Cytokine-induced viral purging--role in viral pathogenesis.

The control of viral infections was previously thought to rely exclusive ly on the antigen-specific destruction of infected cells by the antigen-specific destruction of infected cells by the immune system; however, recent studies have shown that several viral infections can be primarily controlled by noncytopathic, cytokine- dependent 'curative' mechanisms (i.e. viral purging). The relative sensitivity of viruses to such curative mechanisms depends not only on the virus but also on the capacity of the specific infected cell to produce the appropriate intracellular antiviral factors.     

Identification of monoclonal antibody defined epitopes on human leukocyte antigens utilizing phage display peptide libraries

Summary Utilizing phage display peptide libraries, we have identified and mapped the antigenic determinants recognized by mouse monoclonal antibodies (mAb) on two sets of immunologically important molecules, HLA class I and class II antigens. Anti-HLA class I mAb TP25.99 recognizes a conformational and a linear determinant on distinct regions of the HLA class I α3 domain. Anti-HLA class I mAb HO-4 recognizes a conformational determinant on the α2 domain of HLA-A2 and A28 allospecificities. Anti-HLA-DR1,-DR4,-DR6,-DR8,-DR9 mAb SM/549 recognizes a conformational determinant on the β chain of HLA class II antigens. These results indicate the versatility of phage display peptide libraries to characterize antigenic determinants recognized by anti-HLA mAb.     

Synthesis, in vitro, and in vivo biological evaluation and molecular docking simulations of chiral alcohol and ether derivatives of the 1,5-diarylpyrrole scaffold as novel anti-inflammatory and analgesic agents

Following our previous research on anti-inflammatory drugs (NSAIDs), we report here the synthesis of chiral 1,5-diarylpyrroles derivatives that were characterized for their in vitro inhibitory effects toward cyclooxygenase (COX) isozymes. Analysis of enzymatic affinity and COX-2 selectivity led us to the selection of one compound (+/-)-10b that was further tested in vitro in the human whole blood (HWB) and in vivo for its anti-inflammatory activity in mice. The affinity data have been rationalized through docking simulations.     

HPLC enantioseparation and absolute configuration of novel anti-inflammatory pyrrole derivatives

The assignment of the absolute configuration of novel anti-inflammatory pyrrole derivatives has been accomplished by a combined strategy based on independent physical methods. The key step of our stereochemical characterization approach is the production at mg-scale of enantiomerically pure forms by HPLC on Chiralpak IA stationary phase.     

Rep-PCR of tropical rhizobia for strain fingerprinting, biodiversity appraisal and as a taxonomic and phylogenetic tool

With more than 30 million doses of rhizobial inoculants marketed per year, it is probable that Brazilian agriculture benefits more than any other country from symbiotic N₂ fixation. As a result of strain-selection programs, 142 strains of rhizobia are officially recommended for use in commercial inoculants for ninety-six leguminous crops. In this study, sixty-eight of these elite strains were characterized by rep-PCR with the BOX-primer. Reproducibility of the DNA profiles was confirmed, suggesting efficacy of BOX-PCR both for control of quality of inoculants and for preliminary characterization of rhizobial culture collections. Strains of different species never showed similarity higher than 70% in the BOX-PCR analysis, however, some strains of the same species fit into more than one cluster, and correlation between BOX-PCR products and l6S rRNA sequences was low (7.6%). On the other hand, a polyphasic approach — 20%∶80% of BOX-PCR:16S rRNA which correlated well with the l6S rRNA analysis (95%), and provided higher definition of the genotypes, resulting in clearer indications of the taxonomic groups — might expedite rhizobial diversity studies.     

The Size of the Viral Inoculum Contributes to the Outcome of Hepatitis B Virus Infection

The impact of virus dose on the outcome of infection is poorly understood. In this study we show that, for hepatitis B virus (HBV), the size of the inoculum contributes to the kinetics of viral spread and immunological priming, which then determine the outcome of infection. Adult chimpanzees were infected with a serially diluted monoclonal HBV inoculum. Unexpectedly, despite vastly different viral kinetics, both high-dose inocula (10¹⁰ genome equivalents [GE] per animal) and low-dose inocula (10° GE per animal) primed the CD4 T-cell response after logarithmic spread was detectable, allowing infection of 100% of hepatocytes and requiring prolonged immunopathology before clearance occurred. In contrast, intermediate (10⁷ and 10⁴ GE) inocula primed the T-cell response before detectable logarithmic spread and were abruptly terminated with minimal immunopathology before 0.1% of hepatocytes were infected. Surprisingly, a dosage of 10¹ GE primed the T-cell response after all hepatocytes were infected and caused either prolonged or persistent infection with severe immunopathology. Finally, CD4 T-cell depletion before inoculation of a normally rapidly controlled inoculum precluded T-cell priming and caused persistent infection with minimal immunopathology. These results suggest that the relationship between the kinetics of viral spread and CD4 T-cell priming determines the outcome of HBV infection.The hepatitis B virus (HBV) is a noncytopathic DNA virus that causes acute and chronic hepatitis and hepatocellular carcinoma (5). Viral clearance and disease pathogenesis during acute HBV infection require the induction of a vigorous CD8⁺ T-cell response and the induction of significant hepatic immunopathology (12, 28). In contrast, chronic HBV infection is associated with a markedly diminished CD8⁺ T-cell response to HBV (23, 24) for reasons that are not well defined.We have previously studied the immunobiology and pathogenesis of HBV infection in chimpanzees that we inoculated with a single (10⁸ genome equivalents [GE]) dose of a monoclonal inoculum of HBV (12, 28, 33). In all of these animals, the infection pursued a reproducible, almost stereotypical course irrespective of the age, size, sex, and genetics of the animals, and it spread to 100% of the hepatocytes before it was terminated by the CD8 T-cell response. The reproducibility of these results suggested that the course and outcome of infection were dominated by the impact of the virus on the kinetics and magnitude of the infection and on the kinetics and magnitude of the immune response that it elicited.Because a high viral load has a negative impact on the outcome of other virus infections (reviewed in references 19 and 32), we examined in the present study the impact of the size of the viral inoculum on the outcome of HBV infection in HBV-naive, immunocompetent adult chimpanzees using a wide dose range of the same monoclonal inoculum that we used in our earlier studies.In contrast to the highly reproducible outcome to the 10⁸ GE dose in our previous experiments, we observed a wide range of outcomes to the various dosages used here, including the development of chronic HBV infection, that we could relate to the kinetics of the CD4 T-cell response in each animal. Furthermore, depletion of CD4⁺ cells before infection with a dose of virus that is otherwise rapidly cleared led to persistent infection. These results suggested that the size of the viral inoculum may contribute to the outcome of infection by altering the balance between the kinetics and magnitude of infection versus the kinetics and magnitude of the immune response. Similar results have been recently published based on in situ analysis of the ratio of virus-infected cells to immune effector cells in the tissues of simian immunodeficiency virus-infected macaques and lymphocytic choriomeningitis virus-infected mice (20).Collectively, these results suggest that the kinetics of T-cell priming relative to the kinetics of viral spread determines the outcome of HBV infection. Specifically, they suggest that early priming of the CD4⁺ T-cell response before or during viral spread initiates a vigorous, synchronized, and functionally efficient CD8⁺ T-cell response and the accompanying immunopathology that ultimately terminates HBV infection. In contrast, the virus persists when CD4⁺ T-cell priming is delayed until after all of the hepatocytes are infected.