Peter Scholz

Structural Evidence for Ligand Specificity in the Binding Domain of the Human Androgen Receptor

The crystal structures of the human androgen receptor (hAR) and human progesterone receptor ligand-binding domains in complex with the same ligand metribolone (R1881) have been determined. Both three-dimensional structures show the typical nuclear receptor fold. The change of two residues in the ligand-binding pocket between the human progesterone receptor and hAR is most likely the source for the specificity of R1881 to the hAR. The structural implications of the 14 known mutations in the ligand-binding pocket of the hAR ligand-binding domains associated with either prostate cancer or the partial or complete androgen receptor insensitivity syndrome were analyzed. The effects of most of these mutants could be explained on the basis of the crystal structure.

Induction of RANTES expression by astrocytes and astrocytoma cell lines

The cellular infiltrate found during the acute phase of multiple sclerosis (MS) consists of monocytes and activated T cells, suggesting the presence of cell-specific chemotactic signals during the inflammatory response. We examined the ability of human astrocytoma cell lines, as well as primary human and rat astrocytes, to generate a specific member of the intercrine/chemokine family of cytokines, RANTES, when exposed to TNF-alpha, IL-1 beta and IFN-gamma. Astrocytoma cells as well as primary astrocytes produced RANTES upon incubation with TNF-alpha or IL-1 beta. IFN-gamma alone did not induce RANTES production by astrocytes, but it potentiated the effects of either TNF-alpha or IL-1 beta. Induction of RANTES by TNF-alpha was mediated by the p55 receptor since a specific anti-p55 antiserum mimicked the effect of TNF-alpha. These results indicate that human astrocytes are capable of generating a cell-specific chemokine that can account for the inflammatory cellular infiltrate observed during the acute phase of MS, in a process that is regulated by cytokines.

Pneumococcal cell wall components induce nitric oxide synthase and TNF-α in astroglial-enriched cultures

Astroglia and microglia, the most numerous cells in the central nervous system (CNS), have been shown to produce the inducible nitric oxide synthase (iNOS) and tumor necrosis factor‐α (TNF‐α) upon stimulation with the cytokines IFN‐γ, IL‐1β, or bacterial lipopolysaccharides (LPS). However, it is not known whether gram‐positive bacteria like Streptococcus pneumoniae cause astroglial cells to release nitric oxide (NO) and TNF‐α. S. pneumoniae meningitis still has a high incidence and mortality in spite of antibiotic therapy. Cell wall components from S. pneumoniae (pneumococcal cell‐wall components, PCW) and TNF‐α have been shown to cause meningeal inflammation and cerebrovascular changes in experimental meningitis.

Supernatants of HIV-infected immune cells affect the barrier function of human HT-29/B6 intestinal epithelial cells.

Objectives Characterization of the diarrhoea-inducing effect of altered cytokine production in HIV infection. Methods Monocyte-derived macrophages (MDM) were infected with macrophagetropic (SF162) and lymphocytotropic (IIIB) HIV-1 strains and cocultured with autologous peripheral blood mononuclear cells (PBMC). After 24 h the supernatants were collected and tested for their immunoreactive levels of cytokines by enzyme-linked immunosorbent assay. The effects of the supernatants and the respective recombinant human cytokines on barrier function of HT-29/B6 cells were determined. Results Infection of MDM with HIV-1 SF162 or IIIB led to increased production of tumour necrosis factor-alpha (TNFα), interleukin-1-beta, interferon-alpha and interferon-gamma after cell–cell contact with PBMC. Supernatants of infected cells decreased transepithelial resistance (Rt), with higher effects on Rt in HIV IIIB infection, which was due to higher cytokine concentrations. The effect was not due to cytotoxicity (negative LDH assay) or epithelial monolayer disruption [zonula occludens protein-1 (ZO-1) immunofluorescence staining]. The effect of HIV-1 IIIB coculture supernatants could be mimicked by the respective recombinant human cytokines. TNFα is an effector cytokine, because inhibition of TNFα by its soluble receptor decreased the effect of the supernatants on transepithelial resistance. Conductance scanning indicated the cytokine-induced barrier defect to be due to both, induction of epithelial apoptoses and tight junction alterations. Conclusions Cell–cell interaction of HIV-infected macrophages with PBMC leads to a release of cytokines sufficient to alter intestinal epithelial barrier function. The main effect was mediated by TNFα inducing a leak-flux which may contribute to the diarrhoea by HIV per se (HIV-enteropathy).

PsiB, an anti-SOS protein, is transiently expressed by the F sex factor during its transmission to an Escherichia coli K-12 recipient

PsiB, an anti‐SOS protein, shown previously to prevent activation of RecA protein, was purified from the crude extract of PsiB overproducing cells. PsiB is probably a tetrameric protein, whose subunit has a sequence‐deduced molecular mass of 15741 daltons. Using an immuno‐assay with anti‐PsiB antibodies, we have monitored PsiB cell concentrations produced by F and R6‐5 piasmids: the latter type produces a detectable level of PsiB protein while the former does not. The discrepancy can be assigned to a Tn 10 outgoing promoter located upstream of psiB. When we inserted a Tn 10 promoter upstream of F psiB, the F PsiB protein concentration reached the level of R6‐5 PsiB.

Mechanisms of Epithelial Barrier Impairment in HIV Infection

Abstract: Diarrhea and malabsorption due to intestinal dysfunction are common symptoms in HIV infection. The pathophysiologic mechanisms of these alterations are often not known, and the role of HIV per se is still controversially discussed. We measured the epithelial transport and barrier function by means of a miniaturized Ussing chamber system in the duodenum of HIV‐infected patients in different disease stages, determined by the CD4 cell count in the serum as well as symptoms in patients with and without diarrhea. We could show that diarrhea induced by HIV per se is caused by a leak flux mechanism due to impaired epithelial barrier function. Antisecretory therapy does not seem to be useful in these patients, because we did not find increased active ion secretion. Along the course of the HIV infection, the epithelial transport and barrier function varies with HIV disease stage (expressed by CD4 cell status). In addition, an in vitro model was studied to characterize the effect of HIV‐infected human immune cells on the epithelial barrier function using the human colonic epithelial cell line HT‐29/B6. HIV infection of human immune cells induced an increase in cytokine release‐for example, TNF‐α, IL‐1β, IFN‐α, and IFN‐γ‐downregulating the epithelial barrier function of the human colonic epithelial cell line HT‐29/B6. Taken together we postulate a specific stage‐dependent cytokine pattern released from HIV‐infected immune cells in the mucosa, which, corresponding to the HIV disease stage, is responsible for the variation in epithelial function.

Immunological and biological identification of tumour necrosis-like factor in sponges: Endotoxin that mediates necrosis formation in xenografts

Xenografts of the sponge Geodia cydonium in its closely related species G. rovinjensis resulted in a rapid rejection of the graft within a period of 5 days. We identified an immunoreactive tumour necrosis factor (TNF)-like activity in the xenograft (Mr of 30,000) two days after grafting. In-vivo injection of 5 micrograms human recombinant TNF-alpha induced cytotoxicity in sponge cells in the same pattern and time course as during natural xenograft rejection. Anti-TNF-alpha polyclonals were found to react with xenograft extracts, by Western blot analysis, as from day 2 after grafting. Using ELISA we detected the TNF-like activity from day 2 after grafting with peak levels at days 4 and 5, where the amount was 0.72 ng/micrograms tissue DNA. By day 1, gp27 (inhibitory aggregation factor) is already formed in the xenograft. In-vitro experiments on isolated G. cydonium cells showed that addition of purified gp27 induced the production of the TNF-like activity (up to 13.5 ng/ml). Evidence is presented that gp27 is a product of the gp180 lectin receptor. We conclude that gp27 induces TNF-like factor production, resulting in destruction and dissolution of the xenograft after 5 days.

Investigation of the binding interactions of progesterone using muteins of the human progesterone receptor ligand binding domain designed on the basis of a three-dimensional protein model.

The aim of this study was to investigate the binding interactions of the human progesterone receptor (hPR) with its natural ligand. Therefore, a homology-derived model of the hPR ligand binding domain has been constructed and used to predict residues potentially involved in interactions with progesterone. These residues and the free cysteines have been mutated (in total 13 residues with 15 mutations). All exchanges have been designed to preserve the three-dimensional structure of the protein. With respect to the binding characteristics towards progesterone, the muteins fall into three groups displaying no, reduced, or wildtype-like binding activity.

Production of human interleukin-8 expressed in Escherichia coli: From a laboratory scale for in vitro tests via a technical scale for animal studies to a process scale for a GMP-compatible production

An Escherichia coli K 12 strain has been constructed for efficient expression of recombinant biologically active human IL-8 (Interleukin-8). The development of a fermentation and purification process from the laboratory scale (cells from 15 l fermentation broth) to a production scale (cells from 200 l fermentation broth) is described. Material obtained from the laboratory scale was used for initial in vitro studies and for the development of a biological assay. An upscale purification process starting from 80 l fermentation broth resulted in larger amounts of IL-8 needed for preclinical studies. This process includes a fully automated control of the initial affinity chromatography step. Finally, a production process which differed markedly from the small-scale processes was tailor-made for GMP conformity and economic considerations. It consists of a cell disruption step followed by two crossflow diafiltrations with different molecular weight cut offs and filtration rates, one cation exchange chromatography and a final dialysis step. In order to enhance the overall yield of biologically active IL-8, conditions for a resolubilisation of insoluble IL-8 present in the remaining pellet after cell disruption were worked out.

Inhibition of the effects of rheumatoid synovial fluid cells on chondrogenesis and cartilage breakdown in vitro: possible therapeutical conclusions. A morphological--biochemical study.

SummaryShort-term co-cultivation of blastemal cells from 12-day-old mouse limb buds and human rheumatoid synovial fluid cells in high density cultures (Trowell culture system) resulted, depending on when co-cultivation started, either in (1) an inhibition of chondrogenesis (co-cultivation right from the start) or in (2) an extensive breakdown of cartilaginous matrix (co-cultivation after formation of embryonic cartilage). These synovial effects were markedly impeded if Avarol (a dioxygenase inhibitor) was applied singly or in combination with PAI-2 (a u-PA-inhibitor). PAI-2 alone, however, had no effect on the synovial-induced inhibition of chondrogenesis, but produced a pronounced inhibitory effect on matrix breakdown. The effects of both inhibitors were studied electron microscopically and biochemically (determination of sulfated-glycosaminoglycans in the high density cultures by Alcian Blue binding assay). The results of this study are consistent with the presumption that rheumatoid synovial cells are capable of inhibiting chondrogenesis and enhancing the breakdown of the cartilaginous matrix. Amongst others, the possible mediators involved are prostaglandins and plasminogen activators. The response to the inhibitors Avarol and PAI-2 is compatible with their mode of action. The chondroprotective action of these substances may be useful in developing potential antirheumatic drugs.