Mechanisms regulating IgA class-specific immunoglobulin production in murine gut-associated lymphoid tissues. I. T cells derived from Peyer's patches that switch sIgM B Cells to sIgA B cells in vitro

To explore mechanisms of T cell regulation governing mucosal IgA immune response, concanavalin A-induced cloned T cell lines from Peyer's patches (PP) as well as spleen were established. The cloned cell lines expressed Thy- 1.2(+), Lyt-l(+)2(-) and were radioresistant (1,500 rad). The capacity of the cloned T cells to regulate Ig synthesis was determined by measuring their effect on lipopolysaccharide (LPS)-induced polyclonal Ig synthesis by PP B cells. In initial studies Ig secreted by B cells was determined by double antibody radioimmunoassay. LPS in the absence of cloned T cells induced abundant amounts of IgM (average 8,860 ng/2 × 10(5) B cells) and IgG (average 1,190 ng/2 × 10(5) B cells), but little or no IgA. The addition of PP cloned T cells markedly suppressed production of IgM (88 percent at the highest T/B cell ratio, 4:1), but the addition of spleen cloned T cells suppressed only a little or not at all. IgG production was inhibited by both PP and spleen T clone cells (70 percent at the 4:1 T/B ratio), wheras IgA synthesis was enhanced by both clones, but only to a limited degree. In subsequent studies the expression of class-specific surface Ig (sIg) and cytoplasmic Ig (cIg) on/in unseparated PP B cells as well as Ig class- specific PP B cells and spleen B cells during culture with or without the cloned T cells was determined by immunofluorescence. The major findings were as follows: (a) Compared with unseparated B cell cultures and cultures of purified sIgM B cells derived from PP containing LPS alone, cultures containing LPS and PP cloned T cells showed a marked decrease in cIgM-, sIgG-, and cIgG-expressing cells that was accompanied by a striking increase in sIgA-bearing, but not cIgA-containing, cells. In contrast, unseparated B cell cultures and cultures of purified sIgM B cells derived from PP containing LPS and spleen cloned T cells did not show any increase in sIgA- bearing cells. (b) Compared with purified sIgG-bearing PP B cell cultures containing LPS alone, purified sIgG-bearing PP B cell cultures containing both LPS and PP cloned T cells showed no substantial change in sIgG- or cIgG- expressing cells, and no sIgA- or cIgA- expressing cells appeared. (c) Compared with sIgA-bearing PP B cell cultures containing LPS alone, purified sIgA-bearing PP B cell cultures containing both LPS and PP cloned T cells showed no increased proliferation, and cIgA cells did not occur. Cultures of purified sIgM B cells derived from spleen containing LPS and PP cloned T cells showed qualitatively similar changes. From these results we conclude that PP cloned T cells induced class-specific switching from sIgM- to sIgA- bearing B cells, whereas spleen cloned T cells lacked this property, although they may have induced an IgM {arrow} IgG or intersubclass IgG switch. These processes seem to be in part tissue dependent. Furthermore, the PP switch T cells appear to operate as true switch cells, which govern the pathway of DNA recombination events, rather than as classical helper cells, which act to expand already differentiated cells. Finally, these switch T cells probably account for the fact that PP are an important source of IgA B cells and also a major site of IgA heavy chain class switching during gut-associated mucosal B cell proliferation and differentation

Development of an in vitro cell system from zebrafish suitable to study bone cell differentiation and extracellular matrix mineralization

Mechanisms of bone formation and skeletal development have been successfully investigated in zebrafish using a variety of in vivo approaches, but in vitro studies have been hindered due to a lack of homologous cell lines capable of producing an extracellular matrix (ECM) suitable for mineral deposition. Here we describe the development and characterization of a new cell line termed ZFB1, derived from zebrafish calcified tissues. ZFB1 cells have an epithelium-like phenotype, grow at 28 degrees C in a regular L-15 medium supplemented with 15% of fetal bovine serum, and are maintained and manipulated using standard methods (e.g., trypsinization, cryopreservation, and transfection). They can therefore be propagated and maintained easily in most cell culture facilities. ZFB1 cells show aneuploidy with 2n=78 chromosomes, indicative of cell transformation. Furthermore, because DNA can be efficiently delivered into their intracellular space by nucleofection, ZFB1 cells are suitable for gene targeting approaches and for assessing gene promoter activity. ZFB1 cells can also differentiate toward osteoblast or chondroblast lineages, as demonstrated by expression of osteoblast- and chondrocyte-specific markers, they exhibit an alkaline phosphatase activity, a marker of bone formation in vivo, and they can mineralize their ECM. Therefore, they represent a valuable zebrafish-derived in vitro system for investigating bone cell differentiation and extracellular matrix mineralization.FISHCELL project [PTDC/MAR/105313/2008]; Portuguese Science and Technology Foundation (FCT); European Regional Development Fund (ERDF) through the COMPETE Program; National Fund through FCT [PEst-C/MAR/LA0015/2011]; FCT [SFRH/BPD/39189/2007]; Association of European Marine Biological Laboratories through the ASSEMBLE project [FP7/227799]info:eu-repo/semantics/publishedVersio

Evidence for the role of EPHX2 gene variants in anorexia nervosa.

Anorexia nervosa (AN) and related eating disorders are complex, multifactorial neuropsychiatric conditions with likely rare and common genetic and environmental determinants. To identify genetic variants associated with AN, we pursued a series of sequencing and genotyping studies focusing on the coding regions and upstream sequence of 152 candidate genes in a total of 1205 AN cases and 1948 controls. We identified individual variant associations in the Estrogen Receptor-ß (ESR2) gene, as well as a set of rare and common variants in the Epoxide Hydrolase 2 (EPHX2) gene, in an initial sequencing study of 261 early-onset severe AN cases and 73 controls (P=0.0004). The association of EPHX2 variants was further delineated in: (1) a pooling-based replication study involving an additional 500 AN patients and 500 controls (replication set P=0.00000016); (2) single-locus studies in a cohort of 386 previously genotyped broadly defined AN cases and 295 female population controls from the Bogalusa Heart Study (BHS) and a cohort of 58 individuals with self-reported eating disturbances and 851 controls (combined smallest single locus P<0.01). As EPHX2 is known to influence cholesterol metabolism, and AN is often associated with elevated cholesterol levels, we also investigated the association of EPHX2 variants and longitudinal body mass index (BMI) and cholesterol in BHS female and male subjects (N=229) and found evidence for a modifying effect of a subset of variants on the relationship between cholesterol and BMI (P<0.01). These findings suggest a novel association of gene variants within EPHX2 to susceptibility to AN and provide a foundation for future study of this important yet poorly understood condition

A preliminary study of genetic factors that influence susceptibility to bovine tuberculosis in the British cattle herd

Associations between specific host genes and susceptibility to Mycobacterial infections such as tuberculosis have been reported in several species. Bovine tuberculosis (bTB) impacts greatly the UK cattle industry, yet genetic predispositions have yet to be identified. We therefore used a candidate gene approach to study 384 cattle of which 160 had reacted positively to an antigenic skin test (‘reactors’). Our approach was unusual in that it used microsatellite markers, embraced high breed diversity and focused particularly on detecting genes showing heterozygote advantage, a mode of action often overlooked in SNP-based studies. A panel of neutral markers was used to control for population substructure and using a general linear model-based approach we were also able to control for age. We found that substructure was surprisingly weak and identified two genomic regions that were strongly associated with reactor status, identified by markers INRA111 and BMS2753. In general the strength of association detected tended to vary depending on whether age was included in the model. At INRA111 a single genotype appears strongly protective with an overall odds ratio of 2.2, the effect being consistent across nine diverse breeds. Our results suggest that breeding strategies could be devised that would appreciably increase genetic resistance of cattle to bTB (strictly, reduce the frequency of incidence of reactors) with implications for the current debate concerning badger-culling

Primary vs. Secondary Antibody Deficiency: Clinical Features and Infection Outcomes of Immunoglobulin Replacement

<div><p>Secondary antibody deficiency can occur as a result of haematological malignancies or certain medications, but not much is known about the clinical and immunological features of this group of patients as a whole. Here we describe a cohort of 167 patients with primary or secondary antibody deficiencies on immunoglobulin (Ig)-replacement treatment. The demographics, causes of immunodeficiency, diagnostic delay, clinical and laboratory features, and infection frequency were analysed retrospectively. Chemotherapy for B cell lymphoma and the use of Rituximab, corticosteroids or immunosuppressive medications were the most common causes of secondary antibody deficiency in this cohort. There was no difference in diagnostic delay or bronchiectasis between primary and secondary antibody deficiency patients, and both groups experienced disorders associated with immune dysregulation. Secondary antibody deficiency patients had similar baseline levels of serum IgG, but higher IgM and IgA, and a higher frequency of switched memory B cells than primary antibody deficiency patients. Serious and non-serious infections before and after Ig-replacement were also compared in both groups. Although secondary antibody deficiency patients had more serious infections before initiation of Ig-replacement, treatment resulted in a significant reduction of serious and non-serious infections in both primary and secondary antibody deficiency patients. Patients with secondary antibody deficiency experience similar delays in diagnosis as primary antibody deficiency patients and can also benefit from immunoglobulin-replacement treatment.</p></div

The effect of cigarette smoke exposure on the development of inflammation in lungs, gut and joints of TNFΔARE mice

The inflammatory cytokine TNF-alpha is a central mediator in many immune-mediated diseases, such as Crohn's disease (CD), spondyloarthritis (SpA) and chronic obstructive pulmonary disease (COPD). Epidemiologic studies have shown that cigarette smoking (CS) is a prominent common risk factor in these TNF-dependent diseases. We exposed TNF Delta ARE mice; in which a systemic TNF-alpha overexpression leads to the development of inflammation; to 2 or 4 weeks of air or CS. We investigated the effect of deregulated TNF expression on CS-induced pulmonary inflammation and the effect of CS exposure on the initiation and progression of gut and joint inflammation. Upon 2 weeks of CS exposure, inflammation in lungs of TNF Delta ARE mice was significantly aggravated. However, upon 4 weeks of CS-exposure, this aggravation was no longer observed. TNF Delta ARE mice have no increases in CD4+ and CD8+ T cells and a diminished neutrophil response in the lungs after 4 weeks of CS exposure. In the gut and joints of TNF Delta ARE mice, 2 or 4 weeks of CS exposure did not modulate the development of inflammation. In conclusion, CS exposure does not modulate gut and joint inflammation in TNF Delta ARE mice. The lung responses towards CS in TNF Delta ARE mice however depend on the duration of CS exposure