Lack of a small animal model of the human hepatitis C virus (HCV) has impeded development of antiviral therapies against this epidemic infection. By transplanting normal human hepatocytes into SCID mice carrying a plasminogen activator transgene (Alb-uPA), we generated mice with chimeric human livers. Homozygosity of Alb-uPA was associated with significantly higher levels of human hepatocyte engraftment, and these mice developed prolonged HCV infections with high viral titers after inoculation with infected human serum. Initial increases in total viral load were up to 1950-fold, with replication confirmed by detection of negative-strand viral RNA in transplanted livers. HCV viral proteins were localized to human hepatocyte nodules, and infection was serially passaged through three generations of mice confirming both synthesis and release of infectious viral particles. These chimeric mice represent the first murine model suitable for studying the human hepatitis C virus in vivo.

Correlates of immune-mediated protection to most viral and cancer vaccines are still unknown. This impedes the development of novel vaccines to incurable diseases such as HIV and cancer. In this study, we have used functional genomics and polychromatic flow cytometry to define the signature of the immune response to the yellow fever (YF) vaccine 17D (YF17D) in a cohort of 40 volunteers followed for up to 1 yr after vaccination. We show that immunization with YF17D leads to an integrated immune response that includes several effector arms of innate immunity, including complement, the inflammasome, and interferons, as well as adaptive immunity as shown by an early T cell response followed by a brisk and variable B cell response. Development of these responses is preceded, as demonstrated in three independent vaccination trials and in a novel in vitro system of primary immune responses (modular immune in vitro construct [MIMIC] system), by the coordinated up-regulation of transcripts for specific transcription factors, including STAT1, IRF7, and ETS2, which are upstream of the different effector arms of the immune response. These results clearly show that the immune response to a strong vaccine is preceded by coordinated induction of master transcription factors that lead to the development of a broad, polyfunctional, and persistent immune response that integrates all effector cells of the immune system.

Background: Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common

A cyclophilin-related protein has recently been found to be involved in tumor recognition by natural killer cells. The N-terminal end of this 150-kDa surface molecule (NK-TR) is homologous to cyclophilin/peptidylprolyl cis-trans-isomerase. We have constructed a soluble bacterial fusion protein between the cyclophilin-homologous domain of the NK-TR molecule and glutathione S-transferase (GST) to test for the presence of peptidylprolyl cis-trans-isomerase and chaperone activities and for cyclosporin A binding. The purified NK-cyp-GST fusion protein is shown to accelerate the isomerization of the prolyl peptide bond of the substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide with a kcat/KM value of 7.4 x 10(5) M-1 s-1. The isomerase activity of the NK-TR cyclophilin homolog has been determined to be relatively insensitive to inhibition by the immunosuppressive drug cyclosporin A, with an IC50 value of 770 nM as compared to 19 nM for human cyclophilin. Furthermore, the NK-cyp-GST fusion protein has been found to participate in the protein folding process as a chaperone by preventing the aggregation of early folding intermediates of carbonic anhydrase. The implications of the finding of both peptidylprolyl cis-trans-isomerase and chaperone activities within the N-terminal domain of a large, cell type-restricted, surface molecule are discussed.

A simple method, primer specific and mispair extension analysis (PSMEA) with pfu DNA polymerase was developed for genotyping. PSMEA is based on the unique properties of 3'-->5' exonuclease proofreading activity. In the presence of an incomplete set of dNTPs, pfu was found to be extremely discriminative in nucleotide incorporation and proofreading at the initiation step of DNA synthesis, completely preventing primer extension when mispair(s) are found adjacent to the 3'-end of the primer. This has allowed us to accurately detect nucleotide variations, deletions and insertions for fast genotyping.

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Background and objectives: Certain clinical conditions are related to the presence of platelet-specific alloantibodies in the patient’s serum. We studied the molecular diversity of HPA-1a antibodies to analyze some peculiarities of this antibody response. Materials and methods: Human antibody Fab fragments that bind to the platelet alloantigen HPA-la on glycoprotein Ilb-IIIa (GPIIbllla) were generated by using a recombinant phage display system. We established an immunoglobulin Gl, kappa combinatorial library from the peripheral blood lymphocytes of a person undergoing a severe posttransfusion purpura. Results: Characterization of Fab clones selected from the fifth round of antigen-specific panning of this library demonstrates a highly specific reactivity to the HPA-1a alloantigen. The nucleotide sequence analysis of representative HPA-1a-specific clones reveals at least 3 distinct VL and 3 V(H) gene segments that present an extensive degree of mutation as demonstrated by comparison of gene usage and homologies to the nearest germline genes. Conclusions: These human HPA-laspecific Fab reagents should allow us to better understand the molecular mechanism involved in HPA-1a alloimmunization.