A proportion of proteinase 3 (PR3)-specific anti-neutrophil cytoplasmic antibodies (ANCA) only react with PR3 after cleavage of its N-terminal activation dipeptide

ANCA directed against PR3 are highly specific for Wegener's granulomatosis and microscopic polyangiitis, and have been implicated in the pathogenesis of small vessel vasculitis. Most PR3-ANCA are directed against conformational epitopes on PR3. This study was designed to determine whether the cleavage of the N-terminal activation dipeptide of PR3 is required for the binding of PR3-ANCA. Recombinant PR3 (rPR3) variants were expressed in the epithelial cell line, 293. As confirmed by radiosequencing, the rPR3 secreted into the 293 cell culture supernatant is N-terminally unprocessed. Two enzymatically inactive rPR3 mutants were expressed in 293 cells: rPR3-S176A and δ -rPR3-S176A. rPR3-S176A contains the N-propetide Ala-2-Glu-1, δ -rPR3-S176A does not. Culture supernatants of rPR3-S176A and δ -rPR3-S176A expressing 293 cells were used as sources of target antigen for PR3-ANCA testing by capture ELISA. Forty unselected consecutive PR3-ANCA⁺ sera were tested. With δ -rPR3-S176A as antigen all 40 were recognized, compared with only 34 of 40 when rPR3-S176A served as target antigen. The majority of the serum samples contained a mixture of antibodies reacting with epitopes accessible on the mature and on the proform of PR3. In conclusion, the cleavage of the N-terminal activation dipeptide of PR3 is not an absolute requirement for recognition by all PR3-ANCA. However, a substantial proportion of PR3-ANCA recognize (a) target antigen(s) exposed only after the conformational change of PR3 associated with the N-terminal processing. In 15% of sera this PR3-ANCA subset occurred exclusively. PR3-ANCA subtypes can be differentiated using specifically designed rPR3 variants as target antigens, and non-haematopoietic mammalian cells without regulated secretory pathway can be used for their expression.     

微小RNA-23b-3p通过靶向TGFBR3促进人心房肌成纤维细胞纤维化相关基因表达

目的:探究微小RNA-23b-3p(mi R-23b-3p)对人心房肌成纤维细胞中纤维化相关基因表达的作用及其可能作用靶基因。方法:分离并体外培养房颤患者心耳中原代心房肌成纤维细胞,并用细胞免疫荧光染色实验鉴定;双萤光素酶报告基因实验检测mi R-23b-3p与潜在靶基因转化生长因子β受体3(TGFBR3) 3'端非翻译区(3'-UTR)的结合作用; CCK-8、Ed U染色及Transwell实验检测细胞活力、增殖及迁移能力,RT-qPCR和Western blot法检测TGFBR3及纤维化相关基因的m RNA和蛋白表达。结果:在人心房肌成纤维细胞中过表达mi R-23b-3p不影响细胞的活力、增殖及迁移能力,但可显著增强细胞中纤维化相关基因COL1A1、COL3A1和ACTA2的表达(P 0. 05或P 0. 01)。双萤光素酶报告基因实验显示mi R-23b-3p与TGFBR3 3'-UTR有结合作用。RT-qPCR和Western blot结果证实mi R-23b-3p可在转录水平抑制TGFBR3表达。过表达mi R-23b-3p和沉默TGFBR3均能显著促进人心房肌成纤维细胞中Smad3激活和纤维化相关基因表达(P 0. 05或P 0. 01)。结论:TGFBR3是mi R-23b-3p的作用靶基因,并介导mi R-23b-3p促进心房肌成纤维细胞中纤维化相关基因表达。     

Immunogenicity and T cell recognition in swine of foot-and-mouth disease virus polymerase 3D

Immunization of domestic pigs with a vaccinia virus (VV) recombinant expressing foot-and-mouth disease virus (FMDV) 3D protein conferred partial protection against challenge with infectious virus. The severity reduction of the clinical symptoms developed by the challenged animals occurred in the absence of significant levels of anti-3D circulating antibodies. This observation suggested that the partial protection observed was mediated by the induction of a 3D-specific cellular immune response. To gain information on the T cell recognition of FMDV 3D protein, we conducted in vitro proliferative assays using lymphocytes from outbred pigs experimentally infected with FMDV and 90 overlapping peptides spanning the complete 3D sequence. The use of pools of two to three peptides allowed the identification of T cell epitopes that were efficiently recognized by lymphocytes from at least four of the five animals analyzed. This recognition was heterotypic because anti-peptide responses increased upon reinfection of animals with a FMDV isolate from a different serotype. The results obtained with individual peptides confirmed the antigenicity observed with peptide pools. Detection of cytokine mRNAs by RT-PCR in lymphocytes stimulated in vitro by individual 3D peptides revealed that IFN-gamma mRNA was the most consistently induced, suggesting that the activated T cells belong to the Th 1 subset. These results indicate that 3D protein contains epitopes that can be efficiently recognized by porcine T lymphocytes from different infected animals, both upon primary and secondary (heterotypic) FMDV infection. These epitopes can extend the repertoire of viral T cell epitopes to be included in subunit and synthetic FMD vaccines.     

Structural and functional complexity of the humoral response against the Trypanosoma cruzi ribosomal P2 beta protein in patients with chronic Chagas' heart disease

High levels of antibodies against the C-terminus of the Trypanosoma cruzi TcP2 beta ribosomal protein, defined by the peptide EEEDDDMGFGLFD, named R13, have been measured in sera from patients with chronic Chagas' Heart Disease (cChHD). These antibodies also recognize an epitope on the second extracellular loop of the beta 1-adrenergic receptor, inducing a functional response on cardiomyocytes. The aim of this study was to gain novel insights into the structural basis of this cross-reactivity as well as to evaluate the origin of anti-M2- cholinergic receptor antibodies, which are also commonly found in cChHD patients. To address these questions we immunopurified anti-R13 antibodies and studied the structural requirements of epitope recognition. Results showed that the immunopurified antibodies recognized a conformation of R13 in which the third Glu residue was essential for binding, explaining their low affinity for the mammalian homologue (peptide H13: EESDDDMGFGLFD). Alanine mutation scanning showed individual variations in epitope recognition in each of the studied patients. The importance of a negatively charged residue at position 3 for the recognition of anti-R13 antibodies was further confirmed by competition experiments using a Ser3-phosphorylated H13 analogue, which had 10 times more affinity for the anti-R13 antibody than the native H13 peptide. Moreover, anti-R13 antibodies stimulated either the beta 1-adrenergic or the M2-cholinergic receptor, in strict agreement with the functional properties of the IgG fractions from which they derived, demonstrating that the same parasite antigen may generate antibody specificities with different functional properties. This may be a clue to explain the high variability of electrophysiological disturbances found in cChHD.     

NMDA receptor mediated dendritic plasticity in cortical cultures after oxygen-glucose deprivation

Dendrites and spines undergo dynamic changes in physiological and pathological conditions. Dendritic outgrowth has been observed in surviving neurons months after ischemia, which is associated with the functional compensation. It remains unclear how dendrites in surviving neurons are altered shortly after ischemia, which might reveal the mechanisms underlying neuronal survival. Using primary cortical cultures, we monitored the dendritic changes in individual neurons after oxygen-glucose deprivation (OGD). Two to four hours of OGD induced approximately 30–50% cell death in 24 h. However, the total dendritic length in surviving neurons was significantly increased after OGD with a peak at 6 h after re-oxygenation. The increase of dendritic length after OGD was mainly due to the sprouting rather than the extension of the dendrites. The dendritic outgrowth after 2 h of OGD was greater than that after 4 h of OGD. Application of NMDA receptor blocker MK-801 abolished OGD-induced dendritic outgrowth, whereas application of AMPA receptor antagonist CNQX had no significant effects. These results demonstrate a NMDA receptor-dependent dendritic plasticity shortly after OGD, which provides insights into the early response of surviving neurons after ischemia.     

Lower expression of Th1-related cytokines and inducible nitric oxide synthase in mice with streptozotocin-induced diabetes mellitus infected with Mycobacterium tuberculosis

Diabetes mellitus is an important predisposing factor for tuberculosis. The aim of this study was to investigate the mechanism underlying this association using a murine model. Mice with streptozotocin-induced diabetes mellitus were prone to Mycobacterium tuberculosis infection, as indicated by increased numbers of live bacteria in lung, liver and spleen. In diabetic mice, the levels of IL-12 and IFN-gamma in the lung, liver and spleen were lower than those in control animals on day 14 postinfection, while the opposite was true for IL-4 levels in the lung and liver. The expression pattern of inducible nitric oxide synthase (iNOS), in the two mice types was as for IL-12 and IFN-gamma. In addition, peritoneal exudate cells obtained from diabetic mice produced lower amounts of IL-12 and NO than those from control mice, when stimulated in vitro with M. bovis BCG. Spleen cells from diabetic mice infected with M. tuberculosis produced a significantly lower amount of IFN-gamma upon restimulation with purified protein derivatives (PPD) than those from infected nondiabetic mice. Interestingly, addition of high glucose levels (33 mM) to the cultures of PPD-restimulated spleen cells reduced the synthesis of IFN-gamma only in diabetic mice, and not in nondiabetic mice. Finally, control of blood glucose levels by insulin therapy resulted in improvement of the impaired host protection and Th1-related cytokine synthesis. Our results suggest that the reduced production of Th1-related cytokines and NO account for the hampered host defense against M. tuberculosis infection under diabetic conditions.     

T cell receptor γδ repertoire is skewed in cerebrospinal fluid of multiple sclerosis patients: molecular and functional analyses of antigen-reactive γδ clones

To study the relevance of γδ T cells in multiple sclerosis (MS) we analyzed the T cell receptor (TCR) γδ repertoire and the antigen reactivity of γδ clones isolated from cerebrospinal fluid (CSF). In T cell cultures derived from CSF we found an increased percentage of Vδ1⁺ cells as compared to peripheral blood of the same donors. Phenotypic analysis of cells from MS CSF with Vγ- and Vγ-specific monoclonal antibodies (mAb) showed that the Vγ1 chain is most frequently associated with γ chains belonging to the VγI family. Sequence analysis of TCR genes revealed heterogeneity of junctional regions in both δ and γ genes indicating polyclonal expansion. γδ clones were established and some recognized glioblastoma, astrocytoma or monocytic cell lines. Stimulation with these targets induced serine esterase release and lymphokine expression characteristic of the T_H0-like phenotype. Remarkably, these tumor-reactive γδ cells were not detected in the peripheral blood using PCR oligotyping, but were found in other CSF lines independently established from the same MS patient. Altogether, these results demonstrate that in the CSF there is a skewed TCR γδ repertoire and suggest that γδ cells reacting against brain-derived antigens might have been locally expanded.     

Expression of a G-protein {beta} subunit-related gene during lymphocyte activation

Using a subtractlve strategy, we have cloned an activation-relatedgene from a human B cell cDNA library. Sequence analysis revealedthat this gene was identical to H12.3, a gene belonging to anexpanding family of guanlne nucleotlde-blndlng protein 3;subunlts. The expression of H12.3 was induclble in the latephase of mltogen-stlmulated T and B cells. In T cells, IL-2and IL-4 by themselves had no direct effect on the expressionof H12.3, but they could augment the level of steady-state H12.3mRNA stimulated by phytohemagglutlnln. On the other hand, IFN-and IL-6 had no obvious effect on the expression in B cellswith or without Staphytococcus aureus Cowan l-stlmulatlon. CyclosporinA, a strong immunosuppressant, Inhibited the mltogen-stlmulatedexpression of H12.3, but rapamycin, another such agent, didnot. In synchronized Jurkat cells, the expression of H12.3 hadno cell cycle-dependent decrease in S and G₂/M phase, whilecyclin E, which controls the progression of the cell cycle unlate G₁ phase, did show a periodic expression pattern. The resultssuggest that H12.3 might be involved in regulation of lymphocyteactivation.     

CD3 antigen-mediated calcium signals and protein kinase C activation are higher in CD45R0+ than in CD45RA+ human T lymphocyte subsets

T lymphocytes may be separated into subsets according to their expression of CD45 isoforms. The CD45R0⁺ T cell subset has been reported to proliferate in response to recall antigen and to mitogenic mAb to a much greater extent than the CD45RA⁺ subset. This difference could be due to more efficient coupling of the T cell antigen receptor complex to mitogenic signaling pathways. To investigate this possibility, CD3 antigen-induced calcium signals, diacylglycerol (DAG) production and protein kinase C (PKC) activation levels were compared in CD45RA⁺ and CD45R0⁺ human T lymphocyte subsets derived from peripheral blood. The mean CD3-induced rise in intracellular calcium was 80% greater in CD45R0⁺ than in CD45RA⁺ cells. Basal DAG levels in CD45R0⁺ cells were found to be, on average, 60% higher than in CD45RA⁺ cells (p = 0.002), but the CD3-induced production of DAG over background was not different in the two subsets (p = 0.4). Basal PKC activity, and CD3-induced PKC activation levels over background, were found to be 50% and 140% higher, respectively, in CD45R0⁺ cells than in CD45RA⁺ cells (p = 0.015 and 0.023). The CD45R0⁺ subset contained a higher proportion of cells expressing activation markers, such as CD25, CD71 and major histocompatibility complex class II, when compared to the CD45RA⁺ subset. Our results suggest that the elevated basal DAG levels observed in the CD45R0⁺ subset may reflect the recent activation of these cells. Both the higher basal DAG and CD3-induced elevation in intracellular calcium observed in the CD45R0⁺ cells may contribute to the greater PKC activation signals triggered by CD3 mAb in this subset. These findings elucidate the greater response of CD45R0⁺ T cells to mitogenic stimuli compared to CD45RA⁺ cells.     

Ontogeny of 3H-muscimol binding to membranes of chick forebrain

Summary A potent GABA agonist, ³H-muscimol, was used to investigate the development of GABA receptors in left and right hemispheres of chick forebrain from day 12 in ovo to day 21 post-hatch. Total specific ³H-muscimol binding (p mol mg^ȁ3;1 protein) increases rapidly in ovo, reaching a peak at around day one post-hatch and then showing a slow decline to approximately 50% of the maximal level at day 21 post-hatch. Despite the considerable evidence from previous studies of lateralization of avain brain function, no significant hemispheric differences were found in ³H-muscimol binding (either of p mol hemisphere^ȁ3;1 or p mol mg protein^ȁ3;1) at any of the developmental ages examined.