Identification and characterization of the carboxy-terminal region of Sip-1, a novel autoantigen in Behçet's disease

Given the lack of a serological test specific for Behçet's disease, its diagnosis rests upon clinical criteria. The clinical diagnosis is nevertheless difficult because the disease manifestations vary widely, especially at the onset of disease. The aim of this study was to identify molecules specifically recognized by serum autoantibodies in patients with Behçet's disease and to evaluate their diagnostic value. We screened a cDNA library from human microvascular endothelial cells with serum IgG from two patients with Behçet's disease and isolated a reactive clone specific to the carboxy-terminal subunit of Sip1 (Sip1 C-ter). Using ELISA, we measured IgG, IgM and IgA specific to Sip1 C-ter in patients with various autoimmune diseases characterized by the presence of serum anti-endothelial cell antibodies, such as Behçet's disease, systemic lupus erythematosus, systemic sclerosis and various forms of primary vasculitis, as well as in patients with diseases that share clinical features with Behçet's disease, such as inflammatory bowel disease and uveitis. IgM immunoreactivity to Sip1 C-ter was significantly higher in patients with Behçet's disease and in patients with primary vasculitis than in the other groups of patients and healthy subjects tested (P < 10^-4 by Mann-Whitney test). ELISA detected IgG specific to Sip1 C-ter in sera from 11/56 (20%) patients with Behçet's disease, IgM in 23/56 (41%) and IgA in 9/54 (17%). No sera from patients with systemic lupus erythematosus, systemic sclerosis, inflammatory bowel disease, uveitis or healthy subjects but 45% of sera from patients with primary vasculitis contained IgM specific to Sip1 C-ter. Serum levels of soluble E-selectin, a marker of endothelial activation and inflammation, correlated with levels of serum IgM anti Sip-1 C-ter in patients with Behçet's disease (r = 0.36, P = 0.023). In conclusion, Sip1 C-ter is a novel autoantigen in Behçet's disease. IgM specific to Sip1 C-ter might be useful in clinical practice as an immunological marker of endothelial dysfunction in vasculitis.     

A protein phosphorylation module patterns the Bacillus subtilis spore outer coat

Assembly of the Bacillus subtilis spore coat involves over 80 proteins which self-organize into a basal layer, a lamellar inner coat, a striated electrodense outer coat and a more external crust. CotB is an abundant component of the outer coat. The C-terminal moiety of CotB, SKR^B, formed by serine-rich repeats, is polyphosphorylated by the Ser/Thr kinase CotH. We show that another coat protein, CotG, with a central serine-repeat region, SKR^G, interacts with the C-terminal moiety of CotB and promotes its phosphorylation by CotH in vivo and in a heterologous system. CotG itself is phosphorylated by CotH but phosphorylation is enhanced in the absence of CotB. Spores of a strain producing an inactive form of CotH, like those formed by a cotG deletion mutant, lack the pattern of electrondense outer coat striations, but retain the crust. In contrast, deletion of the SKR^B region, has no major impact on outer coat structure. Thus, phosphorylation of CotG by CotH is a key factor establishing the structure of the outer coat. The presence of the cotB/cotH/cotG cluster in several species closely related to B. subtilis hints at the importance of this protein phosphorylation module in the morphogenesis of the spore surface layers.     

Antagonistic Role of CotG and CotH on Spore Germination and Coat Formation in Bacillus subtilis

Spore formers are bacteria able to survive harsh environmental conditions by differentiating a specialized, highly resistant spore. In Bacillus subtilis, the model system for spore formers, the recently discovered crust and the proteinaceous coat are the external layers that surround the spore and contribute to its survival. The coat is formed by about seventy different proteins assembled and organized into three layers by the action of a subset of regulatory proteins, referred to as morphogenetic factors. CotH is a morphogenetic factor needed for the development of spores able to germinate efficiently and involved in the assembly of nine outer coat proteins, including CotG. Here we report that CotG has negative effects on spore germination and on the assembly of at least three outer coat proteins. Such negative action is exerted only in mutants lacking CotH, thus suggesting an antagonistic effect of the two proteins, with CotH counteracting the negative role of CotG.     

A Regulatory Polymorphism in HAVCR2 Modulates Susceptibility to HIV-1 Infection

The HAVCR2 gene encodes TIM-3, an immunoglobulin superfamily member expressed by exhausted CD8+ T cells during chronic viral infection. We investigated whether genetic variation at HAVCR2 modulates the susceptibility to HIV-1 acquisition; specifically we focused on a 3′ UTR variant (rs4704846, A/G) that represents a natural selection target. We genotyped rs4704846 in three independent cohorts of HIV-1 exposed seronegative (HESN) individuals with different geographic origin (Italy and Spain) and distinct route of exposure to HIV-1 (sexual and injection drug use). Matched HIV-1 positive subjects and healthy controls were also analyzed. In all case-control cohorts the minor G allele at rs4704846 was more common in HIV-1 infected individuals than in HESN, with healthy controls showing intermediate frequency. Results from the three association analyses were combined through a random effect meta-analysis, which revealed no heterogeneity among samples (Cochrane''s Q, p value =  0.89, I² =  0) and yielded a p value of 6.8 ×10⁻⁴. The minor G allele at rs4704846 was found to increase HAVCR2 expression after in vitro HIV-1 infection. Thus, a positively selected polymorphism in the 3′ UTR, which modulates HAVCR2 expression, is associated with the susceptibility to HIV-1 infection. These data warrant further investigation into the role of TIM-3 in the prevention and treatment of HIV-1/AIDS.     

The Flavonoid Quercetin Reverses Pulmonary Hypertension in Rats

Quercetin is a dietary flavonoid which exerts vasodilator, antiplatelet and antiproliferative effects and reduces blood pressure, oxidative status and end-organ damage in humans and animal models of systemic hypertension. We hypothesized that oral quercetin treatment might be protective in a rat model of pulmonary arterial hypertension. Three weeks after injection of monocrotaline, quercetin (10 mg/kg/d per os) or vehicle was administered for 10 days to adult Wistar rats. Quercetin significantly reduced mortality. In surviving animals, quercetin decreased pulmonary arterial pressure, right ventricular hypertrophy and muscularization of small pulmonary arteries. Classic biomarkers of pulmonary arterial hypertension such as the downregulated expression of lung BMPR2, Kv1.5, Kv2.1, upregulated survivin, endothelial dysfunction and hyperresponsiveness to 5-HT were unaffected by quercetin. Quercetin significantly restored the decrease in Kv currents, the upregulation of 5-HT_2A receptors and reduced the Akt and S6 phosphorylation. In vitro, quercetin induced pulmonary artery vasodilator effects, inhibited pulmonary artery smooth muscle cell proliferation and induced apoptosis. In conclusion, quercetin is partially protective in this rat model of PAH. It delayed mortality by lowering PAP, RVH and vascular remodeling. Quercetin exerted effective vasodilator effects in isolated PA, inhibited cell proliferation and induced apoptosis in PASMCs. These effects were associated with decreased 5-HT_2A receptor expression and Akt and S6 phosphorylation and partially restored Kv currents. Therefore, quercetin could be useful in the treatment of PAH.     

Identification of a new susceptibility variant for multiple sclerosis in OAS1 by population genetics analysis

Contrasting results have been reported concerning the association of a splice-site polymorphism (rs10774671) in OAS1 with multiple sclerosis (MS). We analysed two OAS1 regions encompassing alternatively spliced exons. While the region carrying the splice-site variant is neutrally evolving, a signature of long-standing balancing selection was observed across an alternative exon 7. Analysis of variants in this exon identified an insertion/deletion polymorphism (rs11352835, A/?) that originates predicted products with distinct C termini. This variant is located along the major branch of the haplotype genealogy, suggesting that it may represent the selection target. A case/control study for MS indicated that rs11352835 is associated with disease susceptibility (for an allelic model with the deleted allele predisposing to MS, OR 1.27, 95% CI 1.072?C1.513, p?=?0.010). No association was found between rs10774671 and MS. As the two SNPs are in linkage disequilibrium in Europeans, the previously reported association between rs10774671 and MS susceptibility might be driven by rs11352835, possibly explaining the contrasting results previously observed for the splice-site polymorphism. Thus, we describe a novel susceptibility variant for MS in OAS1 and show that population genetic analyses can be instrumental to the identification of selection targets and, consequently, of functional polymorphisms with an effect on phenotypic traits.     

An aspartate residue in the external vestibule of GLYT2 (glycine transporter 2) controls cation access and transport coupling

Synaptic glycine levels are controlled by GLYTs (glycine transporters). GLYT1 is the main regulator of synaptic glycine concentrations and catalyses Na+-Cl--glycine co-transport with a 2:1:1 stoichiometry. In contrast, neuronal GLYT2 supplies glycine to the presynaptic terminal with a 3:1:1 stoichiometry. We subjected homology models of GLYT1 and GLYT2 to molecular dynamics simulations in the presence of Na+. Using molecular interaction potential maps and in silico mutagenesis, we identified a conserved region in the GLYT2 external vestibule likely to be involved in Na+ interactions. Replacement of Asp471 in this region reduced Na+ affinity and Na+ co-operativity of transport, an effect not produced in the homologous position (Asp295) in GLYT1. Unlike the GLYT1-Asp295 mutation, this Asp471 mutant increased sodium leakage and non-stoichiometric uncoupled ion movements through GLYT2, as determined by simultaneously measuring current and [3H]glycine accumulation. The homologous Asp471 and Asp295 positions exhibited distinct cation-sensitive external accessibility, and they were involved in Na+ and Li+-induced conformational changes. Although these two cations had opposite effects on GLYT1, they had comparable effects on accessibility in GLYT2, explaining the inhibitory and stimulatory responses to lithium exhibited by the two transporters. On the basis of these findings, we propose a role for Asp471 in controlling cation access to GLYT2 Na+ sites, ion coupling during transport and the subsequent conformational changes.     

Immunomodulatory mechanisms during Echinococcus granulosus infection

The pathologic events that ensue after humans ingest the eggs of Echinococcus granulosus and continue while cystic echinococcosis develops, provide an excellent example illustrating the evasive strategies helminth parasites use to develop, progress and cause chronic disease. The hydatid cyst secretes and exposes numerous immunomodulatory molecules to the host’s immune system. By characterizing these molecules we can understand the mechanisms that E. granulosus uses for increasing the efficiency and persistency of infection in the host. These molecules modulate both the innate and adaptive arms of the immune response and appear to target cellular and humoral responses. In this review, we discuss recent advances in the immunobiology of host-E. granulosus interactions that provide intriguing insights into the complex interplay between host and parasite that ultimately facilitates parasite survival.