Genes coding for enterotoxins and verotoxins in porcine Escherichia coli strains belonging to different O:K:H serotypes: Relationship with toxic phenotypes

Seventy-four E. coli strains isolated from piglets with diarrhea or edema disease in Spain were serotyped and examined for production of heat-labile (LT) and heat-stable (ST) enterotoxins (LT-I, LT-II, STaH, STaP, and STb) and verotoxins (VT1, VT2, and VT2v VTe) by phenotypic (Vero cell assay and infant mouse test) and genotypic (colony hybridization and PCR) methods. In general, an excellent correlation was found between the results obtained with a PCR approach and those determined with biological assays. DNA probes used in the hybridization also showed a very good agreement with phenotypic results, with the exception of a VT1 probe that initially produced 10 false-positive reactions. The gene coding for STb (58 strains) was the most prevalent gene detected by PCR, followed by those coding for STa (46 strains), LT (19 strains), VT2v (11 strains), and VT1 (1 strain). Apparently, in Spain three seropathotypes are predominant: (i) 0149:K91:H10 K88(+) LT-I+ STb+ (ii) 0141:K85ab:H- P987(+) STaP+, and (iii) 0138:K81:H14 or H-STaP+ VT2v(+). We conclude that PCR is a fast, specific, and practical method for the identification of enterotoxin and VT genes in clinical and epidemiological studies.35112958296

Combinatorial Effect of Non-Steroidal Anti-inflammatory Drugs and NF-κB Inhibitors in Ovarian Cancer Therapy

Several epidemiological studies have correlated the use of non-steroidal anti-inflammatory drugs (NSAID) with reduced risk of ovarian cancer, the most lethal gynecological cancer, diagnosed usually in late stages of the disease. We have previously established that the pro-apoptotic cytokine melanoma differentiation associated gene-7/Interleukin-24 (mda-7/IL-24) is a crucial mediator of NSAID-induced apoptosis in prostate, breast, renal and stomach cancer cells. In this report we evaluated various structurally different NSAIDs for their efficacies to induce apoptosis and mda-7/IL-24 expression in ovarian cancer cells. While several NSAIDs induced apoptosis, Sulindac Sulfide and Diclofenac most potently induced apoptosis and reduced tumor growth. A combination of these agents results in a synergistic effect. Furthermore, mda-7/IL-24 induction by NSAIDs is essential for programmed cell death, since inhibition of mda-7/IL-24 by small interfering RNA abrogates apoptosis. mda-7/IL-24 activation leads to upregulation of growth arrest and DNA damage inducible (GADD) 45 α and γ and JNK activation. The NF-κB family of transcription factors has been implicated in ovarian cancer development. We previously established NF-κB/IκB signaling as an essential step for cell survival in cancer cells and hypothesized that targeting NF-κB could potentiate NSAID-mediated apoptosis induction in ovarian cancer cells. Indeed, combining NSAID treatment with NF-κB inhibitors led to enhanced apoptosis induction. Our results indicate that inhibition of NF-κB in combination with activation of mda-7/IL-24 expression may lead to a new combinatorial therapy for ovarian cancer

Gadd45α activity is the principal effector of Shigella mitochondria-dependent epithelial cell death in vitro and ex vivo

Modulation of death is a pathogen strategy to establish residence and promote survival in host cells and tissues. Shigella spp. are human pathogens that invade colonic mucosa, where they provoke lesions caused by their ability to manipulate the host cell responses. Shigella spp. induce various types of cell death in different cell populations. However, they are equally able to protect host cells from death. Here, we have investigated on the molecular mechanisms and cell effectors governing the balance between survival and death in epithelial cells infected with Shigella. To explore these aspects, we have exploited both, the HeLa cell invasion assay and a novel ex vivo human colon organ culture model of infection that mimics natural conditions of shigellosis. Our results definitely show that Shigella induces a rapid intrinsic apoptosis of infected cells, via mitochondrial depolarization and the ensuing caspase-9 activation. Moreover, for the first time we identify the eukaryotic stress-response factor growth arrest and DNA damage 45α as a key player in the induction of the apoptotic process elicited by Shigella in epithelial cells, revealing an unexplored role of this molecule in the course of infections sustained by invasive pathogens

Factors associated with delayed diagnosis of tuberculosis in hospitalized patients in a high TB and HIV burden setting: a cross-sectional study

<p>Abstract</p> <p>Background</p> <p>The most essential components of TB control are early diagnosis and adequate treatment. Delay in the diagnosis and treatment of tuberculosis may result in more extensive disease and more complications, increase severity of the disease and is associated with higher risk of mortality. The purpose of this study was to identify factors associated with delayed diagnosis of TB in hospitalized patients.</p> <p>Methods</p> <p>We conducted a cross-sectional study in a general, tertiary care, university-affiliated hospital. Adult patients with TB that were hospitalized were identified retrospectively, and risk factors for delayed diagnosis were collected.</p> <p>Results</p> <p>The median delay until diagnosis was 6 days (IQR: 2-12 days). One hundred and sixty six (54.4%) patients were diagnosed ≤ 6 days, and 139 (45.6%) > 6 days after admission. The main factors associated with diagnostic delay (> 6 days) were extra-pulmonary TB and negative sputum smear.</p> <p>Conclusions</p> <p>Although hospitalization permits a rapid management of the patient and favors a faster diagnosis, we found an unacceptable time delay before the diagnosis of pulmonary TB was made. Future studies should focus on attempt to explain the reasons of diagnostic retard in the patients with the characteristics related to delay in this study.</p

Mitogen-Activated Protein Kinases Regulate Susceptibility to Ventilator-Induced Lung Injury

Background: Mechanical ventilation causes ventilator-induced lung injury in animals and humans. Mitogen-activated protein kinases have been implicated in ventilator-induced lung injury though their functional significance remains incomplete. We characterize the role of p38 mitogen-activated protein kinase/mitogen activated protein kinase kinase-3 and c-jun-NH2-terminal kinase-1 in ventilator-induced lung injury and investigate novel independent mechanisms contributing to lung injury during mechanical ventilation. Methodology and Principle Findings: C57/BL6 wild-type mice and mice genetically deleted for mitogen-activated protein kinase kinase-3 (mkk-3-/-) or c-Jun-NH2-terminal kinase-1 (jnk1-/-) were ventilated, and lung injury parameters were assessed. We demonstrate that mkk3-/- or jnk1-/- mice displayed significantly reduced inflammatory lung injury and apoptosis relative to wild-type mice. Since jnk1-/- mice were highly resistant to ventilator-induced lung injury, we performed comprehensive gene expression profiling of ventilated wild-type or jnk1-/- mice to identify novel candidate genes which may play critical roles in the pathogenesis of ventilator-induced lung injury. Microarray analysis revealed many novel genes differentially expressed by ventilation including matrix metalloproteinase-8 (MMP8) and GAFF45α. Functional characterization of MMP8 revealed that mmp8-/- mice were sensitized to ventilator-induced lung injury with increased lung vascular permeability. Conclusion: We demonstrate that mitogen-activated protein kinase pathways mediate inflammatory lung injury during ventilator-induced lung injury. C-Jun-NH2-terminal kinase was also involved in alveolo-capillary leakage and edema formation, whereas MMP8 inhibited alveolo-capillary protein leakage. © 2008 Dolinay et al

The Prostate Specific Membrane Antigen Regulates the Expression of IL-6 and CCL5 in Prostate Tumour Cells by Activating the MAPK Pathways1

The interleukin-6 (IL-6) and the chemokine CCL5 are implicated in the development and progression of several forms of tumours including that of the prostate. The expression of the prostate specific membrane antigen (PSMA) is augmented in high-grade and metastatic tumors. Observations of the clinical behaviour of prostate tumors suggest that the increased secretion of IL-6 and CCL5 and the higher expression of PSMA may be correlated. We hypothesized that PSMA could be endowed with signalling properties and that its stimulation might impact on the regulation of the gene expression of IL-6 and CCL5. We herein demonstrate that the cross-linking of cell surface PSMA with specific antibodies activates the small GTPases RAS and RAC1 and the MAPKs p38 and ERK1/2 in prostate carcinoma LNCaP cells. As downstream effects of the PSMA-fostered RAS-RAC1-MAPK pathway activation we observed a strong induction of NF-κB activation associated with an increased expression of IL-6 and CCL5 genes. Pharmacological blockade with specific inhibitors revealed that both p38 and ERK1/2 participate in the phenomenon, although a major role exerted by p38 was evident. Finally we demonstrate that IL-6 and CCL5 enhanced the proliferative potential of LNCaP cells synergistically and in a dose-dependent manner and that CCL5 functioned by receptor-mediated activation of the STAT5-Cyclin D1 pro-proliferative pathway. The novel functions attributable to PSMA which are described in the present report may have profound influence on the survival and proliferation of prostate tumor cells, accounting for the observation that PSMA overexpression in prostate cancer patients is related to a worse prognosis

In vivo glioblastoma growth is reduced by apyrase activity in a rat glioma model

BACKGROUND: ATP is an important signalling molecule in the peripheral and central nervous system. Both glioma growth and tumor resection induces cell death, thus liberating nucleotides to the extracellular medium. Nucleotides are hydrolyzed very slowly by gliomas when compared with astrocytes and induce neuronal cell death and glioma proliferation. The objective of the present study was to test the involvement of extracellular ATP in glioblastoma growth in a rat glioma model. METHODS: To deplete the extracellular ATP, the enzyme apyrase was tested on the treatment of gliomas implanted in the rats CNS. One million glioma C6 cells in 3 microliters of DMEM/FCS were injected in the right striata of male Wistar rats, 250–270 g. After 20 days, the rats were decapitated and the brain sectioning and stained with hematoxylin and eosine. We performed immunohistochemical experiments with Ki67, CD31 and VEGF. Total RNA was isolated from cultured glioma C6 cells and the cDNA was analyzed by Real Time-PCR with primers for the NTPDase family. RESULTS: C6 glioma cells effectively have a low expression of all NTPDases investigated, in comparison with normal astrocytes. The implanted glioma co-injected with apyrase had a significant reduction in the tumor size (p < 0.05) when compared with the rats injected only with gliomas or with gliomas plus inactivated apyrase. According to the pathological analysis, the malignant gliomas induced by C6 injection and co-injected with apyrase presented a significant reduction in the mitotic index and other histological characteristics that indicate a less invasive/proliferative tumor. Reduction of proliferation induced by apyrase co-injection was confirmed by counting the percentage of Ki67 positive glioma cell nuclei. According to counts with CD31, vessel density and neoformation was higher in the C6 group 20 days after implantation. Confirming this observation, rats treated with apyrase presented less VEGF staining in comparison to the control group. CONCLUSION: These results indicate that the participation of extracellular ATP and the ecto-nucleotidases may be associated with the development of this type of brain tumor in an in vivo glioma model

Onconase responsive genes in human mesothelioma cells: implications for an RNA damaging therapeutic agent

<p>Abstract</p> <p>Background</p> <p>Onconase represents a new class of RNA-damaging drugs. Mechanistically, Onconase is thought to internalize, where it degrades intracellular RNAs such as tRNA and double-stranded RNA, and thereby suppresses protein synthesis. However, there may be additional or alternative mechanism(s) of action.</p> <p>Methods</p> <p>In this study, microarray analysis was used to compare gene expression profiles in untreated human malignant mesothelioma (MM) cell lines and cells exposed to 5 μg/ml Onconase for 24 h. A total of 155 genes were found to be regulated by Onconase that were common to both epithelial and biphasic MM cell lines. Some of these genes are known to significantly affect apoptosis (IL-24, TNFAIP3), transcription (ATF3, DDIT3, MAFF, HDAC9, SNAPC1) or inflammation and the immune response (IL-6, COX-2). RT-PCR analysis of selected up- or down-regulated genes treated with varying doses and times of Onconase generally confirmed the expression array findings in four MM cell lines.</p> <p>Results</p> <p>Onconase treatment consistently resulted in up-regulation of IL-24, previously shown to have tumor suppressive activity, as well as ATF3 and IL-6. Induction of ATF3 and the pro-apoptotic factor IL-24 by Onconase was highest in the two most responsive MM cell lines, as defined by DNA fragmentation analysis. In addition to apoptosis, gene ontology analysis indicated that pathways impacted by Onconase include MAPK signaling, cytokine-cytokine-receptor interactions, and Jak-STAT signaling.</p> <p>Conclusions</p> <p>These results provide a broad picture of gene activity after treatment with a drug that targets small non-coding RNAs and contribute to our overall understanding of MM cell response to Onconase as a therapeutic strategy. The findings provide insights regarding mechanisms that may contribute to the efficacy of this novel drug in clinical trials of MM patients who have failed first line chemotherapy or radiation treatment.</p

Aryl hydrocarbon receptor (AhR) agonists suppress interleukin-6 expression by bone marrow stromal cells: an immunotoxicology study

BACKGROUND: Bone marrow stromal cells produce cytokines required for the normal growth and development of all eight hematopoietic cell lineages. Aberrant cytokine production by stromal cells contributes to blood cell dyscrasias. Consequently, factors that alter stromal cell cytokine production may significantly compromise the development of normal blood cells. We have shown that environmental chemicals, such as aromatic hydrocarbon receptor (AhR) agonists, suppress B lymphopoiesis by modulating bone marrow stromal cell function. Here, we extend these studies to evaluate the potential for two prototypic AhR agonists, 7,12-dimethylbenz [a]anthracene (DMBA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), to alter stromal cell cytokine responses. METHODS: Bone marrow stromal cells were treated with AhR agonists and bacterial lipopolysaccharide (LPS) to mimic innate inflammatory cytokine responses and to study the effects of AhR ligands on those responses. Steady state cytokine RNA levels were screened by RNAse protection assays (RPA) and quantified by real-time PCR. Cytokine (IL-6) protein production was measured by ELISA. NF-κB EMSAs were used to study IL-6 transcriptional regulation. RESULTS: RPAs indicated that AhR(+ )bone marrow stromal cells consistently up-regulated genes encoding IL-6 and LIF in response to LPS, presumably through activation of Toll-like receptor 4. Pre-treatment with low doses of DMBA or TCDD selectively abrogated IL-6 gene induction but had no effect on LIF mRNA. Real-time-PCR indicated a significant inhibition of IL-6 mRNA by AhR ligands within 1 hour of LPS challenge which was reflected in a profound down-regulation of IL-6 protein induction, with DMBA and TCDD suppressing IL-6 levels as much as 65% and 88%, respectively. This potent inhibitory effect persisted for at least 72 hours. EMSAs measuring NF-κB binding to IL-6 promoter sequences, an event known to induce IL-6 transcription, indicated a significant decrease in the LPS-mediated induction of DNA-binding RelA/p50 and c-Rel/p50 heterodimers in the presence of DMBA. CONCLUSIONS: Common environmental AhR agonists can suppress the response to bacterial lipopolysaccharide, a model for innate inflammatory responses, through down-regulation of IL-6, a cytokine critical to the growth of several hematopoietic cell subsets, including early B cells. This suppression occurs at least at the level of IL-6 gene transcription and may be regulated by NF-κB

The molecular basis of genistein-induced mitotic arrest and exit of self-renewal in embryonal carcinoma and primary cancer cell lines

<p>Abstract</p> <p>Background</p> <p>Genistein is an isoflavonoid present in soybeans that exhibits anti-carcinogenic properties. The issue of genistein as a potential anti-cancer drug has been addressed in some papers, but comprehensive genomic analysis to elucidate the molecular mechanisms underlying the effect elicited by genistein on cancer cells have not been performed on primary cancer cells, but rather on transformed cell lines. In the present study, we treated primary glioblastoma, rhabdomyosarcoma, hepatocellular carcinoma and human embryonic carcinoma cells (NCCIT) with μ-molar concentrations of genistein and assessed mitotic index, cell morphology, global gene expression, and specific cell-cycle regulating genes. We compared the expression profiles of NCCIT cells with that of the cancer cell lines in order to identify common genistein-dependent transcriptional changes and accompanying signaling cascades.</p> <p>Methods</p> <p>We treated primary cancer cells and NCCIT cells with 50 μM genistein for 48 h. Thereafter, we compared the mitotic index of treated versus untreated cells and investigated the protein expression of key regulatory self renewal factors as OCT4, SOX2 and NANOG. We then used gene expression arrays (Illumina) for genome-wide expression analysis and validated the results for genes of interest by means of Real-Time PCR. Functional annotations were then performed using the DAVID and KEGG online tools.</p> <p>Results</p> <p>We found that cancer cells treated with genistein undergo cell-cycle arrest at different checkpoints. This arrest was associated with a decrease in the mRNA levels of core regulatory genes, <it>PBK</it>, <it>BUB1</it>, and <it>CDC20 </it>as determined by microarray-analysis and verified by Real-Time PCR. In contrast, human NCCIT cells showed over-expression of <it>GADD45 A </it>and <it>G </it>(growth arrest- and DNA-damage-inducible proteins 45A and G), as well as down-regulation of OCT4, and NANOG protein. Furthermore, genistein induced the expression of apoptotic and anti-migratory proteins p53 and p38 in all cell lines. Genistein also up-regulated steady-state levels of both <it>CYCLIN A </it>and <it>B</it>.</p> <p>Conclusion</p> <p>The results of the present study, together with the results of earlier studies show that genistein targets genes involved in the progression of the M-phase of the cell cycle. In this respect it is of particular interest that this conclusion cannot be drawn from comparison of the individual genes found differentially regulated in the datasets, but by the rather global view of the pathways influenced by genistein treatment.</p