Matrix metalloproteinase-9 in pneumococcal meningitis: activation via an oxidative pathway

In experimental bacterial meningitis, matrix metalloproteinases (MMPs) and reactive oxygen species (ROS) contribute to brain damage. MMP-9 increases in cerebrospinal fluid (CSF) during bacterial meningitis and is associated with the brain damage that is a consequence of the disease. This study assesses the origin of MMP-9 in bacterial meningitis and how ROS modulate its activity. Rat brain-slice cultures and rat polymorphonuclear cells (PMNs) that had been challenged with capsule-deficient heat-inactivated Streptococcus pneumoniae R6 (hiR6) released MMP-9. Coincubation with either catalase, with the myeloperoxidase inhibitor azide, or with the hypochlorous acid scavenger methionine almost completely prevented activation, but not the release, of MMP-9, in supernatants of human PMNs stimulated with hiR6. Thus, in bacterial meningitis, both brain-resident cells and invading PMNs may act as sources of MMP-9, and stimulated PMNs may activate MMP-9 via an ROS-dependent pathway. MMP-9 activation by ROS may represent a target for therapeutic intervention in bacterial meningitis.

Systemsklerose

Die Systemsklerose ist charakterisiert durch autoimmune Phänomene und eine progressive Fibrose. Klinische Charakteristika sind neben der Sklerodermie ein ausgeprägtes Raynaud-Phänomen und Abnormitäten der Nagelfalzkapillaren, ferner pulmonale, renale, kardiale und intestinale Veränderungen. In der Regel wird eine diffuse von einer limitierten Form unterschieden. Die diffuse Form führt zu einer Sklerose des gesamten Integumentes, ist assoziiert mit dem Auto-antikörper Scl-70 und zeichnet sich durch eine rasche Krankheitsprogression aus. Die limitierte Form betrifft die distalen Extremitäten und das Gesicht, ist assoziiert mit dem centromer Autoantikörper und führt schleichend, über mehrere Jahre zu viszeralen Problemen. Neue therapeutische Möglichkeiten können zu einer besseren Lebensqualität verhelfen und verpflichten zu einem systematischen Krankheitsmonitoring.

CD69 upregulation on T cells as an in vitro marker for delayed-type drug hypersensitivity

BACKGROUND: T cells play a key role in delayed-type drug hypersensitivity reactions. Their reactivity can be assessed by their proliferation in response to the drug in the lymphocyte transformation test (LTT). However, the LTT imposes limitations in terms of practicability, and an alternative method that is easier to implement than the LTT would be desirable. METHODS: Four months to 12 years after acute drug hypersensitivity reactions, CD69 upregulation on T cells of 15 patients and five healthy controls was analyzed by flow cytometry. RESULTS: All 15 LTT-positive patients showed a significant increase of CD69 expression on T cells after 48 h of drug-stimulation exclusively with the drugs incriminated in drug-hypersensitivities. A stimulation index of 2 as cut-off value allowed discrimination between nonreactive and reactive T cells in LTT and CD69 upregulation. T cells (0.5-3%) showed CD69 up-regulation. The reactive cell population consisted of a minority of truly drug reactive T cells secreting cytokines and a higher number of bystander T cells activated by IL-2 and possibly other cytokines. CONCLUSIONS: CD69 upregulation was observed after 2 days in all patients with a positive LTT after 6 days, thus appearing to be a promising tool to identify drug-reactive T cells in the peripheral blood of patients with drug-hypersensitivity reactions.

Severe hepatotoxicity following ingestion of Herbalife nutritional supplements contaminated with Bacillus subtilis

BACKGROUND/AIMS: Nutritional supplements are widely used. Recently, liver injury after consumption of Herbalife preparations was reported but the underlying pathogenesis remained cryptic. METHODS: Two patients presented with cholestatic hepatitis and pruritus, and cirrhosis, respectively. Viral, alcoholic, metabolic, autoimmune, neoplastic, vascular liver diseases and synthetic drugs as the precipitating causes of liver injury were excluded. However, both patients reported long-term consumption of Herbalife products. All Herbalife products were tested for contamination with drugs, pesticides, heavy metals, and softeners, and examined for microbial contamination according to standard laboratory procedures. Bacteria isolated from the samples were identified as Bacillus subtilis by sequencing the 16S rRNA and gyrB genes. RESULTS: Causality between consumption of Herbalife products and disease according to CIOMS was scored "probable" in both cases. Histology showed cholestatic and lobular/portal hepatitis with cirrhosis in one patient, and biliary fibrosis with ductopenia in the other. No contamination with chemicals or heavy metals was detected, and immunological testing showed no drug hypersensitivity. However, samples of Herbalife products ingested by both patients showed growth of Bacillus subtilis of which culture supernatants showed dose- and time-dependent hepatotoxicity. CONCLUSIONS: Two novel incidents of severe hepatic injury following intake of Herbalife products contaminated with Bacillus subtilis emphasize its potential hepatotoxicity.

Functional regeneration of intraspinal connections in a new in vitro model

Abstract—Regeneration in the adult mammalian spinal cord is limited due to intrinsic properties of mature neurons and a hostile environment, mainly provided by central nervous system myelin and reactive astrocytes. Recent results indicate that propriospinal connections are a promising target for intervention to improve functional recovery. To study this functional regeneration in vitro we developed a model consisting of two organotypic spinal cord slices placed adjacently on multi-electrode arrays. The electrodes allow us to record the spontaneously occurring neuronal activity, which is often organized in network bursts. Within a few days in vitro (DIV), these bursts become synchronized between the two slices due to the formation of axonal connections. We cut them with a scalpel at different time points in vitro and record the neuronal activity 3 weeks later. The functional recovery ability was assessed by calculating the percentage of synchronized bursts between the two slices. We found that cultures lesioned at a young age (7–9 DIV) retained the high regeneration ability of embryonic tissue. However, cultures lesioned at older ages (>19 DIV) displayed a distinct reduction of synchronized activity. This reduction was not accompanied by an inability for axons to cross the lesion site. We show that functional regeneration in these old cultures can be improved by increasing the intracellular cAMP level with Rolipram or by placing a young slice next to an old one directly after the lesion. We conclude that co-cultures of two spinal cord slices are an appropriate model to study functional regeneration of intraspinal connections.

Delay discounting task in pigs reveals response strategies related to dopamine metabolite.

We developed a novel delay discounting task to investigate outcome impulsivity in pigs. As impulsivity can affect aggression, and might also relate to proactive and reactive coping styles, eight proactive (HR) and eight reactive (LR) pigs identified in a manual restraint test ("Backtest", after Bolhuis et al., 2003) were weaned and mixed in four pens of four unfamiliar pigs, so that each pen had two HR and two LR pigs, and aggression was scored in the 9h after mixing. In the delay discounting task, each pig chose between two levers, one always delivering a small immediate reward, the other a large delayed reward with daily increasing delays, impulsive individuals being the ones discounting the value of the large reward quicker. Two novel strategies emerged: some pigs gradually switched their preference towards the small reward ('Switchers') as predicted, but others persistently preferred the large reward until they stopped making choices ('Omitters'). Outcome impulsivity itself was unrelated to these strategies, to urinary serotonin metabolite (5-HIAA) or dopamine metabolite (HVA) levels, aggression at weaning, or coping style. However, HVA was relatively higher in Omitters than Switchers, and positively correlated with behavioural measures of indecisiveness and frustration during choosing. The delay discounting task thus revealed two response strategies that seemed to be related to the activity of the dopamine system and might indicate a difference in execution, rather than outcome, impulsivity.

Multiple greenhouse-gas feedbacks from the land biosphere under future climate change scenarios

Atmospheric concentrations of the three important greenhouse gases (GHGs) CO2, CH4 and N2O are mediated by processes in the terrestrial biosphere that are sensitive to climate and CO2. This leads to feedbacks between climate and land and has contributed to the sharp rise in atmospheric GHG concentrations since pre-industrial times. Here, we apply a process-based model to reproduce the historical atmospheric N2O and CH4 budgets within their uncertainties and apply future scenarios for climate, land-use change and reactive nitrogen (Nr) inputs to investigate future GHG emissions and their feedbacks with climate in a consistent and comprehensive framework1. Results suggest that in a business-as-usual scenario, terrestrial N2O and CH4 emissions increase by 80 and 45%, respectively, and the land becomes a net source of C by AD 2100. N2O and CH4 feedbacks imply an additional warming of 0.4–0.5 °C by AD 2300; on top of 0.8–1.0 °C caused by terrestrial carbon cycle and Albedo feedbacks. The land biosphere represents an increasingly positive feedback to anthropogenic climate change and amplifies equilibrium climate sensitivity by 22–27%. Strong mitigation limits the increase of terrestrial GHG emissions and prevents the land biosphere from acting as an increasingly strong amplifier to anthropogenic climate change.

Genetic Analyses Reveal a Role for Vitamin D Insufficiency in HCV-Associated Hepatocellular Carcinoma Development

Background Vitamin D insufficiency has been associated with the occurrence of various types of cancer, but causal relationships remain elusive. We therefore aimed to determine the relationship between genetic determinants of vitamin D serum levels and the risk of developing hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). Methodology/Principal Findings Associations between CYP2R1, GC, and DHCR7 genotypes that are determinants of reduced 25-hydroxyvitamin D (25[OH]D3) serum levels and the risk of HCV-related HCC development were investigated for 1279 chronic hepatitis C patients with HCC and 4325 without HCC, respectively. The well-known associations between CYP2R1 (rs1993116, rs10741657), GC (rs2282679), and DHCR7 (rs7944926, rs12785878) genotypes and 25(OH)D3 serum levels were also apparent in patients with chronic hepatitis C. The same genotypes of these single nucleotide polymorphisms (SNPs) that are associated with reduced 25(OH)D3 serum levels were found to be associated with HCV-related HCC (P = 0.07 [OR = 1.13, 95% CI = 0.99–1.28] for CYP2R1, P = 0.007 [OR = 1.56, 95% CI = 1.12–2.15] for GC, P = 0.003 [OR = 1.42, 95% CI = 1.13–1.78] for DHCR7; ORs for risk genotypes). In contrast, no association between these genetic variations and liver fibrosis progression rate (P>0.2 for each SNP) or outcome of standard therapy with pegylated interferon-α and ribavirin (P>0.2 for each SNP) was observed, suggesting a specific influence of the genetic determinants of 25(OH)D3 serum levels on hepatocarcinogenesis. Conclusions/Significance Our data suggest a relatively weak but functionally relevant role for vitamin D in the prevention of HCV-related hepatocarcinogenesis.

Prevalence of paunch calf syndrome carriers in Italian Romagnola cattle.

The term 'paunch calf syndrome' encompasses the multi-organic lethal developmental dysplasia reported in the Romagnola breed of cattle and is characterised by facial deformities, an enlarged and floating abdomen containing considerable abdominal effusion, and hepatic fibrosis. Paunch calf syndrome is caused by a missense mutation in the KDM2B gene (c.2503G>A) that is thought to lead to an amino acid exchange (p.D835N). In this study, the prevalence of carriers of the mutant KDM2B allele (and thus the frequency of the allele) was assessed in selected subpopulations of Romagnola cattle. The prevalence of carriers within top-ranked Romagnola sires over the years 2007-2012 was 29.3% (allele frequency 14.6%). In young bull calves, 30.9% were carriers with an allele frequency of 15.4%.

Evaluation of genome-wide loci of iron metabolism in hereditary hemochromatosis identifies PCSK7 as a host risk factor of liver cirrhosis

Genome-wide association studies (GWAS) have revealed genetic determinants of iron metabolism, but correlation of these with clinical phenotypes is pending. Homozygosity for HFE C282Y is the predominant genetic risk factor for hereditary hemochromatosis (HH) and may cause liver cirrhosis. However, this genotype has a low penetrance. Thus, detection of yet unknown genetic markers that identify patients at risk of developing severe liver disease is necessary for better prevention. Genetic loci associated with iron metabolism (TF, TMPRSS6, PCSK7, TFR2 and Chr2p14) in recent GWAS and liver fibrosis (PNPLA3) in recent meta-analysis were analyzed for association with either liver cirrhosis or advanced fibrosis in 148 German HFE C282Y homozygotes. Replication of associations was sought in additional 499 Austrian/Swiss and 112 HFE C282Y homozygotes from Sweden. Only variant rs236918 in the PCSK7 gene (proprotein convertase subtilisin/kexin type 7) was associated with cirrhosis or advanced fibrosis (P = 1.02 × 10(-5)) in the German cohort with genotypic odds ratios of 3.56 (95% CI 1.29-9.77) for CG heterozygotes and 5.38 (95% CI 2.39-12.10) for C allele carriers. Association between rs236918 and cirrhosis was confirmed in Austrian/Swiss HFE C282Y homozygotes (P = 0.014; ORallelic = 1.82 (95% CI 1.12-2.95) but not in Swedish patients. Post hoc combined analyses of German/Swiss/Austrian patients with available liver histology (N = 244, P = 0.00014, ORallelic = 2.84) and of males only (N = 431, P = 2.17 × 10(-5), ORallelic = 2.54) were consistent with the premier finding. Association between rs236918 and cirrhosis was not confirmed in alcoholic cirrhotics, suggesting specificity of this genetic risk factor for HH. PCSK7 variant rs236918 is a risk factor for cirrhosis in HH patients homozygous for the HFE C282Y mutation.

The role of adenosine in pulmonary angiogenesis

Angiogenesis is a feature of chronic lung diseases such as asthma and pulmonary fibrosis; however, the pathways controlling pathological angiogenesis during lung disease are not completely understood. Adenosine is a signaling nucleoside that accumulates as a result of tissue hypoxia and damage. Adenosine has been implicated in the exacerbation of chronic lung disease and in the regulation of angiogenesis; however, the relationship between these factors has not been investigated. The work presented in this dissertation utilized adenosine deaminase (ADA)-deficient mice to determine whether chronic elevations of adenosine in vivo result in pulmonary angiogenesis, and to identify factors that could potentially mediate this process. Results demonstrate that there is substantial angiogenesis in the tracheas of ADA-deficient mice in association with adenosine elevations. Replacement enzyme therapy with pegylated ADA resulted in a lowering of adenosine levels and reversal of tracheal angiogenesis, indicating that the increases in vessel number are dependent on adenosine elevations. Levels of the ELR+ angiogenic chemokine CXCL1 were found to be elevated in an adenosine-dependent manner in the lungs of ADA-deficient mice. Neutralization of CXCL1 and its putative receptor, CXCR2, in ADA-deficient lung lysates resulted in the inhibition of angiogenic activity suggesting that CXCL1 signaling through the CXCR2 receptor is responsible for mediating the observed increases in angiogenesis. Taken together, these findings suggest that adenosine plays an important role, via CXCL1, in the induction of pulmonary angiogenesis and may therefore represent an important therapeutic target for the treatment of pathological angiogenesis. ^

Deciphering the C-Type Lectin Receptor Signaling Pathway in Macrophages in Response to Candida albicans

Candida albicans causes opportunistic fungal infections in humans and is a significant cause of mortality and morbidity in immune-compromised individuals. Dectin-2, a C-type lectin receptor, is required for recognition of C. albicans by innate immune cells and is required for initiation of the anti-fungal immune response. We set out to identify components of the intracellular signaling cascade downstream of Dectin-2 activation in macrophages and to understand their importance in mediating the immune response to C. albicans in vivo. Using macrophages derived from Phospholipase-C-gamma 1 and 2 (PLCγ1and PLCγ2) knockout mice, we demonstrate that PLCγ2, but not PLCγ1, is required for activation of NF-κB and MAPK signaling pathways after C. albicans stimulation, resulting in impaired production of pro-inflammatory cytokines and reactive oxygen species. PLCγ2-deficient mice are highly susceptible to infections with C. albicans, indicating the importance of this pathway to the anti-fungal immune response. TAK1 and TRAF6 are critical nodes in NF-κB and MAPK activation downstream of immune surveillance and may be critical to the signaling cascade initiated by C-type lectin receptors in response to C. albicans. Macrophages derived from both TAK1 and TRAF6-deficient mice were unable to activate NF-κB and MAPK and consequently failed to produce inflammatory cytokines characteristic of the response to C. albicans. In this work we have identified PLCγ2, TAK1 and TRAF6 as components of a signaling cascade downstream of C. albicans recognition by C-type lectin receptors and as critical mediators of the anti-fungal immune response. A mechanistic understanding of the host immune response to C. albicans is important for the development of anti-fungal therapeutics and in understanding risk-factors determining susceptibility to C. albicans infection.

BIOCHEMICAL CHARACTERIZATION OF BINDING PARTNERS OF TWO HSP70 CO-CHAPERONES IN SACCHAROMYCES CEREVISIAE

Cells are exposed to a variety of environmental and physiological changes including temperature, pH and nutrient availability. These changes cause stress to cells, which results in protein misfolding and altered cellular protein homeostasis. How proteins fold into their three-dimensional functional structure is a fundamental biological process with important relevance to human health. Misfolded and aggregated proteins are linked to multiple neurodegenerative diseases, cardiovascular disease and cystic fibrosis. To combat proteotoxic stress, cells deploy an array of molecular chaperones that assist in the repair or removal of misfolded proteins. Hsp70, an evolutionarily conserved molecular chaperone, promotes protein folding and helps maintain them in a functional state. Requisite co-chaperones, including nucleotide exchange factors (NEFs) strictly regulate and serve to recruit Hsp70 to distinct cellular processes or locations. In yeast and human cells, three structurally non-related cytosolic NEFs are present: Sse1 (Hsp110), Fes1 (HspBP1) and Snl1 (Bag-1). Snl1 is unique among the cytosolic NEFs as it is localized at the ER membrane with its Hsp70 binding (BAG) domain exposed to the cytosol. I discovered that Snl1 distinctly interacts with assembled ribosomes and several lines of evidence indicate that this interaction is both independent of and concurrent with binding to Hsp70 and is not dependent on membrane localization. The ribosome-binding site is identified as a short lysine-rich motif within the amino terminus of the Snl1 BAG domain distinct from the Hsp70 interaction region. In addition, I demonstrate ribosome association with the Snl1 homolog in the pathogenic fungus, Candida albicans and localize this putative NEF to a perinuclear/ER membrane, suggesting functional conservation in fungal BAG domain-containing proteins. As a first step in determining specific domain architecture in fungal BAG proteins, I present the preliminary steps of protein purification and analysis of the minimal Hsp70 binding region in in both S.cerevisiae and C. albicans Snl1. Contrary to previous in vitro evidence which showed the Fes1 NEF to interact with both cytosolic Hsp70s, Ssa and Ssb, Fes1 is shown to interact specifically with Ssa when expressed under normal cellular conditions in S. cerevisiae. This is the first reported evidence of Hsp70 binding selectivity for a cytosolic NEF, and suggests a possible mechanism to achieve specificity in Hsp70-dependent functions. Taken together, the work presented in this dissertation highlights the striking divergence among Hsp70 co-chaperones in selecting binding partners, which may correlate with their specific roles in the cell.