Epstein-Barr virus promotes human monocyte survival and maturation through a paracrine induction of IFN-alpha

The role of monocytes and macrophages during EBV infection is not clear. The interaction of EBV with human monocytes was investigated in terms of cell survival and morphological and phenotypic changes to gain a better understanding of the role of these cells during EBV infection. We show that EBV infection of PBMCs rescues monocytes from undergoing spontaneous apoptosis and dramatically enhances their survival. Results obtained with heat-inactivated virus, neutralizing anti-EBV mAb 72A1 and recombinant gp350, suggest that enhancement of viability by EBV requires both infectious virus and interaction between gp350 and its receptor. IFN-alpha either secreted within 24 h from PBMCs upon infection with EBV or exogenously added to unstimulated monocytes inhibited spontaneous apoptosis, indicating that induction of IFN-alpha is an early important survival signal responsible for the delay in the apoptosis of monocytes. EBV infection also induced acute maturation of monocytes to macrophages with morphological and phenotypic characteristics of potent APCs. Monocytes exposed to EBV became larger in size with increased granularity and expressed considerably higher levels of membrane HLA classes I and II, ICAM-1, CD80, CD86, and CD40 compared with uninfected cultures. These observations provide the first immunoregulatory links among EBV, IFN-alpha, and monocyte survival and maturation and importantly raise the possibility that these cells may serve as a vehicle for the dissemination of the virus as well as being active participants in eliciting anti-EBV T cell responses during acute infection.     

Structural and functional analysis of human cytomegalovirus US3 protein

Human cytomegalovirus (HCMV) unique short region 3 (US3) protein, a type I membrane protein, prevents maturation of class I major histocompatibility complex (MHC) molecules by retaining them in the endoplasmic reticulum (ER) and thus helps inhibit antigen presentation to cytotoxic T cells. US3 molecules bind to class I MHC molecules in a transient fashion but retain them very efficiently in the ER nonetheless. The US3 luminal domain is responsible for ER retention of US3 itself, while both the US3 luminal and transmembrane domains are necessary for retaining class I MHC in the ER. We have expressed the luminal domain of US3 molecule in Escherichia coli and analyzed its secondary structure by using nuclear magnetic resonance. We then predicted the US3 tertiary structure by modeling it based on the US2 structure. Unlike the luminal domain of US2, the US3 luminal domain does not obviously interact with class I MHC molecules. The luminal domain of US3 dynamically oligomerizes in vitro and full-length US3 molecules associate with each other in vivo. We present a model depicting how dynamic oligomerization of US3 may enhance its ability to retain class I molecules within the ER.     

Conjugation of tetanus toxoid with Salmonella typhimurium PTCC 1735 O-specific polysaccharide and its effects on production of opsonizing antibodies in a mouse model

Serious enteric and extra-intestinal infections with Salmonella typhimurium are very common in many human populations. Phagocytosis is the main defense mechanism against this bacterium; however, the unique structure of S. typhimurium lipopolysaccharide (LPS) makes it resistant to opsonization by complement components. In the present study, the S. typhimurium LPS O-chain was purified and conjugated with tetanus toxoid and the effects of the conjugated vaccine (O-specific polysaccharide tetanus toxoid (O-SP-TT)) on induction of specific antibodies were investigated in a mouse model. In vitro assays measuring phagocytosis in the presence of opsonizing antibodies were performed. Three subcutaneous injections of the O-SP-TT conjugate conferred protection against the intraperitoneal challenge with S. typhimurium and the LD50 was greater for immunized animals than for controls. The mean number of ingested S. typhimirium / mouse peritoneal cell in the presence of sera obtained from immunized mice with purified O-chain, O-SP-TT conjugate, heat-killed bacteria, and negative control were 6.96, 14.24, 15.96, and 6.67, respectively. In summary, we developed an O-SP-TT conjugate that induced opsonizing antibodies that increased phagocytosis, as determined by in vitro assays. In addition, chemiluminescence results, an indicator of oxidative burst, indicated that peritoneal cells respond better to live S. typhimurium cells in the presence of sera obtained from O-SP-TT conjugate immunized mice.     

Pathophysiological characterization of congenital myasthenic syndromes: the example of mutations in the MUSK gene

Congenital myasthenic syndromes (CMS) are rare genetic diseases affecting the neuromuscular junction (NMJ) and are characterized by a dysfunction of the neurotransmission. They are heterogeneous at their pathophysiological level and can be classified in three categories according to their presynaptic, synaptic and postsynaptic origins. We report here the first case of a human neuromuscular transmission dysfunction due to mutations in the gene encoding a postsynaptic molecule, the muscle-specific receptor tyrosine kinase (MuSK). Gene analysis identified two heteroallelic mutations, a frameshift mutation (c.220insC) and a missense mutation (V790M). The muscle biopsy showed dramatic pre- and postsynaptic structural abnormalities of the neuromuscular junction and severe decrease in acetylcholine receptor (AChR) epsilon-subunit and MuSK expression. In vitro and in vivo expression experiments were performed using mutant MuSK reproducing the human mutations. The frameshift mutation led to the absence of MuSK expression. The missense mutation did not affect MuSK catalytic kinase activity but diminished expression and stability of MuSK leading to decreased agrin-dependent AChR aggregation, a critical step in the formation of the neuromuscular junction. In electroporated mouse muscle, overexpression of the missense mutation induced, within a week, a phenotype similar to the patient muscle biopsy: a severe decrease in synaptic AChR and an aberrant axonal outgrowth. These results strongly suggest that the missense mutation, in the presence of a null mutation on the other allele, is responsible for the dramatic synaptic changes observed in the patient.     

Evaluating the role of autologous mesenchymal stem cell seeded on decellularized pericardium in the treatment of myocardial infarction: an animal study

Inappropriate left ventricular remodeling following myocardial infarction (MI) can result in subsequent severe dysfunction. In this study, we tested the hypothesis that decellularized pericardium (DP) or seeded pericardial patch with autologous adipose-derived mesenchymal stem cells (ADMSCs) could be safely used in a MI scar and could improve heart function. Twelve rabbits were randomly divided into three equal groups. Four weeks after MI induction by ligation of the left anterior descending artery in 12 rabbits, animals of G1 (n = 4) received DP patch with labeled ADMSCs. DP patch was implanted in animals of G2 (n = 4). Rabbits of G3 (n = 4) remained without any intervention after MI induction (control group). Serial examinations including echocardiography, electrocardiography (ECG), scanning electron microscopy, histology and immunohistochemistry (IHC) were performed to evaluate the efficacy of the implanted scaffolds on recovery of the infracted myocardium. The results demonstrated that left ventricular contractile function and myocardial pathological changes were significantly improved in rabbits implanted with either DP or ADMSC-seeded pericardium. However, the seeded pericardium was more effective in scar repairing 2 months after the operation, IHC staining with Desmin and CD34 and positive immunofluorescence staining verified the differentiation of ADMSCs to functional cardiomyocytes. This approach may involve the application of autologous ADMSCs seeded on pericardial patch in an attempt to regenerate a contractible myocardium in an animal model of MI.     

Intestinal Barrier Dysfunction Develops at the Onset of Experimental Autoimmune Encephalomyelitis,and Can Be Induced by Adoptive Transfer of Auto-Reactive T Cells

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system with a pathogenesis involving a dysfunctional blood-brain barrier and myelin-specific, autoreactive T cells. Although the commensal microbiota seems to affect its pathogenesis, regulation of the interactions between luminal antigens and mucosal immune elements remains unclear. Herein, we investigated whether the intestinal mucosal barrier is also targeted in this disease. Experimental autoimmune encephalomyelitis (EAE), the prototypic animal model of MS, was induced either by active immunization or by adoptive transfer of autoreactive T cells isolated from these mice. We show increased intestinal permeability, overexpression of the tight junction protein zonulin and alterations in intestinal morphology (increased crypt depth and thickness of the submucosa and muscularis layers). These intestinal manifestations were seen at 7 days (i.e., preceding the onset of neurological symptoms) and at 14 days (i.e., at the stage of paralysis) after immunization. We also demonstrate an increased infiltration of proinflammatory Th1/Th17 cells and a reduced regulatory T cell number in the gut lamina propria, Peyer''s patches and mesenteric lymph nodes. Adoptive transfer to healthy mice of encephalitogenic T cells, isolated from EAE-diseased animals, led to intestinal changes similar to those resulting from the immunization procedure. Our findings show that disruption of intestinal homeostasis is an early and immune-mediated event in EAE. We propose that this intestinal dysfunction may act to support disease progression, and thus represent a potential therapeutic target in MS. In particular, an increased understanding of the regulation of tight junctions at the blood-brain barrier and in the intestinal wall may be crucial for design of future innovative therapies.     

Cyclic Strain Alters the Expression and Release of Angiogenic Factors by Human Tendon Cells

Angiogenesis is associated with the tissue changes underlying chronic overuse tendinopathy. We hypothesized that repetitive, cyclic loading of human tendon cells would lead to increased expression and activity of angiogenic factors. We subjected isolated human tendon cells to overuse tensile loading using an in vitro model (1 Hz, 10% equibiaxial strain). We found that mechanically stimulated human tendon cells released factors that promoted in vitro proliferation and tube formation by human umbilical vein endothelial cells (HUVEC). In response to cyclic strain, there was a transient increase in the expression of several angiogenic genes including ANGPTL4, FGF-2, COX-2, SPHK1, TGF-alpha, VEGF-A and VEGF-C, with no change in anti-angiogenic genes (BAI1, SERPINF1, THBS1 and 2, TIMP1-3). Cyclic strain also resulted in the extracellular release of ANGPTL4 protein by tendon cells. Our study is the first report demonstrating the induction of ANGPTL4 mRNA and release of ANGPTL4 protein in response to cyclic strain. Tenocytes may contribute to the upregulation of angiogenesis during the development of overuse tendinopathy.     

Characterization of the major histocompatibility complex locus association with Beh?et’s disease in Iran

IntroductionThe aim of this study was to characterize the association of human leukocyte antigen (HLA) B alleles and major histocompatibility complex (MHC) single nucleotide polymorphisms (SNPs) with Behçet’s disease (BD) in an Iranian dataset.MethodsThe association of three SNPs in the MHC region previously identified as the most associated in high-density genotyping studies was tested in a case–control study on 973 BD patients and 825 controls from Iran, and the association of HLA-B alleles was tested in a subset of 681 patients and 414 controls.ResultsWe found that HLA-B*51 (P = 4.11 × 10⁻⁴¹, OR [95% CI] = 4.63[3.66-5.85]) and B*15 confer risk for BD (P = 2.83 × 10⁻², OR [95% CI] = 1.75[1.08-2.84]) in Iranian, and in B*51 negative individuals, only the B*15 allele is significantly associated with BD (P = 2.51 × 10⁻³, OR [95% CI] = 2.40[1.37-4.20]). rs76546355, formerly known as rs116799036, located between HLA-B and MICA (MHC class I polypeptide-related sequence A), demonstrated the same level of association with BD as HLA-B*51 (P_adj = 1.78 × 10⁻⁴⁶, OR [95% CI] = 5.46[4.21-7.09], and P_adj = 8.34 × 10⁻⁴⁸, OR [95% CI] = 5.44[4.20-7.05], respectively) in the HLA-B allelotyped subset, while rs2848713 was less associated (P_adj = 7.14 × 10⁻³⁵, OR [95% CI] = 3.73[2.97-4.69]) and rs9260997 was not associated (P_adj = 1.00 × 10⁻¹). Additionally, we found that B*51 genotype-phenotype correlations do not survive Bonferroni correction, while carriers of the rs76546355 risk allele predominate in BD cases with genital ulcers, positive pathergy test and positive BD family history (2.31 × 10⁻⁴ ≤ P ≤ 1.59 × 10⁻³).ConclusionsWe found that the HLA-B*51 allele and the rs76546355/rs116799036 MHC SNP are independent genetic risk factors for BD in Iranian, and that positivity for the rs76546355/rs116799036 risk allele, but not for B*51, does correlate with specific demographic characteristics or clinical manifestations in BD patients.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0585-6) contains supplementary material, which is available to authorized users.     

The effects of a stimulating intron on the expression of heterologous genes in Arabidopsis thaliana

Introns are often added to transgenes to increase expression, although the mechanism through which introns stimulate gene expression in plants and other eukaryotes remains mysterious. While introns vary in their effect on expression, it is unknown whether different genes respond similarly to the same stimulatory intron. Furthermore, the degree to which gene regulation is preserved when expression is increased by an intron has not been thoroughly investigated. To test the effects of the same intron on the expression of a range of genes, GUS translational fusions were constructed using the promoters of eight Arabidopsis genes whose expression was reported to be constitutive (GAE1, CNGC2 and ROP10), tissue specific (ADL1A, YAB3 and AtAMT2) or regulated by light (ULI3 and MSBP1). For each gene, a fusion containing the first intron from the UBQ10 gene was compared to fusions containing the gene's endogenous first intron (if the gene has one) or no intron. In every case, the UBQ10 intron increased expression relative to the intronless control, although the magnitude of the change and the level of expression varied. The UBQ10 intron also changed the expression patterns of the CNGC2 and YAB3 fusions to include strong activity in roots, indicating that tissue specificity was disrupted by this intron. In contrast, the regulation of the ULI3 and MSBP1 genes by light was preserved when their expression was stimulated by the intron. These findings have important implications for biotechnology applications in which a high level of transgene expression in only certain tissues is desired.