Computer-based design of an HLA-haplotype and HIV-clade independent cytotoxic T-lymphocyte assay for monitoring HIV-specific immunity

BACKGROUND: Human immunodeficiency virus (HIV)- specific CD8-positive cytotoxic T-lymphocytes (CTL) play a key role in controlling HIV infection. Monitoring CTL response could be clinically relevant during structured therapy interruption (STI), HIV exposure, and vaccine trials. However, HLA patients' restriction and HIV variability limited the development of a CTL assay with broad specificity. MATERIALS AND METHODS: We designed an HLA-class I/HIV-1 clade independent assay for assessing HIV- specific CTL by using a computer-assisted selection ofthe CTL epitopes. Twenty-eight 15-mers were selected by peptide-binding motifs analysis using different databases (HIV-Immunology Database, SYFPEITHI, BIMAS). Altogether they putatively bind to more than 90% of HLA haplotypes in different populations, with an overall HIV-1 variability below 9%. The peptide pool was used as an antigen in an intracellular cytokine staining (ICS) assay for quantifying HIV-specific CTL response. RESULTS: The test can be performed using both fresh and cryopreserved peripheral blood mononuclear cells (PBMC), whereas GAG protein as antigen works only on fresh PBMC. A significantly higher CTL response with respect to HIV-negative controls was detected in all HIV-1 infected subjects of two groups of patients with different ethnicities (Caucasians and Africans) and coming from areas with different HIV-1 clade prevalences (clade B and A/G, respectively). In Caucasian patients, after month of STI, the number of HIV-1 specific CTL (2,896 +/- 2,780 IFN-gamma specific CD8 cells/ml) was significantly higher than that found at enrolment (2,125 +/- 4,426 IFN-gamma specific CD8 cells/ml, p< 0.05). CONCLUSIONS: These data indicate that this CTL assay is broadly specific and could represent a useful clinical tool for HIV immunodiagnostic independent of HLA-haplotype and HIV-clade variabilities.     

The Gam protein of bacteriophage Mu is an orthologue of eukaryotic Ku

Mu bacteriophage inserts its DNA into the genome of host bacteria and is used as a model for DNA transposition events in other systems. The eukaryotic Ku protein has key roles in DNA repair and in certain transposition events. Here we show that the Gam protein of phage Mu is conserved in bacteria, has sequence homology with both subunits of Ku, and has the potential to adopt a similar architecture to the core DNA-binding region of Ku. Through biochemical studies, we demonstrate that Gam and the related protein of Haemophilus influenzae display DNA binding characteristics remarkably similar to those of human Ku. In addition, we show that Gam can interfere with Ty1 retrotransposition in Saccharomyces cerevisiae. These data reveal structural and functional parallels between bacteriophage Gam and eukaryotic Ku and suggest that their functions have been evolutionarily conserved.     

Enantiomerically pure tetrahydroquinoline derivatives as in vivo potent antagonists of the glycine binding site associated to the NMDA receptor

To identify neuroprotective agents after stroke, new substituted tetrahydroquinoline derivatives were designed as antagonists of the glycine binding site associated to the NMDA receptor, satisfying the key pharmacophoric requirements. In particular, the racemate 3c exhibited outstanding in vivo activity in the MCAo model in rats, when given iv both pre- and post-ischemia. Pure enantiomers 3c-(+) and 3c-(-) have been prepared following an original synthetic route. Despite the significant difference of activity observed in vitro, they shown similar neuroprotective profile in the MCAo model in rats.     

Autologous and Heterologous Neutralization Analyses of Primary Feline Immunodeficiency Virus Isolates

Feline immunodeficiency virus (FIV) provides a model system with which the significance of neutralizing antibody (NA) in immunosuppressive lentivirus infections may be studied. To date, no detailed analysis of the neutralization properties of primary FIV isolates has been reported. In this study, we have conducted the first comprehensive study of the sensitivity to autologous and heterologous neutralization in a lymphoid cell-based assay of 15 primary FIV isolates and, for comparison, of one tissue culture-adapted strain. Primary isolates in general proved highly NA resistant, although there was considerable individual variation. Variation was also observed in the capacity of immune sera to neutralize heterologous FIV isolates. The ability of sera to neutralize isolates or for isolates to be neutralized by sera did not correlate with epidemiological and genetic relatedness or with the quasispecies complexity of the isolates. From the study of specific-pathogen-free cats experimentally infected with viral isolates associated with NA of different breadths, it appears that the development of FIV vaccines cannot rely on the existence of viral strains inherently capable of inducing especially broad NA responses.Feline immunodeficiency virus (FIV) is a lentivirus that is regarded as the feline counterpart of human immunodeficiency virus (HIV) because it produces persistent infections of domestic cats which, after an incubation period of several years, progress to clinical manifestations of immunodeficiency and neurological damage that closely resemble those observed in HIV-infected humans. FIV is therefore a valuable model for investigating many aspects of AIDS pathobiology and control, including vaccination (4, 11, 39, 56).Based on DNA phylogenesis, FIV isolates worldwide have been classified into at least five distinct genetic subtypes, designated A to E, with uneven geographical distributions (2). While there is little hope of developing a monovalent vaccine capable of protecting across different FIV subtypes, a more reasonable goal is the development of one or several protective immunogens for each subtype and subsequent selection of the immunogens on the basis of the subtypes prevalent in the area where the vaccine is to be used (56). However, because genetic diversity is also high within a subtype, especially in the env region (2, 42), successful vaccines will have to induce immune responses effective against a wide range of antigenically diverse strains. Mapping the immunological relatedness of FIV strains belonging to the same genetic subtype therefore represents a prerequisite for identifying shared critical protective epitopes and an essential step for ongoing vaccine development efforts. Similar problems exist for HIV vaccine development (33).Although the humoral and cell-mediated immune responses that will eventually prove important for vaccine-induced protection against lentiviruses are unresolved (3, 7, 17), the ability to evoke a broadly reactive neutralizing-antibody (NA) response would seem to be an advantageous feature of candidate immunogens because it would at least contrast the dissemination of the initial viral inoculum from the site of entry (8, 9). In previous studies, we found that cats immunized with a fixed-cell vaccine were protected against FIV challenge in the apparent absence of NA (27, 28), but it is possible that a detectable NA response could be elicited with improved vaccines, adjuvants, and immunization regimens.FIV vaccines must be designed to protect against strains of FIV as they circulate in nature. For this reason, it is important to learn more about the immunobiological properties of fresh clinical isolates, including their ability to evoke and interact with NA and their neutralizing determinant(s). Here we report on the sensitivity of 15 FIV isolates subjected to minimal passage in culture to neutralization by autologous and heterologous immune sera. Primary FIV isolates proved only slightly prone to inhibition by immune sera. However, certain isolates were more neutralizable by heterologous sera than others and certain infected cat sera neutralized fairly large numbers of primary isolates. A relationship was also sought between neutralization properties of the isolates and immune sera and a number of factors that theoretically might influence the induction or the activity of cross-reactive NA, including epidemiological and genetic relatedness and quasispecies complexity of the isolates. Finally, to ascertain whether the cross-neutralizing potency of anti-FIV antibody was dependent on properties of the viruses that had induced their formation, we studied the NA response of specific-pathogen-free (SPF) cats inoculated with selected FIV isolates.     

Synthesis,biological evaluation,and SAR study of novel pyrazole analogues as inhibitors of Mycobacterium tuberculosis: Part 2. Synthesis of rigid pyrazolones

Two series of novel rigid pyrazolone derivatives were synthesized and evaluated as inhibitors of Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis. Two of these compounds showed a high activity against MTB (MIC = 4 μg/mL). The newly synthesized pyrazolones were also computationally investigated to analyze if their properties fit the pharmacophoric model for antitubercular compounds previously built by us. The results are in agreement with those reported by us previously for a class of pyrazole analogues and confirm the fundamental role of the p-chlorophenyl moiety at C4 in the antimycobacterial activity.     

Effective Coverage and Systems Effectiveness for Malaria Case Management in Sub-Saharan African Countries

Scale-up of malaria preventive and control interventions over the last decade resulted in substantial declines in mortality and morbidity from the disease in sub-Saharan Africa and many other parts of the world. Sustaining these gains will depend on the health system performance. Treatment provides individual benefits by curing infection and preventing progression to severe disease as well as community-level benefits by reducing the infectious reservoir and averting emergence and spread of drug resistance. However many patients with malaria do not access care, providers do not comply with treatment guidelines, and hence, patients do not necessarily receive the correct regimen. Even when the correct regimen is administered some patients will not adhere and others will be treated with counterfeit or substandard medication leading to treatment failures and spread of drug resistance. We apply systems effectiveness concepts that explicitly consider implications of health system factors such as treatment seeking, provider compliance, adherence, and quality of medication to estimate treatment outcomes for malaria case management. We compile data for these indicators to derive estimates of effective coverage for 43 high-burden Sub-Saharan African countries. Parameters are populated from the Demographic and Health Surveys and other published sources. We assess the relative importance of these factors on the level of effective coverage and consider variation in these health systems indicators across countries. Our findings suggest that effective coverage for malaria case management ranges from 8% to 72% in the region. Different factors account for health system inefficiencies in different countries. Significant losses in effectiveness of treatment are estimated in all countries. The patterns of inter-country variation suggest that these are system failures that are amenable to change. Identifying the reasons for the poor health system performance and intervening to tackle them become key priority areas for malaria control and elimination policies in the region.     

Analysis of the Role of Interleukin 6 Receptor Haplotypes in the Regulation of Circulating Levels of Inflammatory Biomarkers and Risk of Coronary Heart Disease

Variants at the interleukin 6 receptor (IL6R) gene regulate inflammation and are associated with risk of coronary heart disease (CHD). The aim of the present study was to investigate the effects of IL6R haplotypes on circulating levels of inflammatory biomarkers and risk of CHD. We performed a discovery analysis in SHEEP, a myocardial infarction (MI) case control study (n = 2,774) and replicated our results in two large, independent European populations, PROCARDIS, a CHD case control study (n = 7,998), and IMPROVE (n = 3,711) a prospective cardiovascular cohort study. Two major haplotype blocks (rs12083537A/G and rs4075015A/T—block 1; and rs8192282G/A, rs4553185T/C, rs8192284A/C, rs4240872T/C and rs7514452T/C—block 2) were identified in the IL6R gene. IL6R haplotype associations with C-reactive protein (CRP), fibrinogen, IL6, soluble IL6R (sIL6R), IL6, IL8 and TNF-α in SHEEP, CRP and fibrinogen in PROCARDIS and CRP in IMPROVE as well as association with risk of MI and CHD, were analyzed by THESIAS. Haplotypes in block 1 were associated neither with circulating inflammatory biomarkers nor with the MI/CHD risk. Haplotypes in block 2 were associated with circulating levels of CRP, in all three study populations, with fibrinogen in SHEEP and PROCARDIS, with IL8 and sIL6Rin SHEEP and with a modest, non significant, increase (7%) in MI/CHD risk in the three populations studied. Our results indicate that IL6R haplotypes regulate the circulating levels of inflammatory biomarkers. Lack of association with the risk of CHD may be explained by the combined effect of SNPs with opposite effect on the CHD risk, the sample size as well as by structural changes affecting sIL6R stability in the circulation.     

Preventive study in subjects at risk of fatal familial insomnia: Innovative approach to rare diseases

The text describes a preventive clinical trial with drug treatment in a very rare neurodegenerative disease (Fatal familial Insomnia, FFI) designed with the help of individuals at genetic risk of developing the disease, asymptomatic carriers, who have agreed to be exposed over a 10-year period to doxycycline, an antibiotic with anti-prion activity. At least 10 carriers of the FFI mutation over 42 y old will be treated with doxycycline (100 mg/die) and the incidence of the disease will be compared to that of an historical dataset. For ethical reasons a randomized, double-blind, placebo-controlled trial was not feasible, however the study design and the statistical analysis ensure the scientific value of the results. This approach might represent an important breakthrough in terms of potential therapy and knowledge of rare diseases that could give some hopes to these neglected patients.