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Pathophysiology of acute myocardial infarction (AMI) has been described in mice and is based on a three-stage model involving temporal modifications of immune cells and fibroblasts. The acute inflammatory phase is followed by a reparative phase and fibrous scar maturation phase. Purinergic signaling, particularly P2Y11 receptor, has been reported to be involved in the regulation of inflammation after ischemia and could be an actor of resolution of inflammation after AMI. The aims of our study were: (1) to characterize the immune and P2Y11R profiles of AMI patients and (2) to analyze interactions between patients' immune cells and cardiac fibroblasts in vitro. We collected peripheral blood mononuclear cells (PBMC) of 182 patients at various times (H0, H4, H24, H48, D3, M1, M6, M12) after reperfused ST-segment elevation AMI, and of 30 healthy donors. Expression level of genes involved in tolerogenicity profile of dendritic cells (HMOX1, STAT3, IDO1), in T cell polarization (CD4, FOXP3, TBX21/GATA3) as well as P2RY11 were evaluated by RT-PCR. P2Y11R expression was analyzed using flow cytometry. PBMC and human cardiac fibroblasts (HCF) were co-cultured during one day (5PBMC/1 HCF) and gene level expression (ACTA2/VIM, COL1A1), phenotype (α-SMA/vimentin) and secretory (soluble collagen) profiles were analyzed by RT-PCR, flow cytometry and Sircol assay. In the first 48 hours after AMI, the expression level of HMOX1 (fold-change = 8.478 at H48, P < 0.0001), STAT3 (2.856 at H0, P = 0.0027) and CD4 increased (2.451 at H48, P = 0.0052); IDO1 (0.2055 at H24, P < 0.0001) and TBX21/GATA3 ratio decreased (0.4498 at H48, P = 0.0026); FOXP3 did not vary significantly. In the same time, the expression level of P2RY11 increased (2.124 at H0, P = 0.0015) as well as protein expression in T cells (median MFI = 845.5 at H0 vs. 229.5 for healthy donors, P = 0.0375). In vitro, we observed a non-significant increase of ACTA2/VIM ratio in HCF co-cultured with H24, M1, M6 PBMC; of α-SMA/vimentin with H48, M12 PBMC; of COL1A1 with H24, H48, M12 PBMC; and of soluble collagen in coculture supernatant with M1 PBMC. Our results suggest that P2Y11R could be involved in evolution of immune response after AMI, and that, in the first 2 days, circulating immune cells have a reparative profile and could participate in the transformation of fibroblast into myofibroblasts. [ABSTRACT FROM AUTHOR]

Proteinase 3 (PR3) is the main target antigen of antineutrophil cytoplasmic antibodies (ANCAs) in PR3-ANCA-associated vasculitis. A small fraction of PR3 is constitutively exposed on the surface of quiescent blood neutrophils in a proteolytically inactive form. When activated, neutrophils expose an induced form of membrane-bound PR3 (PR3mb) on their surface as well, which is enzymatically less active than unbound PR3 in solution due to its altered conformation. In this work, our objective was to understand the respective role of constitutive and induced PR3mb in the immune activation of neutrophils triggered by murine anti-PR3 mAbs and human PR3-ANCA. We quantified immune activation of neutrophils by the measurement of the production of superoxide anions and secreted protease activity in the cell supernatant before and after treatment of the cells by alpha-1 protease inhibitor that clears induced PR3mb from the cell surface. Incubation of TNFa-primed neutrophils with anti-PR3 antibodies resulted in a significant increase in superoxide anion production, membrane activation marker exposition, and secreted protease activity. When primed neutrophils were first treated with alpha-1 protease inhibitor, we observed a partial reduction in antibody-induced neutrophil activation, suggesting that constitutive PR3mb is sufficient to activate neutrophils. The pretreatment of primed neutrophils with purified antigenbinding fragments used as competitor significantly reduced cell activation by whole antibodies. This led us to the conclusion that PR3mb promoted immune activation of neutrophils. We propose that blocking and/or elimination of PR3mb offers a new therapeutic strategy to attenuate neutrophil activation in patients with PR3-ANCA-associated vasculitis. [ABSTRACT FROM AUTHOR]

Abstract: Mycophenolic acid (MPA) is an immunosuppressive drug which induces resistance to several maturation signals in human dendritic cells (DC) by unknown mechanisms. As mitogen-activated protein kinases (MAPK) are involved in the maturation process, we studied whether MPA affected p38MAPK and extracellular signal-regulated kinase (ERK1/2) in human DC. We first showed that MPA reduced TNFα-induced phenotype maturation, whereas it had no effect after LPS activation, suggesting that MPA preferentially affects the signaling pathway used by TNFα. We found that TNFα preferentially used p38MAPK to induce phenotype maturation in DC, whereas LPS preferentially activated NF-κB. Importantly, we showed that MPA more strongly inhibited p38MAPK phosphorylation induced by TNFα than by LPS. This difference in inhibition may therefore explain its different effect on DC phenotype. Interestingly, MPA inhibited the inflammatory cytokine synthesis and allostimulatory capacity induced by both stimuli. Exogenous guanosine antagonized the effect of MPA on the phenotype of TNFα-matured-DC as well as the IL-12p70 and IFNγ secretion induced by both stimuli, without affecting p38MAPK phosphorylation. The action of MPA on human DC phenotype maturation appears mainly to be due to its ability to inhibit p38MAPK. Furthermore, the difference between LPS and TNFα emphasizes that the DC microenvironment strongly influences DC sensitivity to MPA. [Copyright &y& Elsevier]

Background Inflammatory bowel disease (IBD) is one of the most common chronic gastrointestinal diseases, but the underlying molecular mechanisms remain largely unknown. Studies of monogenic diseases can provide insight into the pathogenesis of IBD. Objective We thought to determine the underlying molecular causes of IBD occurring in 2 unrelated families in association with an immune deficiency. Methods We performed genetic linkage analysis and candidate gene sequencing on 13 patients from a large consanguineous family affected by early-onset IBD, progressive immune deficiency, and, in some cases, autoimmunity and alopecia, a condition we named enteropathy-lymphocytopenia-alopecia. The candidate gene was also sequenced in an unrelated patient with a similar phenotype. We performed histologic analysis of patients' intestinal biopsy specimens and carried out functional assays on PBMCs. Gut organoids derived from a patient's biopsy specimen were analyzed. Results We identified biallelic missense mutations in tetratricopeptide repeat domain 7A (TTC7A) in all patients from both families. The resulting TTC7A depletion modified the proliferation, adhesion, and migratory capacities of lymphocytes through inappropriate activation of the RhoA signaling pathway. Normal function was restored by wild-type TTC7A expression or addition of a RhoA kinase inhibitor. The growth and polarity of gut epithelial organoids were also found to be dependent on the RhoA signaling pathway. Conclusions We show that TTC7A regulates the actin cytoskeleton dynamics in lymphocytes through the RhoA signaling pathway and is required in both lymphocytes and epithelial cells for maintaining equilibrium between cell proliferation, migration, polarization, and cell death. Our study highlights variability in the phenotypic expression resulting from TTC7A deficiency and outlines that impairment of both epithelial cells and lymphocytes cooperatively causes IBD. [ABSTRACT FROM AUTHOR]

Rabbit antithymocyte globulin (rATG; Thymoglobulin®) is currently used to prevent acute rejection in kidney transplantation. The dose and regimen of rATG have not been optimized. Moreover, the impact of different treatment regimens on T-cell phenotype reconstitution remains unknown. We conducted a prospective randomized study of 17 renal transplant patients to determine the pharmacokinetics of total and active (bound to human cells) rATG and T-cell phenotype reconstitution after rATG administration. Patients received rATG at a total dose of 6 mg/kg, administered either as 1.5 mg/kg/day on days 0-3 (Group 1, n = 8) or 3 mg/kg on days 0 and 3 (Group 2, n = 9). All patients received tacrolimus, mycophenolate mofetil and steroids. Blood samples were assayed for total rATG by enzyme linked immunosorbent assay and active tATG by flow cytometry. Maximum concentrations and terminal half-lives were similar between the two groups but at month 3 Group 1 had significantly lower values for total rATG (concentration was 6.2 ± 1.1 ug/mL versus 10.2 ± 2.9 ug/mL in Group 2, p = 0.027) and total rATG dose-normalized AUC (374 ± 83 day g/mL versus 508 ± 149 day g/mL in Group 2, p = 0.046). Time to sub-therapeutic levels (<1 µg/mL) of active rATG was significantly shorter in Group 1 (18.75 ± 6.9 days versus 20 ± 7.5 days in Group 2, p < 0.001). rATG induced significant depletion followed by slow reconstitution of CD3+, CD4+ and CD8+ cells, with no marked differences between groups. B-ceII count was unaffected, whereas CD3~CD56+ NK-cell depletion was observed in both groups. rATG induced a significant decrease in the proportion of naive CD4+ T-cells, which plateaued after month 1 in Group 1 and after month 6 in Group 2. The proportion of central memory CD4+ T-cells increased to a similar extent in both groups (Group 1:38 ± 18% at baseline, 74 ± 23% at one year, p = 0.009; Group 2:32 ± 14% at baseline, 65 ± 14% at one year, p = 0.001 ). In conclusion, our results suggest that the dosing regimen for rATG induction influences pharmacokinetic parameters without affecting the quality of immune reconstitution. [ABSTRACT FROM AUTHOR]

In HIV, the polyprotein precursor Gag orchestrates the formation of the viral capsid. In the current view of this viral assembly, Gag forms low-order oligomers that bind to the viral genomic RNA triggering the formation of high-ordered ribonucleoprotein complexes. However, this assembly model was established using biochemical or imaging methods that do not describe the cellular location hosting Gag-gRNA complex nor distinguish gRNA packaging in single particles. Here, we studied the intracellular localization of these complexes by electron microscopy and monitored the distances between the two partners by morphometric analysis of gold beads specifically labeling Gag and gRNA. We found that formation of these viral clusters occurred shortly after the nuclear export of the gRNA. During their transport to the plasma membrane, the distance between Gag and gRNA decreases together with an increase of gRNA packaging. Point mutations in the zinc finger patterns of the nucleocapsid domain of Gag caused an increase in the distance between Gag and gRNA as well as a sharp decrease of gRNA packaged into virions. Finally, we show that removal of stem loop 1 of the 5'-untranslated region does not interfere with gRNA packaging, whereas combined with the removal of stem loop 3 is sufficient to decrease but not abolish Gag-gRNA cluster formation and gRNA packaging. In conclusion, this morphometric analysis of Gag-gRNA cluster formation sheds new light on HIV-1 assembly that can be used to describe at nanoscale resolution other viral assembly steps involving RNA or protein-protein interactions. [ABSTRACT FROM AUTHOR]

HBc is a small protein essential for the formation of the icosahedral HBV capsid. Its multiple roles in the replication cycle make this protein a promising target for the development of antiviral molecules. Based on the structure of HBc, a series of HBV assembly inhibitors, also known as capsid assembly modulators, were identified. We investigated the effect of BAY 41–4109, a heteroaryldihydropyrimidine derivative that promotes the assembly of a non-capsid polymer. We showed, by confocal microscopy, that BAY 41–4109 mediated HBc aggregation, mostly in the cytoplasm of Huh7 cells. Image analysis revealed that aggregate size depended on BAY 41–4109 concentration and treatment duration. Large aggregates in the vicinity of the nucleus were enclosed by invaginations of the nuclear envelope. This deformation of the nuclear envelope was confirmed by transmission electron microscopy (TEM) and immuno-TEM. These two techniques also revealed that the HBc aggregates were accumulations of capsid-like shells with an electron-dense material consisting of HBV core fragments. These findings, shedding light on the ultrastructural organization of HBc aggregates, provide insight into the mechanisms of action of BAY 41–4109 against HBV and will serve as a basis for comparison with other HBV capsid assembly inhibitors. • Low concentration of BAY 41-4109 incubated for short time induce large HBc aggregates. • HBc aggregates are mostly in the cytoplasm, partly surrounded by the nuclear envelop. • HBc aggregates correspond to accumulations of capsid-like shells compressed with HBc core fragments. [ABSTRACT FROM AUTHOR]