors, in various positions and orientations. However, the stability of these promoters in the reverse orientation has not been rigorously evaluated. RESULTS: A series of retroviral vectors was generated carrying the U6+1 promoter with 3 different HIV-1 RT-specific RNA aptamers and one control aptamer, all in the reverse orientation. After shuttle packaging, the CD4+ cell line CEMx174 was transduced with each vector, selected for expression of GFP, and challenged with HIV-1. We did not observe inhibition of HIV-1 replication in these transduced populations. PCR amplification of the U6+1 promoter-RNA aptamer inhibitor cassette from transduced CEMx174 cells and RT-PCR amplification from transfected Phoenix (amphotropic) packaging cells showed two distinct products: a full-length product of the expected size as well as a truncated product. The sequence of the full-length PCR product was identical to the predicted amplicon sequence. However, sequencing of the truncated product revealed a 139 bp deletion in the U6 promoter. This deletion decreased transcriptional activity of the U6 promoter. Analysis of the deleted sequences from the U6 promoter in the antisense direction indicated consensus splice donor, splice acceptor and branch point sequences. CONCLUSION: The existence of a cryptic splice site in the U6 promoter when expressed in a retroviral vector in the reverse orientation generates deletions during packaging and may limit the utility of this promoter for expression of small RNA inhibitors.

    Title Comparison of Fecal Versus Rectoanal Mucosal Swab Sampling for Detecting Escherichia Coli O157:h7 in Experimentally Inoculated Cattle Used in Assessing Bacteriophage As a Mitigation Strategy.
    Date June 2008
    Journal Journal of Food Protection
    Excerpt

    This study was conducted to compare fecal grab (FEC) and rectoanal mucosal swab (RAMS) techniques as sampling methods for surveillance of Escherichia coli O157:H7 in conjunction with administration of a mitigation therapy. The study was nested within a larger experiment that investigated bacteriophage as a preharvest strategy for controlling E. coli O157:H7 in feedlot steers. Samples (FEC and RAMS) were collected from 16 of the 32 feedlot steers (control and oral bacteriophage treatment; n = 8) involved in the mitigation study. All steers had been inoculated on day 0 with 10(10) CFU of nalidixic acid-resistant E. coli O157:H7, and samples were collected on 16 occasions over the next 83 days. FEC samples were assessed by direct plating of serial dilutions in PBS, plus a 6-h enrichment and immunomagnetic separation when E. coli O157:H7 concentrations were below limits detectable by direct plating (i.e., <1 log CFU/g). All RAMS samples were assessed by enrichment and immunomagnetic separation. E. coli O157:H7 was detected more frequently (P < 0.01) by FEC than by RAMS. Overall, 213 of 256 samples were positive either by FEC or RAMS. Discrepancies between sampling techniques were observed in 63 of the 213 positive samples; FEC missed 11 samples that were positive by RAMS, and RAMS missed 52 of those positive by FEC (miss rates of 5.16 and 24.41%, respectively). Kappa values (0.36 to 0.45) indicated only fair to moderate agreement between FEC and RAMS results, but this agreement was higher at lower levels of E. coli O157:H7 shedding (later in the experimental period). Selection of sampling procedure could significantly influence the assessed merit during testing of potential strategies for controlling E. coli O157:H7 on the farm.

    Title How Hiv Guts the Immune System.
    Date June 2008
    Journal The New England Journal of Medicine
    Title Vaccine Protection by Live, Attenuated Simian Immunodeficiency Virus in the Absence of High-titer Antibody Responses and High-frequency Cellular Immune Responses Measurable in the Periphery.
    Date April 2008
    Journal Journal of Virology
    Excerpt

    An attenuated derivative of simian immunodeficiency virus strain 239 deleted of V1-V2 sequences in the envelope gene (SIV239DeltaV1-V2) was used for vaccine/challenge experiments in rhesus monkeys. Peak levels of viral RNA in plasma of 10(4) to 10(6.5) copies/ml in the weeks immediately following inoculation of SIV239DeltaV1-V2 were 10- to 1,000-fold lower than those observed with parental SIV239 ( approximately 10(7.3) copies/ml). Viral loads consistently remained below 200 copies/ml after 8 weeks of infection by the attenuated SIV239DeltaV1-V2 strain. Viral localization experiments revealed large numbers of infected cells within organized lymphoid nodules of the colonic gut-associated lymphoid tissue at 14 days; double-labeling experiments indicated that 93.5% of the virally infected cells at this site were positive for the macrophage marker CD68. Cellular and humoral immune responses measured principally by gamma interferon enzyme-linked immunospot and neutralization assays were variable in the five vaccinated monkeys. One monkey had responses in these assays comparable to or only slightly less than those observed in monkeys infected with parental, wild-type SIV239. Four of the vaccinated monkeys, however, had low, marginal, or undetectable responses in these same assays. These five vaccinated monkeys and three naïve control monkeys were subsequently challenged intravenously with wild-type SIV239. Three of the five vaccinated monkeys, including the one with strong anti-SIV immune responses, were strongly protected against the challenge on the basis of viral load measurements. Surprisingly, two of the vaccinated monkeys were strongly protected against SIV239 challenge despite the presence of cellular anti-SIV responses of low-frequency and low-titer anti-SIV antibody responses. These results indicate that high-titer anti-SIV antibody responses and high-frequency anti-SIV cellular immune responses measurable by standard assays from the peripheral blood are not needed to achieve strong vaccine protection, even against a difficult, neutralization-resistant strain such as SIV239.

    Title Escherichia Coli O157:h7 and Other Shiga Toxin-producing E. Coli in White Veal Calves.
    Date February 2008
    Journal Veterinary Microbiology
    Excerpt

    The aims of the study were to determine the prevalence of enterohemorrhagic Escherichia coli O157:H7 (EHEC O157) and other Shiga toxin-producing E. coli (STEC) in feces of white veal calves in an operation in Ontario, to evaluate exposure of the calves to EHEC O157, and to investigate the milk replacer diet and antimicrobial resistance as factors that might influence the prevalence of EHEC O157. Feces from three cohorts of 20-21 calves were collected weekly for 20 weeks and processed for isolation of EHEC O157:H7 and detection of STEC by an ELISA. Exposure to EHEC O157 was also investigated by measuring IgG and IgM antibodies to the O157 lipopolysaccharide (O157 Ab) in sera by ELISA. The prevalences of EHEC O157 were 0.17% of 1151 fecal samples and 3.2% of 62 calves, and for STEC were 68% of 1005 fecal samples and 100% of 62 calves. Seroconversion to active IgG and IgM O157 Ab responses in some calves was not associated with isolation of EHEC O157. The milk replacer contained low levels of antibodies to EHEC antigens and without antimicrobial drugs, it did not inhibit the growth of EHEC O157 in vitro. Two E. coli O157:H7 that were isolated were totally drug sensitive whereas 60 commensal E. coli isolates that were examined were highly resistant. Antibodies in milk replacer that might be protective in vivo, and susceptibility to antimicrobial agents in the milk replacer may contribute to the low prevalence of EHEC O157 in white veal calves.

    Title Dynamics of T- and B-lymphocyte Turnover in a Natural Host of Simian Immunodeficiency Virus.
    Date February 2008
    Journal Journal of Virology
    Excerpt

    Increased lymphocyte turnover is a hallmark of pathogenic lentiviral infection. To investigate perturbations in lymphocyte dynamics in natural hosts with nonpathogenic simian immunodeficiency virus (SIV) infection, the nucleoside analog bromodeoxyuridine (BrdU) was administered to six naturally SIV-infected and five SIV-negative sooty mangabeys. As a measure of lymphocyte turnover, we estimated the mean death rate by fitting a mathematical model to the fraction of BrdU-labeled cells during a 2-week labeling and a median 10-week delabeling period. Despite significantly lower total T- and B-lymphocyte counts in SIV-infected sooty mangabeys than in SIV-negative mangabeys, the turnover rate of B lymphocytes and CD4(+) and CD8(+) T lymphocytes was not increased in the SIV-infected animals. A small, rapidly proliferating CD45RA(+) memory subset and a large, slower-proliferating CD45RA(-) central memory subset of CD4(+) T lymphocytes identified in the peripheral blood of sooty mangabeys also did not show evidence of increased turnover in the context of SIV infection. Independently of SIV infection, the turnover of CD4(+) T lymphocytes in sooty mangabeys was significantly higher (P < 0.01) than that of CD8(+) T lymphocytes, a finding hitherto not reported in rhesus macaques or humans. The absence of aberrant T-lymphocyte turnover along with an inherently high rate of CD4(+) T-lymphocyte turnover may help to preserve the pool of central memory CD4(+) T lymphocytes in viremic SIV-infected sooty mangabeys and protect against progression to AIDS.

    Title Complete Genomic Sequence of Bacteriophage Phiecom-gj1, a Novel Phage That Has Myovirus Morphology and a Podovirus-like Rna Polymerase.
    Date January 2008
    Journal Applied and Environmental Microbiology
    Excerpt

    The complete genome of phiEcoM-GJ1, a lytic phage that attacks porcine enterotoxigenic Escherichia coli of serotype O149:H10:F4, was sequenced and analyzed. The morphology of the phage and the identity of the structural proteins were also determined. The genome consisted of 52,975 bp with a G+C content of 44% and was terminally redundant and circularly permuted. Seventy-five potential open reading frames (ORFs) were identified and annotated, but only 29 possessed homologs. The proteins of five ORFs showed homology with proteins of phages of the family Myoviridae, nine with proteins of phages of the family Podoviridae, and six with proteins of phages of the family Siphoviridae. ORF 1 encoded a T7-like single-subunit RNA polymerase and was preceded by a putative E. coli sigma(70)-like promoter. Nine putative phage promoters were detected throughout the genome. The genome included a tRNA gene of 95 bp that had a putative 18-bp intron. The phage morphology was typical of phages of the family Myoviridae, with an icosahedral head, a neck, and a long contractile tail with tail fibers. The analysis shows that phiEcoM-GJ1 is unique, having the morphology of the Myoviridae, a gene for RNA polymerase, which is characteristic of phages of the T7 group of the Podoviridae, and several genes that encode proteins with homology to proteins of phages of the family Siphoviridae.

    Title Erythematous Annular Lesions.
    Date December 2007
    Journal American Family Physician
    Title Isolation and Characterization of Nine Bacteriophages That Lyse O149 Enterotoxigenic Escherichia Coli.
    Date December 2007
    Journal Veterinary Microbiology
    Excerpt

    The goal of this study was to isolate and characterize phages that might be used in prevention and treatment of porcine post-weaning diarrhea due to O149 enterotoxigenic E. coli (ETEC). Serotype O149:H10:F4 was especially targeted because this is the dominant ETEC serotype. Mixtures of 10 strains of O149:H10:F4 ETEC and of 10 O149:H43:F4 ETEC were used as hosts for isolation of phages in sewage from 38 Ontario pig farms. Six phages (GJ1-GJ6) that lysed O149:H10:F4 ETEC and three (GJ7-GJ9) that lysed O149:H43:F4 ETEC were isolated. All phages produced large, clear plaques. All nine phages had necks and contractile tails and therefore belonged to the Myoviridae. Their estimated genome sizes were 48.3-50.7kb and their restriction enzyme fragments suggested that they were closely related. Phages GJ1-GJ6 lysed 99-100% of 85 O149:H10:F4 ETEC, 0-12% of 42 O149:H43:F4 ETEC, 3-35% of 37 non-O149 porcine ETEC, and 6-68% of the 72 strains of the ECOR collection. Phages GJ7-GJ9 lysed 86-98% of the O149:H43:F4 ETEC, 2-53% of the O149:H10:F4 ETEC, and 24-41% of the non-O149 porcine ETEC. Titres of the nine phages were unaffected by exposure for 16h to pH 5-9. Among phages GJ1-GJ6, resistance of O149:H10:F4 ETEC to one phage was generally not accompanied by resistance to other phages. It is concluded that the nine phages are suitable candidates for prophylaxis and therapy of porcine post-weaning diarrhea due to O149 ETEC.

    Title Potent Inhibition of Simian Immunodeficiency Virus (siv) Replication by an Siv-based Lentiviral Vector Expressing Antisense Env.
    Date December 2007
    Journal Human Gene Therapy
    Excerpt

    In light of findings demonstrating that the macaque TRIM5alpha protein inhibits infection of cells by human immunodeficiency virus (HIV)-1, simian immunodeficiency virus (SIV)-based lentiviral vectors may have distinct advantages over HIV-1 vectors for the transduction of macaque hematopoietic stem cells. We evaluated the ability of an SIV vector (VRX859) encoding an antisense SIV envelope sequence and enhanced green fluorescent protein (GFP) to inhibit viral replication and to transduce rhesus CD34(+) lymphoid progenitor cells. After infection with homologous SIV strains, CD4(+) cell lines transduced with VRX859 exhibited more than 600-fold inhibition of viral replication compared with control cells. Less inhibition was observed with the divergent SIV strain SIVsmE660. Partial inhibition of a chimeric simian-human immunodeficiency virus, which contains an HIV-1 envelope in an SIV backbone, was observed, suggesting that the SIV vector also contributes to viral inhibition independent of the antisense envelope inhibitor. Transduction of rhesus CD34(+) cells with VRX859 at various multiplicities of infection resulted in transduction efficiencies comparable to those obtained with the HIV vector VRX494. However, when we evaluated transduction of rhesus T lymphocyte progenitors by examining GFP expression in CD4(+) T cells derived from transduced CD34(+) cells, we observed more efficient transduction with the SIV-based vector. GFP(+)CD4(+) T cells derived from VRX859-transduced CD34(+) cells strongly inhibited SIVmac239 replication as compared with control CD4(+) T cells. The ability of this SIV-based vector to mediate potent inhibition of SIV replication, coupled with its efficient transduction of rhesus hematopoietic progenitor cells, make it an important candidate for proof-of-principle experiments of stem cell gene therapy in the SIV-macaque model.

    Title Short Pulse Laser Train for Laser Plasma Interaction Experiments.
    Date November 2007
    Journal The Review of Scientific Instruments
    Excerpt

    A multiframe, high-time resolution pump-probe diagnostic consisting of a consecutive train of ultrashort laser pulses (approximately ps) has been developed for use with a chirped pulse amplification (CPA) system. A system of high quality windows is used to create a series of 1054 nm picosecond-laser pulses which are injected into the CPA system before the pulse stretcher and amplifiers. By adding or removing windows in the pulse train forming optics, the number of pulses can be varied. By varying the distance and thickness of the respective optical elements, the time in between the pulses, i.e., the time in between frames, can be set. In our example application, the CPA pulse train is converted to 527 nm using a KDP crystal and focused into a preformed plasma and the reflected laser light due to stimulated Raman scattering is measured. Each pulse samples different plasma conditions as the plasma evolves in time, producing more data on each laser shot than with a single short pulse probe. This novel technique could potentially be implemented to obtain multiple high-time resolution measurements of the dynamics of physical processes over hundreds of picoseconds or even nanoseconds with picosecond resolution on a single shot.

    Title Strain Estimates for Small-ring Cyclic Allenes and Butatrienes.
    Date June 2007
    Journal The Journal of Organic Chemistry
    Excerpt

    Isodesmic and homodesmic equations at the B3LYP/6-311+G(d,p)+ZPVE level of theory have been used to estimate strain for the homologous series of cyclic allenes and cyclic butatrienes. A simple fragment deformation approach also has been applied and appears to work better for the larger rings. For the cyclic allene series, estimates for allene functional group strain (kcal/mol) include: 1,2-cyclobutadiene, 65; 1,2-cyclopentadiene, 51; 1,2-cyclohexadiene, 32; 1,2-cycloheptadiene, 14; 1,2-cyclooctadiene, 5; 1,2-cyclononadiene, 2; 1,2,4-cyclohexatriene, 34; and bicyclo[3.2.1]octa-2,3-diene, 39. For cyclic butatrienes, functional group strain estimates include: 1,2,3-cyclobutatriene, >100; 1,2,3-cyclopentatriene, 80; 1,2,3-cyclohexatriene, 50; 1,2,3-cycloheptatriene, 26; 1,2,3-cyclooctatriene, 17; and 1,2,3-cyclononatriene, 4. Barriers to interconversion of enantiomers in cyclic allenes are reduced with increasing strain. Newly predicted values include: 1,2-cyclopentadiene <1 kcal/mol and bicyclo[3.2.1]octa-2,3-diene, 7.4 kcal/mol. Estimated levels of strain parallel the known reactivity of these substances.

    Title Mycobacterium Marinum Infection: a Case Report and Review of the Literature.
    Date April 2007
    Journal Cutis; Cutaneous Medicine for the Practitioner
    Excerpt

    Mycobacterium marinum is a nontuberculous mycobacteria that is often acquired via contact with contaminated salt or fresh water. We present a case of a 67-year-old man who developed several solitary nontender nodules on his hands and forearm after working on the underside of his boat. In addition, we provide a review of the literature and discuss how this infection is acquired, the underlying pathogenesis, the cutaneous and histologic findings, the differential diagnosis, the diagnostic methods, and the various treatment options.

    Title Induction of a Virus-specific Effector-memory Cd4+ T Cell Response by Attenuated Siv Infection.
    Date February 2007
    Journal The Journal of Experimental Medicine
    Excerpt

    We investigated simian immunodeficiency virus (SIV)-specific CD4+ T cell responses in rhesus macaques chronically infected with attenuated or pathogenic SIV strains. Analysis of SIVDeltanef-infected animals revealed a relatively high frequency of SIV-specific CD4+ T cells representing 4-10% of all CD4+ T lymphocytes directed against multiple SIV proteins. Gag-specific CD4+ T cells in wild-type SIV-infected animals were 5-10-fold lower in frequency and inversely correlated with the level of plasma viremia. SIV-specific CD4+ cells from SIVDeltanef animals were predominantly CD27-CD28-CD45RAlowCCR7-CCR5-, consistent with an effector-memory subset, and included a fully differentiated CD45RA+CCR7- subpopulation. In contrast, SIV-specific CD4+ T cells from SIV-infected animals were mostly CD27+CD28+CD45RA-CCR7+CCR5+, consistent with an early central memory phenotype. The CD45RA+CCR7-CD4+ subset from SIVDeltanef animals was highly enriched for effector CD4+ T cells, as indicated by the perforin expression and up-regulation of the lysosomal membrane protein CD107a after SIV Gag stimulation. SIV-specific CD4+ T cells in attenuated SIV-infected animals were increased in frequency in bronchioalveolar lavage and decreased in lymph nodes, consistent with an effector-memory T cell population. The ability of SIVDeltanef to induce a high frequency virus-specific CD4+ T cell response with direct effector function may play a key role in protective immunity produced by vaccination with attenuated SIV strains.

    Title Expression of Cd8alpha Identifies a Distinct Subset of Effector Memory Cd4+ T Lymphocytes.
    Date November 2006
    Journal Immunology
    Excerpt

    Circulating CD4+ CD8+ T lymphocytes have been described in the peripheral blood of humans and several animal species. However, the origin and functional properties of these cells remain poorly understood. In the present study, we evaluated the frequency, phenotype and function of peripheral CD4+ CD8+ T cells in rhesus macaques. Two distinct populations of CD4+ CD8+ T cells were identified: the dominant one was CD4hi CD8lo and expressed the CD8alphaalpha homodimer, while the minor population was CD4lo CD8hi and expressed the CD8alphabeta heterodimer. The majority of CD4hi CD8alphalo T cells exhibited an activated effector/memory phenotype (CCR5lo CD7- CD28- HLA-DR+) and expressed relatively high levels of granzyme B. Intracellular cytokine staining assays demonstrated that the frequency of cytomegalovirus-specific T cells was enriched five-fold in CD4hi CD8alphalo T cells compared to single-positive CD4+ T cells, whereas no consistent enrichment was observed for simian immunodeficiency virus (SIV)-specific T cells. Cross-sectional studies of SIV-infected animals demonstrated that the frequency of CD4hi CD8alphalo T cells was lower in wild-type SIV-infected animals compared to uninfected controls, although prospective studies of SIV-infected animals demonstrated depletion of CD4hi CD8alphalo lymphocytes only in a subset of animals. Taken together, these data suggest that CD4+ T cells expressing CD8alpha represent an effector/memory subset of CD4+ T cells and that this cell population can be depleted during the course of SIV infection.

    Title C. Elegans Dystroglycan Dgn-1 Functions in Epithelia and Neurons, but Not Muscle, and Independently of Dystrophin.
    Date July 2006
    Journal Development (cambridge, England)
    Excerpt

    The C. elegans dystroglycan (DG) homolog DGN-1 is expressed in epithelia and neurons, and localizes to basement membrane (BM) surfaces. Unlike vertebrate DG, DGN-1 is not expressed in muscle or required for muscle function. dgn-1 null mutants are viable but sterile owing to severe disorganization of the somatic gonad epithelium, and show defects in vulval and excretory cell epithelia and in motoneuron axon guidance. The defects resemble those of epi-1 laminin alphaB mutants, suggesting that DGN-1 serves as a receptor for laminin. dgn-1(0)/+ animals are fertile but show gonad migration defects in addition to the defects seen in homozygotes, indicating that DGN-1 function is dosage sensitive. Phenotypic analyses show that DGN-1 and dystrophin-associated protein complex (DAPC) components have distinct and independent functions, in contrast to the situation in vertebrate muscle. The DAPC-independent functions of DGN-1 in epithelia and neurons suggest that vertebrate DG may also act independently of dystrophin/utrophin in non-muscle tissues.

    Title Hiv Pathogenesis and Vaccine Development.
    Date July 2006
    Journal Topics in Hiv Medicine : a Publication of the International Aids Society, Usa
    Excerpt

    Cautious optimism was a recurring theme of many of the AIDS vaccine-related presentations at the 13th annual Conference on Retroviruses and Opportunistic Infections. Several investigators suggested that the ability of HIV to escape cytotoxic T-lymphocyte responses may be more limited than previously thought, and encouraging results were presented regarding the ability of consensus ancestral sequences or polyvalent vaccines to increase the breadth of induced immune responses. A number of studies highlighted the potential efficacy of neutralizing antibodies: data from 2 groups suggested that neutralizing antibodies may play a role in preventing superinfection and previously unrecognized neutralizing epitopes were identified in the membrane proximal external region of envelope. Two studies documented that immunization with polyvalent simian immunodeficiency virus vaccines can induce sustained control of viremia following repeated low-dose mucosal challenge with pathogenic SIVmac strains and provided hope for the potential of T-cell-based vaccines to slow disease progression.

    Title Setting the Stage for Bench-to-bedside Movement of Anti-hiv Rna Inhibitors-gene Therapy for Aids in Macaques.
    Date June 2006
    Journal Frontiers in Bioscience : a Journal and Virtual Library
    Excerpt

    Despite significant progress over the last two decades, treatment of HIV infection remains a tremendous challenge. Although antiretroviral therapy has proved quite effective in most HIV-infected patients, increasing recognition of toxicity and the emergence of multidrug resistant HIV strains has fueled the development of alternative therapeutic approaches. Introduction of genes to inhibit HIV replication into CD4+ T lymphocytes or hematopoietic stem cells represents a potentially attractive but still unproven strategy. Despite the availability of a diverse range of molecular strategies that are able to provide potent inhibition of HIV replication in the laboratory, translation of these in vitro successes to in vivo therapies has been difficult. Fundamental challenges facing AIDS gene therapy at the present time includes the need to increase the efficiency of gene transfer in vivo, to confer upon genetically-modified T cells the ability to have a selective growth advantage in vivo, and the development of additional techniques to decrease the probability of emergence of resistant viruses. As one of the leading animal models for AIDS and for hematopoietic stem cell gene therapy, nonhuman primates are ideally suited to help address many of these basic questions. This review will provide a general overview of RNA-based genetic strategies for inhibition of HIV and SIV replication, criteria to be considered in the selection of promising inhibitory strategies for in vivo use, and key questions that can be addressed in the macaque model.

    Title Genomic Sequence of Rhesus Cytomegalovirus 180.92: Insights into the Coding Potential of Rhesus Cytomegalovirus.
    Date April 2006
    Journal Journal of Virology
    Excerpt

    A pathogenic isolate of rhesus cytomegalovirus (rhCMV 180.92) was cloned, sequenced, and annotated. Comparisons with the published rhCMV 68.1 genome revealed 8 open reading frames (ORFs) in isolate 180.92 that are absent in 68.1, 10 ORFs in 68.1 that are absent in 180.92, and 34 additional ORFs that were not previously annotated. Most of the differences appear to be due to genetic rearrangements in both isolates from a region that is frequently altered in human CMV (hCMV) during in vitro passage. These results indicate that the rhCMV ORF repertoire is larger than previously recognized. Like hCMV, understanding of the complete coding capacity of rhCMV is complicated by genomic instability and may require comparisons with additional isolates in vitro and in vivo.

    Title Inhibition of Simian/human Immunodeficiency Virus Replication in Cd4+ T Cells Derived from Lentiviral-transduced Cd34+ Hematopoietic Cells.
    Date April 2006
    Journal Molecular Therapy : the Journal of the American Society of Gene Therapy
    Excerpt

    We examined the ability of a HIV-1-based vector (VRX494) encoding a 937-bp antisense HIV-1 envelope sequence to inhibit the replication of chimeric SIV/HIV-1 viruses encoding the HIV-1 envelope. Challenge of VRX494-transduced CEMx174 cells resulted in potent inhibition of HIV-1 and several SHIV strains. To evaluate the potential efficacy of the VRX494 vector for stem cell gene therapy, rhesus CD34(+) bone marrow cells were transduced with VRX494 and then cultured on thymus stroma to induce T cell differentiation. Transduction conditions for CD34(+) cells were optimized to yield high transduction efficiency with minimal effective multiplicity of infection. Purified CD4(+) GFP(+) T cells derived from VRX494-transduced CD34(+) cells strongly inhibited SHIV HXBC2P 3.2 and SHIV 89.6P replication compared to controls. Southern blot analysis of VRX494-transduced T cell clones revealed a subset of cells with multiple proviral copies per cell. Expression of GFP and the antisense inhibitor in VRX494-transduced cells was upregulated by Tat. Analysis of HIV-1 envelope sequences in VRX494-transduced cells revealed modifications consistent with those mediated by double-stranded RNA-dependent adenosine deaminase. These results indicate that the macaque/SHIV model should serve as a useful preclinical model to evaluate this lentiviral vector expressing an HIV-1 antisense inhibitor for stem cell gene therapy for AIDS.

    Title Quasi-isentropic Compression by Ablative Laser Loading: Response of Materials to Dynamic Loading on Nanosecond Time Scales.
    Date October 2005
    Journal Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics
    Excerpt

    The TRIDENT laser was used to induce quasi-isentropic compression waves to approximately 15 GPa in samples of Si, by ablative loading using a laser pulse whose intensity increased smoothly over 2.5 ns. The intensity history of the pulse and the velocity history at the opposite surface of the sample were recorded. Experiments were performed using samples of two different thicknesses simultaneously, in which the evolution of the compression wave was clearly visible. Isentropic stress states deduced were consistent with the previously investigated response of Si to uniaxial loading. The ablative loading was simulated using radiation hydrodynamics, with different equations of state in the plasma and condensed regions and including elasticity in the solid Si. These calculations reproduced the measured velocity histories quite well, demonstrating that quasi-isentropic compression was induced with no preheat from the laser drive. Normal continuum behavior was demonstrated to hold below nanosecond time scales for isentropic compression waves, with no evidence for nonequilibrium effects in the crystal lattice. Details of the velocity history over about 10 GPa were reproduced less well, suggesting a deficiency in the model used for compressed Si, which may be consistent with recent theoretical predictions of uniaxial compression at high strain rates.

    Title Observation of a Transition from Fluid to Kinetic Nonlinearities for Langmuir Waves Driven by Stimulated Raman Backscatter.
    Date October 2005
    Journal Physical Review Letters
    Excerpt

    Thomson scattering is used to measure Langmuir waves (LW) driven by stimulated Raman scattering (SRS) in a diffraction limited laser focal spot. For SRS at wave numbers klambda(D) less similar 0.29, where k is the LW number and lambda(D) is the Debye length, multiple waves are detected and are attributed to the Langmuir decay instability (LDI) driven by the primary LW. At klambda(D) greater similar 0.29, a single wave, frequency-broadened spectrum is observed. The transition from the fluid to the kinetic regime is qualitatively consistent with particle-in-cell simulations and crossing of the LDI amplitude threshold above that for LW self-focusing.

    Title Isolation of Viable Antigen-specific Cd4 T Cells by Cd40l Surface Trapping.
    Date October 2005
    Journal Journal of Immunological Methods
    Excerpt

    A number of techniques have recently been developed for the identification of antigen-specific cells, yet the ability of these techniques to identify all subclasses of memory T cells has often been overlooked. Here we describe a novel approach for the isolation of live antigen-specific CD4 T cells using CD40L and CD69 surface staining and demonstrate its utility for isolating antigen-specific rhesus macaque CD4 T cells. Critical to the success of the technique was staining for CD40L concurrent with antigen stimulation. Isolation of CD4 T cells based on CD40L/CD69 surface marker upregulation identified both effector and central memory CD4 T cells. In contrast, the majority of central memory CD4 T cells did not secrete TNFalpha or IFNgamma and thus would not be identified by techniques based on their secretion. The methodology described here therefore complements existing approaches for isolating viable antigen-specific CD4 T cells, opens new avenues for investigating human diseases in nonhuman primate animal models and may prove beneficial in instances where the induced response is largely T cell central memory restricted.

    Title Effect of Cd8+ Lymphocyte Depletion on Virus Containment After Simian Immunodeficiency Virus Sivmac251 Challenge of Live Attenuated Sivmac239delta3-vaccinated Rhesus Macaques.
    Date August 2005
    Journal Journal of Virology
    Excerpt

    Although live attenuated vaccines can provide potent protection against simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus challenges, the specific immune responses that confer this protection have not been determined. To test whether cellular immune responses mediated by CD8+ lymphocytes contribute to this vaccine-induced protection, we depleted rhesus macaques vaccinated with the live attenuated virus SIVmac239Delta3 of CD8+ lymphocytes and then challenged them with SIVmac251 by the intravenous route. While vaccination did not prevent infection with the pathogenic challenge virus, the postchallenge levels of virus in the plasmas of vaccinated control animals were significantly lower than those for unvaccinated animals. The depletion of CD8+ lymphocytes at the time of challenge resulted in virus levels in the plasma that were intermediate between those of the vaccinated and unvaccinated controls, suggesting that CD8+ cell-mediated immune responses contributed to protection. Interestingly, at the time of challenge, animals expressing the Mamu-A*01 major histocompatibility complex class I allele showed significantly higher frequencies of SIV-specific CD8+ T-cell responses and lower neutralizing antibody titers than those in Mamu-A*01- animals. Consistent with these findings, the depletion of CD8+ lymphocytes abrogated vaccine-induced protection, as judged by the peak postchallenge viremia, to a greater extent in Mamu-A*01+ than in Mamu-A*01- animals. The partial control of postchallenge viremia after CD8+ lymphocyte depletion suggests that both humoral and cellular immune responses induced by live attenuated SIV vaccines can contribute to protection against a pathogenic challenge and that the relative contribution of each of these responses to protection may be genetically determined.

    Title Limited Sequence Evolution Within Persistently Targeted Cd8 Epitopes in Chronic Human Immunodeficiency Virus Type 1 Infection.
    Date August 2005
    Journal Journal of Virology
    Excerpt

    Studies in acute human immunodeficiency virus type 1 (HIV-1) infection indicate viral evolution under CD8 T-cell immune selection pressure, but the effects of ongoing immune pressure on epitope evolution during chronic infection are not well described. In this study, we performed a detailed longitudinal analysis of viral sequence variation within persistently targeted cytotoxic T-lymphocyte (CTL) epitopes in two HIV-1-infected persons during 6 years of persistent viremia. Responses were quantitated using freshly isolated peripheral blood lymphocytes in direct lytic assays as well as by gamma interferon (IFN-gamma) Elispot assays on cryopreserved cells. Seven targeted epitopes were identified in each person. In the majority of cases, the dominant epitope sequence did not change over time, even in the presence of responses of sufficient magnitude that they were detectable using fresh peripheral blood mononuclear cells in direct lytic assays. Only 4 of the 14 autologous epitopes tested represented potential CTL escape variants; however, in most cases strong responses to these epitopes persisted for the 6 years of study. Although persistent IFN-gamma responses were detected to all epitopes, direct lytic assays demonstrated declining responses to some epitopes despite the persistence of the targeted sequence in vivo. These data indicate limited viral evolution within persistently targeted CD8 T-cell epitopes during the chronic phase of infection and suggest that these regions of the virus are either refractory to sequence change or that persistently activated CD8 T-cell responses in chronic infection exert little functional selection pressure.

    Title Delineation of Multiple Subpopulations of Natural Killer Cells in Rhesus Macaques.
    Date July 2005
    Journal Immunology
    Excerpt

    Natural killer (NK) cells in rhesus macaques have been variably defined as CD3- CD16+ or CD3- CD8+, although only limited efforts have been made to validate these definitions rigorously. To better understand the role of NK cells in macaque disease models, we undertook a multiparameter analysis of macaque NK cells employing four-colour flow cytometry and a panel of lineage-specific and non-lineage-specific lymphocyte markers. Using this approach, we identified two distinct populations of candidate NK cells: a major CD8bright CD16+ population and a minor CD8bright CD16- population. Further analysis of the major and minor NK cell populations revealed the expression of multiple markers characteristic of NK cells, including CD2, CD7, CD16, CD161, NKG2A and granzyme B. In addition, a CD56+ subset of cells within the minor rhesus NK population was identified which expressed chemokine and lymph node homing receptors similar to those expressed by the CD56bright NK cell population identified in humans. Cytolytic assays confirmed that the phenotypically defined rhesus NK cells lysed NK-susceptible target cells. Our observations support the existence of several distinct subpopulations of rhesus macaque NK cells, which have significant phenotypic and functional similarities to their human counterparts. These improved immunophenotypic definitions of macaque NK cells should facilitate future analysis of innate immune responses in rhesus macaques and the role of NK cells in AIDS pathogenesis in Simian immunodeficiency virus (SIV)-infected macaques.

    Title Hiv Pathogenesis and Vaccine Development.
    Date June 2005
    Journal Topics in Hiv Medicine : a Publication of the International Aids Society, Usa
    Excerpt

    New information on the crystal structures of the HIV and the simian immunodeficiency virus (SIV) envelopes represented one of the scientific highlights of the 12th Annual Conference on Retroviruses and Opportunistic Infections. Numerous presentations also underscored the increasing recognition of the central role of gut-associated lymphoid tissue in AIDS pathogenesis and helped reveal a better understanding of the multiple mechanisms underlying CD4+ T lymphocyte depletion in AIDS. Progress on vaccine development was largely incremental but was strongly influenced by the impact of an expanding array of flow cytometric assays that have revealed significant functional and phenotypic differences in virus-specific CD8+ cells. The interplay between host cellular and humoral immune responses and virus evolution was another prominent theme, and it underscored the challenge facing host immune responses and vaccine developers in attempting to thwart an ever-mutating virus.

    Title Two Distinct Head-tail Interfaces Cooperate to Suppress Activation of Vinculin by Talin.
    Date June 2005
    Journal The Journal of Biological Chemistry
    Excerpt

    Vinculin is autoinhibited by an intramolecular interaction that masks binding sites for talin and F-actin. Although a recent structural model explains autoinhibition solely in terms of the interaction between vinculin tail (Vt) and residues 1-258 (D1), we find an absolute requirement for an interface involving the D4 domain of head (Vh residues 710-836) and Vt. Charge-to-alanine mutations in Vt revealed a class of mutants, T12 and T19, distal to the V-(1-258) binding site, which showed increases in their Kd values for head binding of 100- and 42-fold, respectively. Reciprocal mutation of residues in the D4 domain that contact Vt yielded a head-tail interaction mutant of comparable magnitude to T19. These findings account for the approximately 120-fold difference in Kd values between Vt binding to V-(1-258), as opposed to full-length Vh-(1-851). The significance of a bipartite autoinhibitory site is evidenced by its effects on talin binding to Vh. Whereas Vt fails to compete with the talin rod domain for binding to V-(1-258), competition occurs readily with full-length Vh, and this requires the D4 interface. Moreover in intact vinculin, mutations in the D4-Vt interface stabilize association of vinculin and talin rod. In cells, these head-tail interaction mutants induce hypertrophy and elongation of focal adhesions. Definition of a second autoinhibitory site, the D4-Vt interface, supports the competing model of vinculin activation that invokes cooperative action of ligands at two sites. Together the D1-Vt and D4-Vt interfaces provide the high affinity (approximately 10(-9)) autoinhibition observed in full-length vinculin.

    Title Hiv: Viral Blitzkrieg.
    Date May 2005
    Journal Nature
    Title Kinetics of Expansion of Siv Gag-specific Cd8+ T Lymphocytes Following Challenge of Vaccinated Macaques.
    Date April 2005
    Journal Virology
    Excerpt

    The ability of memory T cells to mount a recall response plays a key role in the ability of vaccinated animals to contain viral challenge. In this study, we intensively monitored the expansion of SIV Gag-specific CD8+ T cells in peripheral blood and tissues of rhesus macaques vaccinated with the attenuated strain SIVmac239Delta3 and challenged with the pathogenic viruses SIVmac239 or SIVsmE660. Although all vaccinated animals were infected with challenge virus, peak levels of plasma viremia in vaccinees were decreased by 1.5 to 2 logs as compared with naive controls. Decreased levels of plasma viremia in vaccinated animals were evident as early as 7 days post-challenge, well before the expansion of SIV-specific CD8+ T cells. Expansion of SIV-specific CD8+ T cells was not observed in peripheral blood or tissues until at least 14 days after infection and did not occur in most animals until after the initial peak of viral replication. The observation that expansion of SIV-specific CD8+ T cells is delayed until 7 days or more after initial detection of viremia highlights fundamental limitations in the ability of lentivirus-specific CD8+ T cells to mediate protection against challenge.

    Title Amyloid Beta Peptides Mediate Physiological Remodelling of the Acute O2 Sensitivity of Adrenomedullary Chromaffin Cells Following Chronic Hypoxia.
    Date February 2005
    Journal Cardiovascular Research
    Excerpt

    The non-neurogenic response of the neonatal adrenal medulla is vital in cardiovascular and respiratory development and to the survival of newborns exposed to hypoxic stress. Here, we examined the acute hypoxic response of immortalised rat adrenomedullary chromaffin cells following exposure to chronic hypoxia (CH; 6% O(2) for 24 h).

    Title Impact of Nef-mediated Downregulation of Major Histocompatibility Complex Class I on Immune Response to Simian Immunodeficiency Virus.
    Date December 2004
    Journal Journal of Virology
    Excerpt

    Functional activities that have been ascribed to the nef gene product of simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) include CD4 downregulation, major histocompatibility complex (MHC) class I downregulation, downregulation of other plasma membrane proteins, and lymphocyte activation. Monkeys were infected experimentally with SIV containing difficult-to-revert mutations in nef that selectively eliminated MHC downregulation but not these other activities. Monkeys infected with these mutant forms of SIV exhibited higher levels of CD8(+) T-cell responses 4 to 16 weeks postinfection than seen in monkeys infected with the parental wild-type virus. Furthermore, unusual compensatory mutations appeared by 16 to 32 weeks postinfection which restored some or all of the MHC-downregulating activity. These results indicate that nef does serve to limit the virus-specific CD8 cellular response of the host and that the ability to downregulate MHC class I contributes importantly to the totality of nef function.

    Title Influence of Animal Origin and Lineage on Survival of Escherichia Coli O157:h7 Strains in Strong and Weak Acid Challenges.
    Date November 2004
    Journal Journal of Food Protection
    Excerpt

    Twenty-five strains of Escherichia coli O157:H7 isolated from humans, cattle, and pigs were maintained in HCl (pH 2.5) and in a volatile fatty acid (VFA) mixture (pH 4.0) for up to 6 h at 37 degrees C to assess their ability to survive in acidic conditions that simulate those of the stomach and ileum, respectively. In HCl, the average group survival of bovine strains was significantly higher than that of porcine and human strains, whereas in VFAs, porcine strains were significantly more resistant than bovine and human strains. Bovine strains exhibited significantly higher average survival in HCl than in VFAs. The average survival of strains classified as octamer-based genome scanning (OBGS) lineage II was significantly superior to that of strains classified as OBGS lineage I in HCl. The group of lineage I strains was more resistant in VFAs compared with lineage II, but only after 6 h of challenge. The possible involvement of urease in acid resistance of E. coli O157:H7 was also examined. Although the strains possessed the ureC gene, as shown by PCR, this gene did not appear to contribute to acid resistance under the conditions tested. The data indicate that there is a relationship between acid resistance and source or lineage of O157:H7 strains.

    Title An Shiv Dna/mva Rectal Vaccination in Macaques Provides Systemic and Mucosal Virus-specific Responses and Protection Against Aids.
    Date October 2004
    Journal Aids Research and Human Retroviruses
    Excerpt

    We explored the use of a simian-human immunodeficiency virus (SHIV) DNA vaccine as an effective mucosal priming agent to stimulate a protective immune response for AIDS prevention. Rhesus macaques were vaccinated rectally with a DNA construct producing replication-defective SHIV particles, and boosted with either the same DNA construct or recombinant modified vaccinia virus Ankara (MVA) expressing SIV Gag, SIV Pol, and HIV Env (MVA-SHIV). Virus-specific mucosal and systemic humoral and cell-mediated immune responses could be stimulated by this approach but were present inconsistently among the vaccinated animals. Rectal vaccination with either SHIV DNA alone or SHIV DNA followed by MVA-SHIV induced SIV Gag/Pol- or HIV gp120-specific IgA in rectal secretions of four of seven animals. However, the gp120-specific rectal IgA antibody responses were not durable and had become undetectable in all but one animal shortly before rectal challenge with pathogenic SHIV 89.6P. Only the macaques primed with SHIV DNA and boosted with MVA-SHIV demonstrated SHIV-specific IgG in plasma. In addition, these animals developed more consistent antiviral cell-mediated responses and had better preservation of CD4 T cells following challenge with SHIV 89.6P. Our study demonstrates the utility of a rectal DNA/MVA vaccination protocol for the induction of diverse responses in different immunological compartments. In addition, the immunity achieved with this mucosal vaccination regimen is sufficient to delay progression to AIDS.

    Title Observation of the Parametric Two-ion Decay Instability with Thomson Scattering.
    Date October 2004
    Journal Physical Review Letters
    Excerpt

    We present the first direct experimental observation of the parametric two-ion decay instability of ion-acoustic waves driven by a high intensity (5 x 10(15) W cm(-2)) laser beam in a laser produced high-Z plasma. Using two separate Thomson scattering diagnostics simultaneously, we directly measure the scattering from thermal ion-acoustic fluctuations, the primary ion waves that are driven to large amplitudes by the high intensity beam, and the two-ion decay products. The decay products are shown to be present only where the interaction takes place and their k spectrum is broad.

    Title Optimization of Intracellular Cytokine Staining for the Quantitation of Antigen-specific Cd4+ T Cell Responses in Rhesus Macaques.
    Date August 2004
    Journal Journal of Immunological Methods
    Excerpt

    Standard proliferation assays used for analysis of CD4+ T cell function have significant shortcomings, including limited sensitivity, lack of truly quantitative readouts and significant variability. We have optimized an intracellular cytokine staining (ICS) assay in rhesus macaques which allows us to identify virus-specific CD4+ T cells at the single-cell level with high sensitivity while reducing background staining to a minimum. A variety of parameters were tested to determine the optimal experimental conditions necessary for the detection of antigen-specific CD4+ T cells in macaques. Central to our optimized protocol was the addition of cross-linked costimulatory anti-CD28 and anti-CD49d Mabs, a modification which resulted in up to threefold enhancement of the frequency of TNF-alpha-secreting CD4+ T cells following superantigen- or antigen-specific stimulation. The ICS protocol was also optimized with respect to antigen concentration and duration of antigenic stimulation. These modifications resulted in a convenient and highly reproducible assay with intra- and inter-assay variability of less than 10%. Although cryopreservation of PBMC generally led to a 40% to 80% decrease in the frequency of antigen-specific CD4+ T cells detected by ICS using stimulation with viral proteins, the use of overlapping peptide pools minimized the effects of cryopreservation on ICS responses. The use of more sensitive techniques such as ICS permits delineation of antigen-specific cells at the single cell level and should provide new insights into pathogen-specific immune responses in the rhesus macaque model.

    Title Total Synthesis of the Ubiquitin-activating Enzyme Inhibitor (+)-panepophenanthrin.
    Date March 2004
    Journal Angewandte Chemie (international Ed. in English)
    Title Elevated Interleukin-7 Levels Not Sufficient to Maintain T-cell Homeostasis During Simian Immunodeficiency Virus-induced Disease Progression.
    Date March 2004
    Journal Blood
    Excerpt

    Elevated levels of interleukin 7 (IL-7) have been correlated with various T-cell depletion conditions, including HIV infection, and suggested as an indicator of HIV disease progression (AIDS and death). Here, the assessment of pathogenic simian immunodeficiency virus (SIVmac239) infection in rhesus macaques demonstrated a clear association between a significant elevation in IL-7 levels and disease progression. In 5 macaques that progressed to simian AIDS and death, elevated IL-7 levels were unable to restore T-cell homeostasis. In contrast, increased IL-7 levels were followed by relatively high and stable T-cell numbers in the SIV-infected macaques with a slow-progressing phenotype. Further, studies in sooty mangabeys that do not progress to simian AIDS and that maintain stable T-cell numbers despite high levels of viral replication support the importance of IL-7 and T-cell homeostasis in disease progression. These data suggest that during pathogenic SIV infection with high viral replication, elevated IL-7 levels are unable to recover T-cell homeostasis, thereby leading to disease progression. The utility of IL-7 as a potential immunotherapeutic agent to improve HIV/SIV-related T-cell depletion may therefore depend on controlling the pathogenic effects of viral replication prior to the administration of IL-7.

    Title System-specific O2 Sensitivity of the Tandem Pore Domain K+ Channel Task-1.
    Date February 2004
    Journal American Journal of Physiology. Cell Physiology
    Excerpt

    Hypoxic inhibition of TASK-1, a tandem pore domain background K+ channel, provides a critical link between reduced O2 levels and physiological responses in various cell types. Here, we examined the expression and O2 sensitivity of TASK-1 in immortalized adrenomedullary chromaffin (MAH) cells. In physiological (asymmetrical) K+ solutions, 3 microM anandamide or 300 microM Zn2+ inhibited a strongly pH-sensitive current. Under symmetrical K+ conditions, the anandamide- and Zn2+-sensitive K+ currents were voltage independent. These data demonstrate the functional expression of TASK-1, and cellular expression of this channel was confirmed by RT-PCR and Western blotting. At concentrations that selectively inhibit TASK-1, anandamide and Zn2+ were without effect on the magnitude of the O2-sensitive current or the hypoxic depolarization. Thus TASK-1 does not contribute to O2 sensing in MAH cells, demonstrating the failure of a known O2-sensitive K+ channel to respond to hypoxia in an O2-sensing cell. These data demonstrate that, ultimately, the sensitivity of a particular K+ channel to hypoxia is determined by the cell, and we propose that this is achieved by coupling distinct hypoxia signaling systems to individual channels. Importantly, these data also reiterate the indirect O2 sensitivity of TASK-1, which appears to require the presence of an intracellular mediator.

    Title Gaba(b) Receptor Activation Augments Task-1 in Mah Cells and Mediates Autoreceptor Feedback During Hypoxia.
    Date February 2004
    Journal Biochemical and Biophysical Research Communications
    Excerpt

    Previously, we demonstrated an autoregulatory feedback loop in the rat carotid body (CB), involving presynaptic GABA(B) receptor-mediated activation of the background K(+) channel TASK-1. Here, we examined the effects of the selective GABA(B) receptor agonist baclofen on K(+) currents in immortalised adrenomedullary chromaffin (MAH) cells, which share the same sympathoadrenal lineage as CB type I cells. Under symmetrical K(+) conditions, 50 microM baclofen enhanced a K(+) current which was linear and reversed close to 0 mV. Under physiological K(+) conditions, baclofen enhanced outward K(+) current and caused membrane hyperpolarisation, effects inhibited by 100 nM CGP 55845. Current enhancement was virtually abolished in the presence of 300 microM Zn(2+), a selective inhibitor of TASK-1. When recording membrane potential from MAH cells in clusters, hypoxic depolarisation was augmented by 100 nM CGP 55845. These data demonstrate that GABA(B) receptors mediate autoreceptor feedback in the adrenal medulla presumably via TASK-1, demonstrating a common autoregulatory feedback pathway in neurosecretory, chemosensitive cells.

    Title Impacts of Avidity and Specificity on the Antiviral Efficiency of Hiv-1-specific Ctl.
    Date January 2004
    Journal Journal of Immunology (baltimore, Md. : 1950)
    Excerpt

    Although CD8(+) CTLs are presumed to be an important mediator of protective immunity in HIV-1 infection, the factors that determine CTL antiviral efficiency are poorly understood. Two factors that have been proposed to influence CTL antiviral function are antigenic avidity and epitope specificity. In this study we evaluate these by examining the activity of HIV-1-specific CTL against acutely infected cells. The ability of CTL to kill infected cells is variable and depends more on epitope specificity than functional avidity within the range for the tested clones (50% of maximal killing, 50 pg/ml to 100 ng/ml); killing efficiency is similar for different clones recognizing the same epitope, despite their variation in avidity. When CTL clones are tested for their ability to suppress viral replication, similar results are observed. Inhibition is more dependent on epitope specificity than functional avidity among the tested clones (50% of maximal killing, 20 pg/ml to 20 ng/ml). Thus, CTL specificity can be an overriding factor in the ability of CTL to interact with HIV-1-infected cells, indicating that factors determining the process of epitope presentation on infected cells have a key influence on CTL efficiency. These results suggest that CTL specificity may have a pivotal role in the immunopathogenesis of infection, and that simple quantitative measures of CTL may be insufficient indicators of the CTL response to HIV-1.

    Title Prevalence of Escherichia Coli O157 and Other Shiga-toxin-producing E. Coli in Lambs at Slaughter.
    Date September 2003
    Journal Journal of Veterinary Diagnostic Investigation : Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc
    Title Hiv Pathogenesis and Vaccine Development.
    Date August 2003
    Journal Topics in Hiv Medicine : a Publication of the International Aids Society, Usa
    Title Identification and Simian Immunodeficiency Virus Infection of Cd1d-restricted Macaque Natural Killer T Cells.
    Date August 2003
    Journal Journal of Virology
    Excerpt

    Natural killer T (NKT) cells express a highly conserved T-cell receptor (TCR) and recognize glycolipids in the context of CD1d molecules. We recently demonstrated that CD4+ NKT cells are highly susceptible to human immunodeficiency virus type 1 (HIV-1) infection and are selectively depleted in HIV-infected individuals. Here, we identified macaque NKT cells using CD1d tetramers and human Valpha24 antibodies. Similar to human NKT cells, alpha-galactosylceramide (alpha-GalCer)-pulsed dendritic cells activate and expand macaque NKT cells. Upon restimulation with alpha-GalCer-pulsed CD1d(+) cells, macaque NKT cells secreted high levels of cytokines, a characteristic of these T cells. Remarkably, the majority of resting and activated macaque NKT cells expressed CD8, and a smaller portion expressed CD4. Macaque NKT cells also expressed the HIV-1/simian immunodeficiency virus (SIV) coreceptor CCR5, and the CD4+ subset was susceptible to SIV infection. Identification of macaque NKT cells has major implications for delineating the role of these cells in nonhuman primate disease models of HIV as well as other pathological conditions, such as allograft rejection and autoimmunity.

    Title Total Synthesis of the Quinone Epoxide Dimer (+)-torreyanic Acid: Application of a Biomimetic Oxidation/electrocyclization/diels-alder Dimerization Cascade.
    Date June 2003
    Journal Journal of the American Chemical Society
    Excerpt

    An asymmetric synthesis of the quinone epoxide dimer (+)-torreyanic acid (48) has been accomplished employing [4 + 2] dimerization of diastereomeric 2H-pyran monomers. Synthesis of the related monomeric natural product (+)-ambuic acid (2) has also been achieved which establishes the biosynthetic relationship between these two natural products. A tartrate-mediated nucleophilic epoxidation involving hydroxyl group direction facilitated the asymmetric synthesis of a key chiral quinone monoepoxide intermediate. Thermolysis experiments have also been conducted on a model dimer based on the torreyanic acid core structure and facile retro Diels-Alder reaction processes and equilibration of diastereomeric 2H-pyrans have been observed. Theoretical calculations of Diels-Alder transition states have been performed to evaluate alternative transition states for Diels-Alder dimerization of 2H-pyran quinone epoxide monomers and provide insight into the stereocontrol elements for these reactions.

    Title Direct Relationship Between Suppression of Virus-specific Immunity and Emergence of Cytomegalovirus Disease in Simian Aids.
    Date June 2003
    Journal Journal of Virology
    Excerpt

    Although opportunistic infections like cytomegalovirus (CMV) are common sequelae of end-stage AIDS, the immune events leading to CMV reactivation in human immunodeficiency virus (HIV)-infected individuals are not well defined. The role of cellular and humoral CMV-specific immune responses in immune control of latent CMV infection was evaluated prospectively in a cohort of 11 simian immunodeficiency virus (SIV)-infected CMV-seropositive rhesus macaques, 6 of whom had histologic evidence of CMV disease at death. Macaques with CMV disease differed from macaques without CMV disease in having significantly higher levels of plasma SIV RNA and CMV DNA and significantly lower titers of anti-CMV binding antibodies (Abs) at the time of death. A significant decline in anti-CMV Abs and CMV-specific CD4(+) and CD8(+) T lymphocytes over time was observed in the macaques with CMV disease, but not in the macaques without CMV disease. Reduction in CMV-specific CD8(+) T lymphocytes and anti-CMV neutralizing Abs was significantly correlated with a decline in CMV-specific CD4(+) T lymphocytes. Although declines in CMV-specific T lymphocytes alone were sufficient for reactivation of low-level CMV viremia, high-level viremia (>1,000 copies of CMV DNA per ml of plasma) was observed when anti-CMV neutralizing and binding Abs had also declined. Thus, the occurrence of CMV reactivation-associated disease in AIDS is associated with suppression of both cellular and humoral CMV-specific immune responses. The underlying mechanism may be a dysfunction of memory B and CD8(+) T lymphocytes associated with SIV-induced impairment of CMV-specific CD4(+) T-cell help.

    Title Optimization of Ex Vivo Activation and Expansion of Macaque Primary Cd4-enriched Peripheral Blood Mononuclear Cells for Use in Anti-hiv Immunotherapy and Gene Therapy Strategies.
    Date April 2003
    Journal Journal of Acquired Immune Deficiency Syndromes (1999)
    Excerpt

    The rhesus macaque model is a useful experimental system to evaluate effects of T-cell autotransfusion and gene therapies for HIV-1 infection and AIDS prior to a clinical trial. To obtain sufficient numbers of primary macaque CD4 T lymphocytes for this purpose, we examined the culture conditions that were needed to optimize ex vivo activation and expansion of macaque primary CD4-enriched peripheral blood mononuclear cells (PBMCs). In this report, we compared the effects of various stimulants on cell expansion, surface expression of CCR5 and CXCR4, and levels of transduction with a Moloney leukemia virus (MoLV) vector encoding the phenotypic selection marker truncated human nerve growth factor receptor (deltaNGFR) alone or with the human anti-HIV-1 tat intrabody sFvhutat2. The use of feeder cells strikingly increased the proliferation rate of macaque CD4-enriched PBMCs in vitro. In the presence of an irradiated rhesus macaque B-lymphoblastoid cell line (BLCL), the highest cell expansion over 21 days was achieved with cells activated by Con A (9648-fold), in turn, from high to low, phytohemagglutinin (PHA) (4855-fold), and anti-CD3/CD28-coated beads (2367-fold). Further studies showed that BLCL feeder cells were more effective than human PBMCs (hPBMCs) in promoting proliferation of macaque CD4-enriched PBMCs activated with Con A and anti-CD3/CD28, respectively. The combined use of both BLCL and hPBMC feeder cells did not further increase cell expansion when compared with the use of BLCL cells alone. In addition, the addition of BLCL-conditioned medium (CM) and hPBMC-CM induced cell growth at a rate higher than did the culture medium alone but not as high as with feeder cells. Con A-activated macaque CD4-enriched PBMCs retained 88% of CXCR4 and 39% of CCR5 expression over 17 days compared with PHA-activated cells (50% for CXCR4, 16% for CCR5) and anti-CD3/CD28-activated cells (34% for CXCR4, 37% for CCR5). Finally, PHA, Con A, and CD3/CD28-coated beads supported comparable levels of MoLV transduction. The results should improve the utility of the rhesus macaque model for the testing of T-cell autotransfusion and gene therapies for HIV-1 infection/AIDS.

    Title Mucosal Priming of Simian Immunodeficiency Virus-specific Cytotoxic T-lymphocyte Responses in Rhesus Macaques by the Salmonella Type Iii Secretion Antigen Delivery System.
    Date March 2003
    Journal Journal of Virology
    Excerpt

    Nearly all human immunodeficiency virus (HIV) infections are acquired mucosally, and the gut-associated lymphoid tissues are important sites for early virus replication. Thus, vaccine strategies designed to prime virus-specific cytotoxic T lymphocyte (CTL) responses that home to mucosal compartments may be particularly effective at preventing or containing HIV infection. The Salmonella type III secretion system has been shown to be an effective approach for stimulating mucosal CTL responses in mice. We therefore tested DeltaphoP-phoQ attenuated strains of Salmonella enterica serovar Typhimurium and S. enterica serovar Typhi expressing fragments of the simian immunodeficiency virus (SIV) Gag protein fused to the type III-secreted SopE protein for the ability to prime virus-specific CTL responses in rhesus macaques. Mamu-A*01(+) macaques were inoculated with three oral doses of recombinant Salmonella, followed by a peripheral boost with modified vaccinia virus Ankara expressing SIV Gag (MVA Gag). Transient low-level CTL responses to the Mamu-A*01 Gag(181-189) epitope were detected following each dose of SALMONELLA: After boosting with MVA Gag, strong Gag-specific CTL responses were consistently detected, and tetramer staining revealed the expansion of Gag(181-189)-specific CD8(+) T-cell responses in peripheral blood. A significant percentage of the Gag(181-189)-specific T-cell population in each animal also expressed the intestinal homing receptor alpha4beta7. Additionally, Gag(181-189)-specific CD8(+) T cells were detected in lymphocytes isolated from the colon. Yet, despite these responses, Salmonella-primed/MVA-boosted animals did not exhibit improved control of virus replication following a rectal challenge with SIVmac239. Nevertheless, this study demonstrates the potential of mucosal priming by the Salmonella type III secretion system to direct SIV-specific cellular immune responses to the gastrointestinal mucosa in a primate model.

    Title Evaluation of Pcr and Pcr-rflp Protocols for Identifying Shiga Toxins.
    Date January 2003
    Journal Research in Microbiology
    Excerpt

    This study evaluated two generic polymerase chain reaction (PCR) protocols, and nine subtyping protocols and three PCR-restriction fragment length polymorphism (RFLP) protocols for detection of stx genes. The PCR protocols were evaluated by testing 12 reference isolates and 496 field strains of Shiga toxin-producing Escherichia coli (STEC). Both generic methods detected all stx genes. In tests with the reference isolates, all methods detected stx1 and stx2, seven subtyping methods detected stx2v(EH250), seven detected stx2e and only two detected stx2f. Four of the subtyping protocols identified stx genes in all of the field isolates. The PCR-RFLP protocols gave contradictory results for approximately 20% of the strains tested. The observed limitations of the protocols were shown to be due to nucleotide sequence variation in the region of the PCR primers. One subtyping protocol that detected the virulence-related genes, eae and ehxA, and all stx except for the stx2f gene, was modified by newly designed primers so that it identified all stx genes. This modified protocol provides comprehensive characterization of STEC in a single multiplex reaction.

    Title Importance of B-cell Responses for Immunological Control of Variant Strains of Simian Immunodeficiency Virus.
    Date January 2003
    Journal Journal of Virology
    Excerpt

    The properties of three variants of cloned simian immunodeficiency virus strain 239 (SIV239) were compared. One strain (M5) lacked five sites for N-linked carbohydrate attachment in variable regions 1 and 2 (V1 and V2) of the gp120 envelope protein, one strain (DeltaV1-V2) completely lacked V1 and V2 sequences, and another (316) had nine mutations in the envelope that impart high replicative capacity for tissue macrophages. All three strains were capable of significant levels of fusion independent of CD4, and all three were considerably more sensitive to antibody-mediated neutralization than the parent strain from which they were derived. Upon experimental infection of rhesus monkeys, these three variant strains replicated to viral loads at peak height around day 14 that were indistinguishable from or only slightly less than those observed in monkeys infected with the parental SIV239 strain. Viral loads at the set point 20 to 50 weeks after infection, however, were more than 400- to 10,000-fold lower with the variant strains. Depletion of B cells around the time of infection with M5 resulted in less effective immunological control and much higher viral loads at the set point in two of three monkeys. The differences between SIV239 infection, where there is not effective immunological control, and SIVM5 infection, where there is effective immunological control, cannot be easily explained by differences in the inherent replicative capacity of the viruses; rather, they are more readily explained by differences in the effectiveness of the antibody response. These results suggest that resistance of SIV239 to antibody-mediated neutralization is very important for evading effective immunological control, for allowing continuous viral replication, for maintenance of moderate-to-high viral loads at set point, and for disease progression.

    Title Mechanisms of Protection Against Simian Immunodeficiency Virus Infection.
    Date November 2002
    Journal Vaccine
    Excerpt

    One of the obstacles to the development of an effective AIDS vaccine has been the limited information on the mechanisms of protective immunity to HIV. In macaques, immunization with attenuated simian immunodeficiency viruses (SIV) has proved to be one of the most effective strategies to induce protection against infection or disease with pathogenic lentiviruses. Infection with attenuated SIV strains induces a broad range of SIV-specific immune responses, including relatively potent cytotoxic T lymphocyte (CTL) and antibody responses. Several studies of macaques vaccinated with attenuated SIV have demonstrated correlations between CTL responses or antibody responses and protection but more detailed studies are needed to document the relative importance of these responses in protective immunity.

    Title Decreased Levels of Recent Thymic Emigrants in Peripheral Blood of Simian Immunodeficiency Virus-infected Macaques Correlate with Alterations Within the Thymus.
    Date September 2002
    Journal Journal of Virology
    Excerpt

    The thymus is responsible for de novo production of CD4(+) and CD8(+) T cells and therefore is essential for T-cell renewal. The goal of this study was to assess the impact of simian immunodeficiency virus (SIV) infection on the production of T cells by the thymus. Levels of recent thymic emigrants within the peripheral blood were assessed through quantification of macaque T-cell receptor excision circles (TREC). Comparison of SIV-infected macaques (n = 15) to uninfected macaques (n = 23) revealed stable or increased TREC levels at 20 to 34 weeks postinfection. Further assessment of SIV-infected macaques (n = 4) determined that TREC levels decreased between 24 and 48 weeks postinfection. Through the assessment of longitudinal time points in three additional SIVmac239-infected macaques, the SIV infection was divided into two distinct phases. During phase 1 (16 to 30 weeks), TREC levels remained stable or increased within both the CD4 and CD8 T-cell populations. During phase 2 (after 16 to 30 weeks), TREC levels declined in both T-cell populations. As has been described for human immunodeficiency virus (HIV)-infected patients, this decline in TREC levels did at times correlate with an increased level of T-cell proliferation (Ki67(+) cells). However, not all TREC decreases could be attributed to increased T-cell proliferation. Further evidence for thymic dysfunction was observed directly in a SIVmac239-infected macaque that succumbed to simian AIDS at 65 weeks postinfection. The thymus of this macaque contained an increased number of memory/effector CD8(+) T cells and an increased level of apoptotic cells. In summary, reduced levels of TREC can be observed beginning at 16 to 30 weeks post-SIV infection and correlate with changes indicative of dysfunction within the thymic tissue. SIV infection of macaques will be a useful model system to elucidate the mechanisms responsible for the thymic dysfunction observed in HIV-infected patients.

    Title Escherichia Coli O157:h7 Infection in Cows and Calves in a Beef Cattle Herd in Alberta, Canada.
    Date September 2002
    Journal Epidemiology and Infection
    Excerpt

    Escherichia coli O157:H7 infection of cows and calves in a naturally-infected beef cattle herd in Alberta, Canada, was investigated over 2 years, encompassing two calf production cycles. In both years of the study, E. coli O157:H7 was isolated from the faeces of cows shortly after but not before parturition in late winter: 6/38 (16%) in 1996 and 13/50 (26%) in 1997. At < 1 week post-partum, 13/52 (25%) calves born in 1997 were shedding the organism. Faecal shedding of E. coli O157:H7 by cows and calves continued over the 7 weeks that they were in the calving pens, with the organism being isolated from the faeces of 2-18% of cows and 23-26% of calves during this period. Five weeks after they were moved onto a native grass pasture, all the calves and all but one cow in 1997 had ceased shedding the organism. When the calves were weaned in the fall, E. coli O157:H7 was isolated from the faeces of 0-1.5% of the calves 1 week prior to weaning and from 6-14% of the calves within 2 weeks after weaning. Parturition, calving pens and weaning appear to be important factors in maintaining E. coli O157:H7 infections in this beef cattle herd. Isolates from cows and calves during the immediate post-partum period were mostly of the same pulsed-field gel electrophoresis (PFGE) type of E. coli O157:H7. Similarly, at weaning a common PFGE type of E. coli O157:H7, which differed slightly from the post-partum PFGE type, was isolated from the calves. These typing data suggest a common source of infection for the animals as well as demonstrate clonal turnover of resident populations of this pathogen.

    Title Beyond the Roger Brown Rearrangement: Long-range Atom Topomerization in Conjugated Polyynes.
    Date August 2002
    Journal Journal of the American Chemical Society
    Excerpt

    Long-range carbon atom topomerization in a 1,3-diyne has been demonstrated for the first time. 1-Phenyl-4-p-tolyl-1,3-butadiyne, (13)C-enriched at C-1, was synthesized and subjected to flash vacuum pyrolysis. At 800 degrees C and 0.01 Torr, this resulted in nearly complete (13)C label equilibration between C-1 and C-2, as seen by NMR analysis. Pyrolysis at 900 degrees C further led to ca. 35% of the label migrating about equally to C-3 and C-4. These results demonstrate that both intrabond and interbond atom exchange processes are operative, with the former having a lower activation barrier. DFT and Moller-Plesset calculations support a mechanism that passes through Brown rearrangement (1,2-shift), closure to trialene (bicyclo[1.1.0]-1,3-butadiene), bond-shift isomerization to exchange C-2 and C-3, and ring opening. The resulting vinylidene can rearrange to a butadiyne with the isotopic label at C-3 or C-4. Consistent with earlier calculations, trialene is predicted to have alternating peripheral bonds, with a weak central sigma bond and significant diradical character. Trialene is predicted [(B3LYP/6-311+G(2d,p)] to lie 64.6 kcal/mol above butadiyne, with barriers of 2.2 and 4.4 kcal/mol, respectively, for ring opening or bond-shift isomerization. Other potential rearrangement mechanisms which pass through tetrahedrene (E(rel) = 167.2 kcal/mol) or 1,2,3-cyclobutatriene (E(rel) = 161.1 kcal/mol) lie at much higher energies.

    Title Measurement of B --> K*gamma Branching Fractions and Charge Asymmetries.
    Date May 2002
    Journal Physical Review Letters
    Excerpt

    The branching fractions of the exclusive decays B0-->K(*0)gamma and B+-->K(*+)gamma are measured from a sample of (22.74+/-0.36)x10(6) BB decays collected with the BABAR detector at the PEP-II asymmetric e(+)e(-) collider. We find B (B0-->K(*0)gamma) = [4.23+/-0.40(stat)+/-0.22(syst)]x10(-5), B(B+-->K(*+)gamma) = [3.83+/-0.62(stat)+/-0.22(syst)]x10(-5) and constrain the CP-violating charge asymmetry to be -0.170<A(CP)(B-->K(*)gamma)<0.082 at 90% C.L.

    Title Decreased Frequency of Cytomegalovirus (cmv)-specific Cd4+ T Lymphocytes in Simian Immunodeficiency Virus-infected Rhesus Macaques: Inverse Relationship with Cmv Viremia.
    Date April 2002
    Journal Journal of Virology
    Excerpt

    The frequency of cytomegalovirus (CMV)-specific CD4+ T lymphocytes was determined in CMV-seropositive rhesus macaques with or without simian immunodeficiency virus (SIV) infection by using the sensitive assays of intracellular cytokine staining and gamma interferon ELISPOT. Both techniques yielded 3- to 1,000-fold-higher frequencies of CMV-specific CD4+ T lymphocytes than traditional proliferative limiting dilution assays. The median frequency of CMV-specific CD4+ T lymphocytes in 23 CMV-seropositive SIV-negative macaques was 0.63% (range, 0.16 to 5.8%). The majority of CMV-specific CD4+ T lymphocytes were CD95(pos) and CD27(lo) but expressed variable levels of CD45RA. A significant reduction (P < 0.05) in the frequency of CMV-specific CD4+ T lymphocytes was observed in pathogenic SIV-infected macaques but not in macaques infected with live attenuated strains of SIV. CMV-specific CD4+ T lymphocytes were not detected in six of nine pathogenic SIV-infected rhesus macaques. CMV DNA was detected in the plasma of four of six of these macaques but in no animal with detectable CMV-specific CD4+ T lymphocytes. In pathogenic SIV-infected macaques, loss of CMV-specific CD4+ T lymphocytes was not predicted by the severity of CD4+ T lymphocytopenia. Neither was it predicted by the pre-SIV infection frequencies of CD45RA(neg) or CCR5(pos) CMV-specific CD4+ T lymphocytes. However, the magnitude of activation, as evidenced by the intensity of CD40L expression on CMV-specific CD4+ T lymphocytes pre-SIV infection, was three- to sevenfold greater in the two macaques that subsequently lost these cells after SIV infection than in the two macaques that retained CMV-specific CD4+ T lymphocytes post-SIV infection. Future longitudinal studies with these techniques will facilitate the study of CMV pathogenesis in AIDS.

    Title Measurement of the B--> J/psik*(892) Decay Amplitudes.
    Date March 2002
    Journal Physical Review Letters
    Excerpt

    We present a measurement of the decay amplitudes in B-->J/psiK*(892) channels using 20.7 fb(-1) of data collected at the Upsilon(4S) resonance with the BABAR detector at PEP-II. We measure a P-wave fraction R(perpendicular) = (16.0 +/- 3.2 +/- 1.4)% and a longitudinal polarization fraction (59.7 +/- 2.8 +/- 2.4)%. The measurement of a relative phase that is neither 0 nor pi, phi = 2.50 +/- 0.20 +/-0.08 radians, favors a departure from the factorization hypothesis. Although the decay B-->/psiK(pi) proceeds mainly via K*(892), there is also evidence for K2*(1430) and K(pi) S-wave contributions.

    Title Search for the Decay B0-->gammagamma.
    Date March 2002
    Journal Physical Review Letters
    Excerpt

    We present a limit on the branching fraction for the decay B0-->gammagamma using data collected at the Upsilon(4S) resonance with the BABAR detector at the PEP-II asymmetric energy e+e- collider. Based on the observation of one event in the signal region, out of a sample of 21.3x10(6) e+e--->Upsilon(4S)-->BB decays, we establish an upper limit on the branching fraction of B(B0-->gammagamma)<1.7x10(-6) at the 90% confidence level. This result substantially improves upon existing limits.

    Title Increasing Insulin Dose for Olanzapine-related Diabetes.
    Date January 2002
    Journal The American Journal of Psychiatry
    Title Measurements of the Branching Fractions of Exclusive Charmless B Meson Decays with Eta(') or Omega Mesons.
    Date January 2002
    Journal Physical Review Letters
    Excerpt

    We present the results of searches for B decays to charmless two-body final states containing eta(') or omega mesons, based on 20.7 fb(-1) of data collected with the BABAR detector. We find the branching fractions Beta(B(+)-->eta(')K(+)) = (70+/-8+/-5) x 10(-6), Beta(B(0)-->eta(')K(0)) = (42(+13)(-11) +/- 4) x 10(-6), and Beta(B(+)-->omega pi(+)) = (6.6(+2.1)(-1.8) +/- 0.7) x 10(-6), where the first error quoted is statistical and the second is systematic. We give measurements of four additional modes for which the 90% confidence level upper limits are Beta(B(+)-->eta(')pi(+)) < 12 x 10(-6), Beta(B(+)-->omega K(+)) < 4 x 10(-6), Beta(B(0)-->omega K(0)) < 13 x 10(-6), and Beta(B(0)-->omega pi(0)) < 3 x 10(-6).

    Title Dna Priming and Recombinant Pox Virus Boosters for an Aids Vaccine.
    Date January 2002
    Journal Developments in Biologicals
    Excerpt

    Eight different protocols for immunization have been compared for the ability to raise protection against immunodeficiency virus challenges in rhesus macaques. The most promising containment of challenge infections was achieved by intradermal DNA priming followed by recombinant fowl pox virus booster immunizations. This containment did not require neutralizing antibody and was active for a series of challenges ending with a highly virulent virus with a primary isolate envelope heterologous to the immunizing strain.

    Title Phenotypic and Genotypic Characterization of Escherichia Coli Verotoxin-producing Isolates from Humans and Pigs.
    Date January 2002
    Journal Journal of Food Protection
    Excerpt

    The aim of this study was to characterize verotoxin-producing Escherichia coli (VTEC) isolates obtained from humans and pigs in the same geographic areas and during the same period of time in order to determine whether porcine VTEC isolates could be related to human cases of diarrhea and also to detect the presence of virulence factors in these isolates. From 1,352 human and 620 porcine fecal samples, 11 human and 18 porcine verotoxin-positive isolates were obtained by the VT immunoblot or the individual colony testing technique. In addition, 52 porcine VTEC strains isolated from diseased pigs at the Faculté de médecine vétérinaire during the same period or from fecal samples collected previously isolated at slaughterhouses were characterized in this study. Antimicrobial resistance profiles were different between human and porcine isolates. In general, the serotypes observed in the two groups were different. No porcine isolate was of serotype O157:H7; however, one isolate was O91:NM, a serotype that has been associated with hemorrhagic colitis in humans. Also, one serotype (O8:H19) was found in isolates from both species; however, the O8:H19 isolates of the two groups were of different pathotypes. The pathotypes observed in the human and porcine isolates were different, with the exception of VT2vx-positive isolates; the serotypes of these isolates from the two groups were nevertheless different. Pulsed-field gel electrophoresis analysis indicated no relatedness between the human and porcine isolates. In conclusion, these results suggest that the porcine and human isolates of the present study were not genetically related. Most porcine VTEC isolates did not possess known virulence factors required to infect humans. However, certain non-O157:H7 porcine VTECs may potentially infect humans.

    Title Measurement of the B(0) and B(+) Meson Lifetimes with Fully Reconstructed Hadronic Final States.
    Date January 2002
    Journal Physical Review Letters
    Excerpt

    The B(0) and B(+) meson lifetimes have been measured in e(+)e(-) annihilation data collected in 1999 and 2000 with the BABAR detector at center-of-mass energies near the Upsilon(4S) resonance. Events are selected in which one B meson is fully reconstructed in a hadronic final state while the second B meson is reconstructed inclusively. A combined fit to the B(0) and the B(+) decay time difference distributions yields tau(B(0)) = 1.546+/-0.032(stat)+/-0.022(syst) ps, tau(B(+)) = 1.673+/-0.032(stat)+/-0.023(syst) ps, and tau(B(+))/tau(B(0)) = 1.082+/-0.026(stat)+/-0.012(syst).

    Title Emergence of Cytotoxic T Lymphocyte Escape Mutations in Nonpathogenic Simian Immunodeficiency Virus Infection.
    Date January 2002
    Journal European Journal of Immunology
    Excerpt

    Although CTL escape has been well documented in pathogenic simian immunodeficiency virus (SIV) infection, there is no information on CTL escape in nonpathogenic SIV infection in nonhuman primate hosts like the sooty mangabeys. CTL responses and sequence variation in the SIV nef gene were evaluated in one sooty mangabey and one rhesus macaque inoculated together with the same stock of cloned SIVmac239. Each animal developed an immunodominant response to a distinct CTL epitope in Nef, aa 157-167 in the macaque and aa 20-28 in the mangabey. Nonsynonymous mutations in their respective epitopes were observed in both animals and resulted in loss of CTL recognition. These mutations were present in the majority of proviral DNA sequences at 16 weeks post infection in the macaque and >2 years post infection in the mangabey. These results document the occurrence of CTL escape in a host that does not develop AIDS, and adds to the growing body of evidence that CTL exert significant selective pressure in SIV infection.

    Title T-cell Differentiation of Human and Non-human Primate Cd34+ Hematopoietic Progenitor Cells Using Porcine Thymic Stroma.
    Date December 2001
    Journal Xenotransplantation
    Excerpt

    Transplantation of swine thymic tissue has been proposed as an approach to reconstitute the immune system of HIV-infected individuals. This is an attractive strategy because miniature swine are readily available as donors and porcine tissue is resistant to infection with HIV-1. Demonstration that porcine thymus tissue supports primate T-cell differentiation is critical to the ultimate utility of this approach. Using a thymic stroma culture system we have previously described [Rosenzweig M, Marks DF, Zhu H et al. In vitro T lymphopoiesis of human and rhesus CD34+ progenitor cells. Blood 1996; 87: 4040], we demonstrate that porcine thymus tissue is able to promote the in vitro T-lymphocyte differentiation of both human and non-human primate hematopoietic progenitor cells. CD34+ hematopoietic progenitors differentiated into both double positive (CD4+CD8+) and single positive thymocytes expressing CD4 or CD8 alone. A polyclonal T-cell repertoire was evident. In addition, the T cells responded appropriately to mitogen and were permissive to infection with simian immunodeficiency virus (SIV). These data demonstrate the ability of porcine thymus to support T-cell differentiation of both human and non-human hematopoietic progenitor cells and support in vivo studies of transplantation of swine thymic tissue as a strategy for immune reconstitution in AIDS.

    Title Measurement of J/psi Production in Continuum E(+)e(-) Annihilations Near Square Root of S = 10.6 Gev.
    Date December 2001
    Journal Physical Review Letters
    Excerpt

    The production of J/psi mesons in continuum e(+)e(-) annihilations has been studied with the BABAR detector at energies near the Upsilon(4S) resonance. The mesons are distinguished from J/psi production in B decays through their center-of-mass momentum and energy. We measure the cross section e(+)e(-)-->J/psi X to be 2.52+/-0.21+/-0.21 pb. We set a 90% C.L. upper limit on the branching fraction for direct Upsilon(4S)-->J/psi X decays at 4.7 x 10(-4).

    Title Measurement of the Decays B--> Phik and B--> Phik*.
    Date December 2001
    Journal Physical Review Letters
    Excerpt

    We have observed the decays B--> phiK and phiK(*) in a sample of over 45 million B mesons collected with the BABAR detector at the PEP-II collider. The measured branching fractions are B(B+--> phiK+) = (7.7(+1.6)(-1.4)+/-0.8)x10(-6), B(B0--> phiK0) = (8.1(+3.1)(-2.5)+/-0.8)x10(-6), B(B+--> phiK(*+)) = (9.7(+4.2)(-3.4)+/-1.7)x10(-6), and B(B0--> phiK(*0)) = (8.7(+2.5)(-2.1)+/-1.1)x10(-6). We also report the upper limit B(B+--> phipi(+))<1.4x10(-6) ( 90% C.L.).

    Title Measurement of Branching Fractions and Search for Cp-violating Charge Asymmetries in Charmless Two-body B Decays into Pions and Kaons.
    Date December 2001
    Journal Physical Review Letters
    Excerpt

    We present measurements, based on a sample of approximately 23x10(6) BB pairs, of the branching fractions and a search for CP-violating charge asymmetries in charmless hadronic decays of B mesons into two-body final states of kaons and pions. We find the branching fractions B(B0-->pi(+)pi(-)) = (4.1+/-1.0+/-0.7)x10(-6), B(B0-->K+pi(-)) = (16.7+/-1.6+/-1.3)x10(-6), B(B+-->K+pi(0)) = (10.8(+2.1)(-1.9)+/-1.0)x10(-6), B(B+-->K0pi(+)) = (18.2(+3.3)(-3.0)+/-2.0)x10(-6), B(B0-->K0pi(0)) = (8.2(+3.1)(-2.7)+/-1.2)x10(-6). We also report 90% confidence level upper limits for B meson decays to the pi(+)pi(0), K+K-, and K0K+ final states. In addition, charge asymmetries have been found to be consistent with zero, where the statistical precision is in the range of +/-0.10 to +/-0.18, depending on the decay mode.

    Title Observation of Stimulated Electron-acoustic-wave Scattering.
    Date December 2001
    Journal Physical Review Letters
    Excerpt

    A diffraction-limited laser interacts with a plasma whose conditions are uniform on the scale of the focused laser spot. Two distinct, narrow waves are observed in the backscattered spectrum with phase velocities of v(phi)/v(e) = 1.4+/-0.08 and 4.2+/-0.1, where v(e) is the electron thermal speed. The high-velocity wave is ordinary stimulated Raman scattering (SRS) from a Langmuir wave. The low-velocity wave corresponds to stimulated scattering from an electron-acoustic wave (SEAS), and implies strong electron trapping. Previous SRS data from low-density plasmas are reinterpreted in terms of SEAS.

    Title Emergence and Kinetics of Simian Immunodeficiency Virus-specific Cd8(+) T Cells in the Intestines of Macaques During Primary Infection.
    Date October 2001
    Journal Journal of Virology
    Excerpt

    In this report, three Mamu-A*01(+) rhesus macaques were examined to compare the emergence of simian immunodeficiency virus (SIV)-specific CD8(+) T cells in the intestines and blood in early SIV infection using a major histocompatibility complex class I tetramer complexed with the Gag(181-189) peptide. Fourteen days after intravenous inoculation with SIVmac251, large numbers of SIV Gag(181-189)-specific CD8(+) T cells were detected in the intestinal mucosa (3.1 to 11.5% of CD3(+) CD8(+) lymphocytes) as well as in the blood (3.1 to 13.4%) of all three macaques. By 21 days postinoculation, levels of tetramer-binding cells had dropped in both the intestines and blood. At day 63, however, levels of SIV Gag(181-189)-specific CD8(+) T cells in the intestines had rebounded in all three macaques to levels that were higher (8.6 to 18.7%) than those at day 21. In contrast, percentages of tetramer-binding cells in the peripheral blood remained comparatively stable (2.5 to 4.5%) at this time point. In summary, SIV Gag(181-189)-specific CD8(+) T cells appeared in both the intestinal mucosa and peripheral blood at a comparable rate and magnitude in primary SIV infection. Given that the intestine is a major site of early viral replication as well as the site where most of the total body lymphocyte pool resides, these data indicate that it is also an early and important site of development of antiviral immune responses.

    Title Identification of Primitive Hematopoietic Progenitor Cells in the Rhesus Macaque.
    Date October 2001
    Journal Journal of Medical Primatology
    Excerpt

    The close phylogenetic relationship of macaques to humans has resulted in their widespread use as a preclinical model for bone marrow transplantation and stem cell gene therapy. To facilitate further use of this model, we undertook analysis of hematopoietic cells using multiparametric flow cytometric analysis. Rhesus CD34+CD38- cells displayed a number of characteristics of primitive hematopoietic cells, including low forward and orthogonal scatter and the lack of expression of lineage-specific markers or human lymphocyte antigen-DR. Four-color flow cytometric analysis demonstrated that rhesus CD34+CD38- cells were heterogenous with respect to Thy-1 expression and were CD59dim. Quantitative limiting dilution long-term culture-initiating cell (LTC-IC) analysis demonstrated that CD34+CD38- cells were approximately 150-fold enriched for LTC-IC as compared with unfractionated bone marrow, and occurred at a frequency similar to that previously reported in humans. Thus, as in humans, the CD34+38- population of rhesus macaque bone marrow is enriched for primitive, multipotent hematopoietic progenitor cells.

    Title Familial Cerebellar Ataxia with Hydrocephalus in Bull Mastiffs.
    Date October 2001
    Journal Veterinary Radiology & Ultrasound : the Official Journal of the American College of Veterinary Radiology and the International Veterinary Radiology Association
    Excerpt

    Familial cerebellar ataxia with concurrent hydrocephalus has previously been described in a family of bull mastiff pups, and recently has been identified in a litter from Louisiana. The 4 affected pups had ataxia, hypermetria, conscious proprioceptive deficits, behavioral abnormalities, and a visual deficit. In magnetic resonance imaging of the brain of two of the pups, there were symmetric hydrocephalus and two focal areas of increased signal intensity within the central nuclei of the cerebellum. Histopathologically there was vacuolization and mild astrogliosis within the deep cerebellar nuclei (dentate, interpositus, fastigial), caudal colliculi, and lateral vestibular nuclei. Although the postmortem results were not exactly the same as in the previously published report, the clinical features and histopathologic findings strongly support the diagnosis. This disorder is most likely inherited in an autosomal recessive manner.

    Title Observation of Cp Violation in the B(0) Meson System.
    Date October 2001
    Journal Physical Review Letters
    Excerpt

    We present an updated measurement of time-dependent CP-violating asymmetries in neutral B decays with the BABAR detector at the PEP-II asymmetric B Factory at SLAC. This result uses an additional sample of Upsilon(4S) decays collected in 2001, bringing the data available to 32 x 10(6) BB macro pairs. We select events in which one neutral B meson is fully reconstructed in a final state containing charmonium and the flavor of the other neutral B meson is determined from its decay products. The amplitude of the CP-violating asymmetry, which in the standard model is proportional to sin2 beta, is derived from the decay time distributions in such events. The result sin2 beta = 0.59+/-0.14(stat)+/-0.05(syst) establishes CP violation in the B(0) meson system. We also determine absolute value of lambda = 0.93+/-0.09(stat)+/-0.03(syst), consistent with no direct CP violation.

    Title Sv40-derived Vectors Provide Effective Transgene Expression and Inhibition of Hiv-1 Using Constitutive, Conditional,and Pol Iii Promoters.
    Date July 2001
    Journal Gene Therapy
    Excerpt

    Vectors based on recombinant SV40 viruses (rSV40) are highly effective in delivering transgene expression driven by constitutive promoters. We tested here whether these vectors could be used with conditional promoters and promoters using RNA polymerase III transcription, with inhibition of HIV-1 by Tat activation response (TAR) decoys as a functional measure of effective transgene delivery and activity. TAR decoys inhibit HIV-1 Tat, a trans-activator of HIV-1 transcription. Tat acts early in the viral replicative cycle and is essential for efficient viral replication. We evaluated rSV40 gene delivery using two different inhibitors of Tat. One was a dual function polyTAR gene encoding 25 sequential TAR elements (TAR(25)), plus an antisense tat, driven either by HIV-1 long terminal repeat (HIV-LTR) as a conditional promoter, or by cytomegalovirus immediate-early promoter (CMV-IEP) as a constitutive promoter. The other inhibitor was a single TAR decoy, driven by the U6 small nuclear RNA promoter (U6-P). These decoys were delivered to unselected cells in two different human T lymphocyte lines and to unstimulated primary human peripheral blood mononuclear cells (pbmc). Gene delivery was confirmed by PCR, and expression by RT-PCR. By in situ hybridization analysis, >95% of cells were transduced. These transgene constructs protected all cell types tested from HIV-1, as measured by syncytia formation and p24 antigen release. Somewhat better inhibition of HIV-1 replication was achieved with HIV-1 long terminal repeat (HIV-1 LTR) as a conditional promoter than with the constitutive CMV-IEP. The U6-P was also very effective, driving a TAR(1) transcript. Cell viability was not detectably affected by TAR decoy expression. Thus, rSV40 vectors effectively deliver HIV-1-inhibitory RNAs using either constitutive or conditional pol II promoters, or using a pol III promoter. The versatility of this gene delivery system may prove to be useful in anti-HIV-1 therapeutics.

    Title The Dynamics of T-lymphocyte Turnover in Aids.
    Date June 2001
    Journal Aids (london, England)
    Title Measurement of Cp-violating Asymmetries in B0 Decays to Cp Eigenstates.
    Date June 2001
    Journal Physical Review Letters
    Excerpt

    We present measurements of time-dependent CP-violating asymmetries in neutral B decays to several CP eigenstates. The measurement uses a data sample of 23x10(6) Upsilon(4S)-->BbarB decays collected by the BABAR detector at the PEP-II asymmetric B Factory at SLAC. In this sample, we find events in which one neutral B meson is fully reconstructed in a CP eigenstate containing charmonium and the flavor of the other neutral B meson is determined from its decay products. The amplitude of the CP-violating asymmetry, which in the standard model is proportional to sin2beta, is derived from the decay time distributions in such events. The result is sin2beta = 0.34+/-0.20 (stat)+/-0.05 (syst).

    Title Induction of Cytotoxic T Lymphocyte and Antibody Responses to Enhanced Green Fluorescent Protein Following Transplantation of Transduced Cd34(+) Hematopoietic Cells.
    Date April 2001
    Journal Blood
    Excerpt

    Genetic modification of hematopoietic stem cells often results in the expression of foreign proteins in pluripotent progenitor cells and their progeny. However, the potential for products of foreign genes introduced into hematopoietic stem cells to induce host immune responses is not well understood. Gene marking and induction of immune responses to enhanced green fluorescent protein (eGFP) were examined in rhesus macaques that underwent nonmyeloablative irradiation followed by infusions of CD34(+) bone marrow cells transduced with a retroviral vector expressing eGFP. CD34(+) cells were obtained from untreated animals or from animals treated with recombinant human granulocyte colony-stimulating factor (G-CSF) alone or G-CSF and recombinant human stem cell factor. Levels of eGFP-expressing cells detected by flow cytometry peaked at 0.1% to 0.5% of all leukocytes 1 to 4 weeks after transplantation. Proviral DNA was detected in 0% to 17% of bone marrow--derived colony-forming units at periods of 5 to 18 weeks after transplantation. However, 5 of 6 animals studied demonstrated a vigorous eGFP-specific cytotoxic T lymphocyte (CTL) response that was associated with a loss of genetically modified cells in peripheral blood, as demonstrated by both flow cytometry and polymerase chain reaction. The eGFP-specific CTL responses were MHC-restricted, mediated by CD8(+) lymphocytes, and directed against multiple epitopes. eGFP-specific CTLs were able to efficiently lyse autologous CD34(+) cells expressing eGFP. Antibody responses to eGFP were detected in 3 of 6 animals. These data document the potential for foreign proteins expressed in CD34(+) hematopoietic cells and their progeny to induce antibody and CTL responses in the setting of a clinically applicable transplantation protocol. (Blood. 2001;97:1951-1959)

    Title Definition of the Mamu A*01 Peptide Binding Specificity: Application to the Identification of Wild-type and Optimized Ligands from Simian Immunodeficiency Virus Regulatory Proteins.
    Date January 2001
    Journal Journal of Immunology (baltimore, Md. : 1950)
    Excerpt

    Single amino acid substitution analogs of the known Mamu A*01 binding peptide gag 181-190 and libraries of naturally occurring sequences of viral or bacterial origin were used to rigorously define the peptide binding motif associated with Mamu A*01 molecules. The presence of S or T in position 2, P in position 3, and hydrophobic or aromatic residues at the C terminus is associated with optimal binding capacity. At each of these positions, additional residues are also tolerated but associated with significant decreases in binding capacity. The presence of at least two preferred and one tolerated residues at the three anchor positions is necessary for good Mamu A*01 binding; optimal ligand size is 8-9 residues. This detailed motif has been used to map potential epitopes from SIVmac239 regulatory proteins and to engineer peptides with increased binding capacity. A total of 13 wild type and 17 analog candidate epitopes were identified. Furthermore, our analysis reveals a significantly lower than expected frequency of epitopes in early regulatory proteins, suggesting a possible evolutionary- and/or immunoselection directed against variants of viral products that contain CTL epitopes.

    Title Characterization of Lymphocyte Subsets in Rhesus Macaques During the First Year of Life.
    Date November 2000
    Journal European Journal of Haematology
    Excerpt

    Establishing reliable phenotypic data sets from the analysis of peripheral blood lymphocytes of normal animals is required to assess disease states. The rhesus macaque animal model is well established with respect to adult animals, but limited data are available that characterizes lymphocyte subsets in normal neonates. To address this, we used four-color flow cytometric analysis to follow phenotypic changes in 29 normal rhesus animals through their first ten months of life. From birth to 44 wk of age, the white cell count and absolute lymphocyte count were both elevated compared to adults. CD4+ cells constituted over 80% of all T cells at birth, a percentage that declined gradually over the first 12 wk of life, coincidental with increases in the percentages of CD8+ T cells, CD3-8+ natural killer cells and CD20+ B cells. This difference in relative frequency of CD4 and CD8 results in a significant skewing of CD4:CD8 ratio from 0.7:1 in adults to 3.5:1 in neonates. In addition, the predominant population of T lymphocytes consisted of CD45RA+CD62L+ naive cells. This subset continues to be the predominant phenotype for at least the first year of age. After birth the expression of activation markers (CD25) increased particularly on CD4+ T cells, although these levels generally reached a frequency similar to that observed in adults between 12 and 20 weeks after birth. These results are similar to those seen in humans and further confirm the reliability of the rhesus macaque animal model to study human diseases.

    Title Effective Induction of Simian Immunodeficiency Virus-specific Systemic and Mucosal Immune Responses in Primates by Vaccination with Proviral Dna Producing Intact but Noninfectious Virions.
    Date November 2000
    Journal Journal of Virology
    Excerpt

    We report a pilot evaluation of a DNA vaccine producing genetically inactivated simian immunodeficiency virus (SIV) particles in primates, with a focus on eliciting mucosal immunity. Our results demonstrate that DNA vaccines can be used to stimulate strong virus-specific mucosal immune responses in primates. The levels of immunoglobulin A (IgA) detected in rectal secretions of macaques that received the DNA vaccine intradermally and at the rectal mucosa were the most striking of all measured immune responses and were higher than usually achieved through natural infection. However, cytotoxic T lymphocyte responses were generally low and sporadically present in different animals. Upon rectal challenge with cloned SIVmac239, resistance to infection was observed, but some animals with high SIV-specific IgA levels in rectal secretions became infected. Our results suggest that high levels of IgA alone are not sufficient to prevent the establishment of chronic infection, although mucosal IgA responses may have a role in reducing the infectivity of the initial viral inoculum.

    Title Inhibition of Natural Killer Cell-mediated Cytotoxicity by Kaposi's Sarcoma-associated Herpesvirus K5 Protein.
    Date October 2000
    Journal Immunity
    Excerpt

    Kaposi's sarcoma-associated herpesvirus (KSHV) K3 and K5 proteins dramatically downregulate MHC class I molecules. However, although MHC class I downregulation may protect KSHV-infected cells from cytotoxic T lymphocyte recognition, these cells become potential targets for natural killer (NK) cell-mediated lysis. We now show that K5 also downregulates ICAM-1 and B7-2, which are ligands for NK cell-mediated cytotoxicity receptors. As a consequence, K5 expression drastically inhibits NK cell-mediated cytotoxicity. Conversely, de novo expression of B7-2 and ICAM-1 resensitizes the K5-expressing cells to NK cell-mediated cytotoxicity. This is a novel viral immune evasion strategy where KSHV K5 achieves immune avoidance by downregulation of cellular ligands for NK cell-mediated cytotoxicity receptors.

    Title Mechanisms Associated with Thymocyte Apoptosis Induced by Simian Immunodeficiency Virus.
    Date October 2000
    Journal Journal of Immunology (baltimore, Md. : 1950)
    Excerpt

    Despite considerable research, the mechanisms by which HIV disrupts thymic function remain controversial. We have described the phenotypic changes that occur in the thymus of SIV-infected macaques during acute SIV infection. In this study, we analyzed the effects of SIV infection on apoptotic pathways in thymic tissue from newborn macaques infected with SIV. Thymocyte apoptosis was accompanied by a modest increase in surface Fas expression, a profound decrease in the frequency of bcl-2-positive cells, as well as the amount of bcl-2 per cell. With control of viral replication, levels of bcl-2 and Fas returned to baseline together with a return to basal levels of apoptosis. In the thymus, SIV infection resulted in depletion of CD4+CD8+ thymocytes, an increase in apoptosis of thymocytes, and a down-regulation of MHC class I molecules. These changes peaked 14-21 days after infection at or just after peak viremia. This data further suggests disruption of the antiapoptotic pathway regulated by bcl-2 plays a critical role in SIV-induced apoptosis of thymocytes.

    Title Differential Dynamics of Cd4(+) and Cd8(+) T-lymphocyte Proliferation and Activation in Acute Simian Immunodeficiency Virus Infection.
    Date September 2000
    Journal Journal of Virology
    Excerpt

    Although lymphocyte turnover in chronic human immunodeficiency virus and simian immunodeficiency virus (SIV) infection has been extensively studied, there is little information on turnover in acute infection. We carried out a prospective kinetic analysis of lymphocyte proliferation in 13 rhesus macaques inoculated with pathogenic SIV. A short-lived dramatic increase in circulating Ki-67(+) lymphocytes observed at 1 to 4 weeks was temporally related to the onset of SIV replication. A 5- to 10-fold increase in Ki-67(+) CD8(+) T lymphocytes and a 2- to 3-fold increase in Ki-67(+) CD3(-) CD8(+) natural killer cells accounted for >85% of proliferating lymphocytes at peak proliferation. In contrast, there was little change in the percentage of Ki-67(+) CD4(+) T lymphocytes during acute infection, although transient increases in Ki-67(-) and Ki-67(+) CD4(+) T lymphocytes expressing CD69, Fas, and HLA-DR were observed. A two- to fourfold decline in CD4(+) T lymphocytes expressing CD25 and CD69 was seen later in SIV infection. The majority of Ki-67(+) CD8(+) T lymphocytes were phenotypically CD45RA(-) CD49d(hi) Fas(hi) CD25(-) CD69(-) CD28(-) HLA-DR(-) and persisted at levels twofold above baseline 6 months after SIV infection. Increased CD8(+) T-lymphocyte proliferation was associated with cell expansion, paralleled the onset of SIV-specific cytotoxic T-lymphocyte activity, and had an oligoclonal component. Thus, divergent patterns of proliferation and activation are exhibited by CD4(+) and CD8(+) T lymphocytes in early SIV infection and may determine how these cells are differentially affected in AIDS.

    Title Induction of Mucosal Homing Virus-specific Cd8(+) T Lymphocytes by Attenuated Simian Immunodeficiency Virus.
    Date September 2000
    Journal Journal of Virology
    Excerpt

    Induction of virus-specific T-cell responses in mucosal as well as systemic compartments of the immune system is likely to be a critical feature of an effective AIDS vaccine. We investigated whether virus-specific CD8(+) lymphocytes induced in rhesus macaques by immunization with attenuated simian immunodeficiency virus (SIV), an approach that is highly effective in eliciting protection against mucosal challenge, express the mucosa-homing receptor alpha4beta7 and traffic to the intestinal mucosa. SIV-specific CD8(+) T cells expressing alpha4beta7 were detected in peripheral blood and intestine of macaques infected with attenuated SIV. In contrast, virus-specific T cells in blood of animals immunized cutaneously by a combined DNA-modified vaccinia virus Ankara regimen did not express alpha4beta7. These results demonstrate the selective induction of SIV-specific CD8(+) T lymphocytes expressing alpha4beta7 by a vaccine approach that replicates in mucosal tissue and suggest that induction of virus-specific lymphocytes that are able to home to mucosal sites may be an important characteristic of a successful AIDS vaccine.

    Title Evaluation of the Reveal and Safepath Rapid Escherichia Coli O157 Detection Tests for Use on Bovine Feces and Carcasses.
    Date September 2000
    Journal Journal of Food Protection
    Excerpt

    The Reveal (Neogen Corp., Lansing, Mich.) and SafePath (SafePath Laboratories LLC, St. Paul, Minn.) tests were evaluated for their performance as beef fecal and beef carcass Escherichia coli O157:H7 monitoring tests. Agreement between these tests and a reference test system was determined using naturally contaminated bovine feces and beef carcasses. The reference system utilized immunomagnetic separation with plating onto cefixime, tellurite, sorbitol MacConkey agar, followed by colony testing using a serum agglutination test for the O157 antigen. Relative to this reference method, the Reveal test showed a sensitivity of 46% and a specificity of 82% on bovine feces and a specificity of 99% on carcass samples. The SafePath test, demonstrated a significantly higher sensitivity at 79% and a similar specificity of 79%. On carcass samples the SafePath test performed similarly to the Reveal test, demonstrating a specificity of 100% relative to the reference system. There was an insufficient number of E. coli O157-positive carcass samples to estimate precisely the sensitivity of these two methods. Both methods show promise as rapid carcass monitoring tests, but further field testing to estimate sensitivity is needed to complete their evaluation. The proportion of positive fecal samples for E. coli O157:H7 by the reference method ranged from 10.2% to 36% in 10 lots of cattle with an overall mean of 17.3% (39/225). Quarter carcass sponging of 125 carcasses revealed 1.6% positive for the pathogen (2/125).

    Title Vaccine Protection Against Simian Immunodeficiency Virus by Recombinant Strains of Herpes Simplex Virus.
    Date September 2000
    Journal Journal of Virology
    Excerpt

    An effective vaccine for AIDS may require development of novel vectors capable of eliciting long-lasting immune responses. Here we report the development and use of replication-competent and replication-defective strains of recombinant herpes simplex virus (HSV) that express envelope and Nef antigens of simian immunodeficiency virus (SIV). The HSV recombinants induced antienvelope antibody responses that persisted at relatively stable levels for months after the last administration. Two of seven rhesus monkeys vaccinated with recombinant HSV were solidly protected, and another showed a sustained reduction in viral load following rectal challenge with pathogenic SIVmac239 at 22 weeks following the last vaccine administration. HSV vectors thus show great promise for being able to elicit persistent immune responses and to provide durable protection against AIDS.

    Title Inhibition of Human Immunodeficiency Virus Type 1 Replication in Primary Cd4(+) T Lymphocytes, Monocytes, and Dendritic Cells by Cytotoxic T Lymphocytes.
    Date August 2000
    Journal Journal of Virology
    Excerpt

    We demonstrate that human immunodeficiency virus type 1 (HIV-1)-specific CD8(+) cytotoxic T lymphocytes (CTL) suppress HIV-1 replication in primary lymphocytes, monocytes, and dendritic cells individually. Viral inhibition is significantly diminished in lymphocyte-dendritic cell clusters, suggesting that these clusters in vivo could be sites where viral replication is more difficult to control by CTL.

    Title Efficient Gene Transfer to Hematopoietic Progenitor Cells Using Sv40-derived Vectors.
    Date August 2000
    Journal Gene Therapy
    Excerpt

    We used recombinant SV40 (rSV40)-derived vectors to deliver transgenes to human and simian hematopoietic progenitor cells in culture, and in vivo after transduction ex vivo. rSV40 are highly efficient vectors that are made in very high titers. They infect almost all cells, whether resting or dividing. Two rSV40s were used: SV(HBS), carrying hepatitis B surface antigen as a marker; and SV(Aw) carrying IN#33, a single chain Fv antibody against HIV-1 integrase. CD34+ cells derived from human fetal bone marrow (HFBM) and rhesus macaque bone marrow were transduced once with SV(HBS) without selection. On average 60% of colonies derived from transduced CD34+ cells carried and expressed HBsAg, as assessed by PCR and immunochemistry. Transgene carriage persisted following differentiation of transduced rhesus CD34+ cells into T lymphocytes. In an effort to increase the percentage of gene-marked cells, three sequential treatments of CD34+ cells were done using SV(Aw), without selection. Two weeks later, >95% of colonies expressed IN#33. Unselected SV(Aw)-transduced CD34+ cells from HFBM were transplanted into sublethally irradiated SCID mice. Bone marrow harvested 3 months later showed that >50% of bone marrow cells expressed IN#33. This is comparable with the percentage of human cells in these animals' bone marrow as judged by immunostaining for human CD45. The stability and longevity of transduction in this setting suggests that rSV40 vectors integrate into the cellular genome. This possibility was supported by finding that PCR of genomic DNA using primer pairs with one cellular and one viral primer yielded PCR products only in transduced, but not control, cells. These PCR products hybridized with an SV40 DNA fragment. Thus, rSV40 vectors transduce normal human and primate bone marrow progenitor cells effectively without selection, and maintain transgene expression in vivo following reimplantation. Such high efficiency transduction may be useful in treating diseases of CD34+ cells and their derivatives.

    Title Immunological Memory and Acquired Immunodeficiency Syndrome Pathogenesis.
    Date June 2000
    Journal Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences
    Excerpt

    Infection with the human immunodeficiency virus results in profound perturbations in immunological memory, ultimately resulting in increased susceptibility to opportunistic infections and acquired immunodeficiency syndrome (AIDS). We have used rhesus macaques infected with the simian immunodeficiency virus (SIV) as a model to understand better the effects of AIDS virus infection on immunological memory. Acute infection with SIV resulted in significant deficits in CD4+ helper responses to cytomegalovirus (CMV) as well as CMV-specific cytotoxic T-lymphocyte and neutralizing antibody responses. Reactivation of CMV was associated with high levels of SIV replication and suppression of both T-helper and cytotoxic responses to CMV. We have also studied the effects of SIV infection on T-cell turnover in non-human primates. T-cell turnover was evaluated using the nucleoside analogue bromodeoxyuridine (BrdU) in combination with five-colour flow cytometric analysis. T cells in normal animals turned over at relatively rapid rates, with memory cells turning over more quickly than naive cells. In SIV-infected animals, the labelling and elimination rates of both CD4+ and CD8+ BrdU-labelled cells were increased by two- to threefold compared with normal controls. Further analysis of immunological memory in non-human primates should offer the opportunity to extend immunological insights from murine models to the pathogenesis and prevention of AIDS.

    Title Quantification of Thymic Function by Measuring T Cell Receptor Excision Circles Within Peripheral Blood and Lymphoid Tissues in Monkeys.
    Date May 2000
    Journal European Journal of Immunology
    Excerpt

    The thymus is the primary organ responsible for the production of mature TCR alpha / beta T cells. Quantification of a DNA excision circle that is produced during TCR rearrangement, termed a signal joint TCR rearrangement excision circle (sjTREC) can be used as a measure of thymic function. Here sjTREC measurement has been applied to two monkey species used as animal models of human disease, rhesus macaques (Asian origin) and sooty mangabeys (African origin). Initial PCR analysis determined that the TCR deltaRec-PsiJalpha rearrangement leading to sjTREC formation occurs in both species. Primers to a DNA sequence conserved in macaques, mangabeys and humans were used in a quantitative competitive PCR assay to quantify sjTREC. We found that as in humans, sjTREC in these two monkey species decline with age. sjTREC are first generated in thymocytes during the early stages of TCR rearrangement. Lymph node CD4(+) and CD8(+) T cells contain more sjTREC than peripheral blood T cell populations, suggesting that recent thymic emigrants home to the lymphoid tissues. The sjTREC level is significantly higher within the peripheral blood CD4(+) and CD8(+) T cells of mangabeys compared to macaques. Removal of the thymus in four macaques led to a profound decrease in peripheral blood sjTREC level by 1 year post-thymectomy, indicating the lack of a significant extra-thymic source of peripheral naive T cells in macaques. Our results indicate that production, trafficking, and proliferation of recent thymic emigrants in these two monkey species represents a useful animal model system for understanding human immunological disorders.

    Title Polyoxometalate Oxidation of Chemical Warfare Agent Simulants in Fluorinated Media.
    Date March 2000
    Journal Journal of Applied Toxicology : Jat
    Excerpt

    The aim of this research is to determine if appropriate polyoxometalates (POMs) could be added to perfluoropolyether topical skin protectants (TSPs) currently available or under development to give these TSPs the additional capability of detecting and in some cases catalytically decontaminating sulfur mustard (HD) and perhaps other chemical warfare agents (CWAs) at ambient temperatures. Detection would be based on significant color changes in the POM upon reduction by the CWA whereas catalytic decontamination would be based on the ability of some families of POMs to catalyze O(2)-based oxidations by more than one mechanism. Five POMs (10-25% by weight) were each suspended in ca. 5 g of the perfluoropolyether (PFPE, CF(3)O[-CF(CF(3))CF(2)O-](x)(-CF(2)O-)(y)CF(3)) 'barrier' cream. A stoichiometric amount of HD sulfide simulant was layered on top of each POM-cream mixture. The short reaction times were recorded for each system. Mechanistic studies were conducted using an PFPE oil analog of the barrier cream in a microemulsion with the sulfide simulant, POM, PFPE surfactant and 2,2,2-trifluoroethanol co-surfactant.

    Title Distribution of Core Oligosaccharide Types in Lipopolysaccharides from Escherichia Coli.
    Date March 2000
    Journal Infection and Immunity
    Excerpt

    In the lipopolysaccharides of Escherichia coli there are five distinct core oligosaccharide (core OS) structures, designated K-12 and R1 to R4. The objective of this work was to determine the prevalences of these core OS types within the species. Unique sequences in the waa (core OS biosynthesis) gene operon were used to develop a PCR-based system that facilitated unequivocal determination of the core OS types in isolates of E. coli. This system was applied to the 72 isolates in the E. coli ECOR collection, a compilation of isolates that is considered to be broadly representative of the genetic diversity of the species. Fifty (69. 4%) of the ECOR isolates contained the R1 core OS, 8 (11.1%) were representatives of R2, 8 (11.1%) were R3, 2 (2.8%) were R4, and only 4 (5.6%) were K-12. R1 is the only core OS type found in all four major phylogenetic groups (A, B1, B2, and D) in the ECOR collection. Virulent extraintestinal pathogenic E. coli isolates tend to be closely related to group B2 and, to a lesser extent, group D isolates. All of the ECOR representatives from the B2 and D groups had the R1 core OS. In contrast, commensal E. coli isolates are more closely related to group A, which contains isolates representing each of the five core OS structures. R3 was the only core OS type found in 38 verotoxigenic E. coli (VTEC) isolates from humans and cattle belonging to the common enterohemorrhagic E. coli serogroups O157, O111, and O26. Although isolates from other VTEC serogroups showed more core OS diversity, the R3 type (83.1% of all VTEC isolates) was still predominant. When non-VTEC commensal isolates from cattle were analyzed, it was found that most possessed the R1 core OS type.

    Title Identification of Multiple Simian Immunodeficiency Virus (siv)-specific Ctl Epitopes in Sooty Mangabeys with Natural and Experimentally Acquired Siv Infection.
    Date February 2000
    Journal Journal of Immunology (baltimore, Md. : 1950)
    Excerpt

    Host immune responses to SIV infection in sooty mangabeys are likely to be an important determinant of how such nonhuman primate species maintain asymptomatic lentivirus infection. We have previously described two patterns of asymptomatic SIV infection in sooty mangabeys: low viral loads with vigorous SIV-specific CTL activity in SIVmac239-infected sooty mangabeys, and high viral loads with generally weak or absent SIV-specific CTL activity in naturally infected sooty mangabeys. To define the specificity of the CTL response in SIV-infected mangabeys, we characterized CTL epitopes in two naturally infected and three SIVmac239-infected sooty mangabeys. Compared with that in SIVmac239-infected mangabeys, the yield of SIV-specific CTL clones was significantly lower in naturally infected sooty mangabeys. All CTL clones were phenotypically CD3+ CD8+, and lysis was MHC restricted. Seven SIV CTL epitopes were identified in five sooty mangabeys: one in Gag and three each in Nef and Envelope (Env). The CTL epitopes mapped to conserved regions in the SIV genome and were immunodominant. Several similar or identical CTL epitopes were recognized by both naturally infected and SIVmac239-infected mangabeys that shared class I MHC alleles. To our knowledge, this is the first report of SIV-specific CTL epitopes in sooty mangabeys. Longitudinal studies of viral load and sequence variation in CTL epitopes may provide useful information on the role of CTL in control or persistence of SIV infection in sooty mangabeys.

    Title Actin Activates a Cryptic Dimerization Potential of the Vinculin Tail Domain.
    Date January 2000
    Journal The Journal of Biological Chemistry
    Excerpt

    The tail domain of vinculin (V(t)) is an actin binding module containing two regions that interact with F-actin. Although intact V(t) purified from a bacterial expression system is a globular monomer, each actin binding region dimerizes when expressed individually, suggesting the presence of cryptic self-association sites whose exposure is regulated. We show that actin modulates V(t) self-association by inducing or stabilizing a conformational change in V(t) that allows dimerization. Chemical cross-linking studies implicate one of the actin binding regions in mediating dimerization in the presence of actin. Actin-induced V(t) dimers may play a role in the filament cross-linking activity of this protein. The V(t) dimers induced by actin are biochemically distinct from the V(t) dimers and higher oligomers induced by acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate, suggesting structural differences in V(t) bound to these two ligands that may provide a mechanistic basis for inhibition of F-actin binding by phosphatidylinositol 4,5-bisphosphate. The ability of actin to regulate the dimerization state of an actin binding protein suggests that, rather than serving a passive structural role, actin filaments may directly participate in signal transduction and other cellular events that are known to depend on cytoskeletal integrity.

    Title Immunization with Live Attenuated Simian Immunodeficiency Virus Induces Strong Type 1 T Helper Responses and Beta-chemokine Production.
    Date January 2000
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    Immunization with live attenuated simian immunodeficiency virus (SIV) strains has proved to be one of the most effective strategies to induce protective immunity in the SIV/macaque model. To better understand the role that CD4(+) T helper responses may play in mediating protection in this model, we characterized SIV-specific proliferative and cytokine responses in macaques immunized with live attenuated SIV strains. Macaques chronically infected with live attenuated SIV had strong proliferative responses to SIV proteins, with stimulation indices of up to 74. The magnitude of the proliferative response to SIV Gag varied inversely with the degree of attenuation; Gag-specific but not envelope-specific responses were lower in animals infected with more highly attenuated SIV strains. SIV-specific stimulation of lymphocytes from vaccinated macaques resulted in secretion of interferon-gamma, IL-2, regulated-upon-activation, normal T cells expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta but not IL-4 or IL-10. Intracellular flow cytometric analysis documented that, in macaques vaccinated with SIVmac239Deltanef, up to 2% of all CD4(+)T cells were specific for SIV p55. The ability of live attenuated SIV to induce a strong, sustained type 1 T helper response may play a role in the success of this vaccination approach to generate protection against challenge with wild-type SIV.

    Title Characterization of Siv-specific Cd4+ T-helper Proliferative Responses in Macaques Immunized with Live-attenuated Siv.
    Date December 1999
    Journal Journal of Medical Primatology
    Excerpt

    Analysis of immune responses generated by live-attenuated simian immunodeficiency virus (SIV) strains may provide clues to the mechanisms of protective immunity induced by this approach. We examined SIV-specific T-helper responses in macaques immunized with the live-attenuated SIV strains SIVmac239deltanef and SIVmac239delta3. Optimization of the concentration and duration of antigenic stimulation resulted in the detection of relatively strong SIV-specific proliferative responses, with peak stimulation indices of up to 84. SIV-specific proliferative responses were mediated by CD4+ T cells and were major histocompatibility (MHC) class II restricted. Limiting dilution analysis revealed SIV-specific T-helper precursor frequencies of up to 96 per 10(6) peripheral blood mononuclear cells (PBMC). Intracellular flow-cytometric analysis demonstrated the production of interleukin (IL)-2, interferon (IFN)-gamma, RANTES and macrophage inhibitory protein-1alpha (MIP-1alpha) by T lymphocytes from SIVmac239deltanef-vaccinated animals following SIV p55 stimulation. Induction of strong SIV-specific T-helper responses by live-attenuated SIV vaccines may play a role in their ability to induce protective immunity.

    Title Efficient Processing of the Immunodominant, Hla-a*0201-restricted Human Immunodeficiency Virus Type 1 Cytotoxic T-lymphocyte Epitope Despite Multiple Variations in the Epitope Flanking Sequences.
    Date December 1999
    Journal Journal of Virology
    Excerpt

    Immune escape from cytotoxic T-lymphocyte (CTL) responses has been shown to occur not only by changes within the targeted epitope but also by changes in the flanking sequences which interfere with the processing of the immunogenic peptide. However, the frequency of such an escape mechanism has not been determined. To investigate whether naturally occurring variations in the flanking sequences of an immunodominant human immunodeficiency virus type 1 (HIV-1) Gag CTL epitope prevent antigen processing, cells infected with HIV-1 or vaccinia virus constructs encoding different patient-derived Gag sequences were tested for recognition by HLA-A*0201-restricted, p17-specific CTL. We found that the immunodominant p17 epitope (SL9) and its variants were efficiently processed from minigene expressing vectors and from six HIV-1 Gag variants expressed by recombinant vaccinia virus constructs. Furthermore, SL9-specific CTL clones derived from multiple donors efficiently inhibited virus replication when added to HLA-A*0201-bearing cells infected with primary or laboratory-adapted strains of virus, despite the variability in the SL9 flanking sequences. These data suggest that escape from this immunodominant CTL response is not frequently accomplished by changes in the epitope flanking sequences.

    Title Humoral Response and Protection from Experimental Challenge Following Vaccination of Raccoon Pups with a Modified-live Canine Distemper Virus Vaccine.
    Date December 1999
    Journal Journal of Wildlife Diseases
    Excerpt

    Eight 8-wk-old raccoon pups (Procyon lotor) with maternal canine distemper virus (CDV) neutralizing antibodies (NAb) and 24 8-wk-old seronegative pups were administered a commercial modified-live CDV vaccine (Galaxy, D, Solvay Animal Health, Inc., Kitchener, Ontario, Canada). All 24 seronegative raccoons had detectable serum CDV NAb titers 14 days after the initial dose. Titers rose to maximum levels 4 wk post-vaccination. Mean titers for groups of vaccinated seronegative pups were maintained between 1:256 and 1:2,048 for the remainder of the 3 mo observation period. Geometric means of the serum CDV NAb titer of eight seronegative pups given a single vaccine dose at 8 wk of age did not differ significantly from those of eight pups that were given serial doses at 8, 12, and 16 wk of age, or from those of eight pups vaccinated once at 16 wk of age. Seven unvaccinated 8-wk-old raccoon pups used as controls remained seronegative throughout the trial. Seven out of eight 8-wk-old pups with maternal antibodies, vaccinated at 8, 12, and 16 wk of age, failed to develop a rise in their CDV NAb titers until at least 18 wk of age, 2 wk after the third vaccination. Titers in eight unvaccinated raccoons with maternal antibodies declined steadily to undetectable levels at 20 wk of age. A half-life of 10.55 days was calculated for maternally-derived CDV NAb in raccoon pups. Sixteen vaccinated raccoons were protected from clinical disease following experimental oronasal challenge with a virulent raccoon strain of CDV, 13 to 23 wk after vaccination. Serum CDV NAb titers at the time of challenge ranged from 1:12 to 1:384 and increased during the period of observation. Three of four unvaccinated seronegative raccoons used as controls failed to mount any detectable CDV NAb and were euthanatized after developing clinical signs of canine distemper 26, 29, and 30 days post-challenge (PC). Necropsies confirmed the diagnosis. The fourth control raccoon exhibited transient equivocal clinical signs, mounted a sluggish humoral response, but was clinically normal when euthanatized 42 days PC. In this raccoon, there was focal non-suppurative encephalitis with intranuclear inclusion bodies typical of CDV infection.

    Title Efficient and Durable Gene Marking of Hematopoietic Progenitor Cells in Nonhuman Primates After Nonablative Conditioning.
    Date November 1999
    Journal Blood
    Excerpt

    Optimization of mobilization, harvest, and transduction of hematopoietic stem cells is critical to successful stem cell gene therapy. We evaluated the utility of a novel protocol involving Flt3-ligand (Flt3-L) and granulocyte colony-stimulating factor (G-CSF) mobilization of peripheral blood stem cells and retrovirus transduction using hematopoietic growth factors to introduce a reporter gene, murine CD24 (mCD24), into hematopoietic stem cells in nonhuman primates. Rhesus macaques were treated with Flt3-L (200 microgram/kg) and G-CSF (20 microgram/kg) for 7 days and autologous CD34(+) peripheral blood stem cells harvested by leukapheresis. CD34(+) cells were transduced with an MFGS-based retrovirus vector encoding mCD24 using 4 daily transductions with centrifugations in the presence of Flt3-L (100 ng/mL), human stem cell factor (50 ng/mL), and PIXY321 (50 ng/mL) in serum-free medium. An important and novel feature of this study is that enhanced in vivo engraftment of transduced stem cells was achieved by conditioning the animals with a low-morbidity regimen of sublethal irradiation (320 to 400 cGy) on the day of transplantation. Engraftment was monitored sequentially in the bone marrow and blood using both multiparameter flow cytometry and semi-quantitative DNA polymerase chain reaction (PCR). Our data show successful and persistent engraftment of transduced primitive progenitors capable of giving rise to marked cells of multiple hematopoietic lineages, including granulocytes, monocytes, and B and T lymphocytes. At 4 to 6 weeks posttransplantation, 47% +/- 32% (n = 4) of granulocytes expressed mCD24 antigen at the cell surface. Peak in vivo levels of genetically modified peripheral blood lymphocytes approached 35% +/- 22% (n = 4) as assessed both by flow cytometry and PCR 6 to 10 weeks posttransplantation. In addition, naïve (CD45RA(+) and CD62L(+)) CD4(+) and CD8(+) cells were the predominant phenotype of the marked CD3(+) T cells detected at early time points. A high level of marking persisted at between 10% and 15% of peripheral blood leukocytes for 4 months and at lower levels past 6 months in some animals. A cytotoxic T-lymphocyte response against mCD24 was detected in only 1 animal. This degree of persistent long-lived, high-level gene marking of multiple hematopoietic lineages, including naïve T cells, using a nonablative marrow conditioning regimen represents an important step toward the ultimate goal of high-level permanent transduced gene expression in stem cells.

    Title Protection by Live, Attenuated Simian Immunodeficiency Virus Against Heterologous Challenge.
    Date October 1999
    Journal Journal of Virology
    Excerpt

    We examined the ability of a live, attenuated deletion mutant of simian immunodeficiency virus (SIV), SIVmac239Delta3, which is missing nef and vpr genes, to protect against challenge by heterologous strains SHIV89.6p and SIVsmE660. SHIV89.6p is a pathogenic, recombinant SIV in which the envelope gene has been replaced by a human immunodeficiency virus type 1 envelope gene; other structural genes of SHIV89.6p are derived from SIVmac239. SIVsmE660 is an uncloned, pathogenic, independent isolate from the same primate lentivirus subgrouping as SIVmac but with natural sequence variation in all structural genes. The challenge with SHIV89.6p was performed by the intravenous route 37 months after the time of vaccination. By the criteria of CD4(+) cell counts and disease, strong protection against the SHIV89.6p challenge was observed in four of four vaccinated monkeys despite the complete mismatch of env sequences. However, SHIV89.6p infection was established in all four previously vaccinated monkeys and three of the four developed fluctuating viral loads between 300 and 10,000 RNA copy equivalents per ml of plasma 30 to 72 weeks postchallenge. When other vaccinated monkeys were challenged with SIVsmE660 at 28 months after the time of vaccination, SIV loads were lower than those observed in unvaccinated controls but the level of protection was less than what was observed against SHIV89.6p in these experiments and considerably less than the level of protection against SIVmac251 observed in previous experiments. These results demonstrate a variable level of vaccine protection by live, attenuated SIVmac239Delta3 against heterologous virus challenge and suggest that even live, attenuated vaccine approaches for AIDS will face significant hurdles in providing protection against the natural variation present in field strains of virus. The results further suggest that factors other than anti-Env immune responses can be principally responsible for the vaccine protection by live, attenuated SIV.

    Title Inhibition of Antigen Presentation by the Glycine/alanine Repeat Domain is Not Conserved in Simian Homologues of Epstein-barr Virus Nuclear Antigen 1.
    Date September 1999
    Journal Journal of Virology
    Excerpt

    Most humans and Old World nonhuman primates are infected for life with Epstein-Barr virus (EBV) or closely related gammaherpesviruses in the same lymphocryptovirus (LCV) subgroup. Several potential strategies for immune evasion and persistence have been proposed based on studies of EBV infection in humans, but it has been difficult to test their actual contribution experimentally. Interest has focused on the EBV nuclear antigen 1 (EBNA1) because of its essential role in the maintenance and replication of the episomal viral genome in latently infected cells and because EBNA1 endogenously expressed in these cells is protected from presentation to the major histocompatibility complex class-I restricted cytotoxic T-lymphocyte (CTL) response through the action of an internal glycine-alanine repeat (GAR). Given the high degree of biologic conservation among LCVs which infect humans and Old World primates, we hypothesized that strategies essential for viral persistence would be well conserved among viruses of this subgroup. We show that the rhesus LCV EBNA1 shares sequence homology with the EBV and baboon LCV EBNA1 and that the rhesus LCV EBNA1 is a functional homologue for EBV EBNA1-dependent plasmid maintenance and replication. Interestingly, all three LCVs possess a GAR domain, but the baboon and rhesus LCV EBNA1 GARs fail to inhibit antigen processing and presentation as determined by using three different in vitro CTL assays. These studies suggest that inhibition of antigen processing and presentation by the EBNA1 GAR may not be an essential mechanism for persistent infection by all LCV and that other mechanisms may be important for immune evasion during LCV infection.

    Title Highly Attenuated Vaccine Strains of Simian Immunodeficiency Virus Protect Against Vaginal Challenge: Inverse Relationship of Degree of Protection with Level of Attenuation.
    Date June 1999
    Journal Journal of Virology
    Excerpt

    Three different deletion mutants of simian immunodeficiency virus (SIV) that vary in their levels of attenuation were tested for the ability to protect against mucosal challenge with pathogenic SIV. Four female rhesus monkeys were vaccinated by intravenous inoculation with SIVmac239Delta3, four with SIVmac239Delta3X, and four with SIVmac239Delta4. These three vaccine strains exhibit increasing levels of attenuation: Delta3 < Delta3X <Delta4. The vaccinated monkeys were challenged by vaginal exposure to uncloned, pathogenic SIVmac251 at 61 weeks after the time of vaccination. On the basis of viral RNA loads in plasma, cell-associated virus loads in peripheral blood, and CD4 cell counts, strong protective effects were observed in all three groups of vaccinated monkeys. However, the degree of protection correlated inversely with the level of attenuation; the least-attenuated strain, SIVmac239Delta3, gave the greatest protection. One monkey in the Delta3X group and two in the Delta4 group clearly became superinfected by the challenge virus, but these animals had levels of SIV RNA in plasma that were considerably lower than those of naive animals that were challenged in parallel. Protection against vaginal challenge appears easier to achieve than protection against intravenous challenge, since four other SIVmac239Delta4-vaccinated monkeys showed no protection when challenged intravenously with a much lower inoculum of the same challenge virus stock. Protection against vaginal challenge in the Delta4-vaccinated group occurred in the absence of detectable serum neutralizing activities and appeared to be associated with the development of an early SIV-specific cytotoxic-T-lymphocyte response. Our results demonstrate that mucosal protection can be achieved by systemic immunization with the highly attenuated SIVmac239Delta4 more than 1 year prior to the time of challenge.

    Title Neutralizing Antibody-independent Containment of Immunodeficiency Virus Challenges by Dna Priming and Recombinant Pox Virus Booster Immunizations.
    Date May 1999
    Journal Nature Medicine
    Excerpt

    Eight different protocols were compared for their ability to raise protection against immunodeficiency virus challenges in rhesus macaques. The most promising containment of challenge infections was achieved by intradermal DNA priming followed by recombinant fowl pox virus booster immunizations. This containment did not require neutralizing antibody and was active for a series of challenges ending with a highly virulent virus with a primary isolate envelope heterologous to the immunizing strain.

    Title Processing of Hiv-1 Envelope Glycoprotein for Class I-restricted Recognition: Dependence on Tap1/2 and Mechanisms for Cytosolic Localization.
    Date April 1999
    Journal Journal of Immunology (baltimore, Md. : 1950)
    Excerpt

    Processing of viral proteins for recognition by CTL involves degradation of the proteins in the cytosol of an infected cell followed by transport of the resulting peptides into the endoplasmic reticulum (ER) by the TAP1/2 complex. Uncertainty exists over the site of processing of viral envelope (env) proteins since the extracellular domains of env proteins are not present in the cytosol where the class I Ag-processing pathway begins. Rather, the ectodomains of env proteins are cotranslationally translocated into the ER during biosynthesis. To analyze env protein processing, we used the herpes simplex virus protein ICP47 to block peptide transport by TAP1/2 and examined the effects of TAP blockade on the processing of the HIV-1 env protein. For the majority of env-specific CD8+ CTL, the processing pathway required TAP1/2-mediated transport of cytosolic peptides into the ER. To determine how env peptides are generated in the cytosol, we analyzed the processing of two TAP1/2-dependent epitopes containing N-linked glycosylation sites. In each case, processing involved glycosylation-dependent posttranslational modification of asparagine residues to aspartic acid. These results are consistent with cotranslational translocation of env into the ER, where glycosylation occurs. This is followed by export of a fraction of the newly synthesized protein into the cytosol, where it is deglycosylated, with conversion of the asparagines to aspartic acid residues. Following cytoplasmic proteolysis, env peptides are retransported by TAP1/2 into the ER, where association with class I occurs. Thus, the env protein can enter the class I pathway through multiple distinct processing mechanisms.

    Title Associations Between Virulence Factors of Shiga Toxin-producing Escherichia Coli and Disease in Humans.
    Date March 1999
    Journal Journal of Clinical Microbiology
    Excerpt

    Associations between known or putative virulence factors of Shiga toxin-producing Escherichia coli and disease in humans were investigated. Univariate analysis and multivariate logistic regression analysis of a set of 237 isolates from 118 serotypes showed significant associations between the presence of genes for intimin (eae) and Shiga toxin 2 (stx2) and isolates from serotypes reported in humans. Similar associations were found with isolates from serotypes reported in hemorrhagic colitis and hemolytic-uremic syndrome. The enterohemorrhagic E. coli (EHEC) hemolysin gene was significantly associated with isolates from serotypes found in severe diseases in univariate analysis but not in multivariate logistic regression models. A strong association between the intimin and EHEC-hemolysin genes may explain the lack of statistical significance of EHEC hemolysin in these multivariate models, but a true lack of biological significance of the hemolysin in humans or in disease cannot be excluded. This result warrants further investigations of this topic. Multivariate analysis revealed an interaction between the eae and stx2 genes, thus supporting the hypothesis of the synergism between the adhesin intimin and Shiga toxin 2. A strong statistical association was observed between the stx2 gene and severity of disease for a set of 112 human isolates from eight major serotypes. A comparison of 77 isolates of bovine origin and 91 human isolates belonging to six major serotypes showed significant associations of the genes for Shiga toxin 1 and EspP protease with bovine isolates and an increased adherence on HEp-2 cell cultures for human isolates, particularly from diarrheic patients and healthy persons.

    Title Live Attenuated Aids Vaccines: Hazards and Hopes.
    Date February 1999
    Journal Nature Medicine
    Title Creatine Kinase Level Changes Following Electromyography in the Normal Anaesthetized Dog.
    Date February 1999
    Journal Veterinary Journal (london, England : 1997)
    Title Protective Immunity Induced by Live Attenuated Simian Immunodeficiency Virus.
    Date December 1998
    Journal Current Opinion in Immunology
    Excerpt

    Lack of information on the mechanisms of protective immunity to AIDS virus infection represents a major obstacle to the development of a rational strategy for an effective HIV vaccine. In macaques, immunization with live attenuated simian immunodeficiency viruses has induced the most potent protective immunity and continued study promises a better understanding of the nature of protective immune responses. Recent evidence supports involvement of both cytotoxic T lymphocytes and neutralizing antibodies in protective immunity against infection by simian immunodeficiency virus, but more detailed studies are needed to document their relative importance.

    Title Diverse Host Responses and Outcomes Following Simian Immunodeficiency Virus Sivmac239 Infection in Sooty Mangabeys and Rhesus Macaques.
    Date November 1998
    Journal Journal of Virology
    Excerpt

    Sooty mangabeys naturally infected with simian immunodeficiency virus (SIV) do not develop immunodeficiency despite the presence of viral loads of 10(5) to 10(7) RNA copies/ml. To investigate the basis of apathogenic SIV infection in sooty mangabeys, three sooty mangabeys and three rhesus macaques were inoculated intravenously with SIVmac239 and evaluated longitudinally for 1 year. SIVmac239 infection of sooty mangabeys resulted in 2- to 4-log-lower viral loads than in macaques and did not reproduce the high viral loads observed in natural SIVsmm infection. During acute SIV infection, polyclonal cytotoxic T-lymphocyte (CTL) activity coincident with decline in peak plasma viremia was observed in both macaques and mangabeys; 8 to 20 weeks later, CTL activity declined in the macaques but was sustained and broadly directed in the mangabeys. Neutralizing antibodies to SIVmac239 were detected in the macaques but not the mangabeys. Differences in expression of CD38 on CD8(+) T lymphocytes or in the percentage of naive phenotype T cells expressing CD45RA and CD62L-selection did not correlate with development of AIDS in rhesus macaques. In macaques, the proportion of CD4(+) T lymphocytes expressing CD25 declined during SIV infection, while in mangabeys, CD25-expressing CD4(+) T lymphocytes increased. Longitudinal evaluation of cytokine secretion by flow cytometric analysis of unstimulated lymphocytes revealed elevation of interleukin-2 and gamma interferon in a macaque and only interleukin-10 in a concurrently infected mangabey during acute SIV infection. Differences in host responses following experimental SIVmac239 infection may be associated with the divergent outcome in sooty mangabeys and rhesus macaques.

    Title Recognition of Two Overlapping Ctl Epitopes in Hiv-1 P17 by Ctl from a Long-term Nonprogressing Hiv-1-infected Individual.
    Date November 1998
    Journal Journal of Immunology (baltimore, Md. : 1950)
    Excerpt

    HIV-1 infection has been shown to elicit strong CTL responses in some infected persons, but few data are available regarding the relationship between targeted epitopes and in vivo viral quasispecies. In this study, we examined the CTL response in a person infected for 15 yr with a CD4 count persistently >500 cells/microl. The dominant in vivo activated CTL response was directed against two overlapping Gag CTL epitopes in an area of p17 known to be essential for viral replication. The 9-mer SLYNTVATL (amino acids 77-85) was recognized in conjunction with HLA-A2, whereas the overlapping 8-mer TLYCVHQR (amino acids 83-91) was recognized by HLA-A11-restricted CTL. Analysis of in vivo virus sequences both in PBMC and plasma revealed the existence of sequence variation in this region, which did not affect viral replication in vitro, but decreased recognition by the A11-restricted CTL response, with maintenance of the A2-restricted response. These results indicate that an essential region of the p17 protein can be simultaneously targeted by CTL through two different HLA molecules, and that immune escape from CTL recognition can occur without impairing viral replication. In addition, they demonstrate that Ag processing can allow for presentation of overlapping epitopes in the same infected cell, which can be affected quite differently by sequence variation.

    Title Immune Modulation of Human B Lymphocytes by Gene Transfer with Recombinant Epstein-barr Virus Amplicons.
    Date September 1998
    Journal Journal of Virological Methods
    Excerpt

    We described previously a novel mode of gene transfer by infection of human B lymphocytes with recombinant Epstein-Barr virus (EBV) amplicons. This system was explored for its potential use in expressing various recombinant genes, including the cytokine IL-4, the HIV envelope glycoprotein (gp120) and a suicide and gag gene. Recombinant genes were present as multiple copy episomes and stable, high level recombinant gene expression could be detected by antigenic and functional assays. Amplicon-infected B cells secreted high levels of recombinant cytokine and efficiently presented recombinant antigens through classes I and II MHC-restricted antigen processing pathways. Thus, recombinant EBV amplicons can be used to express components of the immune system or heterologous genes for immune recognition in human B cells. Combining gene transfer with EBV infection may provide unique advantages for in vitro and in vivo gene transfer.

    Title Repeatability of the Petrifilm Hec Test and Agreement with a Hydrophobic Grid Membrane Filtration Method for the Enumeration of Escherichia Coli O157:h7 on Beef Carcasses.
    Date September 1998
    Journal Journal of Food Protection
    Excerpt

    The Petrifilm HEC test (3M Canada Inc., London, Ontario), a quantitative microbiological test for Escherichia coli O157:H7, was evaluated for its performance as a beef-carcass monitoring test. Test repeatability and agreement and agreement with an E. coli O157:H7 detection method using a hydrophobic grid membrane filter (HGMF) overlaid onto cefixime-tellurite-sorbitol MacConkey agar (CT-SMAC) followed by a latex agglutination test for the O157 antigen were determined by using pure cultures of E. coli O157:H7, beef samples experimentally contaminated with bovine feces containing E. coli O157:H7, and naturally contaminated beef carcasses of unknown E. coli O157:H7 status from a local abattoir. The Petrifilm HEC test showed excellent repeatability and excellent agreement with the HGMF-CT-SMAC method when test samples were obtained from pure cultures and experimentally contaminated meat. All 125 naturally contaminated beef carcasses surveyed were negative for E. coli O157:H7 with both microbial methods. The Petrifilm HEC test, however, demonstrated a significantly lower proportion of cross-reactive organisms (false-positive reactions) than the HGMF-CT-SMAC method. Given the performance of this test coupled with its ease of use and compact size, it shows considerable promise for carcass testing where abattoir laboratory facilities are limited and as a substitute for more complex laboratory testing methods used in established laboratories.

    Title T-lymphopoietic Capacity of Cord Blood-derived Cd34+ Progenitor Cells.
    Date September 1998
    Journal Experimental Hematology
    Excerpt

    Umbilical cord blood contains an abundance of CD34+ hematopoietic progenitor cells and has been used in transplantation as an alternative to adult bone marrow or mobilized peripheral blood. Although efficient myelomonocytic, erythroid, and B lymphoid differentiation has been shown in highly purified cord blood CD34+ mononuclear cells lacking expression of lineage-specific antigens, generation of functional T cells has not been previously documented. Exploiting two recently developed, complementary thymic stromal monolayer systems, we show here that immature hematopoietic progenitor cells (CD34+lineage-) from human and rhesus monkey cord blood mononuclear cells undergo T lymphopoiesis in a manner that recapitulates T cell ontogeny in vivo. After 2 weeks of proliferation, cultures contained myeloid [corrected] cells and discrete populations of CD4+CD8+ (double-positive) immature T lymphocytes, followed after an additional 2 weeks by the appearance of single-positive CD4+CD8- and CD4-CD8+ T cells that coexpressed CD3. The T lymphoid phenotype was confirmed at the transcriptional level by the presence of the lymphoid-restricted genes RAG-2 and T cell receptor. T cells generated from cord blood progenitors in these systems exhibited immunofunction as assessed by alloreactive responses in mixed lymphocyte reactions. These findings were comparable between human and rhesus progenitor cells and closely resemble previous data using adult bone marrow CD34+ cells in these models. Together, these observations demonstrate that cord blood contains abundant lymphoid progenitors that undergo T lymphopoiesis in vitro, suggesting the full multipotentiality of this stem cell source and its validity in investigating T lymphoid differentiation.

    Title Cellular Immune Responses to Hiv-1.
    Date September 1998
    Journal Aids (london, England)
    Title A Conserved Motif in the Tail Domain of Vinculin Mediates Association with and Insertion into Acidic Phospholipid Bilayers.
    Date August 1998
    Journal Biochemistry
    Excerpt

    The tail domain of vinculin (Vt) contains a salt-insensitive binding site for acidic phospholipids which is masked by the intramolecular head-tail interaction in native vinculin [Johnson, R. P., and Craig, S. W. (1995) Biochem. Biophys. Res. Commun. 210, 159-164]. To characterize further this phospholipid binding site, we have used hydrophobic photolabeling with a photoactivatable phosphatidylcholine analogue to detect insertion of protein into the lipid bilayer. We show here that, although the properties of binding to acidic phospholipid vesicles and spontaneous insertion into the bilayer are cryptic and inactive in vinculin at physiologic ionic strength, these activities of the purified tail domain can be activated by physical and chemical disruption of the intramolecular interaction between the head and tail domains. By analyzing the lipid binding and insertion activity of a series of GST-Vt fusion proteins, we defined 55 amino acids, comprising vinculin residues 916-970, that mimic the lipid-binding and insertion activity of Vt. Predictions of secondary structure suggest that these 55 amino acids form a basic, amphipathic helical hairpin. This prediction is supported by circular dichroism analysis, which indicates that at least 80% of the residues in residues 916-970 are in a helical conformation. This predicted helical hairpin motif, which is conserved in all vinculins and is present in an acidic phospholipid-binding region of alpha-catenin, is distinct from C2 and PH domains, and likely represents a third type of acidic phospholipid-binding structure.

    Title Inhibition of Simian Immunodeficiency Virus (siv) Replication by Cd8(+) T Lymphocytes from Macaques Immunized with Live Attenuated Siv.
    Date August 1998
    Journal Journal of Virology
    Excerpt

    Characterization of immune responses induced by live attenuated simian immunodeficiency virus (SIV) strains may yield clues to the nature of protective immunity induced by this vaccine approach. We investigated the ability of CD8(+) T lymphocytes from rhesus macaques immunized with the live, attenuated SIV strain SIVmac239Deltanef or SIVmac239Delta3 to inhibit SIV replication. CD8(+) T lymphocytes from immunized animals were able to potently suppress SIV replication in autologous SIV-infected CD4(+) T cells. Suppression of SIV replication by unstimulated CD8(+) T cells required direct contact and was major histocompatibility complex (MHC) restricted. However, CD3-stimulated CD8(+) T cells produced soluble factors that inhibited SIV replication in an MHC-unrestricted fashion as much as 30-fold. Supernatants from stimulated CD8(+) T cells were also able to inhibit replication of both CCR5- and CXCR4-dependent human immunodeficiency virus type 1 (HIV-1) strains. Stimulation of CD8(+) cells with cognate cytotoxic T-lymphocyte epitopes also induced secretion of soluble factors able to inhibit SIV replication. Production of RANTES, macrophage inhibitory protein 1alpha (MIP-1alpha), or MIP-1beta from stimulated CD8(+) T cells of vaccinated animals was almost 10-fold higher than that from stimulated CD8(+) T cells of control animals. However, addition of antibodies that neutralize these beta-chemokines, either alone or in combination, only partly blocked inhibition of SIV and HIV replication by soluble factors produced by stimulated CD8(+) T cells. Our results indicate that inhibition of SIV replication by CD8(+) T cells from animals immunized with live attenuated SIV strains involves both MHC-restricted and -unrestricted mechanisms and that MHC-unrestricted inhibition of SIV replication is due principally to soluble factors other than RANTES, MIP-1alpha, and MIP-1beta.

    Title Risk Factors for Infection with Verocytotoxigenic Escherichia Coli in Cattle on Ontario Dairy Farms.
    Date July 1998
    Journal Preventive Veterinary Medicine
    Excerpt

    Risk factors for prevalent infection with verocytotoxigenic Escherichia coli (VTEC) were studied in a random sample of 886 cows and 592 calves under 3 months of age on 80 randomly selected dairy farms in southern Ontario. Fecal-culture supernatants from each animal were screened for verocytotoxicity using a Vero cell assay (VCA) and for verocytotoxin (VT) genes by a polymerase chain reaction (PCR) procedure. Up to 20 F. colt isolates from positive samples were tested for VT production using VCA and PCR. VTEC isolates were serotyped. Farm managers were interviewed using a standardized questionnaire to obtain information on farm- and individual animal-level management practices and characteristics. There was a significant (P < 0.001) positive association between age of calves and their VTEC infection status, and calves were significantly more likely to be infected than cows. The proportion of calves infected on the farm was positively associated with both the use of regular pails for feeding calves (as opposed to nipple bottles or nipple pails) and bringing new animals into the herd in the previous year.

    Title Characterization of the Peptide Binding Motif of a Rhesus Mhc Class I Molecule (mamu-a*01) That Binds an Immunodominant Ctl Epitope from Simian Immunodeficiency Virus.
    Date July 1998
    Journal Journal of Immunology (baltimore, Md. : 1950)
    Excerpt

    The majority of immunogenic CTL epitopes bind to MHC class I molecules with high affinity. However, peptides longer or shorter than the optimal epitope rarely bind with high affinity. Therefore, identification of optimal CTL epitopes from pathogens may ultimately be critical for inducing strong CTL responses and developing epitope-based vaccines. The SIV-infected rhesus macaque is an excellent animal model for HIV infection of humans. Although a number of CTL epitopes have been mapped in SIV-infected rhesus macaques, the optimal epitopes have not been well defined, and their anchor residues are unknown. We have now defined the optimal SIV gag CTL epitope restricted by the rhesus MHC class I molecule Mamu-A*01 and defined a general peptide binding motif for this molecule that is characterized by a dominant position 3 anchor (proline). We used peptide elution and sequencing, peptide binding assays, and bulk and clonal CTL assays to demonstrate that the optimal Mamu-A*01-restricted SIV gag CTL epitope was CTPYDINQM(181-189). Mamu-A*01 is unique in that it is found at a high frequency in rhesus macaques, and all SIV-infected Mamu-A*01-positive rhesus macaques studied to date develop an immunodominant gag-specific CTL response restricted by this molecule. Identification of the optimal SIV gag CTL epitope will be critical for a variety of studies designed to induce CD8+ CTL responses specific for SIV in the rhesus macaque.

    Title Early Regeneration of Thymic Progenitors in Rhesus Macaques Infected with Simian Immunodeficiency Virus.
    Date June 1998
    Journal The Journal of Experimental Medicine
    Excerpt

    The thymus plays a critical role in the maturation and production of T lymphocytes and is a target of infection by human immunodeficiency virus (HIV) and the related simian immunodeficiency virus (SIV). Using the SIV/macaque model of AIDS, we examined the early effects of SIV on the thymus. We found that thymic infection by SIV resulted in increased apoptosis 7-14 d after infection, followed by depletion of thymocyte progenitors by day 21. A marked rebound in thymocyte progenitors occurred by day 50 and was accompanied by increased levels of cell proliferation in the thymus. Our results demonstrate a marked increase in thymic progenitor activity very early in the course of SIV infection, long before marked declines in peripheral CD4(+) T cell counts.

    Title Lack of Strong Immune Selection Pressure by the Immunodominant, Hla-a*0201-restricted Cytotoxic T Lymphocyte Response in Chronic Human Immunodeficiency Virus-1 Infection.
    Date June 1998
    Journal The Journal of Clinical Investigation
    Excerpt

    Despite detailed analysis of the HIV-1-specific cytotoxic T lymphocyte response by various groups, its relation to viral load and viral sequence variation remains controversial. We analyzed HLA-A*0201 restricted cytotoxic T lymphocyte responses in 17 HIV-1-infected individuals with viral loads ranging from < 400 to 221,000 HIV RNA molecules per milliliter of plasma. In 13 out of 17 infected subjects, CTL responses against the SLYNTVATL epitope (p17 Gag; aa 77-85) were detectable, whereas two other HLA-A*0201 restricted epitopes (ILKEPVHGV, IV9; and VIYQYMDDL, VL9) were only recognized by six and five individuals out of 17 individuals tested, respectively. Naturally occurring variants of the SL9 epitope were tested for binding to HLA-A*0201 and for recognition by specific T cell clones generated from five individuals. Although these variants were widely recognized, they differed by up to 10,000-fold in terms of variant peptide concentrations required for lysis of target cells. A comparison of viral sequences derived from 10 HLA-A*0201-positive individuals to sequences obtained from 11 HLA-A*0201-negative individuals demonstrated only weak evidence for immune selective pressure and thus question the in vivo efficacy of immunodominant CTL responses present during chronic HIV-1 infection.

    Title Increased Rates of Cd4(+) and Cd8(+) T Lymphocyte Turnover in Simian Immunodeficiency Virus-infected Macaques.
    Date June 1998
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    Defining the rate at which T cells turn over has important implications for our understanding of T lymphocyte homeostasis and AIDS pathogenesis, yet little information on T cell turnover is available. We used the nucleoside analogue bromodeoxyuridine (BrdUrd) in combination with five-color flow cytometric analysis to evaluate T lymphocyte turnover rates in normal and simian immunodeficiency virus (SIV)-infected rhesus macaques. T cells in normal animals turned over at relatively rapid rates, with memory cells turning over more quickly than naive cells. In SIV-infected animals, the labeling and elimination rates of both CD4(+) and CD8(+) BrdUrd-labeled cells were increased by 2- to 3-fold as compared with normal controls. In normal and SIV-infected animals, the rates of CD4(+) T cell BrdUrd-labeling and decay were closely correlated with those of CD8(+) T cells. The elimination rate of BrdUrd-labeled cells was accelerated in both naive and memory T lymphocytes in SIV-infected animals. Our results provide direct evidence for increased rates of both CD4(+) and CD8(+) T cell turnover in AIDS virus infection and have important implications for our understanding of T cell homeostasis and the mechanisms responsible for CD4(+) T cell depletion in AIDS.

    Title What is Your Diagnosis? Multiple Epiphyseal Dysplasia of the Femoral Head, Proximal Humerus and Vertebral End Plates.
    Date June 1998
    Journal Journal of the American Veterinary Medical Association
    Title Phenotypic and Genotypic Variation of Feline Calicivirus During Persistent Infection of Cats.
    Date May 1998
    Journal Veterinary Microbiology
    Excerpt

    Amino acid sequence of the capsid protein hypervariable region of nine feline calicivirus (FCV) isolates recovered from cats persistently infected after inoculation with the FCV strain 255 parent virus is reported. Capsid proteins from all the isolates were highly cross reactive by Western blot analysis using polyclonal antisera to FCV. Reverse-transcription PCR was used to obtain sequence information of the FCV capsid protein highly variable E region. Amino acid substitutions occurred between residues 426 and 458 of the FCV capsid protein E region. The sequence data and phylogenetic reconstructions based on the sequence information correlated well with antigenic differences among isolates determined by two-way cross neutralization. These results agree with previous reports using divergent isolates of FCV that correlated amino acid differences with serology. This further supports the hypothesis that the FCV capsid protein E region from residues 426 to 458 contains the serotypic determinants of FCV important to antigenic variation.

    Title Gastrointestinal Tract As a Major Site of Cd4+ T Cell Depletion and Viral Replication in Siv Infection.
    Date May 1998
    Journal Science (new York, N.y.)
    Excerpt

    Human and simian immunodeficiency virus (HIV and SIV) replicate optimally in activated memory CD4(+) T cells, a cell type that is abundant in the intestine. SIV infection of rhesus monkeys resulted in profound and selective depletion of CD4+ T cells in the intestine within days of infection, before any such changes in peripheral lymphoid tissues. The loss of CD4+ T cells in the intestine occurred coincident with productive infection of large numbers of mononuclear cells at this site. The intestine appears to be a major target for SIV replication and the major site of CD4+ T cell loss in early SIV infection.

    Title Follow-up Study of Verocytotoxigenic Escherichia Coli Infection in Dairy Farm Families.
    Date April 1998
    Journal The Journal of Infectious Diseases
    Title Escherichia Coli O157:h7 Diarrhoea Associated with Well Water and Infected Cattle on an Ontario Farm.
    Date April 1998
    Journal Epidemiology and Infection
    Excerpt

    A 16-month old female child living on an Ontario dairy farm was taken to hospital suffering from bloody diarrhoea. Escherichia coli O157:H7 was isolated from her stool. Initial tests of well water samples were negative for E. coli by standard methods but culture of selected coliform colonies on sorbitol-MacConkey agar led to isolation of E. coli O157:H7. E. coli O157:H7 was also isolated from 63% of cattle on the farm. The E. coli O157:H7 isolates from the child, the water and the cattle were phage type 14, produced verotoxins 1 and 2, and were highly related on analysis by pulsed field gel electrophoresis. The child did not have known direct contact with the cattle and did not consume unpasteurized milk. Hydrogeological investigation revealed the design and location of the well would allow manure-contaminated surface water to flow into the well. This investigation demonstrates that cattle farm well water is a potential source of E. coli O157:H7 which may not be identified by standard screening for E. coli in water.

    Title Identification of Highly Attenuated Mutants of Simian Immunodeficiency Virus.
    Date February 1998
    Journal Journal of Virology
    Excerpt

    Deletion mutants of the pathogenic clone of simian immunodeficiency virus isolate 239 (SIVmac239) were derived that are missing nef, vpr, and upstream sequences (US) in the U3 region of the LTR (SIVmac239 delta3), nef, vpx, and US (SIVmac239 delta3x), and nef, vpr, vpx, and US (SIVmac239 delta4). These multiply deleted derivatives replicated well in the continuously growing CEMx174 cell line and were infectious for rhesus monkeys. However, on the basis of virus load measurements, strength of antibody responses, and lack of disease progression, these mutants were highly attenuated. Measurements of cell-associated viral load agreed well with assays of plasma viral RNA load and with the strengths of the antibody responses; thus, these measurements likely reflected the extent of viral replication in vivo. A derivative of SIVmac239 lacking vif sequences (SIVmac239 delta vif) could be consistently grown only in a vif-complementing cell line. This delta vif virus appeared to be very weakly infectious for rhesus monkeys on the basis of sensitive antibody tests only. The weak antibody responses elicited by SIVmac239 delta vif were apparently in response to low levels of replicating virus since they were not elicited by heat-inactivated virus and the anti-SIV antibody responses persisted for greater than 1 year. These results, and the results of previous studies, allow a rank ordering of the relative virulence of nine mutant strains of SIVmac according to the following order: delta vpr > delta vpx > delta vpr delta vpx approximately delta nef > delta3 > delta3x > or = delta4 > delta vif > delta5. The results also demonstrate that almost any desired level of attenuation can be achieved, ranging from still pathogenic in a significant proportion of animals (delta vpr and delta vpx) to not detectably infectious (delta5), simply by varying the number and location of deletions in these five loci.

    Title Dye Efflux Studies Suggest That Hematopoietic Stem Cells Expressing Low or Undetectable Levels of Cd34 Antigen Exist in Multiple Species.
    Date January 1998
    Journal Nature Medicine
    Excerpt

    We previously described a method for isolating murine hematopoietic stem cells capable of reconstituting lethally irradiated recipients, which depends solely on dual-wavelength flow cytometric analysis of murine bone marrow cells stained with the fluorescent DNA-binding dye Hoechst 33342. This method, which appears to rely on the differential ability of stem cells to efflux the Hoechst dye, defines an extremely small and homogeneous population of cells (termed SP cells). We show here that dual-wavelength analysis of Hoechst dye-stained human, rhesus and miniature swine bone marrow cells reveals a small, distinct population of cells that efflux the dye in a manner identical to murine SP cells. Like the murine SP cells, both human and rhesus SP cells are primarily CD34-negative and lineage marker-negative. In vitro culture studies demonstrated that rhesus SP cells are highly enriched for long-term culture-initiating cells (LTC-ICs), an indicator of primitive hematopoietic cells, and have the capacity for differentiation into T cells. Although rhesus SP cells do not initially possess any hematopoietic colony-forming capability, they acquire the ability to form colonies after long-term culture on bone marrow stroma, coincident with their conversion to a CD34-positive phenotype. These studies suggest the existence of a hitherto unrecognized population of hematopoietic stem cells that lack the CD34 surface marker classically associated with primitive hematopoietic cells.

    Title Intracellular Immunization of Rhesus Cd34+ Hematopoietic Progenitor Cells with a Hairpin Ribozyme Protects T Cells and Macrophages from Simian Immunodeficiency Virus Infection.
    Date December 1997
    Journal Blood
    Excerpt

    Evaluation of candidate genes for stem cell gene therapy for acquired immunodeficiency syndrome (AIDS) has been limited by the difficulty of supporting in vitro T-cell differentiation of genetically modified hematopoietic progenitor cells. Using a novel thymic stromal culture technique, we evaluated the ability of a hairpin ribozyme specific for simian immunodeficiency virus (SIV) and human immunodeficiency virus type 2 (HIV-2) to inhibit viral replication in T lymphocytes derived from transduced CD34+ progenitor cells. Retroviral transduction of rhesus macaque CD34+ progenitor cells with a retroviral vector (p9456t) encoding the SIV-specific ribozyme and the selectable marker neomycin phosphotransferase in the presence of bone marrow stroma and in the absence of exogenous cytokines resulted in efficient transduction of both colony-forming units and long-term culture-initiating cells, with transduction efficiencies ranging between 21% and 56%. After transduction, CD34+ cells were cultured on rhesus thymic stromal culture (to support in vitro differentiation of T cells) or in the presence of cytokines (to support differentiation of macrophage-like cells). After expansion and selection with the neomycin analog G418, cells derived from transduced progenitor cells were challenged with SIV. CD4+ T cells derived from CD34+ hematopoietic cells transduced with the ribozyme vector p9456t were highly resistant to challenge with SIV, exhibiting up to a 500-fold decrease in SIV replication, even after high multiplicities of infection. Macrophages derived from CD34+ cells transduced with the 9456 ribozyme exhibited a comparable level of inhibition of SIV replication. These results show that a hairpin ribozyme introduced into CD34+ hematopoietic progenitor cells can retain the ability to inhibit AIDS virus replication after T-cell differentiation and support the feasibility of intracellular immunization of hematopoietic stem cells against infection with HIV and SIV. Protection of multiple hematopoietic lineages with the SIV-specific ribozyme should permit analysis of stem cell gene therapy for AIDS in the SIV/macaque model.

    Title A Herpesvirus of Rhesus Monkeys Related to the Human Kaposi's Sarcoma-associated Herpesvirus.
    Date December 1997
    Journal Journal of Virology
    Excerpt

    A herpesvirus that is related to but distinct from the Kaposi's sarcoma-associated herpesvirus (KSHV, or human herpesvirus 8) was isolated from rhesus monkeys. The sequence of 10.6 kbp from virion DNA revealed the presence of an interleukin-6 homolog similar to what is present in KSHV and a closer relatedness of the DNA polymerase and glycoprotein B reading frames to those of KSHV than to those of any other herpesvirus. This rhesus monkey herpesvirus replicated lytically and to high titers in cultured rhesus monkey fibroblasts. Antibody testing revealed a high prevalence for at least 10 years in our rhesus monkey colony and a high prevalence in two other colonies that were tested. Thus, rhesus monkeys naturally harbor a virus related to KSHV, which we have called RRV, for rhesus monkey rhadinovirus.

    Title The Role of Bone Imaging in Orthopedic Practice.
    Date December 1997
    Journal Seminars in Nuclear Medicine
    Excerpt

    Bone scans fill the void left by anatomical imaging by providing a metabolic assessment of change in local bone turnover associated with many common disorders of the skeleton. Bone scanning is valuable in uncovering skeletal lesions that are obscure, the extent of known lesions, and following their natural evolution. Its high sensitivity and relative ease makes it an extremely useful tool in the diagnosis and management of a wide variety of skeletal disorders.

    Title Persistence of Escherichia Coli O157:h7 in Dairy Cattle and the Dairy Farm Environment.
    Date December 1997
    Journal Epidemiology and Infection
    Excerpt

    The persistence of Escherichia coli O157:H7 in cattle and the farm environment was investigated on eight Ontario dairy farms positive for E. coli O157:H7 in a longitudinal study commenced one year previously. Faecal samples from cows, calves, humans, cats, rodents, wild birds, a composite fly sample and numerous composite and individual environmental samples were cultured and tested for verotoxin-producing E. coli (VTEC). VTEC isolates were serotyped and E. coli O157:H7 isolates were phage typed. E. coli O157:H7 phage type 34 was isolated from one calf on each of two farms. The same phage type had been isolated on one of these farms 12 months earlier. Most E. coli O157:H7-positive animals and farms became culture-negative within 2 and 3 months, respectively. E. coli O157:H7 was not isolated from any environmental samples, although evidence of VTEC was found in composite samples from calf feeders (19.1%), calf barn surfaces (18%), cow feeders (14.9%), flies (12.5%), cow barn surfaces (11.3%), and individual milk filters (12.5%). VTEC belonging to 21 non-O157 serotypes were isolated from 24 cows (8.2%), 21 calves (18.3%), 2 cow feeder samples (3.0%), and 1 calf feeder sample (4.8%). Shedding of E. coli O157:H7 by infected dairy cattle appears to be transient and persistence of E. coli O157:H7 was not demonstrated from the farm environment sites tested.

    Title Lysis of Hiv-1-infected Cells and Inhibition of Viral Replication by Universal Receptor T Cells.
    Date November 1997
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    Increasing evidence suggests that HIV-1-specific cytotoxic T lymphocytes (CTLs) are a key host immune response to HIV-1 infection. Generation of CTL responses for prevention or therapy of HIV-1 infection has several intrinsic technical barriers such as antigen expression and presentation, the varying HLA restrictions between different individuals, and the potential for viral escape by sequence variation or surface molecule alteration on infected cells. A strategy to circumvent these limitations is the construction of a chimeric T cell receptor containing human CD4 or HIV-1-specific Ig sequences linked to the signaling domain of the T cell receptor zeta chain (universal T cell receptor). CD8+ CTLs transduced with this universal receptor can then bind and lyse infected cells that express surface HIV-1 gp120. We evaluated the ability of universal-receptor-bearing CD8+ cells from a seronegative donor to lyse acutely infected cells and inhibit HIV-1 replication in vitro. The kinetics of lysis and efficiency of inhibition were comparable to that of naturally occurring HIV-1-specific CTL clones isolated from infected individuals. Further study will be required to determine the utility of these cells as a therapeutic strategy in vivo.

    Title Enhanced Maintenance and Retroviral Transduction of Primitive Hematopoietic Progenitor Cells Using a Novel Three-dimensional Culture System.
    Date November 1997
    Journal Gene Therapy
    Excerpt

    Current techniques for the in vitro maintenance of hematopoietic stem cells often lead to loss of pluripotency. Overcoming the technical difficulties that result in alterations in stem cells in vitro has important implications for areas of basic science and clinical medicine such as cell expansion, bone marrow transplantation and gene therapy. Recent insights into hematopoietic stem cell biology have demonstrated that the three-dimensional architecture of the culture environment may influence the maintenance of stem cell pluripotency in vitro. An intriguing hypothesis is that the utilization of three-dimensional culture systems may improve the maintenance and manipulation of these cells in vitro. We report that a novel, three-dimensional, tantalum-coated porous biomaterial (TCPB) may be employed effectively as a hematopoietic progenitor cell culture device that offers distinct advantages over conventional culture systems. Specifically, we demonstrate that the use of TCPB for culturing hematopoietic progenitor cells in the absence of exogenous cytokines results in enhanced hematopoietic progenitor cell survival, improved maintenance of the immature CD34+/38- phenotype, and improved retroviral transduction of CD34+ cells and long-term culture initiating cells (LTCIC), without compromising multipotency, as compared with cultures in plastic dishes or bone marrow stroma. These findings suggest that this three-dimensional culture system may be useful in advancing the in vitro culture and transduction of hematopoietic stem and progenitor cells.

    Title Four Color Immunofluorescence Detection Using Two 488-nm Lasers on a Becton Dickinson Facs Vantage Flow Cytometer.
    Date November 1997
    Journal Cytometry
    Excerpt

    Multiparameter flow cytometric analysis is sometimes limited by the availability of directly conjugated monoclonal antibodies or streptavidin-conjugated secondary reagents. While many manufacturers offer a wide variety of monoclonal antibodies or streptavidin reagents directly conjugated to 488-nm excitable fluorochromes, there are not many available that are directly conjugated to 360-nm (UV) or 630-nm (HeNe) excitable fluorochromes. For this reason we attempted to develop a four color immunofluorescence staining protocol on a FACS Vantage using four 488-nm excitable fluorochromes. The fixed configuration of the FACS Vantage limits the feasibility of using four 488-nm excitable fluorochrome simultaneously because of the five fluorescence detectors, two are always electronically delayed. This means that only three signals-FL1, FL2, and FL3-can be detected off the primary 488-nm laser beam. FL4 and FL5 are always delayed, only detecting signals off the second laser beam. Our instrument is configured with an ILT air cooled 488-nm laser in the second position that is used in order to conserve the Coherent Enterprise laser when using only 488-nm excitable fluorochromes. Because of this, we were able to develop a four-color immunofluorescence staining protocol using only 488-nm excitable fluorochromes.

    Title Induction of Vigorous Cytotoxic T-lymphocyte Responses by Live Attenuated Simian Immunodeficiency Virus.
    Date October 1997
    Journal Journal of Virology
    Excerpt

    Although live attenuated vaccine strains of simian immunodeficiency virus (SIV) have proven highly effective in protecting macaques against challenge with pathogenic SIV strains, little is known about the mechanisms of protective immunity induced by these vaccines. We examined cytotoxic T-lymphocyte (CTL) responses against SIV in animals infected with SIVmac239delta nef (deficient in nef) or SIVmac239delta 3 (deficient in nef, vpr, and upstream sequences in U3). To enhance detection of SIV-specific CTL activity, we stimulated peripheral blood mononuclear cells with autologous B-lymphoblastoid cell lines which had been infected with recombinant vaccinia viruses expressing SIV proteins and subsequently inactivated with psoralen and UV light. Animals chronically infected with SIV239delta nef or SIV239delta 3 mounted vigorous CTL responses against the SIV Gag and Env proteins. This CTL activity was major histocompatibility class restricted and mediated by CD8+ T lymphocytes. CTL responses persisted at relatively high levels for more than 6 years after infection. Limiting dilution precursor frequency assays demonstrated that the frequency of SIV-specific CTLs was as high as 234 CTL precursors per 100,000 cells. Animals acutely infected with SIV239delta nef developed CTL activity by day 14 after infection, coincident with decreases in viral load. Animals acutely infected with SIV239delta 3 developed CTL responses within 4 weeks of infection. Thus, vaccination of juvenile or adult animals with SIV239delta nef or SIV239delta 3 results in the induction of a vigorous CTL response which arises early in the course of infection and persists for years after a single inoculation of virus.

    Title Suppression of Human Immunodeficiency Virus Type 1 Replication by Cd8+ Cells: Evidence for Hla Class I-restricted Triggering of Cytolytic and Noncytolytic Mechanisms.
    Date April 1997
    Journal Journal of Virology
    Excerpt

    Although CD8+ lymphocytes in human immunodeficiency virus type 1 (HIV-1)-infected individuals have been demonstrated to suppress viral replication, the mechanisms of inhibition have not been defined precisely. A large body of evidence indicates that these cells act via soluble inhibitory factors, but the potential role of HLA class I-restricted cytolysis has remained controversial. Here we demonstrate that HIV-1-specific cytotoxic T lymphocytes (CTL) mediate antiviral suppression by both cytolytic and noncytolytic mechanisms. The predominant mechanism requires direct contact of CTL with the infected cells, is HLA class I restricted, and can achieve complete elimination of detectable virus in infected cell cultures. Inhibition occurs even at high multiplicities of infection or at ratios of CTL to CD4 cells as low as 1:1,000. The other mechanism is mediated by soluble inhibitory factors which are triggered in an antigen-specific and HLA-restricted fashion but then act without HLA restriction. These include MIP-1alpha, MIP-1beta, and RANTES, as well as a distinct factor(s) capable of inhibiting HIV-1 strains insensitive to these chemokines. These data indicate that HIV-1-specific CTL are potent mediators of HIV-1 suppression at cell ratios existing in vivo and demonstrate an antigen-specific trigger for CD8+ cell-derived soluble inhibitory factors. These results suggest that CTL play an important role in the observed antiviral activity of CD8+ cells from infected individuals.

    Title Identification of Type-specific Cytotoxic T Lymphocyte Responses to Homologous Viral Proteins in Laboratory Workers Accidentally Infected with Hiv-1.
    Date March 1997
    Journal The Journal of Clinical Investigation
    Excerpt

    Characterization of the cytotoxic T lymphocyte (CTL) response against HIV-1 has been limited by the use of target cells expressing viral proteins from laboratory isolates of HIV-1. This approach has favored identification of group-specific CTL responses and precluded assessment of the extent of type-specific CTL responses directed against HIV-1. Using cells expressing viral proteins from the HIV-1 IIIB strain, we performed a detailed characterization of HIV-1-specific CTL response in three laboratory workers accidentally infected with HIV-1 IIIB. Eight of the epitopes identified were group specific, lying in relatively conserved regions of Gag, reverse transcriptase, and envelope. Three type-specific epitopes were identified, two of them in highly variable regions of envelope. In longitudinal studies in one subject, seven different epitopes and five different restricting HLA class I alleles were identified, with a progressive increase in the number of CTL epitopes recognized by this subject over time. Our data demonstrate that type-specific CTL responses make up a significant proportion of the host cellular immune response against HIV-1 and that a broadening of epitope specificity may occur.

    Title Overlapping Epitopes in Human Immunodeficiency Virus Type 1 Gp120 Presented by Hla A, B, and C Molecules: Effects of Viral Variation on Cytotoxic T-lymphocyte Recognition.
    Date February 1997
    Journal Journal of Virology
    Excerpt

    Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) are thought to exert immunologic selection pressure in infected persons, yet few data regarding the effects of this constraint on viral sequence variation in vivo, particularly in the highly variable Env protein, are available. In this study, CD8+ HIV type 1 (HIV-1) envelope-specific CTL clones specific for gp120 were isolated from peripheral blood mononuclear cells of four HIV-infected individuals, all of which recognized the same 25-amino-acid (aa) peptide (aa 371 to 395), which is partially contained in the CD4-binding domain of HIV-1 gp120. Fine mapping studies revealed that two of the clones optimally recognized the 9-aa sequence 375 to 383 (SFNCGGEFF), while the two other clones optimally recognized the epitope contained in the overlapping 9-aa sequence 376 to 384 (FNCGGEFFY). Lysis of target cells by the two clones recognizing aa 375 to 383 was restricted by HLA B15 and Cw4, respectively, whereas both clones recognizing aa 376 to 384 were restricted by HLA A29. Sequence variation, relative to the IIIB strain sequence used to identify CTL clones, was observed in autologous viruses in the epitope-containing region in all four subjects. However, poorly recognized autologous sequence variants were predominantly seen for the A29-restricted clones, whereas the clones specific for SFNCGGEFF continued to recognize the predominant autologous sequences. These results suggest that the HLA profile of an individual may not only be important in determining the specificity of CTL recognition but may also affect the ability to recognize virus variants and suppress escape from CTL recognition. These results also identify overlapping viral CTL epitopes which can be presented by HLA A, B, and C molecules.

    Title Cytotoxic T-lymphocyte Responses to Cytomegalovirus in Normal and Simian Immunodeficiency Virus-infected Rhesus Macaques.
    Date December 1996
    Journal Journal of Virology
    Excerpt

    Disseminated cytomegalovirus (CMV) infection is a frequent occurrence in human immunodeficiency virus-infected humans and in simian immunodeficiency virus (SIV)-infected rhesus macaques. Rhesus macaques are a suitable animal model with which to study in vivo interactions between CMV and AIDS-associated retroviruses. Since cytotoxic T lymphocytes (CTL) play a major role in control of viral infections, we have characterized CMV-specific CTL responses in SIV-infected and uninfected rhesus macaques. Autologous fibroblasts infected with rhesus CMV were used to stimulate freshly isolated peripheral blood mononuclear cells from CMV-seropositive animals. Following in vitro stimulation, specific CTL activity against CMV-infected autologous fibroblasts was detected in CMV-seropositive but not in CMV-seronegative normal macaques. CMV-specific CTL activity comparable to that in normal animals was also detected in two CMV-seropositive macaques infected with a live attenuated SIV strain (SIVdelta3) and in two of three macaques infected with pathogenic SIV strains. The CMV-specific CTL response was class I major histocompatibility complex restricted and mediated by CD8+ cells. An early CMV protein(s) was the dominant target recognized by bulk CTL, although the pattern of CTL recognition of CMV proteins varied among animals. Analysis of CMV-specific CTL responses in macaques should serve as a valuable model for CMV immunopathogenesis and will facilitate prospective in vivo studies of immune interactions between CMV and SIV in AIDS.

    Title Humoral and Cellular Immune Responses of Nonhuman Primates to Long-term Repeated Lung Exposure to Ad2/cftr-2.
    Date November 1996
    Journal Gene Therapy
    Excerpt

    To evaluate the host immune response to long-term repeat administration of adenovirus vector, rhesus monkeys were treated at intervals of approximately 3 weeks with up to 18 instillations of Ad2/CFTR-2, a second generation vector encoding the cystic fibrosis transmembrane conductance regulator (CFTR). All monkeys instilled with Ad2/CFTR-2 developed a significant humoral immune response against adenovirus but not CFTR. Antibodies with virus neutralizing activity were detected in the serum and bronchoalveolar lavage (BAL) of all vector-treated monkeys and included both IgG and secretory IgA. Virus-specific T cells capable of proliferating in response to stimulation with adenovirus antigen were detected in all vector-treated monkeys. No CFTR-specific proliferation of peripheral blood lymphocytes was detected. An increase in the proportion of CD8+ T cells was noted in the BAL of virus-treated monkeys but cells from the BAL displayed little or no cytolytic activity against infected autologous fibroblasts when tested under a variety of culture conditions. However, MHC-restricted cytolytic activity was detected in the tracheobronchial lymph nodes and spleen of one of three virus-treated monkeys tested. MHC-unrestricted killing of infected fibroblasts was also observed with spleen cells from all animals tested. From these results, it appears that both the humoral and cell-mediated arms of the immune response were stimulated by repeated administration of high doses of Ad2/CFTR-2 suggesting that effective, long-term adenovirus gene therapy may require modification of the vector or treatment of the host to allow the virus to evade host immune defenses.

    Title Strong Cytotoxic T Cell and Weak Neutralizing Antibody Responses in a Subset of Persons with Stable Nonprogressing Hiv Type 1 Infection.
    Date October 1996
    Journal Aids Research and Human Retroviruses
    Excerpt

    Some individuals in well-defined cohorts have now been infected with HIV-1 for well over a decade and yet remain clinically asymptomatic with normal CD4 counts. To determine immunologic and virologic parameters in these individuals, we examined 10 persons from the San Francisco City Clinic with firmly documented infection of 11-15 years duration who had maintained stable CD4 counts above 500 cells/microliters. Our results indicate that long-term nonprogressors are a heterogeneous group with respect to viral load and HIV-1-specific immune responses, and that progression can occur even after 15 years of stable infection. However, in a subset of persons with the lowest viral loads and persistent nonprogressive infection, we detected strong CTL responses, whereas neutralizing antibody studies revealed weak to undetectable titers against a panel of 10 primary isolates. This study demonstrates that a vigorous in vivo activated HIV-1-specific CTL response can be part of the host immune response in stable nonprogressive HIV-1 infection, and that circulating activated CTL can be detected in the setting of an extremely low viral load. These results also indicate that long-term nonprogressing HIV-1 infection does not require the presence of broadly cross-reactive neutralizing antibodies.

    Title Cytotoxic T Lymphocytes in Asymptomatic Long-term Nonprogressing Hiv-1 Infection. Breadth and Specificity of the Response and Relation to in Vivo Viral Quasispecies in a Person with Prolonged Infection and Low Viral Load.
    Date September 1996
    Journal Journal of Immunology (baltimore, Md. : 1950)
    Excerpt

    Although vigorous activated and memory CTL have been associated with HIV-1 infection, data are lacking regarding the breadth of epitopes recognized in a given individual and the relationship to the viral quasispecies present in vivo. In this study we performed a detailed analysis of the HIV-1-specific CTL response in a seropositive person with documented HIV-1 infection of 15 yr duration, stable CD4 counts above 500 cells/ml, and viral load persistently below 500 molecules of RNA/ml of plasma. Epitope mapping studies revealed the presence of HLA class I-restricted CTL responses to six different epitopes in p17, p24, RT, Env, and Nef, which conferred broadly cross-reactive recognition of reported HIV-1 variants. Sequence analysis of autologous viruses revealed the absence of immune escape variants within five of the six epitopes. Despite consistently low viral RNA levels in plasma and viral DNA levels in PBMC, in vivo-activated circulating CTL were detected against three of the epitopes. Five of the six epitopes, including the three dominant epitopes, have been detected in persons with progressive disease, suggesting that nonprogressors may not target unique epitopes. This study demonstrates that HIV-1-specific CTL can be highly activated and broadly directed in the setting of an extremely low viral load, and that neither high viral load nor antigenic diversity is required for the generation of a multispecific CTL response. Although the detection of strong CTL responses, low viral load, and lack of immune escape are consistent with the hypothesis that CTL may contribute to lack of disease progression in this individual, the contribution of these responses to maintenance of the asymptomatic state remains to be determined.

    Title Efficient Lysis of Human Immunodeficiency Virus Type 1-infected Cells by Cytotoxic T Lymphocytes.
    Date September 1996
    Journal Journal of Virology
    Excerpt

    Numerous studies of human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T lymphocytes (CTL) have examined their ability to recognize B-cell lines expressing recombinant HIV-1 proteins, but relatively few data regarding the lysis of HIV-1-infected cells by CTL are available. We studied the ability of HIV-1-specific CTL clones of defined epitope specificity and HLA restriction to lyse infected CD4+ cells at serial time points following infection. CD4+ cell lines were acutely infected with HIV-1 IIIB at a high multiplicity of infection, and the kinetics of cell lysis were examined and compared with the kinetics of viral replication. Intracellular HIV-1 p24 expression was detected by 1 to 2 days after infection, reaching over 98% positive cells by day 4. Recognition of the infected cells by HLA A2- and B14-restricted CTL clones closely paralleled intracellular p24 expression and preceded peak virion production. The maximal levels of lysis with Gag-, reverse transcriptase-, and envelope-specific clones were different, however. The Gag- and envelope-specific clones lysed infected cells at levels equivalent to peptide-sensitized controls, whereas lysis by the reverse transcriptase-specific clones plateaued at a lower level. Peptide titration curves indicated that this effect was not due to differences in sensitivity to the cognate epitopes for the different clones. Although HIV-1 infection induced an approximately 50% decrease in class I HLA expression on the surface of infected cells, lysis by CTL clones was unaffected. These studies indicate that HIV-1-specific CTL can efficiently lyse HIV-1-infected CD4+ cells and suggest that the partial downregulation of class I molecules in infected cells does not significantly affect recognition by CTL.

    Title T Cell Receptor Usage and Fine Specificity of Human Immunodeficiency Virus 1-specific Cytotoxic T Lymphocyte Clones: Analysis of Quasispecies Recognition Reveals a Dominant Response Directed Against a Minor in Vivo Variant.
    Date August 1996
    Journal The Journal of Experimental Medicine
    Excerpt

    Numerous virus-specific, class I-restricted cytotoxic T lymphocyte (CTL) epitopes have been identified, yet little information is available regarding the specificity of the CTL response in persons of the same human histocompatibility leukocyte antigen (HLA) type. In this study, the human immunodeficiency virus (HIV) 1 envelope-specific CTL response was evaluated in five HLA-B14-positive persons. CTL responses specific for a previously described nine-amino acid epitope in gp41 (aa 584-592, ERYLKDQQL) could be identified in all subjects, and CTL clones specific for this epitope could be isolated from four persons. Despite heterogeneous T cell receptor usage, the fine specificity of the clones was similar, as defined by recognition of alanine-substituted peptides as well as peptides representing natural HIV-1 sequence variants. Correlation with in vivo virus sequences revealed that the dominant species in two of the subjects represented poorly recognized variants, with a K-->Q substitution at amino acid 588, whereas no variants were observed in the other two subjects. Although clonal type-specific responses to these dominant variants could be identified, the magnitude of these responses remained small, and the dominant CTL response was directed at the minor in vivo variant. These studies indicate that despite similar epitope-specific immunologic pressure in persons of the same HLA type, the in vivo quasispecies may differ, and that the major in vivo immune response to a given CTL epitope can be directed at a minor variant.

    Title In Vitro T Lymphopoiesis of Human and Rhesus Cd34+ Progenitor Cells.
    Date July 1996
    Journal Blood
    Excerpt

    Differentiation of hematopoietic progenitor cells into T lymphocytes generally occurs in the unique environment of the thymus, a feature that has hindered efforts to model this process in the laboratory. We now report that thymic stromal cultures from rhesus macaques can support T-cell differentiation of human or rhesus CD34+ progenitor cells. Culture of rhesus or human CD34+ bone marrow-derived cells depleted of CD34+ lymphocytes on rhesus thymic stromal monolayers yielded CD3+CD4+CD8+, CD3+CD4+CD8-, and CD3+CD4-CD8+ cells after 10 to 14 days. In addition to classical T lymphocytes, a discrete population of CD3+CD8loCD16+CD56+ cells was detected after 14 days in cultures inoculated with rhesus CD34+ cells. CD3+ T cells arising from these cultures were not derived from contaminating T cells present in the CD34+ cells used to inoculate thymic stromal monolayers or from the thymic monolayers, as shown by labeling of cells with the lipophilic membrane dye PKH26. Expression of the recombinase activation gene RAG-2, which is selectively expressed in developing lymphocytes, was detectable in thymic cultures inoculated with CD34+ cells but not in CD34+ cells before thymic culture or in thymic stromal monolayers alone. Reverse transcriptase-polymerase chain reaction analysis of T cells derived from thymic stromal cultures of rhesus and human CD34+ cells showed a polyclonal T-cell receptor repertoire. T-cell progeny derived from rhesus CD34+ cells cultured on thymic stroma supported vigorous simian immunodeficiency virus replication in the absence of exogenous mitogenic stimuli. Rhesus thymic stromal cultures provide a convenient means to analyze T-cell differentiation in vitro and may be useful as a model of hematopoietic stem cell therapy for diseases of T cells, including acquired immunodeficiency syndrome.

    Title Ex Vivo Expansion of Cd4 Lymphocytes from Human Immunodeficiency Virus Type 1-infected Persons in the Presence of Combination Antiretroviral Agents.
    Date July 1995
    Journal The Journal of Infectious Diseases
    Excerpt

    Expansion of CD4 lymphocytes from human immunodeficiency virus type 1 (HIV-1)-infected persons ex vivo has been limited by enhanced virus replication and cell death. The successful expansion of functional CD4 lymphocytes from HIV-1-infected persons has now been accomplished using a bispecific monoclonal antibody to CD3 and CD8 in combination with three antiretroviral agents. CD4 lymphocytes were polyclonally expanded by a factor of 10(3)-10(7) during 4-8 weeks in culture. Supernatants from most cultures were persistently HIV-1 p24 antigen-negative by day 14 and remained negative despite removal of antiretroviral agents at day 28. In such cultures, HIV-1 could not be recovered by cocultivation, and amounts of HIV-1 DNA declined or remained stable at low levels, eventually becoming undetectable in 2 cases. This approach establishes the feasibility of CD4 lymphocyte expansion in persons with HIV disease and may be useful for immune-based or gene therapies.

    Title An Epitope-selective, Transporter Associated with Antigen Presentation (tap)-1/2-independent Pathway and a More General Tap-1/2-dependent Antigen-processing Pathway Allow Recognition of the Hiv-1 Envelope Glycoprotein by Cd8+ Ctl.
    Date June 1995
    Journal Journal of Immunology (baltimore, Md. : 1950)
    Excerpt

    The lysis of virally infected cells by CTLs requires the recognition of processed fragments of viral proteins presented in association with class I MHC molecules on the surfaces of infected cells. Processing begins in the cytosol with the degradation of viral proteins into peptides that are then transported into the endoplasmic reticulum (ER) for association with newly synthesized class I molecules. Transport is mediated by a heterodimer of the MHC-encoded proteins, transporter associated with Ag presentation (TAP)-1 and TAP-2. Uncertainty exists over the site of processing of viral envelope (env) proteins. The extracellular domains of env proteins are not present in the cytosol, the site in which the class I-restricted Ag-processing pathway begins. Rather, the ecto-domains of env proteins are cotranslationally translocated into the ER during biosynthesis. We have analyzed the processing of the HIV-1 env protein by using a large series of env-specific human CD8+ CTL clones. These studies have led to the delineation of two distinct processing pathways. The first pathway permits a subset of class I-restricted epitopes in the ecto-domain of the env protein to be generated efficiently by a TAP-1/2-independent mechanism localized to the ER or a premedial Golgi compartment. A second, more general pathway that is capable of generating all env epitopes uses as a substrate env protein mislocalized to the cytosol and produces peptides that are transported from the cytoplasm to the ER in a TAP-1/2-dependent fashion.

    Title Endogenous Endophthalmitis.
    Date April 1995
    Journal Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America
    Title Hiv-1-specific Cytotoxic T Lymphocyte Response in Healthy, Long-term Nonprogressing Seropositive Persons.
    Date March 1995
    Journal Aids Research and Human Retroviruses
    Title Cytotoxic Cd8+ T Lymphocytes Reactive with Human Immunodeficiency Virus-1 Produce Granulocyte/macrophage Colony-stimulating Factor and Variable Amounts of Interleukins 2, 3, and 4 Following Stimulation with the Cognate Epitope.
    Date January 1995
    Journal Clinical Immunology and Immunopathology
    Excerpt

    Infection with human immunodeficiency virus type 1 (HIV-1) induces vigorous and persistent cytotoxic CD8+ T cell responses. CTL clones were derived from peripheral blood or cerebrospinal fluid of three HIV-1 patients, with depressed CD4+ T cell counts. When stimulated with HLA-compatible target cells (B-LCL) presensitized with cognate HIV-1 peptides, all clones produced GM-CSF, TNF-alpha, and IFN-gamma and most produced low amounts of IL2, IL3, and IL4. After nonspecific stimulation with a phorbol ester and calcium ionophore, the clones secreted cytokines at levels similar to those from CD4+ lines from an HIV-1 infected donor. The ability of supernatants from the stimulated CTL clones to support the formation of granulocyte-macrophage colonies in normal bone marrow suggests that the GM-CSF was biologically active. Release of cytokines by activated CTL may influence the immunopathogenesis of HIV disease.

    Title Endogenous Bacterial Endophthalmitis. Report of a Ten-year Retrospective Study.
    Date June 1994
    Journal Ophthalmology
    Excerpt

    PURPOSE: The purpose of this study is to report the predisposing factors, timing of symptoms, timing of diagnosis, causative organisms, source of infection, and visual outcome in cases of endogenous bacterial endophthalmitis. METHODS: The records of 28 patients with endogenous bacterial endophthalmitis who presented to our combined ophthalmology and medicine services over a 10-year period were reviewed. RESULTS: Ninety percent of the patients had prior medical conditions, including diabetes mellitus, gastrointestinal disorders, hypertension, cardiac disorders, and malignancy. Acute ocular symptoms were the most common reasons why the patient went to the physician (usually an ophthalmologist) rather than systemic symptoms. A correct initial diagnosis was made in 50% of patients, with a delay in diagnosis of 4 days or more in 29% of patients. Organisms were identified in 27 of the 28 patients (96% identification rate), two thirds of which were gram-positive organisms. Streptococcal species were the most common group (32% of patients), although the single most common organism was Staphylococcus aureus (25% of patients). Sources of infection were identified in 93% of patients, with endocarditis and the gastrointestinal tract being the most common. In the majority of patients, visual outcome was poor. However, six eyes that received intravenous and intravitreal antibiotics, as well as therapeutic vitrectomy, achieved visual acuities of 20/50 or better. CONCLUSION: These results provide a further understanding of the manner of presentation, organisms involved, and sources of infection in endogenous bacterial endophthalmitis. They also suggest that improved outcome may be associated with early initiation of combined medical and surgical treatment.

    Title Longitudinal Analysis of T Cell Receptor (tcr) Gene Usage by Human Immunodeficiency Virus 1 Envelope-specific Cytotoxic T Lymphocyte Clones Reveals a Limited Tcr Repertoire.
    Date May 1994
    Journal The Journal of Experimental Medicine
    Excerpt

    Human immunodeficiency virus 1 (HIV-1) infection is associated with a vigorous cellular immune response that allows detection of cytotoxic T lymphocyte (CTL) activity using freshly isolated peripheral blood mononuclear cells (PBMC). Although restricting class I antigens and epitopes recognized by HIV-1-specific CTL have been defined, the effector cells mediating this vigorous response have been characterized less well. Specifically, no studies have addressed the breadth and duration of response to a defined epitope. In the present study, a longitudinal analysis of T cell receptor (TCR) gene usage by CTL clones was performed in a seropositive person using TCR gene sequences as a means of tracking responses to a well-defined epitope in the glycoprotein 41 transmembrane protein. 10 CTL clones specific for this human histocompatibility leukocyte antigen-B14-restricted epitope were isolated at multiple time points over a 31-mo period. All clones were derived from a single asymptomatic HIV-1-infected individual with a vigorous response to this epitope that was detectable using unstimulated PBMC. Polymerase chain reaction amplification using V alpha and V beta family-specific primers was performed on each clone, followed by DNA sequencing of the V-D-J regions. All 10 clones utilized V alpha 14 and V beta 4 genes. Sequence analysis of the TCR revealed the first nine clones isolated to also be identical at the nucleotide level. The TCR-alpha junctional region sequence of the tenth clone was identical to the junctional region sequences of the other nine, but this clone utilized distinct D beta and J beta gene segments. This study provides evidence that the observed high degree of HIV-1-specific CTL activity may be due to monoclonal or oligoclonal expansion of specific effector cells, and that progeny of a particular CTL clone may persist for prolonged periods in vivo in the presence of a chronic productive viral infection. The observed limited TCR diversity against an immunodominant epitope may limit recognition of virus variants with mutations in regions interacting with the TCR, thereby facilitating immune escape.

    Title Viruses Accumulate Spontaneously Near Droplet Surfaces: a Method to Concentrate Viruses for Electron Microscopy.
    Date November 1993
    Journal Journal of Microscopy
    Excerpt

    Virus particles suspended in a drop of water tend to concentrate at or near its surface with the air. The concentrated, and probably more purified, particles may then be collected on a film-coated grid for negative staining and electron microscopy. This is a useful method, simpler than others (e.g. high-speed centrifugation, Lyphogel, or precipitation by (NH4)2SO4) which are used to process clinical specimens for diagnosis where virus particles may be too dilute in the original sample. It is shown, by freeze-fracturing for electron microscopy, that Orf virus particles do accumulate at and just below the surfaces of drops. Various physical effects which may cause the particles to accumulate are considered. Results from a computer model suggest that Brownian motion alone could be adequate to transport a useful quantity of the particles in the body of a 2-mm-diameter hemispherical drop to its surface if the particles do not clump and if they remain trapped at the surface when they reach it. In practice, transport by Brownian motion is likely to be augmented by swirling, convection and other effects within drops.

    Title Recognition of a Highly Conserved Region of Human Immunodeficiency Virus Type 1 Gp120 by an Hla-cw4-restricted Cytotoxic T-lymphocyte Clone.
    Date January 1993
    Journal Journal of Virology
    Excerpt

    Human immunodeficiency virus type 1 (HIV-1) isolates exhibit extensive sequence variation, particularly in the gp120 subunit of the envelope glycoprotein, and the degree of this variation has raised questions as to whether conserved regions of the HIV-1 envelope can be recognized by the host immune response. A CD8+ cytotoxic T-lymphocyte (CTL) clone specific for the HIV-1 envelope was derived by culturing peripheral blood mononuclear cells from an HIV-1 seropositive subject in the presence of a CD3-specific monoclonal antibody, interleukin-2, and irradiated allogeneic peripheral blood mononuclear cells. Lysis of target cells was restricted by an HLA-C molecule, Cw4, which has not been previously shown to present viral antigen to CTL. Mapping of the specificity of this CTL clone by using synthetic HIV-1 peptides localized the epitope to an 8-amino-acid region of gp120 (amino acids 376 to 383) which is conserved among approximately 90% of sequenced viral isolates. Examination of the recognition of variant peptides by this CTL clone demonstrated that a single, nonconservative amino acid substitution within the 8-amino-acid minimal epitope could abrogate lysis of targets incubated with the variant peptide. The identification of a CTL epitope in a highly conserved region of gp120 documents the ability of cellular immune responses of infected persons to respond to relatively invariant portions of this highly variable envelope glycoprotein. However, the ability of even a single-amino-acid change in gp120 to abolish lysis by CTL supports the hypothesis that sequence variation in HIV-1 may serve as a mechanism of immune escape. In addition, the identification of an HLA-C molecule presenting viral antigen to CTL supports a functional role for these molecules.

    Title Identification of Overlapping Hla Class I-restricted Cytotoxic T Cell Epitopes in a Conserved Region of the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein: Definition of Minimum Epitopes and Analysis of the Effects of Sequence Variation.
    Date April 1992
    Journal The Journal of Experimental Medicine
    Excerpt

    Although the immunologic basis of protective immunity in human immunodeficiency virus type 1 (HIV-1) infection has not yet been defined, virus-specific cytotoxic T lymphocytes (CTL) are likely to be an important host defense and may be a critical feature of an effective vaccine. These observations, along with the inclusion of the HIV-1 envelope in the majority of vaccine candidates presently in clinical trials, underscore the importance of the precise characterization of the cellular immune responses to this protein. Although humoral immune responses to the envelope protein have been extensively characterized, relatively little information is available regarding the envelope epitopes recognized by virus-specific CTL and the effects of sequence variation within these epitopes. Here we report the identification of two overlapping CTL epitopes in a highly conserved region of the HIV-1 transmembrane envelope protein, gp41, using CTL clones derived from two seropositive subjects. An eight-amino acid peptide was defined as the minimum epitope recognized by HLA-B8-restricted CTL derived from one subject, and in a second subject, an overlapping nine-amino acid peptide was identified as the minimal epitope for HLA-B14-restricted CTL clones. Selected single amino acid substitutions representing those found in naturally occurring HIV-1 isolates resulted in partial to complete loss of recognition of these epitopes. These data indicate the presence of a highly conserved region in the HIV-1 envelope glycoprotein that is immunogenic for CTL responses. In addition, they suggest that natural sequence variation may lead to escape from immune detection by HIV-1-specific CTL. Since the region containing these epitopes has been previously shown to contain an immunodominant B cell epitope and also overlaps with a major histocompatibility complex class II T cell epitope recognized by CD4+ CTL from HIV-1 rgp160 vaccine recipients, it may be particularly important for HIV-1 vaccine development. Finally, the identification of minimal CTL epitopes presented by class I HLA molecules should facilitate the definition of allele-specific motifs.

    Title Hiv-1 Gag-specific Cytotoxic T Lymphocytes Recognize Multiple Highly Conserved Epitopes. Fine Specificity of the Gag-specific Response Defined by Using Unstimulated Peripheral Blood Mononuclear Cells and Cloned Effector Cells.
    Date September 1991
    Journal Journal of Immunology (baltimore, Md. : 1950)
    Excerpt

    CTL directed at the highly conserved HIV-1 gag protein have been described in HIV-1 seropositive persons and may be an important host defense against this retrovirus. Presently only limited data are available regarding the specific epitopes recognized by these CTL. In this study, we have performed a detailed examination of the gag-specific CTL response in three HIV-1 seropositive subjects, using both unstimulated PBMC and cloned CTL. Lysis of gag-expressing targets was found to be mediated by CD3+CD8+ lymphocytes and restricted by class I Ag. Multiple class I Ag were found to restrict gag epitopes in each subject studied, with as many as three of these Ag involved in presenting gag CTL epitopes in a single subject. The majority of gag-specific CTL activity was found to be directed against epitopes in the p24 subunit of the gag protein, with at least seven different HLA class I-restricted CTL p24 epitopes identified in these three subjects. Less CTL activity was directed against p17 subunit of gag and two CTL epitopes were identified in this protein. Although as many as four different epitopes in gag were recognized using CTL from a single subject, none of the epitopes was recognized by CTL from more than one subject. Analysis of gag epitope recognition using cloned CTL demonstrated heterogeneity and specificity not appreciated using unstimulated PBMC. The identification of multiple relatively conserved epitopes in the HIV-1 gag protein and the heterogeneity of CTL responses to this protein may have important implications for vaccine development and our understanding of AIDS pathogenesis.

    Title Introducing the 'walk-up' Flap.
    Date December 1990
    Journal Oral Surgery, Oral Medicine, and Oral Pathology
    Excerpt

    Reconstructive oral and maxillofacial surgeons often find the need for additional soft tissue even when a myocutaneous flap has been previously placed. This article introduces the principles, technique, and results of 32 "walk-up" muscle flaps derived from existing myocutaneous flaps. The anastomotic vascular ingrowth at the original myocutaneous flap's distal end permits its proximal detachment and axial vessel ligation to rotate the proximal two thirds to a tissue-deficient site. In our experience at the University of Miami, these flaps have been predictable (97% viable transfer rate), have provided vascular soft tissue in deficient areas, and have been skin grafted at their surface to increase oral lining or skin cover, among several other uses.

    Title Update on Antiretroviral Agents Other Than Zidovudine.
    Date March 1990
    Journal Aids (london, England)
    Title Arteriolization of Venous Blood Gases: a Clue to the Diagnosis of Cyanide Poisoning.
    Date March 1989
    Journal The Journal of Emergency Medicine
    Excerpt

    Prompt diagnosis and treatment of cyanide poisoning is essential for a successful therapeutic outcome. We present a patient with acute cyanide poisoning in whom venous blood gases disclosed an abnormally high pO2. Prompt treatment of the patient with the cyanide antidote, sodium nitrite plus sodium thiosulfate, rapidly reversed the cardiovascular and central nervous system manifestations of cyanide toxicity. The pathogenesis of cyanide-induced blood gas abnormalities and their potential value in the recognition of cyanide intoxication are discussed. Current treatment recommendations for cyanide poisoning are also reviewed.

    Title Studies in the Radiobiology of Osteoradionecrosis and Their Clinical Significance.
    Date November 1987
    Journal Oral Surgery, Oral Medicine, and Oral Pathology
    Excerpt

    The radiobiology of osteoradionecrosis is a complex of cellular death and cellular functional impairments from radiation energy transfers. Four studies of irradiated patients and a data base from 536 patients with osteoradionecrosis revealed separate pathophysiologic conditions for osteoradionecrosis induced by early trauma, osteoradionecrosis induced by late trauma, and spontaneous osteoradionecrosis. A large body of data suggested useful clinical guidelines for the management of irradiated patients. The guidelines, in part, include a recommendation for deferring radiation treatment for 21 days after tissue wounding, if possible; a relative contraindication to wounding tissue during a radiation course; a recommendation for the use of hyperbaric oxygen before wounding; and a strong recommendation to provide comprehensive dental care to the irradiated patient.

    Title Use of Retrograde Bone Grafting in the Treatment of Osseous Defects of the Lateral Condyle of the Knee. A Preliminary Report of Three Knees in Two Patients.
    Date May 1987
    Journal Orthopedics
    Title Chronic Capitolunate Instability.
    Date November 1986
    Journal The Journal of Bone and Joint Surgery. American Volume
    Excerpt

    Twelve patients, twelve to thirty-two years old, were evaluated for complaints of chronic vague pain, weakness, and clicking in the wrist that had followed a significant but remote dorsiflexion injury to the wrist. Although a standard orthopaedic examination and plain roentgenograms of the carpus were unremarkable, a dorsal-displacement stress test done under fluoroscopic control with the radius fixed showed dorsal subluxation of the capitate out of the cup of the lunate, accompanied by a marked feeling of apprehension by the patient. This was also associated with a painful snap or click due to a sudden dorsal attitude and ulnar shift of the lunate, best elicited with the wrist in slight ulnar deviation. We believe that this condition is due to attenuation of the radiocapitate ligament resulting from prior trauma. Eleven patients were operated on. The volar radiocapitate ligament was tightened by tethering its central portion to the radiotriquetral ligament, partially obliterating the space of Poirier. Slight extension of the wrist was lost by this procedure, but the capitate could no longer be passively displaced and the lunate became stable. Using both objective and subjective criteria, six patients had an excellent result; three, good; one, fair; and one, poor. There was an average final loss of 15 degrees of extension and 19 degrees of flexion of the wrist. The average length of follow-up was four years and four months (range, twenty-four to 109 months). We concluded that insufficiency of the radiocapitate ligament after trauma to the wrist is one cause of chronic symptomatic capitolunate instability. Shortening of the radiocapitate ligament is recommended to stabilize the lunate and capitate.

    Title Olecranon Fractures Treated with Ao Screw and Tension Bands.
    Date April 1986
    Journal Orthopedics
    Excerpt

    Twenty-eight patients averaging 36.4 years of age underwent open reduction and internal fixation of olecranon fractures. The AO cancellous screw was used alone in 16 and with tension banding in 12. Banding was used when the bone was soft or severely comminuted. The fractures were clinically healed at an average of nine weeks and roentgenographic healing with obliteration of the fracture line occurred at 13 weeks. Sixty-seven percent acquired full motion by the ninth postoperative week. All but two patients regained full supination and pronation. No patient lost greater than 30 degrees of extension and only two lost greater than 30 degrees of flexion. There were few operative difficulties or postoperative complications. We found the AO cancellous screw alone and in severely comminuted cases in combination with tension band wiring to be an excellent fixation device for olecranon fractures. It allows for early range of motion in young patients with excellent healing prospects by 16 weeks.

    Title Chronic Knee Pain Assessed by Spect: Comparison with Other Modalities.
    Date December 1985
    Journal Radiology
    Excerpt

    Twenty-seven patients with chronic knee pain were examined prospectively using conventional radiography, radionuclide angiography, planar bone scintigraphy, and single-photon emission computed tomographic (SPECT) bone scintigraphy. When the results of subsequent arthroscopic examination of all three compartments of the knee were correlated with those of the noninvasive methods, SPECT bone scintigraphy was found to be most sensitive for evaluating the extent of osteoarthritis. Differences in detection sensitivity for articular cartilage damage and synovitis were greatest in the patellofemoral compartment. The frequency with which hyperemia was present in association with cartilage damage and synovitis indicates that osteoarthritis of the knee is capable of producing hyperemia and further implies that increased perfusion cannot be used to distinguish with confidence between osteoarthritis and septic processes involving the knee. SPECT (1.00) and planar (0.91) bone scintigraphy were highly sensitive indicators of torn menisci in a subgroup of 14 patients, each having a prearthroscopic clinical diagnosis of a torn meniscus. This result suggests that for patients with chronic knee pain and clinical suggestion of a torn meniscus, bone scintigraphy has significant potential as a high-sensitivity, prearthroscopic screening examination.

    Title Detection of Femoral Head Avascular Necrosis in Adults by Spect.
    Date October 1985
    Journal Journal of Nuclear Medicine : Official Publication, Society of Nuclear Medicine
    Excerpt

    Twenty-one adult patients with the clinical diagnosis of avascular necrosis (AVN) of the femoral head were examined with radionuclide angiography, planar bone scintigraphy, and single photon emission computed tomography (SPECT). A final diagnosis of AVN was established for 15 symptomatic patients with a total of 20 involved hips. SPECT and planar bone scintigraphy were considered positive for AVN only if a photopenic bony defect could be identified. Using SPECT bone scintigraphy, 12 of 15 symptomatic patients and 17 of 20 involved hips (sensitivity of 0.85) were correctly identified, whereas with planar imaging only eight of 15 patients and 11 of 20 involved hips were detected. There were no false-positive diagnoses on SPECT or planar bone scintigraphy. In addition, hyperemia in the region of the proximal femoral metaphysis was demonstrated in six of 20 involved hips. It is concluded that by identifying a photopenic defect that is not evident on planar views, SPECT can contribute to the diagnosis of AVN of the femoral head. In addition, metaphyseal hyperemia appears to be a promising new scintigraphic sign of AVN worthy of further investigation.

    Title Prevention of Osteoradionecrosis: a Randomized Prospective Clinical Trial of Hyperbaric Oxygen Versus Penicillin.
    Date October 1985
    Journal Journal of the American Dental Association (1939)
    Excerpt

    A prospective randomized trial comparing hyperbaric oxygen and systemic antibiotics in the prevention of osteoradionecrosis was presented. The results indicated, in a high-risk population who required tooth removal in irradiated mandibles, that up-front hyperbaric oxygen produced an incidence of osteoradionecrosis of 5.4% as compared with the antibiotic group of 29.9% (P = .005). Hyperbaric oxygen should be considered a prophylactic measure when post-irradiation dental care involving trauma to tissue is necessary.

    Title The Use of Ender Nails in Fractures of the Tibial Shaft.
    Date April 1985
    Journal The Journal of Bone and Joint Surgery. American Volume
    Excerpt

    Between November 1979 and January 1983, we treated fifty-one severe fractures of the tibial shaft with multiple intramedullary Ender nails. Thirty-six fractures were treated within two weeks after injury. Forty-one fractures united in less than four months and eight, in four to eight months. Only two were not united after eight months. An anatomical reduction was maintained in all but three of the fractures, in which the tibia shortened. Two tibiae united with an angulation of 7 degrees and one with 6 degrees, as measured in two planes. There were two infections, both after an open fracture. It has been our experience that Ender nails provide excellent rotational stability, allow early full weight-bearing, and markedly decrease the duration of need for immobilization. Ender nailing was of value both for the acute management of complicated high-energy fractures of the tibial shaft with extensive soft-tissue damage and as a salvage procedure to maintain reduction of a fracture when other techniques had failed.

    Title Painful Spondylolysis or Spondylolisthesis Studied by Radiography and Single-photon Emission Computed Tomography.
    Date January 1985
    Journal Radiology
    Excerpt

    Planar bone scintigraphy (PBS) and single-photon emission computed tomography (SPECT) were compared in 19 adults with radiographic evidence of spondylolysis and/or spondylolisthesis. SPECT was more sensitive than PBS when used to identify symptomatic patients and sites of "painful" defects in the pars interarticularis. In addition, SPECT allowed more accurate localization than PBS. In 6 patients, spondylolysis or spondylolisthesis was unrelated to low back pain, and SPECT images of the posterior neural arch were normal. The authors conclude that when spondylolysis or spondylolisthesis is the cause of low back pain, pars defects are frequently heralded by increased scintigraphic activity which is best detected and localized by SPECT.

    Title Arc-aggregation: a New Method of Range of Motion Analysis in Hemophilia.
    Date November 1984
    Journal Archives of Physical Medicine and Rehabilitation
    Excerpt

    Arc-aggregation is a new graphic method of analysis of progressive loss of range of motion that can be applied to any diarthrodial joint undergoing a specific degenerative process. It is based on the premise that loss of motion (LOM) for a specific condition occurs in a predictable way or pattern. Using this method to analyze a population of 48 hemophilic patients with various stages of joint involvement, we studied 95 knees, 93 ankles, and 83 elbows for a total of 271 joints. We found a predictable pattern of progressive loss of range of motion for each joint. To detect the early onset of joint involvement, the first LOM is important. The early loss with the knee was in flexion, which gravitated toward fusion at 40 degrees. For the elbow, the early LOM was extension with fixation, occurring at 90 degrees of flexion, the preferred position for fusion. For the ankle, the early LOM was dorsiflexion with final ankylosis in slight equinus.

    Title The Os Trigonum Syndrome: Use of Bone Scan in the Diagnosis.
    Date September 1984
    Journal The Journal of Trauma
    Excerpt

    The os trigonum is an accessory bone of the foot found in 7% of the normal adult population. It is located at the posterolateral projection of the talus, and can occasionally give rise to symptoms of acute and chronic unexplained ankle pain. We report three patients, one with acute fracture and two with chronic ankle symptoms. Technetium 99 methylene diphosphonate showed intense focal uptake at the posterior talus pointing to the os trigonum as the site of symptoms. It was excised in two patients with complete relief. The third went on to develop an asymptomatic nonunion. We recommend bone scanning as a procedure that is helpful in delineating obscure pain in the ankle that may be due to chronic irritative nonunion of the os trigonum.

    Title Biological Response Modifiers and Their Promise in Clinical Medicine.
    Date June 1984
    Journal Pharmacology & Therapeutics
    Title Scintigraphic Diagnosis of Stress-induced Incomplete Fractures of the Proximal Tibia.
    Date March 1984
    Journal The Journal of Trauma
    Excerpt

    Incomplete stress fractures of the proximal tibial diaphysis can be diagnosed by bone scintigraphy. The scintigraphic appearance of incomplete rather than complete tibial stress fractures is apparently reported for the first time in this article. With no treatment other than restricted activity, this injury heals rapidly and completely in 4 to 6 weeks. The major threat to the patient's welfare is unfounded suspicion of tumor or infection which may lead to biopsy or inappropriate therapy.

    Title Predominance of Type Ii Collagen in Synovial Chondromatosis.
    Date October 1982
    Journal Clinical Orthopaedics and Related Research
    Title Calcium-oxalate-crystal-induced Bone Disease.
    Date June 1982
    Journal American Journal of Kidney Diseases : the Official Journal of the National Kidney Foundation
    Excerpt

    A 13-year-old boy with primary hyperoxaluria and a successful renal allograft developed symptomatic bone disease, hypercalcemia, and hypercalciuria. Transiliac bone biopsy revealed calcium oxalate crystals in the marrow within mononuclear phagocytes and multinucleated giant cells. Deep resorption bays were seen adjacent to these crystal-cell aggregates. Serum 1,25-(OH)2-vitamin D (calcitriol) and iPTH concentrations were low or normal. We suggest that hypercalcemia results from macrophage-mediated bone resorption initiated by Ca oxalate crystal deposition.

    Title The Use of Freeze-dried Allogeneic Bone in Oral and Maxillofacial Surgery.
    Date May 1981
    Journal Journal of Oral Surgery (american Dental Association : 1965)
    Excerpt

    Clinical experience with 36 cases using freeze-dried allogeneic bone is reported. Its use in a variety of surgeries, including ridge augmentation, mandibular and maxillary osteotomies, mandibular reconstruction, midfacial advancement and obliteration of cystic cavities was associated with a minimal incidence of complications and excellent host acceptance. The collection of bone under sterile conditions and the rigid criteria for the tissue banking process are shown to account for this host compatibility and low complication rate. The physiochemical and biologic properties of freeze-dried allogeneic bone are discussed. This material provides unique practical advantages leading to long-term stability.

    Title Addicted Physicians. A Closer Look.
    Date March 1981
    Journal Jama : the Journal of the American Medical Association
    Excerpt

    Fifty physicians were evaluated and treated in a psychiatrically oriented, short-term addiction program. Psychopathology ranged from overt schizophrenia to no demonstrable psychiatric syndrome other than the addiction. Physicians experiencing addiction problems before age 40 years were more likely to exhibit serious psychopathology in the borderline range, while physicians older than 40 years were more likely to exhibit organic brain impairment and depression. Treatment outcome had some relation to psychiatric diagnosis but no relation to the addictive agent.

    Title Correction of Hemifacial Microsomia.
    Date March 1981
    Journal Journal of Oral Surgery (american Dental Association : 1965)
    Excerpt

    Hemifacial microsomia is a combination of malformations of the first and second branchial arch derivatives. Reconstruction of three adult cases of hemifacial microsomia with varying degrees of deformity was presented.

    Title Cardiopulmonary Resuscitation: a Teaching Aid.
    Date August 1980
    Journal Journal of Oral Surgery (american Dental Association : 1965)
    Excerpt

    Laboratory dogs are used to demonstrate and practice cardiopulmonary resuscitation (CPR) procedures. Cardiovascular collapse, cardiac arrest, and ventricular fibrillation are produced in anesthesized dogs and then managed by the student. The laboratory exercise focuses and emphasizes CPR material taught in the classroom and on mannequins. With the current emphasis on CPR training and certification, we recommend the concept to the dental profession as an added dimension in teaching CPR.

    Title Myasthenia in Springer Spaniel Littermates.
    Date March 1976
    Journal The Journal of Small Animal Practice
    Title Prompt Electron Production from the Delco Detector at the Slac Storage Ring Pep.
    Date
    Journal Physical Review D: Particles and Fields
    Title Measurement of the Average Lifetime of Hadrons Containing Bottom Quarks.
    Date
    Journal Physical Review D: Particles and Fields
    Title Upper Bound on the Tau -neutrino Mass from the Previously Unobserved Decay Mode Tau -->kk Pi Nu Tau.
    Date
    Journal Physical Review Letters
    Title Study of Three-prong Tau Decays and Determination of the A1 Parameters.
    Date
    Journal Physical Review Letters
    Title Flow-induced Beam Steering in a Single Laser Hot Spot
    Date
    Journal Physical Review Letters
    Excerpt

    The transmitted angular distribution of a 527 nm nearly diffraction-limited laser is measured after it propagates through a plasma with supersonic transverse flow. The laser beam is deflected by as much as 10 degrees and exhibits bowlike features in the flow direction, which is attributed to flow-induced beam steering. The finite interaction volume allows for direct comparison with a 3D hydrodynamic simulation, which is in good agreement with details of the experiment.

    Title Conformational Selectivity in the Diels-alder Cycloaddition: Predictions for Reactions of S-trans-1,3-butadiene
    Date
    Journal The Journal of Organic Chemistry
    Excerpt

    Diels-Alder cycloaddition of s-trans-1,3-butadiene (1) should yield trans-cyclohexene (7), just as reaction of the s-cis conformer gives cis-cyclohexene (9). Investigation of this long-overlooked process with Hartree-Fock, Moller-Plesset, CASSCF, and DFT methods yielded in every case a C(2)-symmetric concerted transition state. At the B3LYP/6-31G (+ZPVE) level, this structure is predicted to be 42.6 kcal/mol above reactants, while the overall reaction is endothermic by 16.7 kcal/mol. A stepwise diradical process has been studied by UBLYP/6-31G theory and found to have barriers of 35.5 and 17.7 kcal/mol for the two steps. Spin correction lowers these values to 30.1 and 13.0 kcal/mol. The barrier to pi-bond rotation in cis-cyclohexene (9) is predicted (B3LYP theory) to be 62.4 kcal/mol, with trans-cyclohexene (7) lying 53.3 kcal/mol above cis isomer 9. Results suggest that pi-bond isomerization and concerted reaction may provide competitive routes for Diels-Alder cycloreversion. It is concluded that full understanding of the Diels-Alder reaction requires consideration of both conformers of 1,3-butadiene.

    Title Triniobium Polytungstophosphates. Syntheses, Structures, Clarification of Isomerism and Reactivity in the Presence of H(2)o(2).
    Date
    Journal Inorganic Chemistry
    Excerpt

    The reaction of K(7)[HNb(6)O(19)], H(2)O(2) and A-Na(9)[PW(9)O(34)] in water followed by treatment with Cs(+) or (n-Bu(4)N)(+) (TBA) affords the corresponding salts of the tris(peroxoniobium) heteropolyanion A,beta-[(NbO(2))(3)PW(9)O(37)](6)(-) (1) in approximately 60% isolated yields. An X-ray structure of the Cs salt, Cs1 (monoclinic P2/c; a = 16.92360(10) Å, b = 13.5721(2) Å, c = 22.31890(10) Å, beta = 92.0460(10) degrees, and Z = 4) confirms the A-type substitution pattern of the three Nb atoms and clarifies the M(3) rotational (Baker-Figgis) isomerism in the Keggin unit as beta. The three terminal eta(2)-O(2)(2)(-) groups on the Nb atoms give 1 an overall symmetry approximating the chiral C(3). These terminal peroxo ligands, and these groups only, thermally decompose when either Cs1 or TBA1 is in solution unless additional H(2)O(2) is present. The peroxo groups can be titrated with triphenylphosphine (2.8 +/- 0.3 peroxide groups found per molecule). Refluxing TBA1 in acetonitrile for 24 h in the presence of base generates the parent heteropolyanion, [Nb(3)PW(9)O(40)](6)(-) (2) in 80% yield after isolation. Treatment of 2 with glacial acetic acid in acetonitrile converts it to [Nb(6)P(2)W(18)O(77)](6)(-) (3) in approximately 100% yield, while treatment of TBA3 with hydroxide converts it back to 2 in high yield. Spectroscopic (FTIR, Raman, (183)W NMR, and (31)P NMR), titrimetric, mass spectrometric (FABMS), and elemental analysis data are all consistent with these formulas. The addition of TBA1 to solutions of alkenes and 33% aqueous peroxide in acetonitrile at reflux results in the generation of the corresponding vicinal diols in high selectivity and yield at high conversion of substrate. Several spectroscopic and kinetics experiments, including a novel one correlating the incubation time of TBA1 under the reaction conditions with the rates of alkene oxidation, establish that TBA1 functions primarily as a catalyst precursor and that much of the catalytic activity is derived from generation of tungstate under the reaction conditions.

    Title Sooty Mangabeys and Rhesus Macaques Exhibit Significant Divergent Natural Killer Cell Responses During Both Acute and Chronic Phases of Siv Infection.
    Date
    Journal Cellular Immunology
    Excerpt

    A correlation between NK cells and rate of disease progression in HIV-1-infected individuals has been documented. The role NK cells play in disease outcome can optimally be studied in SIV-infected disease susceptible rhesus macaques (RM) and SIV-infected disease resistant sooty mangabeys (SM). In this study, three main subsets of CD16(+)CD56(-), CD16(-/dim)CD56(+) and CD16(-)CD56(-) NK cells have been identified with the predominant CD16(+)CD56(-) subset being primarily responsible for cytolytic activity in both species. Cross-sectional studies revealed a significant decline in the frequency and function of this cytolytic subset in SIV-infected RM while an increase occurred in SIV-infected SM. Longitudinal studies revealed that an earlier NK response during acute infection occurred in all SIV-infected SM and in select SIV-infected RM that eventually controlled viral load set point during chronic infection, suggesting that early NK activity and continued maintenance of this cell lineage may indirectly contribute to disease resistance.

    Title The Use of Nonhuman Primate Models in Hiv Vaccine Development.
    Date
    Journal Plos Medicine
    Title Renal Medullary Carcinoma Metastatic to the Scalp.
    Date
    Journal The American Journal of Dermatopathology
    Excerpt

    A 40-year-old woman presented to the Brooke Army Medical Center Emergency Department complaining of 2 episodes of gross hematuria. Computed tomography and intravenous pyelogram revealed a right renal mass. A radical nephrectomy was performed with complete excision of the mass with negative margins on histological examination. The patient was diagnosed with renal medullary carcinoma, Fuhrman grade 4, based upon histological examination. A positron emission tomography scan revealed no other evidence of the disease. The pathologic stage was stage I renal medullary carcinoma. Four months after her nephrectomy, the patient developed a papule on her right frontal scalp. Initially thought to be a cyst, the papule increased in size over the course of 2 months and eventually ulcerated. At that time, she presented to the Wilford Hall Medical Center Dermatology Clinic with a 2.5-cm ulcerated tumor with pink rolled borders. A punch biopsy of the tumor revealed an infiltrating carcinoma with scattered glandular lumina and desmoplastic and mucinous stroma. The carcinoma was completely intradermal. Expert consultation confirmed the diagnosis of metastatic renal medullary carcinoma. Clear cell (conventional) and papillary renal cell carcinomas are known to metastasize to the skin, including the scalp. Renal medullary carcinoma commonly metastasizes to the regional lymph nodes, lung, liver, or adrenals. To our knowledge, this is the first report of a cutaneous metastasis of renal medullary carcinoma.

    Title Vinylogous Addition of Siloxyfurans to Benzopyryliums: a Concise Approach to the Tetrahydroxanthone Natural Products.
    Date
    Journal Journal of the American Chemical Society
    Excerpt

    A concise approach to the tetrahydroxanthone natural products employing vinylogous addition of siloxyfurans to benzopyryliums and a late-stage Dieckmann cyclization has been developed. With this methodology, chiral, racemic forms of the natural products blennolides B and blennolide C have been synthesized in a maximum of four steps from a 5-hydroxychromone substrate. The regio- and diastereoselectivity of the vinylogous additions was probed using computational studies, which suggested the involvement of Diels-Alder-like transition states.

    Similar doctors nearby

    Dr. Peter Block

    Internal Medicine
    48 years experience
    Atlanta, GA

    Dr. Stephanie Grossman

    Internal Medicine
    20 years experience
    Atlanta, GA

    Dr. Robert Cooper

    Clinical Pharmacology
    33 years experience
    Atlanta, GA

    Dr. Susmita Parashar

    Internal Medicine
    Atlanta, GA

    Dr. Jeff Sands

    Internal Medicine
    31 years experience
    Atlanta, GA

    Dr. Nana Arthur

    Internal Medicine
    11 years experience
    Atlanta, GA
    Search All Similar Doctors