Characterization of the antibody response elicited by HIV-1 Env glycomutants in rabbits.

HIV-1 N-glycans are known to shield underlying epitopes towards the protective antibody repertoire. We previously described HIV-1 acute infection Env glycomutants designed from 3D-model in which the removal of clustered N-glycans did not disturb the envelope antigenicity, but increased the neutralization sensitivity. The potential of such immunogens to elicit neutralizing responses was estimated after rabbit immunizations with a DNA/protein protocol. Maturation of the Env-specific antibody response was confirmed by a change in avidity and conformational dependence. For one immunogen, the neutralizing response was increased with a higher breadth compared to the Wild-Type. Our data suggest that Env selective deglycosylation based on 3D data may represent a valuable strategy to improve elicitation of neutralizing antibodies.

Primary and memory T cell responses induced by hepatitis C virus multiepitope long peptides.

To develop a vaccine against hepatitis C virus, we synthesized four long peptides from nonstructural proteins NS3, NS4 and NS5B containing HLA-class I and class II epitopes mainly inducing responses in natural infection. In HLA-A2.1 transgenic mice, the four peptides primed higher CTL responses to 6:7 minimal HLA-A2 epitopes than those induced by the minimal epitopes. HLA-A2.1/HLA-DR1 transgenic mice immunized with one peptide, containing a class II epitope implicated in viral resolution, developed IFNgamma-producing CD4+-T and CD8+-T cells. These peptides recalled HCV-specific IFNgamma-producing cells from HCV-infected patients' PBMC. This support the selection of these domains for inclusion in a vaccine formulation.

Control of heterologous hepatitis C virus infection in chimpanzees is associated with the quality of vaccine-induced peripheral T-helper immune response.

Prophylactic hepatitis C virus (HCV) vaccine trials with human volunteers are pending. There is an important need for immunological end points which correlate with vaccine efficacy and which do not involve invasive procedures, such as liver biopsies. By using a multicomponent DNA priming-protein boosting vaccine strategy, naïve chimpanzees were immunized against HCV structural proteins (core, E1, and E2) as well as a nonstructural (NS3) protein. Following immunization, exposure to the heterologous HCV 1b J4 subtype resulted in a peak of plasma viremia which was lower in both immunized animals. Compared to the naïve infection control and nine additional historical controls which became chronic, vaccinee 2 (Vac2) rapidly resolved the infection, while the other (Vac1) clearly controlled HCV infection. Immunization induced antibodies, peptide-specific gamma interferon (IFN-gamma), protein-specific lymphoproliferative responses, IFN-gamma, interleukin-2 (IL-2), and IL-4 T-helper responses in both vaccinees. However, the specificities were markedly different: Vac2 developed responses which were lower in magnitude than those of Vac1 but which were biased towards Th1-type cytokine responses for E1 and NS3. This proof-of-principle study in chimpanzees revealed that immunization with a combination of nonstructural and structural antigens elicited T-cell responses associated with an alteration of the course of infection. Our findings provide data to support the concept that the quality of the response to conserved epitopes and the specific nature of the peripheral T-helper immune response are likely pivotal factors influencing the control and clearance of HCV infection.

Impaired allostimulatory function of dendritic cells in chronic hepatitis C infection.

BACKGROUND & AIMS: Dendritic cells (DC), which play an essential role in the triggering of primary antiviral immune reactions, may also contribute, in some viral models, to the propagation of viral infection and the pathogenesis of viral disease. During natural infection with hepatitis C virus (HCV), the interactions between the virus and DC may contribute to viral persistence, a general feature of HCV infection. METHODS: We compared the phenotypical and biological functions of monocyte-derived DC from patients with chronic hepatitis C (HCV-DC; n = 6), seronegative individuals (naive-DC; n = 8), long-term responders to antiviral therapy (LTR-DC; n = 8), and a group of patients with non-HCV-hepatic disorders (n = 11). The presence and the nature of HCV sequences during the DC cultures was assessed by reverse transcription-polymerase chain reaction and the analysis of the viral quasispecies distribution. RESULTS: Although HCV-DC displayed a normal morphology, phenotype, and capacity to capture antigen, their ability to stimulate the proliferation of allogeneic T cells was dramatically impaired in comparison with naive-DC (P = 0.0013). Mixing experiments revealed that HCV-DC did not affect the proliferation of T cells induced by naive-DC. Remarkably, the allostimulatory function of LTR-DC or DC from patients with non-HCV-hepatic disorders did not show any impairment. The presence of HCV genomic sequences could be documented for 5 of 6 HCV carriers either in the cells and/or the supernatants of the DC cultures. The presence of HCV sequences was found in the DC cultures from one patient showing a dramatic allostimulation defect. For that patient, extensive analysis of the viral quasispecies distribution revealed the presence, in the DC cultures, of genomic sequences of a unique nature, distinct from those identified in the patient's mononuclear cells, serum, or liver. CONCLUSIONS: Overall, these results indicate that chronic infection by HCV is associated with an allostimulatory defect of monocyte-derived DC, possibly because these cells constitute an extrahepatic reservoir for the virus. Although the exact mechanism responsible for such an alteration remains to be unraveled, our observations argue against an active immunosuppression-based mechanism.

IL-2-resistant hyporesponsiveness induced by CD4 mAbs in OKT3-activated human T cells is reversed by CD45RA triggering.

CD4 monoclonal antibodies (mAbs) are potent immunosuppressive agents which have been shown to induce in vivo tolerance to antigens and alloantigens and are presently evaluated in humans in the treatment of autoimmune diseases. In previous studies, we observed that clinical improvement of CD4 mAb-treated psoriasis patients was achieved without depletion of CD4+ lymphocytes and at nonsaturating CD4 mAb concentrations, suggesting a functional blockade of CD4+ lymphocyte responses. In this study, we demonstrate that priming of normal human CD4+ T cells by immobilized OKT3 (iOKT3) in the presence of CD4 mAbs in soluble phase induces a hyporesponsiveness following subsequent restimulation by iOKT3 in the absence of CD4 mAbs. This hyporesponsiveness was not associated with increased cell death during priming or restimulation cultures and could be reversed by the combination of phorbol ester + ionomycin, demonstrating a functional blockade of viable cells by CD4 mAbs. Following iOKT3 restimulation, hyporesponsive cells showed a lack of blast transformation and CD25 expression and were not able to respond to IL-2 since addition of high doses of exogenous IL-2 +/- CD28 mAbs did not reverse the hyporesponsiveness. However, costimulation with CD45RA mAb completely reversed the hyporesponsiveness, suggesting that CD45 controlled the CD4-mediated hyporesponsiveness.

Variability of recombination frequencies in the Iowa Stiff Stalk Synthetic (Zea mays L.).

Variability in recombination frequency has been reported in several plant populations. The objectives of the present research were to establish the range in variability in recombination among genotypes in the important corn population Iowa Stiff Stalk Synthetic and to identify individual genotypes which produced increased or decreased recombination frequencies. Approximately 150 individual S0 plants were testcrossed to measure male recombination frequency on three chromosomes: 4, sul-c2; 5, a2-btl-pr1; and 9, sh1-bz1-wx1. Although the variance component for individuals accounted for only 20-33% of the total variation, highly significant variability among individuals was present at all chromosome regions. Thus the environmental effects did not prevent measurement of differences between S0 individuals. At each chromosome region, individual genotypes with recombination frequencies at least two standard deviations above or below the population mean were isolated. Reports in the literature suggest that the variability reported here for the BSSS population should be representative of that present in other corn breeding populations. Recombination frequencies were positively correlated between adjacent regions of chromosome 9 and also between adjacent regions of chromosome 5. Recombination frequencies were positively correlated between both regions on chromosome 5 with the su1-c2 region of chromosome 4. Negative correlations were observed between chromosome 9 recombination and recombination in each region of chromosomes 4 and 5. Thus rankings of S0 individual recombination frequencies were not consistent for all three chromosomes.

Determination of fluvoxamine in human plasma by high-performance liquid chromatography with fluorescence detection.

Fluvoxamine and nortriptyline, the assay internal standard, were extracted from plasma with ethyl acetate, then reacted with dansyl chloride. The derivatives were quantitated by isocratic reversed-phase high-performance liquid chromatography with fluorescence detection. The assay calibration range for fluvoxamine was 10-1000 ng/ml using a 1-ml plasma sample. Pooled plasma quality control sample relative recoveries at 25 and 250 ng/ml were 103 and 105%, respectively. Estimates of quality control inter-day precision during validation were less than or equal to 3% relative standard deviation. The assay was cross-validated with a gas chromatographic method and has been employed in therapeutic drug level monitoring.

Liquid chromatographic determination of medroxyprogesterone acetate in tablets.

A reverse-phase liquid chromatographic method is described for the assay of medroxyprogesterone acetate in tablets. An octadecylsilane (C18) column with a mobile phase of methanol-0.01M dibasic ammonium phosphate (80 + 20 v/v, pH 7.2 +/- 0.1) and photometric detection at 254 nm separates medroxyprogesterone acetate from excipients. Detector responses were linear to concentrations of medroxyprogesterone acetate over the range 50-150 micrograms/mL (r = 0.999). Mean recovery of medroxyprogesterone acetate added to tablet excipients was 100.8%. Mean assay results were 101.3% (n = 3). The assay results are comparable to those obtained by the compendial liquid chromatographic method.

Determination of temazepam and related compounds in capsules by high-performance liquid chromatography.

A reversed-phase, high-performance liquid chromatographic assay method is described for temazepam hard gelatin and soft gelatin capsule analysis. The method is simple, specific, accurate, fast, and stability indicating. A reversed-phase octylsilane (C8) column with a mobile phase composed of methanol:1% acetic acid and detection at 254 nm separated sulfanilamide (internal standard), temazepam, synthetic precursor, and possible degradation products. Detector responses showed linearity to temazepam concentrations over the range 0.075-0.60 mg/mL (r = 0.9999). Mean recovery of temazepam added to capsule excipients was 100.3%. Mean assay results for 15- and 30-mg hard gelatin capsules were 101.5 and 101.3%, respectively. Mean assay results for 10- and 20-mg soft elastic gelatin capsules were 101.1 and 101.5%, respectively.

Synthesis of previously inaccessible quinazolines and 1,4-benzodiazepines as potential anticonvulsants.

A series of 4,6,7,8- tetrasubstituted 3,4- dihydroquinazolines , quinazolines, quinazolin -2-ones, 1,2,3,4- tetrahydroquinazolin -2-ones, and 5,7,8,9- tetrasubstituted 1,4-benzodiazepines have been synthesized by utilizing the Diels -Alder reaction between furan o-amino nitriles and various alkyl or aryl vinyl ketone dienophiles to obtain the anthranilic acid precursors. All of the newly synthesized target compounds were evaluated in mice for anticonvulsant activity. Pro- and anticonvulsant action was quantified by the timed intravenous pentylenetetrazol seizure threshold method. Selected compounds were also evaluated for benzodiazepine receptor binding properties and in vivo antagonist potential. Although the compounds lack potency, the data suggest that previously inaccessible substituted analogues may be useful to segregate the proconvulsant , anticonvulsant, and antagonist actions of benzodiazepines and quinazolines.