Genome-Wide Analyses Suggest Mechanisms Involving Early B-Cell Development in Canine IgA Deficiency

Immunoglobulin A deficiency (IgAD) is the most common primary immune deficiency disorder in both humans and dogs, characterized by recurrent mucosal tract infections and a predisposition for allergic and other immune mediated diseases. In several dog breeds, low IgA levels have been observed at a high frequency and with a clinical resemblance to human IgAD. In this study, we used genome-wide association studies (GWAS) to identify genomic regions associated with low IgA levels in dogs as a comparative model for human IgAD. We used a novel percentile groups-approach to establish breed-specific cut-offs and to perform analyses in a close to continuous manner. GWAS performed in four breeds prone to low IgA levels (German shepherd, Golden retriever, Labrador retriever and Shar-Pei) identified 35 genomic loci suggestively associated (p <0.0005) to IgA levels. In German shepherd, three genomic regions (candidate genes include KIRREL3 and SERPINA9) were genome-wide significantly associated (p <0.0002) with IgA levels. A ~20kb long haplotype on CFA28, significantly associated (p = 0.0005) to IgA levels in Shar-Pei, was positioned within the first intron of the gene SLIT1. Both KIRREL3 and SLIT1 are highly expressed in the central nervous system and in bone marrow and are potentially important during B-cell development. SERPINA9 expression is restricted to B-cells and peaks at the time-point when B-cells proliferate into antibody-producing plasma cells. The suggestively associated regions were enriched for genes in Gene Ontology gene sets involving inflammation and early immune cell development.     

Protective Effects of Transforming Growth Factor β2 in Intestinal Epithelial Cells by Regulation of Proteins Associated with Stress and Endotoxin Responses

Transforming growth factor (TGF)-β2 is an important anti-inflammatory protein in milk and colostrum. TGF-β2 supplementation appears to reduce gut inflammatory diseases in early life, such as necrotizing enterocolitis (NEC) in young mice. However, the molecular mechanisms by which TGF-β2 protects immature intestinal epithelial cells (IECs) remain to be more clearly elucidated before interventions in infants can be considered. Porcine IECs PsIc1 were treated with TGF-β2 and/or lipopolysaccharide (LPS), and changes in the cellular proteome were subsequently analyzed using two-dimensional gel electrophoresis-MS and LC-MS-based proteomics. TGF-β2 alone induced the differential expression of 13 proteins and the majority of the identified proteins were associated with stress responses, TGF-β and Toll-like receptor 4 signaling cascades. In particular, a series of heat shock proteins had similar differential trends as previously shown in the intestine of NEC-resistant preterm pigs and young mice. Furthermore, LC-MS-based proteomics and Western blot analyses revealed 20 differentially expressed proteins following treatment with TGF-β2 in LPS-challenged IECs. Thirteen of these proteins were associated with stress response pathways, among which five proteins were altered by LPS and restored by TGF-β2, whereas six were differentially expressed only by TGF-β2 in LPS-challenged IECs. Based on previously reported biological functions, these patterns indicate the anti-stress and anti-inflammatory effects of TGF-β2 in IECs. We conclude that TGF-β2 of dietary or endogenous origin may regulate the IEC responses against LPS stimuli, thereby supporting cellular homeostasis and innate immunity in response to bacterial colonization, and the first enteral feeding in early life.     

Estrogen Receptor β2 Induces Hypoxia Signature of Gene Expression by Stabilizing HIF-1α in Prostate Cancer

The estrogen receptor (ER) β variant ERβ2 is expressed in aggressive castration-resistant prostate cancer and has been shown to correlate with decreased overall survival. Genome-wide expression analysis after ERβ2 expression in prostate cancer cells revealed that hypoxia was an overrepresented theme. Here we show that ERβ2 interacts with and stabilizes HIF-1α protein in normoxia, thereby inducing a hypoxic gene expression signature. HIF-1α is known to stimulate metastasis by increasing expression of Twist1 and increasing vascularization by directly activating VEGF expression. We found that ERβ2 interacts with HIF-1α and piggybacks to the HIF-1α response element present on the proximal Twist1 and VEGF promoters. These findings suggest that at least part of the oncogenic effects of ERβ2 is mediated by HIF-1α and that targeting of this ERβ2 – HIF-1α interaction may be a strategy to treat prostate cancer.     

Characterization of an antiserum against the glucocorticoid receptor

An immunoglobulin (IgG) fraction from serum of a rabbit immunized with a highly purified preparation of glucocorticoid receptor from rat liver cytosol contained specific antibodies to glucocorticoid receptor. This was shown following incubation of the [³H]triamcinolone acetonide-glucocorticoid receptor (TA-GR) complex with the IgG fraction by (I) adsorption of the [³H]TA-GR-antibody complex to protein A linked to Sepharose, (II) an increased sedimentation rate of the [³H]TA-GR-antibody complex compared to that of the [³H]TA-GR complex, and (III) an increased molecular size of the [³H]TA-GR-antibody complex when compared to that of the [³H]TA-GR complex as judged from gel filtration. The antibody fraction was characterized with regard to titer, cross-reactivity and specificity. The antibodies cross-reacted with the glucocorticoid receptor from various rat tissues (liver, thymus and hippocampus), as well as with the glucocorticoid receptor from human normal lymphocytes, chronic lymphatic leukemia cells and human hippocampus. In the rat liver, the antibody bound to both the nuclear and the cytosolic glucocorticoid receptor (Stokes radius 6.1 nm). It did not cross-react with the proteolytic fragments of the glucocorticoid receptor, the 3.6 nm complex or the 1.9 nm complex. Binding of the antibodies was not seen to the androgen, estrogen or progestin receptors in rat to rat serum transcortin. With an indirect competitive ELISA (enzyme-linked immunosorbent assay) combined with various separation techniques, based on different physiocochemical principles, it was shown that the glucocorticoid receptor was the only detectable antibody binding protein from rat liver cytosol using this assay system. These findings also indicate an immunochemical similarity between glucocorticoid receptors in different tissues as well as in different species, but not between glucocorticoid receptors and other steroid hormone receptor proteins. The cytosolic and nuclear glucocorticoid receptors in rat liver were shown to be immunochemically similar.     

Dopamine-beta-hydroxylase in human lymph

The dopamine-β-hydroxylase (DBH) activity in human lymph was examined. In 14 of 16 persons DBH activity could be detected in the lymph taken from the right lymphatic duct or the thoracic duct. There was a significant (0.05 > p > 0.01) correlation (r = 0.61) between the DBH activity in lymph and serum. The ratio between the levels of DBH in lymph and serum was less than that of the albumin levels (0.36 ± 0.06 and 0.66 ± 0.05).     

Structure of sperm,spermatozeugmata and ‘lateral organs’ in the bivalve Arthritica (Galeommatoidea: Leptonidae)

The position and structure of paired ‘lateral organs’ in the foot of Arthritica semen and Arthritica bifurca might indicate a chemosensory function. In both species part of the organ is also glandular. In A. semen the glandular epithelium is detached piecemeal and, probably by means of the foot, is moved to and grafted upon the gills of the same individual. The transferred epithelia appear as disk‐shaped actively secretory ‘gill bodies’ which, attached to the abfrontal side of the inner demibranch, replace the ordinary unciliated gill epithelium. The secretion is liberated into the suprabranchial chamber, which serves as a marsupium, but its function is uncertain. Arthritica semen is a protandric hermaphrodite and produces very large ova that undergo a direct development that results in a non‐planktonic lecithotrophic crawling juvenile stage. The sperm cells have filiform nuclei that are straight in the euspermatozoa and more or less helicoidal in what is considered to represent paraspermatozoa. By a process of aggregation, spermatozeugmata are formed which consist exclusively either of euspermatozoa or paraspermatozoa. Spermatozoa are stored in the oviduct in A. semen but in paired seminal receptacles in A. bifurca.     

Two different mechanisms for modulation of bronchoconstriction in guinea-pigs by cyclooxygenase metabolites

Leukotriene D₄ (LTD₄)-induced bronchoconstriction in guinea-pig airways has a cyclooxygenase (COX)-dependent component. The main objective of this study was to establish if prostaglandin (PG) D₂-induced bronchoconstriction also was modulated by COX products. The effects of non-selective and selective COX-1 and COX-2 inhibitors on bronchoconstriction induced by LTD₄ and PGD₂ were investigated in the perfused and ventilated guinea-pig lung (IPL). Both LTD₄-induced bronchoconstriction and thromboxane (TX) A₂ release was suppressed by COX inhibitors or by TX synthesis inhibition. The release of additional COX products following CysLT₁ receptor activation by LTD₄ was established by measurements of immunoreactive 6-keto PGF_1α (a stable metabolite of PGI₂) and PGE₂. In contrast, TP receptor-mediated bronchoconstriction by PGD₂ was somewhat enhanced by COX inhibitors, and there was no measurable release of COX products after TP receptor activation with U-46619. PGE₂ was bronchoprotective in IPL as it inhibited the histamine-induced bronchoconstriction. In the isolated guinea-pig trachea, neither PGD₂ nor U-46619 actively released PGE₂, but continuous production of PGE₂ and PGI₂ was established, and the response to PGD₂ was enhanced also in the trachea by COX inhibition. The study documented that bronchoconstriction induced by LTD₄ and PGD₂ in IPL was modulated differently by COX products. Whereas bronchoconstriction induced by LTD₄ was amplified predominantly by secondarily released TXA₂, that induced by PGD₂ was attenuated by bronchoprotective PGE₂ and PGI₂, presumably tonically produced in the airways.     

Household Epidemics: Modelling Effects of Early Stage Vaccination

A Markovian susceptible → infectious → removed (SIR) epidemic model is considered in a community partitioned into households. A vaccination strategy, which is implemented during the early stages of the disease following the detection of infected individuals is proposed. In this strategy, the detection occurs while an individual is infectious and other susceptible household members are vaccinated without further delay. Expressions are derived for the influence on the reproduction numbers of this vaccination strategy for equal and unequal household sizes. We fit previously estimated parameters from influenza and use household distributions for Sweden and Tanzania census data. The results show that the reproduction number is much higher in Tanzania (6 compared with 2) due to larger households, and that infected individuals have to be detected (and household members vaccinated) after on average 5 days in Sweden and after 3.3 days in Tanzania, a much smaller difference.     

Proteomic identification of proteins translocated to membrane microdomains upon treatment of fibroblasts with the glycosphingolipid,C8‐β‐D‐lactosylceramide

Plasma membrane (PM) microdomains, including caveolae and other cholesterol‐enriched subcompartments, are involved in the regulation of many cellular processes, including endocytosis, attachment and signaling. We recently reported that brief incubation of human skin fibroblasts with the synthetic glycosphingolipid, D‐erythro‐octanoyl‐lactosylceramide (C8‐D ‐e‐LacCer), stimulates endocytosis via caveolae and induces the appearance of micron‐size microdomains on the PM. To further understand the effects of C8‐D ‐e‐LacCer treatment on PM microdomains, we used a detergent‐free method to isolate microdomain‐enriched membranes from fibroblasts treated ±C8‐D ‐e‐LacCer, and performed 2‐DE and mass spectrophotometry to identify proteins that were altered in their distribution in microdomains. Several proteins were identified in the microdomain‐enriched fractions, including lipid transfer proteins and proteins related to the functions of small GTPases. One protein, Rho‐associated protein kinase 2 (ROCK2), was verified by Western blotting to occur in microdomain fractions and to increase in these fractions after D ‐e‐LacCer treatment. Immunofluorescence revealed that ROCK2 exhibited an increased localization at or near the PM in C8‐D ‐e‐LacCer‐treated cells. In contrast, ROCK2 distribution in microdomains was decreased by treatment of cells with C8‐L ‐threo‐lactosylceramide, a glycosphingolipid with non‐natural stereochemistry. This study identifies new microdomain‐associated proteins and provides evidence that microdomains play a role in the regulation of the Rho/ROCK signaling pathway.     

Manipulation of phospholipid polar headgroup composition in primary cultures of rat hepatocytes

Isolated hepatocytes in suspension or in primary culture were incubated with different phospholipid bases and the effects on the synthesis and composition of phospholipids were studied. After incubation in the presence of 1 mM diethylethanolamine for three days, an unnatural phospholipid, phosphatidyl-diethylethanolamine, constituted more than 20% of total phospholipids. Its fatty acid composition differed from that of other phospholipids. Incubation of hepatocytes with ethanolamine gave smaller effects and in this case the increased synthesis of phosphatidylethanolamine was compensated for by methylation to phosphatidylcholine. This system can be used for studies on the functional significance of phospholipid polar headgroups in a specialized type of cell.