Christian Schwerk

Non-apoptotic functions of caspases in cellular proliferation and differentiation.

The cysteinyl aspartate-specific proteases (caspases) have been identified as key players in the cellular process termed programmed cell death or apoptosis. During apoptosis, activated apoptotic caspases cleave selected target proteins to execute cell death. Additionally to their established function in cell death, a variety of recent publications have provided increasing evidence that apoptotic caspases also participate in several non-apoptotic cellular processes. Activated caspases exhibit functions during T-cell proliferation and cell cycle regulation, but are also involved in the differentiation of a diverse array of cell types. In some cell types, their differentiation can be morphologically viewed as a kind of incomplete apoptosis. Analysis of well-known apoptotic targets of caspases implicates that the cleavage of a limited number of selected substrates plays a major role during non-apoptotic functions of caspases. Selective substrate cleavage might be regulated by activation of anti-apoptotic factors, via a compartmentalized activation of caspases, or through limited activity of caspases during apoptosis-independent functions. The increasing evidence for caspase function in non-apoptotic cellular events suggests that caspases play a much more diverse role than previously assumed.

Polar bacterial invasion and translocation of Streptococcus suis across the blood-cerebrospinal fluid barrier in vitro.

Previous experimental studies in a standard Transwell culture system have shown Streptococcus suis ability to compromise barrier function of porcine choroid plexus epithelial cells (PCPEC). The development of an ‘inverted’ Transwell filter system of PCPEC enables us now for the first time to investigate bacterial invasion and translocation from the physiologically relevant basolateral (blood) to the apical (cerobrospinal fluid) side. Most importantly, we observed specific invasion and translocation of S. suis across the PCPEC exclusively from the basolateral side. During this process, bacterial viability and the presence of a capsule as well as cytoskeletal regulation of PCPEC seemed to play an important role. No loss of barrier function was observed. Bacterial translocation could be significantly inhibited by the phosphatidylinositol 3‐kinase inhibitor LY294002, but not by its inactive analogue Ly303511 or dexamethasone. Apotome imaging as well as electron microscopy revealed intracellular bacteria often in cell vacuoles. Thus, possibly regulated by the presence of a capsule, S. suis induces signals that depend on the lipid kinase phosphatidylinositol 3‐kinase pathway, which paves the way for cellular uptake during the bacterial transcellular translocation process. Taken together, our data underline the relevance of the blood–cerebrospinal fluid barrier as a gate for bacterial entry into the central nervous system.

Identification of a Transactivation Function in the Progesterone Receptor That Interacts with the TAFII110 Subunit of the TFIID Complex

Transcriptional activation of target genes by the human progesterone receptor is thought to involve direct or indirect protein-protein interactions between the progesterone receptor and general transcription factors. A key role in transcription plays the general transcription factor TFIID, a multiprotein complex consisting of the TATA-binding protein and several tightly associated factors (TAFs). TAFs have been shown to be required for activated transcription and are, thus, potential targets of activator proteins. Using in vitro interaction assays, we could identify specific interactions between the progesterone receptor and the TATA-binding protein-associated factor dTAFII110. The dTAFII110 domain responsible for the interaction is distinct from that reported to suffice for binding to Sp1. Somewhat surprisingly, deletion analysis indicated that the previously identified activation functions 1 and 2 of the progesterone receptor are not required for this interaction but pointed to an important role of the DNA binding domain. In cotransfection experiments and an in vitro transcription assay, the DNA binding domain of the progesterone receptor displayed significant activation potential. These findings, taken together, suggest that an interaction between the progesterone receptor and TAFII110 may represent an important step in the mechanism of activation.

Culture models to study leukocyte trafficking across the choroid plexus

BackgroundA critical point during the course of central nervous system infection is the influx of leukocytes from the blood into the brain across the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB). However, experimental in vitro models to investigate leukocyte transmigration across cultured choroid plexus epithelial cells have been lacking so far.MethodsWe have developed a porcine and human “inverted” culture insert system that enables leukocyte transmigration specifically from the physiologically relevant basolateral side. The models use primary porcine choroid plexus epithelial cells (PCPEC) and human choroid plexus papilloma cells (HIBCPP). As a prerequisite for a functional barrier, we optimized culture conditions in which cells are maintained in serum-containing medium until high barrier function is reached. Leukocyte transmigration through the plexus epithelial cells is analysed by three-dimensional Apotome®-imaging and electron microscopy, and the route of transmigration through the plexus epithelial cells, i.e. transcellular as well as paracellular, can be determined.DiscussionAs a functionally relevant porcine and human BCSFB model, PCPEC and HIBCPP respectively, offer a wide range of options for analysis of disease-related mechanisms at the choroid plexus epithelium, especially involving human pathogens. Moreover, our in vitro models facilitate the investigation of leukocyte entry into the CNS via the blood-CSF barrier.

Polar Invasion and Translocation of Neisseria meningitidis and Streptococcus suis in a Novel Human Model of the Blood-Cerebrospinal Fluid Barrier

Acute bacterial meningitis is a life-threatening disease in humans. Discussed as entry sites for pathogens into the brain are the blood-brain and the blood-cerebrospinal fluid barrier (BCSFB). Although human brain microvascular endothelial cells (HBMEC) constitute a well established human in vitro model for the blood-brain barrier, until now no reliable human system presenting the BCSFB has been developed. Here, we describe for the first time a functional human BCSFB model based on human choroid plexus papilloma cells (HIBCPP), which display typical hallmarks of a BCSFB as the expression of junctional proteins and formation of tight junctions, a high electrical resistance and minimal levels of macromolecular flux when grown on transwell filters. Importantly, when challenged with the zoonotic pathogen Streptococcus suis or the human pathogenic bacterium Neisseria meningitidis the HIBCPP show polar bacterial invasion only from the physiologically relevant basolateral side. Meningococcal invasion is attenuated by the presence of a capsule and translocated N. meningitidis form microcolonies on the apical side of HIBCPP opposite of sites of entry. As a functionally relevant human model of the BCSFB the HIBCPP offer a wide range of options for analysis of disease-related mechanisms at the choroid plexus epithelium, especially involving human pathogens.

Dexamethasone prevents alteration of tight junction-associated proteins and barrier function in porcine choroid plexus epithelial cells after infection with Streptococcus suis in vitro

Apart from antibiotic treatment in bacterial meningitis supportive therapy including dexamethasone is widely used. In investigations on the pathogenesis of bacterial meningitis we previously demonstrated that Streptococcus suis (S. suis), a relevant cause of bacterial meningitis in pigs and humans, affects porcine choroid plexus epithelial cell (PCPEC) barrier function. The choroid plexus epithelium constitutes the structural basis of the blood-CSF barrier. Now, we investigated the role of tight junction proteins and the actin cytoskeleton of PCPEC in correlation to barrier function after S. suis infection and analyzed the influence of dexamethasone. S. suis caused massive rearrangement of the tight junction proteins ZO-1, occludin and claudin-1, caused loss of actin at the apical cell pole and induced basolateral stress fiber formation. Moreover, tight junctions were shifted from the Triton X insoluble to the Triton X soluble fraction, and additionally occludin was dephosphorylated and degraded. Infection with S. suis leads to an inflammatory response exemplified by the induction of tumor necrosis factor (TNF) alpha and matrix metalloproteinase (MMP)-3 gene activation, which correlated with phosphorylation of extracellular signal regulated kinases (ERKs). Importantly, dexamethasone significantly prevented S.suis-induced protein and morphological tight junction alterations and attenuated ERK activation and MMP-3 expression. It especially improved the barrier function by preventing tight junction protein reorganization and degradation. In the pathogenesis of bacterial meningitis protection of blood-CSF barrier by dexamethasone may prevent the penetration of bacteria and leukocytes into the CSF.

Loss of Acinus Inhibits Oligonucleosomal DNA Fragmentation but Not Chromatin Condensation during Apoptosis

Chromatin condensation and oligonucleosomal DNA fragmentation are the nuclear hallmarks of apoptosis. A proteolytic fragment of the apoptotic chromatin condensation inducer in the nucleus (Acinus), which is generated by caspase cleavage, has been implicated in mediating apoptotic chromatin condensation prior to DNA fragmentation. Acinus is also involved in mRNA splicing and a component of the apoptosis and splicing-associated protein (ASAP) complex. To study the role of Acinus for apoptotic nuclear alterations, we generated stable cell lines in which Acinus isoforms were knocked down by inducible and reversible RNA interference. We show that Acinus is not required for nuclear localization and interaction of the other ASAP subunits SAP18 and RNPS1; however, knockdown of Acinus leads to a reduction in cell growth. Most strikingly, down-regulation of Acinus did not inhibit apoptotic chromatin condensation either in intact cells or in a cell-free system. In contrast, although apoptosis proceeds rapidly, analysis of nuclear DNA from apoptotic Acinus knockdown cells shows inhibition of oligonucleosomal DNA fragmentation. Our results therefore suggest that Acinus is not involved in DNA condensation but rather point to a contribution of Acinus in internucleosomal DNA cleavage during programmed cell death.

The choroid plexus—a multi-role player during infectious diseases of the CNS

The choroid plexus (CP) is the source of cerebrospinal fluid (CSF) production and location of the blood-CSF barrier (BCSFB), which is constituted by the epithelial cells of the CP. Several infectious pathogens including viruses, bacteria, fungi and parasites cross the BCSFB to enter the central nervous system (CNS), ultimately leading to inflammatory infectious diseases like meningitis and meningoencephalitis. The CP responds to this challenge by the production of chemokines and cytokines as well as alterations of the barrier function of the BCSFB. During the course of CNS infectious disease host immune cells enter the CNS, eventually contributing to the cellular damage caused by the disease. Additional complications, which are in certain cases caused by choroid plexitis, can arise due to the response of the CP to the pathogens. In this review we will give an overview on the multiple functions of the CP during brain infections highlighting the CP as a multi-role player during infectious diseases of the CNS. In this context the importance of tools for investigation of these CP functions and a possible suitability of the CP as therapeutic target will be discussed.

Transmigration of polymorphnuclear neutrophils and monocytes through the human blood-cerebrospinal fluid barrier after bacterial infection in vitro

BackgroundBacterial invasion through the blood-cerebrospinal fluid barrier (BCSFB) during bacterial meningitis causes secretion of proinflammatory cytokines/chemokines followed by the recruitment of leukocytes into the CNS. In this study, we analyzed the cellular and molecular mechanisms of polymorphonuclear neutrophil (PMN) and monocyte transepithelial transmigration (TM) across the BCSFB after bacterial infection.MethodsUsing an inverted transwell filter system of human choroid plexus papilloma cells (HIBCPP), we studied leukocyte TM rates, the migration route by immunofluorescence, transmission electron microscopy and focused ion beam/scanning electron microscopy, the secretion of cytokines/chemokines by cytokine bead array and posttranslational modification of the signal regulatory protein (SIRP) α via western blot.ResultsPMNs showed a significantly increased TM across HIBCPP after infection with wild-type Neisseria meningitidis (MC58). In contrast, a significantly decreased monocyte transmigration rate after bacterial infection of HIBCPP could be observed. Interestingly, in co-culture experiments with PMNs and monocytes, TM of monocytes was significantly enhanced. Analysis of paracellular permeability and transepithelial electrical resistance confirmed an intact barrier function during leukocyte TM. With the help of the different imaging techniques we could provide evidence for para- as well as for transcellular migrating leukocytes. Further analysis of secreted cytokines/chemokines showed a distinct pattern after stimulation and transmigration of PMNs and monocytes. Moreover, the transmembrane glycoprotein SIRPα was deglycosylated in monocytes, but not in PMNs, after bacterial infection.ConclusionsOur findings demonstrate that PMNs and monoctyes differentially migrate in a human BCSFB model after bacterial infection. Cytokines and chemokines as well as transmembrane proteins such as SIRPα may be involved in this process.

Latest developments on Streptococcus suis: an emerging zoonotic pathogen: part 2.

First International Workshop on Streptococcus suis, Beijing, China, 12-13 August 2013 The first international workshop on Streptococcus suis, which is an important swine pathogen and emerging zoonotic agent, took place in Beijing, jointly organized by the Faculty of Veterinary Medicine, University of Montreal, Canada and the National Institute for Communicable Disease Control and Prevention, China CDC. The aim of the meeting was to gather together, for the first time, more than 80 researchers working on S. suis, from countries including China, Canada, Japan, The Netherlands, Germany, Thailand, the UK and Vietnam. This article, the first of a two-part report on this First International Workshop, reviews current aspects of the epidemiology and population genomics of S. suis, covers public health concerns and discusses questions about S. suis serotyping and molecular diagnostics.