Immunogenicity. I. Use of peptide libraries to identify epitopes that activate clonotypic CD4+ T cells and induce T cell responses to native peptide ligands.

Recent studies have demonstrated the utility of synthetic combinatorial libraries for the rapid identification of peptide ligands that stimulate clonotypic populations of T cells. Here we screen a decapeptide combinatorial library arranged in a positional scanning format with two different clonotypic populations of CD4+ T cells to identify peptide epitopes that stimulate proliferative responses by these T cells in vitro. An extensive collection of mimic peptide sequences was synthesized and used to explore the fine specificity of TCR/peptide/MHC interactions. We also demonstrate that many of these deduced ligands are not only effective immunogens in vivo, but are capable of inducing T cell responses to the original native ligands used to generate the clones. These results have significant implications for considerations of T cell specificity and the design of peptide vaccines for infectious disease and cancer using clinically relevant T cell clones of unknown specificity.

Exploring immunological specificity using synthetic peptide combinatorial libraries.

The definition of epitopes for human B and T cells is fundamental for the understanding of the immune response mechanism and its role in the prevention and cause of human disease. This understanding can be applied to the design of diagnostics and synthetic vaccines. In recent years, the understanding of the specificity of B and T cells has been advanced significantly by the development and use of combinatorial libraries made up of thousands to millions of synthetic peptides. The use of this approach has had four major effects: first, the definition of high affinity ligands both for T cells and antibodies; second, the application of alternative means for identifying immunologically relevant peptides for use as potential preventive and therapeutic vaccines; third, a new appreciation of the requirements for TCR interactions with peptide-MHC complexes in immunogenicity; fourth, the establishment of new principles regarding the level of cross-reactivity in immunological recognition.

Rat NKR-P1+ CD3+ T cells: selective proliferation in interleukin-2, diverse T-cell-receptor-Vbeta repertoire and polarized interferon-gamma expression.

Cells expressing markers of both natural killer and T lymphocytes (NK T cells) in humans and mice express a restricted T-cell receptor (TCR) repertoire, are of CD4- CD8- or CD4+ CD8- phenotype, and upon anti-CD3 stimulation secrete large amounts of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma). NK T cells may be the primary source of IL-4-promoting T helper type 2 (Th2) responses and/or they might be involved in regulating the balance between Th1- and Th2-type immune responses, and may consequently affect susceptibility to autoimmune diseases associated with a skewed Th phenotype. We show that rat NK T cells selectively proliferate to IL-2, and use this fact to analyse cytokine production by NK T cells in two rat strains differentially susceptible to Th1- or Th2-type autoimmune diseases. Analysis by reverse transcription-polymerase chain reaction revealed that, in contrast to mouse, rat NK T cells secrete exclusively IFN-gamma and not IL-4 after anti-CD3 stimulation, and use a wider TCR-Vbeta repertoire, suggesting that rat NK T cells are not essential for the development of Th2-type CD4+ T-cell responses.

Antibody testing and RT-PCR results in hepatitis C virus (HCV) infection: HCV-RNA detection in PBMC of plasma viremia-negative HCV-seropositive persons.

To evaluate the concordance between viremia and antibody testing in hepatitis C virus (HCV) diagnosis, 682 serum or plasma samples collected from patients with known or suspected HCV infection were tested. An overall concordance of 77% between serological and PCR results was found, 5% was RNA positive/antibody negative and 18% antibody positive/RNA negative. The relationship between HCV infection, risk group and clinical diagnosis was studied in 116 patients: the presence of anti-HCV antibody without viremia was shown in 72.7% of asymptomatic subjects and 17.6% of chronic hepatitis subjects without interferon treatment. However, the detection of HCV-RNA in peripheral blood mononuclear cells (PBMC) in four out of 38 plasma viremia-negative HCV-seropositive subjects (10.5%), showed that HCV-RNA could persist in PBMC and could begin the viral replication again at different times. The detection of HCV-RNA in PBMC in anti-HCV-positive subjects without viremia could reduce false-negative results of HCV-RNA testing by RT-PCR in serum or plasma.

Construction and characterization of retroviral vectors for interleukin-2 gene therapy.

Several investigators have employed interleukin-2 (IL-2) gene transfer to enhance the immunogenicity of tumor cell vaccines. We describe in this report the construction and characterization of retroviral vectors for IL-2 gene therapy. Human IL-2 cDNA with a chimeric rat preproinsulin/IL-2 DNA leader sequence was subcloned into the pLXSN (long terminal repeat promoter) and pLNCX (cytomegalovirus [CMV] promoter) vectors to generate the plasmids pLXSN-iIL2 and pLNCX-iIL2, respectively. Human IL-2 cDNA with a chimeric human tissue factor/IL-2 DNA leader sequence was utilized to construct the vector pLXSN-tIL2. The levels of IL-2 secreted by transduced tumor cells and fibroblasts were evaluated by enzyme-linked immunosorbent assay (ELISA) of culture supernatants and compared with those of normal peripheral blood mononuclear cells (PBMC) activated in vitro with calcium ionophore and phorbol 12-myristate 13-acetate. The highest levels of IL-2 secreted by transduced tumor cells (760 units/10(6) cells/24 h), adult fibroblasts (625 units/10(6) cells/24 h), and embryonic fibroblasts (3,975 units/10(6) cells/24 h) were 150- to 1,000-fold higher than than secreted by the activated PBMC (4 units/10(6) cells/24 h). Similar levels of IL-2 were expressed by human fibroblasts transduced with pLXSN vectors employing the preproinsulin (pLXSN-iIL2) or tissue factor (pLXSN-tIL2) leader sequences (range in IL-2 units/10(6) cells/24 h pLXSN-iIL2 = 375-625 vs. pLXSN-tIL2 = 90-440). Because IL-2-transduced cells for clinical applications are generally irradiated to prevent cellular proliferation, we evaluated the effects of radiation on IL-2 production. Radiation doses between 1,500 and 10,000 cGy resulted in gradual decreases in IL-2 secretion by transduced cells. The range of the decrease in IL-2 secretion was 7-11% by day 7, 0-29% by day 14, and 25-50% by day 35. For clinical applications, stable production of the vector in high concentrations is an important consideration. The retroviral vector pLXSN-tIL2 produced the highest viral titer and was chosen for further characterization. Southern blot analysis of SacI-digested genomic DNA from the LXSN-tIL2 producer cell line and SacI-digested pLXSN-tIL2 plasmid DNA revealed the expected 3.2-kbp fragment, suggesting the absence of transgene rearrangement and the suitability of this vector as a candidate for clinical applications.

Hepatitis C (HCV) genotype and viral titer distribution among Argentinean hemophilic patients in the presence or absence of human immunodeficiency virus (HIV) co-infection.

Hepatitis C (HCV) infection is frequent among hemophilic patients treated with non-inactivated factor-concentrates. Both HCV genotype and viral load have been suggested to be important prognostic markers of disease progression and treatment outcome. In addition, co-infection with the human immunodeficiency virus (HIV) has been associated with increased level of HCV replication and higher risk of developing liver failure. Thus, HCV genotype, viral load, and HIV co-infection are important factors in HCV infection. Using restriction fragment length polymorphism analysis (RFLP) and the branched-DNA (bDNA) assay, we retrospectively investigated the HCV genotypes and viral loads present in 59 Argentinean hemophiliacs, in the presence or absence of HIV infection. HCV genotype 1 was the predominant viral variant detected among HIV-negative (HIV-) (76%) and HIV-positive (HIV+) (82.5%) patients, followed by genotypes 3 (10.4%), 2 (2%) and a small proportion of multiply co-infected patients including genotypes 4 and 5 (6.25%). HIV+ patients had higher plasma HCV RNA levels than HIV- patients (88.4 +/- 16.5 x 10(5) Eq/ml vs. 24.7 +/- 10(5) Eq/ml) (P < 0.001); however, no correlation between HCV replication and level of immune suppression, evaluated by CD4+ T-cell measurement, was observed among HIV+ patients (r = 0.017). Abnormal and higher ALT levels were more frequently detected among HIV+ (93%; 123.6 +/- 15.7 U/liter) than HIV- (41%; 70.2 +/- 24.2 U/liter) patients (P < 0.001; P < 0.05). Although we were able to confirm previous reports suggesting the existence of increased HCV replication in HIV/HCV co-infected hemophiliacs, our data did not support the conclusion that HIV-induced immune suppression is directly responsible for this phenomena. It is possible that other factors induced by HIV are responsible for the increased levels in HCV replication observed.

PIP: Hepatitis C virus (HCV) infection is widespread among hemophiliacs treated with non-inactivated factor concentrates. The HCV genotypes and viral loads present in 59 hemophiliacs from Argentina were investigated through use of restriction fragment length polymorphism analysis and the branched DNA assay. 30 subjects were also infected with HIV. In both HIV-positive and HIV-negative hemophiliacs, HCV genotype 1 was the predominant viral variant (82.5% and 76%, respectively), followed by genotypes 3 and 2. HIV-positive hemophiliacs had significantly higher mean HCV viral loads than HIV-negative hemophiliacs; however, there was no association between HCV replication and the level of immune suppression as evaluated by CD4 T-cell measurement. Although HCV replication was increased in individuals co-infected with HCV and HIV, the data did not support the hypothesis that HIV-induced immune suppression is directly responsible for this finding. A study currently underway is investigating a possible correlation between infecting HCV genotype or pre-existing viral load and the severity of disease as assessed by liver histology or treatment outcome.

Inhibition of inducible nitric oxide synthase intensifies injury and functional deterioration in autoimmune interstitial nephritis.

T lymphocytes are exquisitely sensitive to the antiproliferative effects of nitric oxide. We examined the effects of oral administration of two nitric oxide synthase inhibitors, Nw-nitro-L-arginine methyl ester (L-NAME) and L-N6-(1-iminoethyl)lysine (L-NIL), on the course of T cell-dependent autoimmune interstitial nephritis in Brown Norway rats. Kidneys from rats immunized to produce interstitial nephritis display a net generation of nitric oxide end products. By immunohistochemical staining, the cytokine-inducible nitric oxide synthase (iNOS) is expressed in cortical tubular epithelial cells. Treatment with either inhibitor results in markedly more severe disease following immunization. Animals receiving L-NAME were hypertensive, while those treated with L-NIL, a highly selective inhibitor of iNOS, were not. Evaluation of the expression of IFN-gamma, IL-2, and IL-4 in diseased kidneys by quantitative reverse transcriptase-PCR demonstrated that L-NAME-treated animals displayed significantly augmented levels of IFN-gamma and IL-2 with preserved ratios of IFN-gamma/IL-4 and IL-2/IL-4, while L-NIL-treated animals had augmented levels of IL-2 and IFN-gamma with augmented IFN-gamma/IL-4 and IL-2/IL-4 ratios. Animals treated with L-NAME or L-NIL both had augmented Ag-specific IgG responses. The L-NAME group demonstrated increases in both the IgG2a and IgG1 subtypes, with a constant IgG2a/IgG1 ratio, while the L-NIL group demonstrated an increase in the ratio of the IgG2a/IgG1 response. These Ab and cytokine data suggest that the L-NIL-treated animals had a skewing of their immune response toward a Th1-like response. We conclude that in autoimmune interstitial nephritis, generation of nitric oxide through the iNOS pathway has host-protective effects, and suggest that this may be broadly applicable to T cell-mediated pathologies.

The potential role of hepatitis C virus in the pathogenesis of the neurological syndrome in chronic hepatitis C.

A 72-year-old man developed chronic sensory neuropathy (CSN) during chronic hepatitis C (HCV) infection. Neurological symptoms began one year after acute HCV hepatitis and slowly worsened over three years. No conventional cause for CSN was found. Circulating antinervous tissue antibodies (including anti-Hu) and inflammatory infiltrates in sural nerve biopsy specimens were absent. However, the presence of anti-HCV antibody and HCV-RNA in cerebrospinal fluid indicated that HCV had reached the intrathecal compartment, suggesting the direct viral involvement in the pathogenesis of CSN.

[Epidemiologico-clinical considerations on a case file of acute hepatitis observed 1980-1983]. / Considerazioni epidemiologico-cliniche su una casistica di epatite acuta osservata negli anni 1980-1983.

Between January 1980 and December 1983, 332 consecutive cases of acute hepatitis were observed in adult patients admitted to the Department of Infectious Disease and Gastroenterology of Siena. Sex and age of the patients, the presence of jaundice, the maximum value of the serum-glutamate-pyruvate-transaminase (SGPT) were considered. Serum specimens were tested for hepatitis B surface antigen (HBsAg), antibody against hepatitis B core antigen (anti-HBc) of the IgM class, antibody against hepatitis A virus (anti-HAV) of the IgM class, antibody against cytomegalovirus (CMV) of the IgM class, Paul Bunnel Davidshon reaction. Hepatitis A was diagnosed in 25 cases (7.5%). Hepatitis B in 167 (50.3%). Hepatitis due to CMV in 2 cases (0.6%). And, by exclusion, hepatitis non A, non B in 138 cases (41.6%). Male patients were affected with significantly higher frequency than female (p less than 0.01); the same was seen for young patients (14-30 years) compared to the older ones (31-50 years, and over 50 years) (p less than 0.01 in both). Biochemical investigation showed that hepatitis A and B had a significantly higher, maximum SGPT value than hepatitis non A non B (p less than 0.01 in both). Icteric patients were significantly more frequently observed among hepatitis A and B cases than hepatitis non A non B cases (p less than 0.01 in both).

[Epidemiological study on the incidence of viral hepatitis in the Province of Siena]. / Indagini epidemiologiche sulla incidenza della virus-epatite in provincia di Siena

The AA. carried out an epidemiological survey on 1,420 cases with viral hepatitis ascertained in the Province of Siena during the period January 1, 1962-December 31,1972. Age, sex, profession, place of origin, annual and seasonal prevalence, therapeutic measures, geographic and social-economic features of several districts, and other characteristics of the affected people were examined. The results obtained were discussed keeping in mind the various parameters examined.