Differential regulation of human 3β-hydroxysteroid dehydrogenase type 2 for steroid hormone biosynthesis by starvation and cyclic AMP stimulation: studies in the human adrenal NCI-H295R cell model

Human steroid biosynthesis depends on a specifically regulated cascade of enzymes including 3β-hydroxysteroid dehydrogenases (HSD3Bs). Type 2 HSD3B catalyzes the conversion of pregnenolone, 17α-hydroxypregnenolone and dehydroepiandrosterone to progesterone, 17α-hydroxyprogesterone and androstenedione in the human adrenal cortex and the gonads but the exact regulation of this enzyme is unknown. Therefore, specific downregulation of HSD3B2 at adrenarche around age 6-8 years and characteristic upregulation of HSD3B2 in the ovaries of women suffering from the polycystic ovary syndrome remain unexplained prompting us to study the regulation of HSD3B2 in adrenal NCI-H295R cells. Our studies confirm that the HSD3B2 promoter is regulated by transcription factors GATA, Nur77 and SF1/LRH1 in concert and that the NBRE/Nur77 site is crucial for hormonal stimulation with cAMP. In fact, these three transcription factors together were able to transactivate the HSD3B2 promoter in placental JEG3 cells which normally do not express HSD3B2. By contrast, epigenetic mechanisms such as methylation and acetylation seem not involved in controlling HSD3B2 expression. Cyclic AMP was found to exert differential effects on HSD3B2 when comparing short (acute) versus long-term (chronic) stimulation. Short cAMP stimulation inhibited HSD3B2 activity directly possibly due to regulation at co-factor or substrate level or posttranslational modification of the protein. Long cAMP stimulation attenuated HSD3B2 inhibition and increased HSD3B2 expression through transcriptional regulation. Although PKA and MAPK pathways are obvious candidates for possibly transmitting the cAMP signal to HSD3B2, our studies using PKA and MEK1/2 inhibitors revealed no such downstream signaling of cAMP. However, both signaling pathways were clearly regulating HSD3B2 expression.

The sequence of addition of IL-3 and IgE-dependent/IgE-independent stimuli regulates RANKL expression in human basophils

Background: Receptor Activator of Nuclear Factor kappaB Ligand (RANKL), a member of the TNF superfamily, contributes to the imbalance of bone resorption and immunoregulation in rheumatoid arthritis. In mice, collagen induced arthritis was exacerbated by IL-3 and anti-IgER antibodies, two mediators activating basophils that are known as effector cells of allergy. Interestingly, our unpublished microarray data revealed that IL-3 induces RANKL mRNA in human basophils. Here we further investigate under which conditions human basophils express surface and/or soluble RANKL. Methods: One part of purified human basophils was co-stimulated with IL-3 and either IgE-dependent or IgE-independent stimuli. The other part of purified basophils was first primed with IL-3 and subsequently triggered with IgE-dependent or IgE-independent stimuli. Expression of surface and soluble RANKL were detected by flow cytometry, ELISA and real-time PCR. Results: By flow cytometry we show that IL-3 induces de novo expression of surface RANKL on human basophils in a time and dose dependent manner. Co-stimulation of basophils with IL-3 and an IgE-dependent stimulus reduces IL-3-induced expression of surface RANKL in a dose dependent manner while IgE-independent stimuli have no effect. In contrast, both IgE-dependent and IgE-independent stimuli enhance expression of surface and soluble RANKL in basophils that were first primed with IL-3 and then triggered. Real-time PCR analysis shows that surface hRANKL1 and soluble hRANKL3 are induced by IL-3 and reduced by co-stimulation with IL-3 and an IgE-dependent stimulus and thus confirms our flow cytometry data. Conclusion: RANKL expression in human basophils is not only dependent on IL-3 and IgE-dependent/IgE-independent stimuli but also on the sequence of their addition to cell culture. Based on our data, we suggest that basophils might have previously unidentified functions in bone resorption or immunoregulation via RANKL.

THE LONG-TERM GROWTH OF NORMAL HUMAN B CELLS

The feasibility of establishment of continuously proliferating growth factor-dependent human B lymphocytes was investigated. Normal B lymphocytes prepared from peripheral venous blood were stimulated with a variety of known polyclonal B cell activators, in the continuous presence of various cytokine preparations. Continuously proliferating growth factor-dependent B cell populations were obtained from cultures activated with either insoluble anti-IgM ((mu)-chain specific), soluble anti-IgM, heat-killed Staphylococcus aureus Cowen I (SAC), or dextran sulphate (DxS), in the continuous presence of exogenously added growth factor preparations containing either IL-1, IL-2 and BCGF, or BCGF alone. Although growth factor-dependent B cell lines were obtained via all three methods of activation, the correlation of mode of activation and growth factor preparation proved to be critical. B cell lines could not be established with anti-(mu) activation in the presence of only BCGF; however, B cell lines were successfully obtained with SAC or DxS activation from those cultures continuously replenished with only BCGF. These cultured B lymphocyte populations were routinely maintained in logarithmic-phase growth in the presence of exogenously added growth factor, and exhibited a population doubling time of approximately 36 hours. They were shown to specifically absorb BCGF, suggesting the presence of membrane receptors for it. Also, these cultured B cells have been utilized for the development of a microassay for the assessment of a M(,r) 12,000-14,000 B cell growth factor activity that is accurate, sensitive, and precise. The pronounced sensitivity of this bioassay beyond that of the conventional peripheral blood B cell assay has aided in the purification to homogeneity of natural product extracellular BCGF (EC-BCGF), and in the determination of the nucleotide sequence for a gene coding for a protein exhibiting BCGF activity. Additionally, these B cell lines specifically absorb, and proliferate in the presence of, an affinity-purified M(,r) 60,000 trypsin-sensitive intracellular protein derived from freshly isolated human T lymphocytes, providing evidence for a putative intracellular precursor of EC-BCGF, or a novel high molecular weight BCGF species. ^

Phenotypic and molecular characterization of hyperpigmented group B Streptococci.

Group B Streptococcus (GBS) causes invasive infections in neonates, older adults and patients with comorbidities. β-hemolysin/cytolysin is an important GBS virulence factor. It is encoded by the cyl operon and confers GBS hemolytic activity. Isolates displaying hyperpigmentation are typically hyperhemolytic. Comparison of clonally identical isolates displaying different levels of pigmentation has shown transcriptional dysregulation due to mutations in components of the control of the virulence S/R (CovS/R) regulatory system. In addition, hyperpigmented isolates show decreased CAMP factor and decreased capsule thickness. In analogy to findings in group A Streptococcus, a pivotal role of CovS/R has been proposed in the host-pathogen interaction of invasive GBS infection. However, corresponding investigations on multiple clinical GBS isolates have not been performed. We prospectively collected hyperpigmented isolates found in a diagnostic laboratory and performed phenotypic, molecular and transcriptional analyses. In the period from 2008 to 2012, we found 10 isolates obtained from 10 patients. The isolates reflected both invasive pathogens and colonizers. In three cases, clonally identical but phenotypically different variants were also found. Hence, the analyses included 13 isolates. No capsular serotype was found to be significantly more frequent. Bacterial pigments were analyzed via spectrophotometry and for their hemolytic activity. Data obtained for typical absorbance spectra peaks correlated significantly with hemolytic activity. Molecular analysis of the cyl operon showed that it was conserved in all isolates. The covR sequence displayed mutations in five isolates; in one isolate, the CovR binding site to cylX was abrogated. Our results on clinical isolates support previous findings on CovR-deficient isogenic mutants, but suggest that - at least in some clinical isolates - for β-hemolysin/cytolysin and CAMP factor production, other molecular pathways may be involved.

Helicobacter pylori Inhibits Dendritic Cell Maturation via Interleukin-10-Mediated Activation of the Signal Transducer and Activator of Transcription 3 Pathway.

Helicobacter pylori infects the human gastric mucosa causing a chronic infection that is the primary risk factor for gastric cancer development. Recent studies demonstrate that H. pylori promotes tolerogenic dendritic cell (DC) development indicating that this bacterium evades the host immune response. However, the signaling pathways involved in modulating DC activation during infection remain unclear. Here, we report that H. pylori infection activated the signal transducer and activator of transcription 3 (STAT3) pathway in murine bone marrow-derived DCs (BMDCs) and splenic DCs isolated ex vivo. Isogenic cagA-, cagE-, vacA- and urease-mutants exhibited levels of phosphoSTAT3 that were comparable to in the wild-type (WT) parent strain. H. pylori-infected BMDCs produced increased immunosuppressive IL-10, which activated STAT3 in an autocrine/paracrine fashion. Neutralization of IL-10 prevented H. pylori-mediated STAT3 activation in both BMDCs and splenic DCs. In addition, anti-IL-10 treatment of infected H. pylori-BMDCs was associated with increased CD86 and MHC II expression and enhanced proinflammatory IL-1β cytokine secretion. Finally, increased CD86 and MHC II expression was detected in H. pylori-infected STAT3 knockout DCs when compared to WT controls. Together, these results demonstrate that H. pylori infection induces IL-10 secretion in DCs, which activates STAT3, thereby modulating DC maturation and reducing IL-1β secretion. These findings identify a host molecular mechanism by which H. pylori can manipulate the innate immune response to potentially favor chronic infection and promote carcinogenesis. © 2014 S. Karger AG, Basel.

A history of the religion of Judaism 500 to 200 b. C.

Archibald Duff

Quantitative depth profiling of Ce 3+ in Pt/CeO 2 by in situ high-energy XPS in a hydrogen atmosphere

The redox property of ceria is a key factor in the catalytic activity of ceria-based catalysts. The oxidation state of well-defined ceria nanocubes in gas environments was analysed in situ by a novel combination of near-ambient pressure X-ray Photoelectron Spectroscopy (XPS) and high-energy XPS at a synchrotron X-ray source. In situ high-energy XPS is a promising new tool to determine the electronic structure of matter under defined conditions. The aim was to quantitatively determine the degree of cerium reduction in a nano-structured ceria-supported platinum catalyst as a function of the gas environment. To obtain a non-destructive depth profile at near-ambient pressure, in situ high-energy XPS analysis was performed by varying the kinetic energy of photoelectrons from 1 to 5 keV, and, thus, the probing depth. In ceria nanocubes doped with platinum, oxygen vacancies formed only in the uppermost layers of ceria in an atmosphere of 1 mbar hydrogen and 403 K. For pristine ceria nanocubes, no change in the cerium oxidation state in various hydrogen or oxygen atmospheres was observed as a function of probing depth. In the absence of platinum, hydrogen does not dissociate and, thus, does not lead to reduction of ceria.

Biochanin A and prunetin improve epithelial barrier function in intestinal CaCo-2 cells via downregulation of ERK, NF-κB, and tyrosine phosphorylation

The single-layered gut epithelium represents the primary line of defense against environmental stressors; thereby monolayer integrity and tightness are essentially required to maintain gut health and function. To date only a few plant-derived phytochemicals have been described as affecting intestinal barrier function. We investigated the impact of 28 secondary plant compounds on the barrier function of intestinal epithelial CaCo-2/TC-7 cells via transepithelial electrical resistance (TEER) measurements. Apart from genistein, the compounds that had the biggest effect in the TEER measurements were biochanin A and prunetin. These isoflavones improved barrier tightness by 36 and 60%, respectively, compared to the untreated control. Furthermore, both isoflavones significantly attenuated TNFα-dependent barrier disruption, thereby maintaining a high barrier resistance comparable to nonstressed cells. In docking analyses exploring the putative interaction with the tyrosine kinase EGFR, these novel modulators of barrier tightness showed very similar values compared to the known tyrosine kinase inhibitor genistein. Both biochanin A and prunetin were also identified as potent reducers of NF-κB and ERK activation, zonula occludens 1 tyrosine phosphorylation, and metalloproteinase-mediated shedding activity, which may account for the barrier-improving ability of these isoflavones.