The role of cell death in the pathogenesis of autoimmune disease: HMGB1 and microparticles as intercellular mediators of inflammation

Cell death is critical to normal homeostasis, although this process, when increased aberrantly, can lead to the production of pro-inflammatory mediators promoting autoimmunity. Two novel intercellular mediators of inflammation generated during cell death are high mobility group box 1 (HMGB1) protein and microparticles (MPs). HMGB1 is a nuclear protein that functions in transcription when inside the nucleus but takes on pro-inflammatory properties when released during cell death. Microparticles are small, membrane-bound structures that extrude from cells when they die and contain cell surface proteins and nuclear material from their parent cells. MPs circulate widely throughout the vasculature and mediate long-distance communication between cells. Both MPs and HMGB1 have been implicated in the pathogenesis of a broad spectrum of inflammatory diseases, including the prototypic autoimmune conditions systemic lupus erythematosus and rheumatoid arthritis. Given their range of activity and association with active disease, both structures may prove to be targets for effective therapy in these and other disorders

Hypoglycemia and the Origin of Hypoxia-Induced Reduction in Human Fetal Growth

The most well known reproductive consequence of residence at high altitude (HA >2700 m) is reduction in fetal growth. Reduced fetoplacental oxygenation is an underlying cause of pregnancy pathologies, including intrauterine growth restriction and preeclampsia, which are more common at HA. Therefore, altitude is a natural experimental model to study the etiology of pregnancy pathophysiologies. We have shown that the proximate cause of decreased fetal growth is not reduced oxygen availability, delivery, or consumption. We therefore asked whether glucose, the primary substrate for fetal growth, might be decreased and/or whether altered fetoplacental glucose metabolism might account for reduced fetal growth at HA.Doppler and ultrasound were used to measure maternal uterine and fetal umbilical blood flows in 69 and 58 residents of 400 vs 3600 m. Arterial and venous blood samples from mother and fetus were collected at elective cesarean delivery and analyzed for glucose, lactate and insulin. Maternal delivery and fetal uptakes for oxygen and glucose were calculated.The maternal arterial – venous glucose concentration difference was greater at HA. However, umbilical venous and arterial glucose concentrations were markedly decreased, resulting in lower glucose delivery at 3600 m. Fetal glucose consumption was reduced by >28%, but strongly correlated with glucose delivery, highlighting the relevance of glucose concentration to fetal uptake. At altitude, fetal lactate levels were increased, insulin concentrations decreased, and the expression of GLUT1 glucose transporter protein in the placental basal membrane was reduced.Our results support that preferential anaerobic consumption of glucose by the placenta at high altitude spares oxygen for fetal use, but limits glucose availability for fetal growth. Thus reduced fetal growth at high altitude is associated with fetal hypoglycemia, hypoinsulinemia and a trend towards lactacidemia. Our data support that placentally-mediated reduction in glucose transport is an initiating factor for reduced fetal growth under conditions of chronic hypoxemia

Paclitaxel alters the expression and specific activity of deoxycytidine kinase and cytidine deaminase in non-small cell lung cancer cell lines

<p>Abstract</p> <p>Background</p> <p>We observed that paclitaxel altered the pharmacokinetic properties of gemcitabine in patients with non-small cell lung cancer (NSCLC) and limited the accumulation of gemcitabine and its metabolites in various primary and immortalized human cells. Therefore, we classified the drug-drug interaction and the effects of paclitaxel on deoxycytidine kinase (dCK) and cytidine deaminase (CDA) in three NSCLC cell lines. These enzymes are responsible for the metabolism of gemcitabine to its deaminated metabolite dFdU (80% of the parent drug) and the phosphorylated metabolites dFdCMP, dFdCDP and dFdCTP. These metabolites appear to relate to sensitivity and tolerability of gemcitabine based on previous animal and laboratory studies.</p> <p>Methods</p> <p>Three immortalized human cells representative of the most common histological subtypes identified in patients with advanced NSCLC were exposed to the individual drugs or combinations to complete a multiple drug effect analysis. These same cell lines were exposed to vehicle-control or paclitaxel and the mRNA levels, protein expression and specific activity of dCK and CDA were compared. Comparisons were made using a two-tailed paired t-test or analysis of variance with a P value of < 0.05 considered significant.</p> <p>Results</p> <p>The multiple drug effect analysis indicated synergy for H460, H520 and H838 cells independent of sequence. As anticipated, paclitaxel-gemcitabine increased the number of G2/M cells, whereas gemcitabine-paclitaxel increased the number of G0/G1 or S cells. Paclitaxel significantly decreased dCK and CDA mRNA levels in H460 and H520 cells (40% to 60%, P < 0.05) and lowered dCK protein (24% to 56%, P < 0.05) without affecting CDA protein. However, paclitaxel increased both dCK (10% to 50%) and CDA (75% to 153%) activity (P < 0.05). Paclitaxel caused substantial declines in the accumulation of the deaminated and phosphorylated metabolites in H520 cells (P < 0.05); the metabolites were not measurable in the remaining two cell lines. The ratio of dCK to CDA mRNA levels corresponded to the combination index (CI) estimated for sequential paclitaxel-gemcitabine.</p> <p>Conclusion</p> <p>In summary, paclitaxel altered the mRNA levels and specific activity of dCK and CDA and these effects could be dependent on histological subtype. More cell and animal studies are needed to further characterize the relationship between mRNA levels and the overall drug-drug interaction and the potential to use histological subtype as a predictive factor in the selection of an appropriate anticancer drug regimen.</p

Detection of antibodies directed at M. hyorhinis p37 in the serum of men with newly diagnosed prostate cancer

<p>Abstract</p> <p>Background</p> <p>Recent epidemiologic, genetic, and molecular studies suggest infection and inflammation initiate certain cancers, including cancers of the prostate. Over the past several years, our group has been studying how mycoplasmas could possibly initiate and propagate cancers of the prostate. Specifically, <it>Mycoplasma hyorhinis </it>encoded protein p37 was found to promote invasion of prostate cancer cells and cause changes in growth, morphology and gene expression of these cells to a more aggressive phenotype. Moreover, we found that chronic exposure of benign human prostate cells to <it>M. hyorhinis </it>resulted in significant phenotypic and karyotypic changes that ultimately resulted in the malignant transformation of the benign cells. In this study, we set out to investigate another potential link between mycoplasma and human prostate cancer.</p> <p>Methods</p> <p>We report the incidence of men with prostate cancer and benign prostatic hyperplasia (BPH) being seropositive for <it>M. hyorhinis</it>. Antibodies to <it>M. hyorhinis </it>were surveyed by a novel indirect enzyme-linked immunosorbent assay (ELISA) in serum samples collected from men presenting to an outpatient Urology clinic for BPH (N = 105) or prostate cancer (N = 114) from 2006-2009.</p> <p>Results</p> <p>A seropositive rate of 36% in men with BPH and 52% in men with prostate cancer was reported, thus leading us to speculate a possible connection between <it>M. hyorhinis </it>exposure with prostate cancer.</p> <p>Conclusions</p> <p>These results further support a potential exacerbating role for mycoplasma in the development of prostate cancer.</p

Marking Embryonic Stem Cells with a 2A Self-Cleaving Peptide: A NKX2-5 Emerald GFP BAC Reporter

Fluorescent reporters are useful for assaying gene expression in living cells and for identifying and isolating pure cell populations from heterogeneous cultures, including embryonic stem (ES) cells. Multiple fluorophores and genetic selection markers exist; however, a system for creating reporter constructs that preserve the regulatory sequences near a gene's native ATG start site has not been widely available.Here, we describe a series of modular marker plasmids containing independent reporter, bacterial selection, and eukaryotic selection components, compatible with both Gateway recombination and lambda prophage bacterial artificial chromosome (BAC) recombineering techniques. A 2A self-cleaving peptide links the reporter to the native open reading frame. We use an emerald GFP marker cassette to create a human BAC reporter and ES cell reporter line for the early cardiac marker NKX2-5. NKX2-5 expression was detected in differentiating mouse ES cells and ES cell-derived mice.Our results describe a NKX2-5 ES cell reporter line for studying early events in cardiomyocyte formation. The results also demonstrate that our modular marker plasmids could be used for generating reporters from unmodified BACs, potentially as part of an ES cell reporter library

GWAS for discovery and replication of genetic loci associated with sudden cardiac arrest in patients with coronary artery disease

<p>Abstract</p> <p>Background</p> <p>Epidemiologic evidence suggests a heritable component to risk for sudden cardiac arrest independent of risk for myocardial infarction. Recent candidate gene association studies for community sudden cardiac arrests have focused on a limited number of biological pathways and yielded conflicting results. We sought to identify novel gene associations for sudden cardiac arrest in patients with coronary artery disease by performing a genome-wide association study.</p> <p>Methods</p> <p>Tagging SNPs (n = 338,328) spanning the genome were typed in a case-control study comparing 89 patients with coronary artery disease and sudden cardiac arrest due to ventricular tachycardia or ventricular fibrillation to 520 healthy controls.</p> <p>Results</p> <p>Fourteen SNPs including 7 SNPs among 7 genes (ACYP2, AP1G2, ESR1, DGES2, GRIA1, KCTD1, ZNF385B) were associated with sudden cardiac arrest (all p < 1.30 × 10^-7), following Bonferroni correction and adjustment for population substructure, age, and sex; genetic variation in ESR1 (p = 2.62 × 10^-8; Odds Ratio [OR] = 1.43, 95% confidence interval [CI]:1.277, 1.596) has previously been established as a risk factor for cardiovascular disease. In tandem, the role of 9 genes for monogenic long QT syndrome (LQT1-9) was assessed, yielding evidence of association with CACNA1C (LQT8; p = 3.09 × 10^-4; OR = 1.18, 95% CI:1.079, 1.290). We also assessed 4 recently published gene associations for sudden cardiac arrest, validating NOS1AP (p = 4.50 × 10^-2, OR = 1.15, 95% CI:1.003, 1.326), CSMD2 (p = 6.6 × 10^-3, OR = 2.27, 95% CI:1.681, 2.859), and AGTR1 (p = 3.00 × 10^-3, OR = 1.13, 95% CI:1.042, 1.215).</p> <p>Conclusion</p> <p>We demonstrate 11 gene associations for sudden cardiac arrest due to ventricular tachycardia/ventricular fibrillation in patients with coronary artery disease. Validation studies in independent cohorts and functional studies are required to confirm these associations.</p

In Vitro Characterization of a Nineteenth-Century Therapy for Smallpox

In the nineteenth century, smallpox ravaged through the United States and Canada. At this time, a botanical preparation, derived from the carnivorous plant Sarracenia purpurea, was proclaimed as being a successful therapy for smallpox infections. The work described characterizes the antipoxvirus activity associated with this botanical extract against vaccinia virus, monkeypox virus and variola virus, the causative agent of smallpox. Our work demonstrates the in vitro characterization of Sarracenia purpurea as the first effective inhibitor of poxvirus replication at the level of early viral transcription. With the renewed threat of poxvirus-related infections, our results indicate Sarracenia purpurea may act as another defensive measure against Orthopoxvirus infections

Bilateral inhibition of HAUSP deubiquitinase by a viral interferon regulatory factor protein

Herpesvirus-associated ubiquitin specific protease (HAUSP) regulates the stability of p53 and MDM2, implicating HAUSP as a therapeutic target for tuning p53-mediated anti-tumor activity. Here, we report the structural analysis of HAUSP with Kaposi’s sarcoma-associated herpesvirus vIRF4 and the discovery of two vIRF4-derived peptides, vif1 and vif2, as potent and selective HAUSP antagonists. This analysis reveals a bilateral belt-type interaction resulting in inhibition of HAUSP. The vif1 peptide binds the HAUSP TRAF domain, competitively blocking substrate binding, while the vif2 peptide binds both the HAUSP TRAF and catalytic domains, robustly suppressing its deubiquitination activity. Consequently, peptide treatments comprehensively blocked HAUSP, leading to p53-dependent cell cycle arrest and apoptosis in culture and tumor regression in xenograft mouse model. Thus, the virus has developed a unique molecular strategy to target the HAUSP-MDM2-p53 pathway, and these virus-derived short peptides represent biologically active HAUSP antagonists

Persistent Exposure to Mycoplasma Induces Malignant Transformation of Human Prostate Cells

Recent epidemiologic, genetic, and molecular studies suggest infection and inflammation initiate certain cancers, including those of the prostate. The American Cancer Society, estimates that approximately 20% of all worldwide cancers are caused by infection. Mycoplasma, a genus of bacteria that lack a cell wall, are among the few prokaryotes that can grow in close relationship with mammalian cells, often without any apparent pathology, for extended periods of time. In this study, the capacity of Mycoplasma genitalium, a prevalent sexually transmitted infection, and Mycoplasma hyorhinis, a mycoplasma found at unusually high frequency among patients with AIDS, to induce a malignant phenotype in benign human prostate cells (BPH-1) was evaluated using a series of in vitro and in vivo assays. After 19 weeks of culture, infected BPH-1 cells achieved anchorage-independent growth and increased migration and invasion. Malignant transformation of infected BPH-1 cells was confirmed by the formation of xenograft tumors in athymic mice. Associated with these changes was an increase in karyotypic entropy, evident by the accumulation of chromosomal aberrations and polysomy. This is the first report describing the capacity of M. genitalium or M. hyorhinis infection to lead to the malignant transformation of benign human epithelial cells and may serve as a model to further study the relationship between prostatitis and prostatic carcinogenesis