Influence of Verapamil and Cyclosporin A on bile acid metabolism and transport in rat liver slices.

Verapamil (V) is a specific inhibitor of the P-glycoprotein (mdr1) in the hepatocyte canalicular membrane. Cyclosporin A (CsA) as an essential immunosuppressive drug has potentially cholestatic adverse effects on the liver, but increases the expression of mdr1. In precision-cut liver slices from 34- to 40-day-old male Wistar rats 26 individual free and conjugated bile acids (BAs) as markers of hepatic transport and synthesis function were analysed after 4 h incubation with V (100 microM) or CsA (5 microM) in Krebs-Henseleit buffer. Some slices were loaded with cholic acid (CA 5 microM) or tauro-ursodeoxycholic acid (T-UDCA 5 microM) to investigate the V and CsA effects under conditions of BA supplementation. BAs were determined in tissue and medium by HPLC with postcolumn derivatisation and fluorescence detection. V and CsA, influencing different targets in BA transport, enhanced slice concentrations of T- and glyco- (G-) conjugated CA only when exogenous CA was given additionally. This BA accumulation in tissue is more reflected at decreased medium concentrations of these BAs after V and CsA incubations. Both V and CsA also inhibited CA uptake into the slices. The acidic chenodeoxycholic acid (CDCA) synthesis pathway is disturbed: T- and G-CDCA concentrations are diminished in slices and medium after V and CsA incubations. T-UDCA plus V or CsA enhanced not only its own slice concentration but also the concentration of the trihydroxylated tauro-muricholic acid (T-beta-MCA), reflecting the conversion of the accumulated dihydroxylated T-UDCA into the T-beta-MCA. The similar effects of V and CsA on BA transport and metabolism can be explained by mdr1 mediated disturbances of cellular ATP transport rather than by inhibition of individual BA transporters.     

Testosterone suppresses protective responses of the liver to blood-stage malaria

Testosterone induces a lethal outcome in otherwise self-healing blood-stage malaria caused by Plasmodium chabaudi. Here, we examine possible testosterone effects on the antimalaria effectors spleen and liver in female C57BL/6 mice. Self-healing malaria activates gating mechanisms in the spleen and liver that lead to a dramatic reduction in trapping activity, as measured by quantifying the uptake of 3-mum-diameter fluorescent polystyrol particles. However, testosterone delays malaria-induced closing of the liver, but not the spleen. Coincidently, testosterone causes an approximately 3- to 28-fold depression of the mRNA levels of nine malaria-responsive genes, out of 299 genes tested, only in the liver and not in the spleen, as shown by cDNA arrays and Northern blotting. Among these are the genes encoding plasminogen activator inhibitor (PAI1) and hydroxysteroid sulfotransferase (STA2). STA2, which detoxifies bile acids, is suppressed 10-fold by malaria and an additional 28-fold by testosterone, suggesting a severe perturbation of bile acid metabolism. PAI1 is protective against malaria, since disruption of the PAI1 gene results in partial loss of the ability to control the course of P. chabaudi infections. Collectively, our data indicate that the liver rather than the spleen is a major target organ for testosterone-mediated suppression of resistance against blood-stage malaria.     

Identification of a novel mycobacterial histone H1 homologue (HupB) as an antigenic target of pANCA monoclonal antibody and serum immunoglobulin A from patients with Crohn's disease

pANCA is a marker antibody associated with inflammatory bowel disease (IBD), including most patients with ulcerative colitis and a subset with Crohn's disease. This study addressed the hypothesis that pANCA reacts with an antigen(s) of microbial agents potentially relevant to IBD pathogenesis. Using a pANCA monoclonal antibody, we have previously identified the C-terminal basic random-coil domain of histone H1 as a pANCA autoantigen. BLAST analysis of the peptide databases revealed H1 epitope homologues in open reading frames of the Mycobacterium tuberculosis genome. Western analysis of extracts from six mycobacterial species directly demonstrated reactivity to a single, conserved approximately 32-kDa protein. Direct protein sequencing, followed by gene cloning, revealed a novel 214-amino-acid protein, an iron-regulated protein recently termed HupB. Sequence analysis demonstrated its homology with the mammalian histone H1 gene family, and recombinant protein expression confirmed its reactivity with the 5-3 pANCA monoclonal antibody. Binding activity of patient serum immunoglobulin G (IgG) to HupB did not correlate with reactivity to histone H1 or pANCA, indicating the complex character of the pANCA antigen. However, anti-HupB IgA was strongly associated with Crohn's disease (P < 0.001). These findings indicate that the 5-3 pANCA monoclonal antibody detects a structural domain recurrent among mycobacteria and cross-reactive with a DNA-binding domain of histone H1. The association of HupB-binding serum IgA with IBD provides new evidence for the association of a mycobacterial species with Crohn's disease.     

Apoptosis in endometrial glandular and stromal cells in women with and without endometriosis

BACKGROUND: The aetiology of endometriosis is unknown. Ectopic dissemination of the endometrial cells gives origin to endometriotic lesions, but occurs in women with and without endometriosis. It has been suggested that increased ectopic cell survival facilitates their implantation. The objectives of this study were to evaluate endometrial apoptosis in women with endometriosis according to: (i) cyclic changes, (ii) glandular and stromal contribution, and (iii) stage of the disease. METHODS: The subjects were women undergoing diagnostic laparoscopy and endometrial biopsies for suspected endometriosis. Spontaneous apoptosis was evaluated using TdT-mediated dUTP-biotin nick end-labelling (TUNEL) assay. Apoptotic cells per 10 mm(2) (apoptotic index) in an area of 10-50 mm(2) in 5 microm endometrial tissue sections were counted and location of these cells was recorded. RESULTS: The apoptotic index in glandular epithelium was lower in endometriosis than controls (26.0 +/- 5.5 versus 51.2 +/- 9.7, P = 0.03) but not in the stroma (36.3 +/- 6.4 versus 48.4 +/- 11.3, NS). In controls, apoptosis was highest during the late secretory/menstrual and early proliferative phases and cyclic variability was apparent. In endometriosis, this cyclic variability was lost. There was a trend toward decreased apoptosis with increasing stage of the disease, but the differences lacked statistical significance. CONCLUSIONS: Spontaneous apoptosis is decreased in the endometrial glands in women with endometriosis, especially during late secretory/menstrual and early proliferative phases of the cycle. This may indicate increased viability of endometrial cells shed during menses, facilitating their ectopic survival and implantation.     

Bipolaris spicifera causes fungus balls of the sinuses and triggers polypoid chronic rhinosinusitis in an immunocompetent patient

We report the rare case of a 19-year-old immunocompetent male suffering both from fungus balls of the sinuses and from chronic rhinosinusitis with massive polyposis. Endoscopic sinus surgery revealed grayish brown necrotic masses embedded in viscous eosinophilic mucus. Inoculated onto petri dishes, these masses as well as the mucus grew a dark pigmented fungus, which was identified as Bipolaris spicifera.     

Analysis of cytokine profiles in synovial T cell clones from chlamydial reactive arthritis patients: predominance of the Th1 subset.

Subpopulations of human T cells (Th0, Th1 and Th2) can be distinguished by their cytokine-secretion pattern. Evidence is increasing from other studies that the outcome of a human disease may depend on the subpopulation of T cells that predominates at the site of inflammation. Reactive arthritis serves as a useful model of chronic inflammatory diseases, because the triggering antigen can be identified. Using this triggering antigen we raised 33 T cell clones reactive with Chlamydia trachomatis and 25 T cell clones that were not reactive, all from the synovial fluid of two patients suffering from Chlamydia-induced arthritis. Their cytokine secretion patterns for interferon-gamma (IFN-gamma), IL-2 and IL-4 were analysed, as also were mRNAs for IFN-gamma and IL-10 by in situ hybridization. Out of the 33 antigen-reactive clones 23 showed a Th1 pattern with IFN-gamma but not IL-4 secretion, while the remaining 10 exhibited a Th0 pattern. The clones that did not react with Chlamydia expressed all patterns of cytokine secretion, including a Th2 pattern, thus providing a control population that excludes bias in the sampling procedure. CD4 and CD8 clones displayed a similar cytokine-secretion pattern. In addition this study demonstrates for the first time the expression of IL-10 mRNA in T cell clones derived from synovial fluid, and this was not confined to the Th2 subset. The Th1 response that Chlamydia provoke can be regarded as appropriate for such an obligate intracellular pathogen.     

Neurotrophins: a link between airway inflammation and airway smooth muscle contractility in asthma?

BACKGROUND: Bronchial asthma (BA) is characterized by a unique type of airway inflammation, epithelial cell damage and increased airway smooth muscle (ASM) contractility. The regulatory network between the immunological events and the neuronal control of ASM contractility remains to be defined. METHODS: Utilizing a well-characterized mouse model of airway inflammation and BA, we analyzed the production and function of neurotrophins in allergic asthma. To confirm these data in humans, segmental allergen provocation was performed in mild asthmatics. RESULTS: Allergen-induced airway inflammation was associated with increased local production of the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in mice as well as in humans. In bronchoalveolar lavage fluid (BALF), NGF levels were increased 4- to 5-fold in men and mice 1 day after allergen provocation. The increase in BDNF was about 2-fold in both models. Treatment of mice with anti-NGF prevented development of airway hyperresponsiveness (AHR). In the human study group, NGF levels in BALF after allergen provocation were correlated significantly with baseline FEV1 levels. CONCLUSION: These data strongly suggest that neurotrophins serve as a link between airway inflammation and neuronal control of ASM constriction in BA.     

Iron regulation of Serratia marcescens hemolysin gene expression.

The hemolytic activity of Serratia marcescens was examined as a function of iron availability. Restriction of iron by the nonmetabolizable chelator 2,2'-dipyridyl or the iron-binding protein transferrin produced a marked increase in hemolytic activity. The hemolytic activity of S. marcescens is determined by two adjacent genes, 5'-shlB-shlA-3', where shlA encodes the hemolysin which requires the ShlB protein for activity. A gene fusion between the promoter-proximal portion of shlA and phoA, the Escherichia coli alkaline phosphatase gene, was subcloned into a medium-copy-number vector, and the recombinant plasmid was introduced into S. marcescens. The expression of shlA was measured as a function of alkaline phosphatase activity, which increased threefold under iron-restricted conditions. Removal of the 5' noncoding region upstream of shlB in the fusion vector resulted in a 10-fold decrease in alkaline phosphatase activity under iron-sufficient conditions, with no effect of iron limitation on this residual activity. This suggested that the site mediating iron regulation of shlA expression occurs upstream of shlB. Consistent with this, we observed iron-regulated synthesis of the ShlB protein in Western immunoblots of isolated outer membranes. The hemolysin determinant was subsequently expressed on a medium-copy-number vector in fur+/fur isogenic strains of E. coli K-12, where a 10-fold-higher activity was observed in the mutant strain compared with the wild type. A sequence exhibiting some homology to the Fur-binding consensus sequence was identified upstream of the shlB coding region, overlapping the -35 region of a putative promoter.     

Oligonucleotide typing is a perfect tool to identify antigens stimulatory in the mixed lymphocyte culture

An important criterion for the selection of donors for bone marrow transplantation is the grade of matching for HLA between donor and recipient. For patients that lack an HLA-identical sibling, an extending pool of unrelated volunteers for bone marrow donation is available. From these donors the best matched candidate can be selected by serological typing, followed by a mixed lymphocyte culture (MLC).Oligonucleotide genotyping for HLA class II antigens is considered to be valuable for the prediction of MLC reactivity. We investigated whether this typing method, in combination with serological typing, would cover the recognition of all MLC stimulatory determinants. One hundred thirty-six combinations of HLA-A, -B, and -DR serologically identical individuals were tested in the MLC. Additional typing for HLA-DRB and HLA-DPB by oligonucleotide genotyping made it possible to evaluate the influence of these genes on MLC reactivity. Combinations that were matched for HLA-DRB gave significantly lower responses than those that were mismatched. Nevertheless, in the matched combinations responses were observed to 94% relative response index. These responses could all be attributed to HLA-DP, since all combinations that were identical by HLA-DPB genotyping were negative in the MLC. In conclusion, with the combined use of serology and oligonucleotide genotyping, responder-stimulator combinations can be selected that are identical for all MLC stimulatory determinats. 245 (1991)     

A European study on the genetics of mite sensitization

BACKGROUND: Sensitization to mite allergens represents a prominent feature of atopy and an important predictor of bronchial asthma. OBJECTIVE: It was the intention of this study to define genetic loci linked to mite sensitization because these could represent the genetic basis of the important atopic component of asthma. METHODS: We studied a multiethnic white population of 99 families, including 224 sib pairs sensitized to Dermatophagoides pteronyssinus. A genome-wide candidate-region search was performed that covered potential asthma and atopy regions. RESULTS: As for nonparametric linkage (NPL) analysis, 14 of the candidate regions showed evidence for linkage (NPL > 2.0), and 4 of them showed prominent linkage (NPL > 3.0). However, there were substantial ethnic differences. Maximizing the LOD score analysis identified candidate regions with suspected dominant and recessive mode of inheritance. Furthermore, genetic imprinting models provided significant evidence for linkage in the 8p23 region and revealed potential maternal imprinting. The regions found are distinct to those in asthma searches that have been found to be linked to asthma, as well to other inflammatory diseases. In addition, we could not find linkage to the HLA region. By different cutoff points of the phenotype definition, the IL cluster showed evidence of being linked to the degree of sensitization rather than to sensitization per se. CONCLUSION: The results indicate that the genetic basis of the atopic component of asthma is different from that of the inflammatory component. Furthermore, it seems reasonable to assume that specific sensitizations are influenced by distinct genetic variants leading to their initiation versus those leading to their enhancement.