Caspase-1 levels in biological fluids from patients with multiple sclerosis and from patients with other neurological and non-neurological diseases

In multiple sclerosis (MS), pathological white matter damage in the central nervous system is sustained by immune-inflammatory response. Caspase-1 plays a pivotal role in immune-mediated inflammation, as it regulates the cellular export of IL-1beta and IL-18. We carried out a preliminary in vitro study of the kinetics of extracellular caspase-1 release. We then measured caspase-1 levels in paired serum and cerebrospinal fluid (CSF) samples of 75 MS patients, 15 healthy subjects, and patients with other neurological diseases. Paired synovial fluid and serum samples of patients with juvenile idiopathic arthritis, and paired sputum and serum samples of asthma patients were also studied. Mean serum caspase-1 concentrations did not differ between groups. Caspase-1 was detected in the CSF of patients with acute, but not stable, MS [7.5 +/- (SEM) 0.9 pg/ml; test's sensitivity, 56% and specificity, 100%]. Its levels correlated with pleocytosis. The highest mean caspase-1 levels were found in the arthritic synovial fluids (945.5 +/- 126.6 pg/ml, which correlated with erythrocyte sedimentation rate), and in the sputum samples (370.1 +/- 71.0 pg/ml, which correlated with the number of macrophages in the sputum). On condition that caspase-1 is determined in the fluids pertaining to the disease-specific inflammatory sites, its level is a reliable marker of ongoing immune-inflammatory response. The enzyme measurement in CSF can also help define state-trait in MS.     

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.     

A novel yeast U2 snRNP protein, Snu17p, is required for the first catalytic step of splicing and for progression of spliceosome assembly

We have isolated and microsequenced Snu17p, a novel yeast protein with a predicted molecular mass of 17 kDa that contains an RNA recognition motif. We demonstrate that Snu17p binds specifically to the U2 small nuclear ribonucleoprotein (snRNP) and that it is part of the spliceosome, since the pre-mRNA and the lariat-exon 2 are specifically coprecipitated with Snu17p. Although the SNU17 gene is not essential, its knockout leads to a slow-growth phenotype and to a pre-mRNA splicing defect in vivo. In addition, the first step of splicing is dramatically decreased in extracts prepared from the snu17 deletion (snu17Delta) mutant. This defect is efficiently reversed by the addition of recombinant Snu17p. To investigate the step of spliceosome assembly at which Snu17p acts, we have used nondenaturing gel electrophoresis. In Snu17p-deficient extracts, the spliceosome runs as a single slowly migrating complex. In wild-type extracts, usually at least two distinct complexes are observed: the prespliceosome, or B complex, containing the U2 but not the U1 snRNP, and the catalytically active spliceosome, or A complex, containing the U2, U6, and U5 snRNPs. Northern blot analysis and affinity purification of the snu17Delta spliceosome showed that it contains the U1, U2, U6, U5, and U4 snRNPs. The unexpected stabilization of the U1 snRNP and the lack of dissociation of the U4 snRNP suggest that loss of Snu17p inhibits the progression of spliceosome assembly prior to U1 snRNP release and after [U4/U6.U5] tri-snRNP addition.     

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.     

CaMADS1, a MADS box gene expressed in the carpel of hazelnut

Hazelnut (Corylus avellana L.) is a species of economic interest that shows a peculiar floral biology. Unlike most of the angiosperms, which produce ovules during floral development such that they are ready for pollen at anthesis, hazelnut ovary development is delayed and triggered by compatible pollination. In order to elucidate the mechanisms regulating this unusual process and the role of the MADS box genes in ovary development, a cDNA library from pollinated styles of hazelnut was screened with a mixture of MADS box genes from different plant species. CaMADS1 (Corylus avellana MADS box), a floral-specific MADS box gene, was isolated, and characterized as belonging to the sub-family of the AGAMOUS genes. Northern blot, RT-PCR analyses and in situ hybridization experiments show a precise correlation between ovary development and CaMADS1 expression, indicating a role of this MADS box gene in the processes of floral organogenesis.