Comparison of HLA-DR1-restricted T cell response induced in HLA-DR1 transgenic mice deficient for murine MHC class II and HLA-DR1 transgenic mice expressing endogenous murine MHC class II molecules

Transgenic mice expressing human HLA class II molecules provide a useful model for identifying HLA-restricted CD4+ epitopes. However, the influence of endogenous murine H-2-restricted T cell responses on HLA-restricted responses is not known. In the present study, we show that HLA-DR1 transgenic mice deficient for H-2 class II expression (HLA-DR1+/+/IAbeta0/0) exhibit an equivalent expression level of the transgene HLA-DR1 and a similar diversity in the TCR repertoire, but a slightly different number of CD4+ peripheral T cells, when compared to HLA-DR1 transgenic mice in which H-2 class II molecules were retained (HLA-DR1+/+/IAbeta+/+). More importantly, a strong antigen-specific HLA-DR1-restricted response was observed in nearly all HLA-DR1+/+/IAbeta0/0 mice immunized with HBV envelope protein (HBs) or capsid protein (HBc), whereas weak HBs- or HBc-specific HLA-DR1-restricted responses were detected in half of the immunized HLA-DR1+/+/IAbeta+/+ mice. Conversely, strong HBs- or HBc-specific H-2-restricted T cell responses were detected in HLA-DR1+/+/IAbeta+/+ mice but not in HLA-DR1+/+/IAbeta0/0 mice. Our results indicate that the coexpression of endogenous H-2 class II molecules reduces the intensity of HLA-DR1-restricted antigen-specific responses in transgenic mice, by favoring murine over human MHC recognition and education. Thus, HLA-DR1+/+/IAbeta0/0 mice represent a better model for identifying and characterizing HLA-DR1-restricted epitopes relevant for human disease.     

Three highly polymorphic microsatellites at the human myelin oligodendrocyte glycoprotein locus, 100 kb telomeric to HLA-F : Characterization and relation to HLA haplotypes

The MOG locus, located on chromosomal bands 6p21.3-p22 and mapped about 100 kb telomeric to HLA-F, was isolated from cosmid ICRFcl09A2434 and shown to contain three microsatellites. These CA-repeat polymorphic markers were characterized in a sample of 173 healthy unrelated individuals and 84 DNAs from the HLA Workshop reference panel, by a method combining fluorescence labeling of PCR products and use of an automated DNA sequencer. For the three markers, frequencies of heterozygotes are well predicted from allele frequencies by the Hardy—Weinberg rule, which suggests that problems of allele nonamplification are unlikely. Typing of cell lines homozygous in the HLA region allowed unambiguous definition of 81 HLA-MOG haplotypes and showed that several HLA ancestral haplotypes extended to the MOG region. The high degree of polymorphism (59%, 51%, and 81% at the three loci, respectively, and 87% at the haplotype level) makes these new markers informative for association or linkage studies with diseases such as hemochromatosis or multiple sclerosis, and for studies aimed at precisely delineating the site of crossover in chromosomes in which recombination occurred in the distal part of the HLA class I region.     

Integration from proteins to organs: the IUPS Physiome Project

The IUPS Physiome Project is an internationally collaborative open source project intended to provide a public domain framework for computational physiology, including the development of modeling standards, computational tools and web-accessible databases of models of structure and function at all spatial scales and across all organ systems. Here, we illustrate the application of this multi-scale modeling approach to three organ systems: the heart, the lungs and the musculo-skeletal system, and in each case we show how the organ level models incorporate tissue and cell-level physiology. Although the computational physiology framework presented here does not yet incorporate models of ageing processes, the model-based approach is certainly capable of describing ageing and disease-related processes both via parameter changes within the models of normal physiological processes and via models of additional processes added to the framework.     

Contrasting roles of DAP10 and KARAP/DAP12 signaling adaptors in activation of the RBL-2H3 leukemic mast cell line

A common feature of hematopoietic activating immunoreceptors resides in their association at the cell surface with transmembrane signaling adaptors. Several adaptors, such as the CD3 molecules, FcRgamma and KARAP/DAP12, harbor intracytoplasmic immunoreceptor tyrosine-based activation motifs (ITAM) that activate Syk-family protein tyrosine kinases. In contrast, another transmembrane adaptor, DAP10, bears a YxxM motif that delivers signals by activation of lipid kinase pathways. We show here that the human signal-regulatory protein SIRPbeta1 can associate with both DAP10 and KARAP/DAP12 in a model of RBL-2H3 cell transfectants. In association with KARAP/DAP12, SIRPbeta1 complexes are capable of inducing serotonin release and tumor necrosis factor (TNF) secretion. By contrast,in the absence of KARAP/DAP12, engagement of SIRPbeta1:DAP10 complexes does not lead to detectable serotonin release or TNF secretion by RBL-2H3 transfectants. However, triggering of SIRPbeta1:DAP10 complexes co-stimulates RBL-2H3 effector function induced by sub-optimal stimulation of the endogenous FcepsilonRI complex. Therefore, we report here a cellular model in which the association of a cell surface receptor with various signaling adaptors dictates the co-stimulatory or the direct stimulatory properties of the complex.     

A microsatellite-based,physically anchored linkage map for the gray,short-tailed Opossum (<Emphasis Type="Italic">Monodelphis domestica</Emphasis>)

The genome of the gray, short-tailed opossum, Monodelphis domestica, will be the first of any marsupial to be fully sequenced. The utility of this sequence will be greatly enhanced by construction and integration of detailed genetic and physical maps. Therefore, it is important to verify the unusual recombinational characteristics that were suggested by the ‘first-generation’ M. domestica linkage map; specifically, very low levels of recombination and severely reduced female recombination, both of which are contrary to patterns in other vertebrates. We constructed a new linkage map based on a different genetic cross, using a new and much larger set of map markers, and physically anchored and oriented the linkage groups onto chromosomes via fluorescence in-situ hybridization mapping. This map includes 150 loci in eight autosomal linkage groups corresponding to the eight autosome pairs, and spans 86–89% of the autosomal genome. The sex-averaged autosomal map covers 715 cM, with a full-length estimate of 866 cM; the shortest full-length linkage map reported for any vertebrate. The sex-specific maps confirmed severely reduced female recombination in all linkage groups, and an overall F/M map ratio =  0.54. These results greatly extend earlier findings, and provide an improved microsatellite-based linkage map for this species. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Autosomal genetics and Y-chromosome haplogroup L1b-M317 reveal Mount Lebanon Maronites as a persistently non-emigrating population

Currently, there are 18 different religious communities living in Lebanon. While evolving primarily within Lebanon, these communities show a level of local isolation as demonstrated previously from their Y-haplogroup distributions. In order to trace the origins and migratory patterns that may have led to the genetic isolation and autosomal clustering in some of these communities we analyzed Y-chromosome STR and SNP sample data from 6327 individuals, in addition to whole genome autosomal sample data from 609 individuals, from Mount Lebanon and other surrounding communities. We observed Y chromosome L1b Levantine STR branching that occurred around 5000 years ago. Autosomal DNA analyses suggest that the North Lebanese Mountain Maronite community possesses an ancestral Fertile Crescent genetic component distinct from other populations in the region. We suggest that the Levantine L1b group split from the Caucasus ancestral group around 7300 years ago and migrated to the Levant. This event was distinct from the earlier expansions from the Caucasus region that contributed to the wider Levantine populations. Differential cultural adaption by populations from the North Lebanese Mountains are clearly aligned with the L1b haplotype STR haplogroup clusters, indicating pre-existing and persistent cultural barriers marked by the transmission of L1b lineages. Our findings highlight the value of uniparental haplogroups and STR haplotype data for elucidating biosocial events among these populations.Subject terms: Population genetics, Computational biology and bioinformatics     

Evaluation of three commercially available hepatitis C virus antibody detection assays under the conditions of a clinical virology laboratory.

BACKGROUND: Most studies evaluating antibody detection assays are conducted on samples from healthy blood donors but not on samples of hospitalized patients which can show non-specific reactions. OBJECTIVES: To compare the performance of three commercial automated assays for the detection of hepatitis C virus (HCV) antibodies, Monolisa anti-HCV Plus version 2, Axsym anti-HCV 3.0 and Vitros anti-HCV, on a population of hospitalized patients. STUDY DESIGN: The specificity of the assays was prospectively evaluated in 2020 routine serum samples. In order to assign the serostatus of each sample, those giving positive or discordant results were further tested by three immunoblots and by RT-PCR (Roche). Moreover, the sensitivity was evaluated on eight commercial HCV seroconversion panels. RESULTS: The Monolisa, Axsym and Vitros assays showed specificities of 99.64%, 99.12% and 99.33%, respectively. Concerning the sensitivity, among 49 samples, the number of positive results was 21, 24 and 24 for the Monolisa, Axsym and Vitros kits, respectively. The differences were not statistically significant at an alpha risk of 5%. CONCLUSIONS: All assays appeared to be reliable for routine screening, but there were a surprising number of indeterminate samples that could not be resolved by confirmatory tests.     

A role for glucose and insulin preprandial profiles to differentiate meals and snacks

A physiological distinction between eating occasions may help account for contradictory findings on the role of eating frequency in energy homeostasis. We assessed this issue using a midafternoon eating occasion known in France as the goûter that often consists of snack foods. Among the 24 male subjects, 8 habitually consumed four meals per day, i.e., were usual goûter eaters (GE) and 16 habitually took 3 meals per day, i.e., usual non-goûter non-snack eaters (NGNSE). All subjects were time blinded from lunchtime and had to request subsequent meals. Blood was continuously withdrawn and collected with a change of tube every 10 min until dinner request. During the session, 8 of the non-goûter eaters (NGE) were offered a snack 210 min after lunch and were designated as non-goûter snack eaters (NGSE) if they ate. Results showed that the goûter was preceded by high hunger scores and a linear decline in plasma glucose (−9.0±3.0%, P<.05) and insulin concentrations (−22.9±6.0%, P<.05). These profiles were not observed before the snack. The dinner of GE was requested later and was smaller compared to NGNSE, whereas the snack altered neither time of request nor energy intake (EI) at dinner. Among blood variables, leptin at the onset of eating was the only factor that was predictive of both intermeal interval and EI. The glucose and insulin profiles indicate that snacks should not be considered as meals in studies on the role of eating frequency in energy homeostasis.     

Development and characterisation of neutralising monoclonal antibody to the SARS-coronavirus

There is a global need to elucidate protective antigens expressed by the SARS-coronavirus (SARS-CoV). Monoclonal antibody reagents that recognise specific antigens on SARS-CoV are needed urgently. In this report, the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mAbs) against the SARS-CoV is presented, based upon their specificity, binding requirements, and biological activity. Initial screening by ELISA, using highly purified virus as the coating antigen, resulted in the selection of 103 mAbs to the SARS virus. Subsequent screening steps reduced this panel to seventeen IgG mAbs. A single mAb, F26G15, is specific for the nucleoprotein as seen in Western immunoblot while five other mAbs react with the Spike protein. Two of these Spike-specific mAbs demonstrate the ability to neutralise SARS-CoV in vitro while another four Western immunoblot-negative mAbs also neutralise the virus. The utility of these mAbs for diagnostic development is demonstrated. Antibody from convalescent SARS patients, but not normal human serum, is also shown to specifically compete off binding of mAbs to whole SARS-CoV. These studies highlight the importance of using standardised assays and reagents. These mAbs will be useful for the development of diagnostic tests, studies of SARS-CoV pathogenesis and vaccine development.