Presence of Escherichia coli O157:H7 in Dairy Farms located in Najaf, Baghdad, Kirkuk, and Erbil, Iraq.

It has been approved that one of the most dangerous foodborne pathogenic bacteria is E. coli O157:H7, which is responsible for several infection and death cases worldwide. It is well documented that in the developing countries E. coli O157:H7 is considered the main causative pathogen of human gastrointestinal infections. Therefore, the current research was aimed to evaluate the prevalence of E. coli O157:H7 in dairy cattle's milk using a rapid method, in Iraq (Najaf, Baghdad, Kirkuk, and Erbil). Over a period of 6 months (During hot months) samples were obtained and investigated by culturing on selective media (CT-SMAC). The multiplex PCR (m-PCR) also used for milk sample direct investigation. Using biochemical tests the recorded data showed that, 2 recognized isolates were E. coli, while the recorded data obtained from m-PCR assay revealed that none of the isolated E. coli was toxigenic E.coli O157:H7. The results of m-PCR on the milk samples revealed that 45 milk samples contained at least one of the following genes: O157, H7, stx1, stx2 genes. Also the results of the m-PCR revealed that 2 samples (raw milk) were toxigenic O157:H7 positive. In conclusion, to the best of authors' knowledge, this investigation was the first report on the prevalence of E. coli O157:H7 in the raw milk samples in Iraq. The results showed that the proportion of contaminated milk samples contaminated with E. coli O157:H7 identified in the current survey were similar to that the results of the previously published research from different dairy products across different countries in the Middle East region.

Willingness of dentists in Jordan to treat HIV-infected patients.

UNLABELLED: Reluctance of dentists to treat human immunodeficiency virus (HIV) positive patients represents a major concern. Many efforts have been extended towards the documentation of the extent of this reluctance and speculation of factors that influence it. OBJECTIVES: Assess the willingness of dentists in Jordan to treat HIV-infected patients. MATERIALS AND METHODS: Two hundred and forty-two general dental practices were surveyed for their willingness to provide treatment of toothache and routine dental care of an HIV-infected individual. RESULTS: Only 15% of the dental practices were willing to provide such care. Willingness to provide treatment did not seem to be influenced by financial factors or the local prevalence of HIV disease. CONCLUSION: Present data suggest that HIV-infected individuals will have difficulty in obtaining dental health care in Jordan.

Epidermal growth factor receptor signaling mediates regranulation of rat nasal goblet cells.

BACKGROUND: Mucus hypersecretion is a common response to inflammation in the lower airways and is a hallmark of chronic rhinitis. OBJECTIVE: The purpose of this study was to elucidate the mechanisms of regranulation (mucus production) of goblet cells in nasal epithelium. METHODS: Because neutrophils induce an epidermal growth factor (EGFR) cascade, we induced degranulation of goblet cells in rat nasal respiratory epithelium by means of intranasal inhalation of N-formyl-methionyl-leucyl-phenylalanine (fMLP), and we examined regranulation of the goblet cells and the role of EGFR inhibitors and neutrophils in the regranulation process. RESULTS: In the control state Alcian blue/periodic acid-Schiff and mucin MUC5AC staining was present. Degranulation was induced in the nasal septal epithelium 4 hours after intranasal inhalation of fMLP (10(-7) mol/L); 48 hours later, goblet-cell regranulation was complete. In the control state EGFR protein staining was absent in the epithelium, but after fMLP-induced degranulation, EGFR protein was expressed. After pretreatment with BIBX1522, a selective EGFR tyrosine kinase inhibitor, fMLP-induced degranulation was unaffected, but goblet-cell regranulation was prevented completely. CONCLUSION: These data suggest a role for the EGFR cascade in neutrophil-dependent production of goblet-cell mucins. Proving this theory will require the use of selective EGFR inhibitors in clinical studies of nasal hypersecretory states.

IL-13 induces mucin production by stimulating epidermal growth factor receptors and by activating neutrophils.

Mucus hypersecretion contributes to the morbidity and mortality in acute asthma. Both T helper 2 (Th2) cytokines and epidermal growth factor receptor (EGFR) signaling have been implicated in allergen-induced goblet cell (GC) metaplasia. Present results show that a cascade of EGFR involving neutrophils is implicated in interleukin (IL)-13-induced mucin expression in GC. Treatment with a selective EGFR tyrosine kinase inhibitor prevented IL-13-induced GC metaplasia dose dependently and completely. Instillation of IL-13 also induced tumor necrosis factor-alpha protein expression, mainly in infiltrating neutrophils. Control airway epithelium contained few leukocytes, but intratracheal instillation of IL-13 resulted in time-dependent leukocyte recruitment by IL-13-induced IL-8-like chemoattractant expression in airway epithelium. Pretreatment with an inhibitor of leukocytes in the bone marrow (cyclophosphamide) or with a blocking antibody to IL-8 prevented both IL-13-induced leukocyte recruitment and GC metaplasia. These findings indicate that EGFR signaling is involved in IL-13-induced mucin production. They suggest a potential therapeutic role for inhibitors of the EGFR cascade in the hypersecretion that occurs in acute asthma.

Alpha-1-antitrypsin stimulates fibroblast proliferation and procollagen production and activates classical MAP kinase signalling pathways.

Connective tissue formation at sites of tissue repair is regulated by matrix protein synthesis and degradation, which in turn is controlled by the balance between proteases and antiproteases. Recent evidence has suggested that antiproteases may also exert direct effects on cell function, including influencing cell migration and proliferation. The antiprotease, alpha1-antitrypsin, is the major circulating serine protease inhibitor which protects tissues from neutrophil elastase attack. Its deficiency is associated with the destruction of connective tissue in the lung and the development of emphysema, whereas accumulation of mutant alpha1-antitrypsin within hepatocytes often leads to liver fibrosis. In this study, we report that alpha1antitrypsin, at physiologically relevant concentrations, promotes fibroblast proliferation, with maximal stimulatory effects of 118 +/- 2% (n=6, P < 0.02) above media controls for cells exposed to 60 microM. We further show that alpha1antitrypsin also stimulates fibroblast procollagen production, independently of its effects on cell proliferation, with values maximally increased by 34 +/- 3% (n = 6, P < 0.01) above media controls at 30 microM. Finally, mechanistic studies to examine the mechanism by which alpha1-antitrypsin acts, showed that alpha1-antitrypsin induced the rapid activation of p42MAPK and p44MAPK (also known as ERK1/2) and that the specific MEK1 inhibitor PD98059 totally blocked alpha1-antitrypsin's mitogenic effects. These results support the hypothesis that alpha1-antitrypsin may play a role in influencing tissue repair in vivo by directly stimulating fibroblast proliferation and extracellular matrix production via classical mitogen-activated signalling pathways.

Local blockade of allergic airway hyperreactivity and inflammation by the poxvirus-derived pan-CC-chemokine inhibitor vCCI.

Allergen-induced asthma is characterized by chronic pulmonary inflammation, reversible bronchoconstriction, and airway hyperreactivity to provocative stimuli. Multiple CC-chemokines, which are produced by pulmonary tissue in response to local allergen challenge of asthmatic patients or experimentally sensitized rodents, chemoattract leukocytes from the circulation into the lung parenchyma and airway, and may also modify nonchemotactic function. To determine the therapeutic potential of local intrapulmonary CC-chemokine blockade to modify asthma, a recombinant poxvirus-derived viral CC-chemokine inhibitor protein (vCCI), which binds with high affinity to rodent and human CC-chemokines in vitro and neutralizes their biological activity, was administered by the intranasal route. Administration of vCCI to the respiratory tract resulted in dramatically improved pulmonary physiological function and decreased inflammation of the airway and the lung parenchyma. In contrast, vCCI had no significant effect on the circulating levels of total or allergen-specific IgE, allergen-specific cytokine production by peripheral lymph node T cells, or peritoneal inflammation after local allergen challenge, indicating that vCCI did not alter systemic Ag-specific immunity or chemoattraction at extrapulmonary sites. Together, these findings emphasize the importance of intrapulmonary CC-chemokines in the pathogenesis of asthma, and the therapeutic potential of generic and local CC-chemokine blockade for this and other chronic diseases in which CC-chemokines are locally produced.

Respiratory infection with influenza A virus interferes with the induction of tolerance to aeroallergens.

Viral respiratory infections have been implicated in influencing allergen sensitization and the development of asthma, but their exact role remains controversial. Because respiratory exposure to Ag normally engenders T cell tolerance and prevents the development of airway hyperreactivity (AHR) and inflammation, we examined the effects of influenza A virus infection on tolerance induced by exposure to intranasal (i.n.) OVA and the subsequent development of AHR. We found that concurrent infection with influenza A abrogated tolerance induced by exposure to i.n. OVA, and instead led to the development of AHR accompanied by the production of OVA-specific IgE, IL-4, IL-5, IL-13, and IFN-gamma. When both IL-4 and IL-5 were neutralized in this system, AHR was still induced, suggesting that influenza-induced cytokines such as IL-13, or mechanisms unrelated to cytokines, might be responsible for the development of AHR. The length of time between influenza A infection and i.n. exposure to OVA was crucial, because mice exposed to i.n. OVA 15-30 days after viral inoculation developed neither AHR nor OVA-specific tolerance. These mice instead acquired Th1-biased OVA-specific immune responses associated with vigorous OVA-induced T cell proliferation, and reduced production of OVA-specific IgE. The protective effect of influenza A on AHR was dependent on IFN-gamma, because protection was abrogated with a neutralizing anti-IFN-gamma mAb. These results suggest that viral respiratory infection interferes with the development of respiratory allergen-induced tolerance, and that the time interval between viral infection and allergen exposure is critical in determining whether viral infection will enhance, or protect against, the development of respiratory allergen sensitization and AHR.

Suplatast tosilate inhibits goblet-cell metaplasia of airway epithelium in sensitized mice.

BACKGROUND: IL-4 and IL-13 play a putative role in mucus hypersecretion in asthma. Suplatast tosilate prevents the synthesis of T(H2) cytokines. OBJECTIVE: Because suplatast tosilate inhibits T(H2) cytokines but does not inhibits IFN-gamma production, we examined the effect of suplatast on IL-4- or IL-13- and ovalbumin (OVA)-induced mucin synthesis in NCI-H292 cells in vitro and in bronchi of pathogen-free BALB/c mice in vivo. METHODS: In vitro, NCI-H292 cells were preincubated with suplatast tosilate (0.1-100 microgram/mL) 1 hour before adding human recombinant IL-4 (10 ng/mL). In vivo, mouse recombinant IL-4 or IL-13 (250 ng per/mouse) was instilled intranasally in mice pretreated with suplatast tosilate (50 mg.kg(-1).d(-1)). Mucous glycoconjugates were stained with Alcian blue (AB)/periodic acid-Schiff (PAS) stain. To evaluate effects of suplatast tosilate on goblet-cell metaplasia in OVA-sensitized mice, animals were pretreated with suplatast tosilate (1-50 mg.kg(-1).d(-1)) intragastrically. IL-4 and IL-13 were measured, and allergic inflammatory cells were analyzed in bronchoalveolar lavage fluid of OVA-sensitized mice. RESULTS: Pretreatment with suplastast did not prevent IL-4- or IL-13-induced increase in mucous glycoconjugate production in NCI-H292 cells or in mice. OVA sensitization increased AB/PAS-stained area of the epithelium (48.1% +/- 2.4%, P <.01 compared with control mice). Suplatast tosilate inhibited OVA-induced goblet-cell metaplasia in airway epithelium in a dose-dependent fashion; 50 mg.kg(-1).d(-1) decreased the AB/PAS area to 22.7% +/- 2.7% (P <.05 compared with OVA sensitization alone). Pretreatment with suplatast tosilate also prevented OVA-induced increase in IL-4 and IL-13 levels and decreased the number of lymphocytes and eosinophils in bronchoalveolar lavage fluid (P <.05 compared with values of mice given OVA alone). CONCLUSION: These results indicate that suplatast tosilate prevents allergen-induced goblet-cell metaplasia and the recruitment of eosinophils and lymphocytes into the airways. These results suggest that this effect is due to the prevention of the production of T(H2) cytokines in airways.

Agarose plug instillation causes goblet cell metaplasia by activating EGF receptors in rat airways.

We hypothesized that foreign bodies in airways cause inflammation leading to goblet cell metaplasia. Instilled agarose plugs lodged in the bronchi of pathogen-free rats caused a time-dependent increase in Alcian blue-periodic acid-Schiff staining that was detected within 24 h and markedly increased at 72 h. Control bronchi contained no pregoblet or goblet cells, but plugged bronchi contained many pregoblet and goblet cells and a decrease in nongranulated secretory cells. In situ hybridization showed no expression of MUC5AC in control airways, but plugged airways showed a marked expression. Control bronchi showed sparse staining for epidermal growth factor receptor (EGFR) protein, but plugged bronchi showed intense EGFR staining in the epithelium. Pretreatment with an EGFR tyrosine kinase inhibitor (BIBX1522) prevented Alcian blue-periodic acid-Schiff staining and MUC5AC gene expression in plugged bronchi. Pretreatment with tumor necrosis factor-alpha neutralizing antibody or pretreatment with cyclophosphamide abolished plug-induced EGFR protein expression and goblet cell metaplasia. Thus instillation of agarose plugs induces profound goblet cell metaplasia by causing EGFR expression and activation.

Oxidative stress causes mucin synthesis via transactivation of epidermal growth factor receptor: role of neutrophils.

Oxidative stress has been implicated in the pathogenesis of inflammatory diseases of airways. Here we show that oxidative stress causes ligand-independent activation of epidermal growth factor receptors (EGFR) and subsequent activation of mitogen-activated protein kinase kinase (MEK)-p44/42 mitogen-activated protein kinase (p44/42mapk), resulting in mucin synthesis in NCI-H292 cells. Exogenous hydrogen peroxide and neutrophils activated by IL-8, FMLP, or TNF-alpha increased EGFR tyrosine phosphorylation and subsequent activation of p44/42mapk and up-regulated the expression of MUC5AC at both mRNA and protein levels in NCI-H292 cells. These effects were blocked by selective EGFR tyrosine kinase inhibitors (AG1478, BIBX1522) and by a selective MEK inhibitor (PD98059), whereas a selective platelet-derived growth factor receptor tyrosine kinase inhibitor (AG1295), a selective p38 MAPK inhibitor (SB203580), and a negative compound of tyrosine kinase inhibitors (A1) were without effect. Neutrophil supernatant-induced EGFR tyrosine phosphorylation, activation of p44/42mapk, and MUC5AC synthesis were inhibited by antioxidants (N-acetyl-cysteine, DMSO, dimethyl thiourea, or superoxide dismutase); neutralizing Abs to EGFR ligands (EGF and TGF-alpha) were without effect, and no TGF-alpha protein was found in the neutrophil supernatant. In contrast, the EGFR ligand, TGF-alpha, increased EGFR tyrosine phosphorylation, activation of p44/42mapk, and subsequent MUC5AC synthesis, but these effects were not inhibited by antioxidants. These results implicate oxidative stress in stimulating mucin synthesis in airways and provide new therapeutic approaches in airway hypersecretory diseases.

IL-4 induces mucin gene expression and goblet cell metaplasia in vitro and in vivo.

Goblet cell metaplasia and mucus hypersecretion are important features in the pathogenesis of asthma. The cytokine IL-4 has been shown to play a role in animal models of asthma, where it induces Th2 lymphocyte differentiation and B lymphocyte IgE class switch. IL-4 has also been implicated in the differentiation of goblet cells via effects on lymphocytes and eosinophils. In this study we hypothesized that IL-4 induces airway epithelial cell mucin gene expression and mucous glycoconjugate production by direct action on these cells. In vitro, cultured airway epithelial cells (NCI-H292) expressed IL-4R constitutively, and IL-4 (10 ng/ml) induced MUC2 gene expression and mucous glycoconjugate production. In vivo, mouse airway epithelial cells expressed IL-4R constitutively, and IL-4 (250 ng) increased MUC5 gene expression and Alcian blue/periodic acid-Schiff-positive staining at 24 h; IL-4 did not increase inflammatory cell numbers in airway tissue or in bronchoalveolar lavage. TNF-alpha and IL-1beta levels in bronchoalveolar lavage were not increased in response to IL-4 instillation. These results indicate that airway epithelial cells express IL-4R constitutively and that IL-4 directly induces the differentiation of epithelium into mucous glycoconjugate-containing goblet cells.

Epidermal growth factor system regulates mucin production in airways.

Goblet-cell hyperplasia is a critical pathological feature in hypersecretory diseases of airways. However, the underlying mechanisms are unknown, and no effective therapy exists. Here we show that stimulation of epidermal growth factor receptors (EGF-R) by its ligands, EGF and transforming growth factor alpha (TGFalpha), causes MUC5AC expression in airway epithelial cells both in in vitro and in vivo. We found that a MUC5AC-inducing epithelial cell line, NCI-H292, expresses EGF-R constitutively; EGF-R gene expression was stimulated further by tumor necrosis factor alpha (TNFalpha). EGF-R ligands increased the expression of MUC5AC at both gene and protein levels, and this effect was potentiated by TNFalpha. Selective EGF-R tyrosine kinase inhibitors blocked MUC5AC expression induced by EGF-R ligands. Pathogen-free rats expressed little EGF-R protein in airway epithelial cells; intratracheal instillation of TNFalpha induced EGF-R in airway epithelial cells, and subsequent instillation of EGF-R ligands increased the number of goblet cells, Alcian blue-periodic acid-Schiff staining (reflecting mucous glycoconjugates), and MUC5AC gene expression, whereas TNFalpha, EGF, or TGFalpha alone was without effect. In sensitized rats, three intratracheal instillations of ovalbumin resulted in EGF-R expression and goblet-cell production in airway epithelium. Pretreatment with EGF-R tyrosine kinase inhibitor, BIBX1522, prevented goblet-cell production both in rats stimulated by TNFalpha-EGF-R ligands and in an asthma model. These findings suggest potential roles for inhibitors of the EGF-R cascade in hypersecretory diseases of airways.

Thrombin.

Thrombin is a multifunctional serine protease which plays a central role in haemostasis by regulating platelet aggregation and blood coagulation. It is formed from its precursor prothrombin following tissue injury and converts fibrinogen to fibrin in the final step of the clotting cascade. It also promotes numerous cellular effects including chemotaxis, proliferation, extracellular matrix turnover and release of cytokines. These actions of thrombin on cells have been implicated in tissue repair processes and in the pathogenesis of inflammatory and fibroproliferative disorders such as pulmonary fibrosis and atherosclerosis. Thrombin mediates its cellular effects by proteolytically activating cell surface receptors. Presently, two such receptors have been described and their roles in regulation of these functions are currently being investigated. The discovery of multiple thrombin receptors creates the possibility of selective receptor blockade of specific thrombin mediated events. New drugs with these actions should add to our current repertoire of thrombin inhibitors used to treat thrombotic diseases.

A novel transforming growth factor beta2 antisense transcript in mammalian lung.

Transforming growth factor (TGF) beta2 gene expression was examined in murine, rat and human lung by in situ hybridization with riboprobes. Hybridization signal was observed in a variety of cells with the sense probe, and Northern-blot analysis with this probe demonstrated the presence of a novel 3.5 kb transcript. This first report suggesting the existance of a natural TGFbeta2 antisense transcript raises the possibility that such a transcript may play a role in regulating TGFbeta2 production.

Thrombin stimulates fibroblast procollagen production via proteolytic activation of protease-activated receptor 1.

Thrombin is a multifunctional serine protease that has a crucial role in blood coagulation. It is also a potent mesenchymal cell mitogen and chemoattractant and might therefore have an important role in the recruitment and local proliferation of mesenchymal cells at sites of tissue injury. We hypothesized that thrombin might also affect the deposition of connective tissue proteins at these sites by directly stimulating fibroblast procollagen production. To address this hypothesis, the effect of thrombin on procollagen production and gene expression by human foetal lung fibroblasts was assessed over 48 h. Thrombin stimulated procollagen production at concentrations of 1 nM and above, with maximal increases of between 60% and 117% at 10 nM thrombin. These effects of thrombin were, at least in part, due to increased steady-state levels of alpha1(I) procollagen mRNA. They could furthermore be reproduced with thrombin receptor-activating peptides for the protease-activated receptor 1 (PAR-1) and were completely abolished when thrombin was rendered proteolytically inactive with the specific inhibitors d-Phe-Pro-ArgCH2Cl and hirudin, indicating that thrombin is mediating these effects via the proteolytic activation of PAR-1. These results suggest that thrombin might influence the deposition of connective tissue proteins during normal wound healing and the development of tissue fibrosis by stimulating fibroblast procollagen production.

Thrombin stimulates smooth muscle cell procollagen synthesis and mRNA levels via a PAR-1 mediated mechanism.

Thrombin is a serine protease involved in haemostasis which exerts a number of cellular effects, including stimulating mesenchymal cell migration, proliferation, and has been implicated both in normal wound healing and pathological conditions associated with hyperproliferation of smooth muscle cells such as atherosclerosis and restenosis. We hypothesize that thrombin, in addition to its proliferative effects, may also influence the deposition of matrix proteins at sites of vascular injury by directly stimulating smooth muscle cell procollagen production. 10 nM thrombin significantly stimulated rat aortic smooth muscle cell procollagen production by 34 +/- 3% compared to media control cells over a 48 h incubation period, and increased steady state alpha1(I) procollagen mRNA levels by up to 104 +/- 22%. These effects are mediated via interaction of thrombin with the PAR-1 receptor since TRAP (Thrombin Receptor Activating Peptide) stimulated procollagen production by 23 +/- 0.5%. In addition, conditioned medium from thrombin-treated cells stimulated procollagen production by 30 +/- 3% suggesting that thrombin is acting via the production and/or release of an autocrine mediator. These data suggest a novel role for thrombin in vascular wound healing and the development of pathological conditions associated with increased connective tissue deposition.