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Contamination of human and animal diets with different mycotoxins have gained significant attention over the past decade. The intestinal barrier is the first site of exposure and a primary target for nutritional contaminants and hazardous substances including mycotoxins. In this study, the potential impact of ochratoxin A (OTA) on intestinal barrier integrity was highlighted using a human intestinal Caco-2 cell line. Cell viability following OTA exposure was determined by lactate dehydrogenase release and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Moreover, markers of barrier integrity, such as transepithelial electrical resistance (TEER) as well as the permeability of Lucifer Yellow (LY) and fluorescein isothiocyanate (FITC)-dextran, were assessed. Furthermore, the protein expression of different tight junction (TJ) proteins, as main constituents of barrier integrity, was evaluated by Western blot. Results show that OTA reduces TEER values in a concentration- and time-dependent manner and increase the permeability of LY through the intestinal epithelial layer, while the cell viability did not change significantly. However, the damage was not severe enough to change the permeability to larger molecules, such as FITC-dextran. OTA exposure down-regulated the expression of TJ proteins claudin-1, -3 and -4 and up-regulated the expression of zona occludens 1. The observation that OTA can disrupt the epithelial barrier is of clinical importance as it may lead to an increased passage of luminal antigens into the systemic circulation.

Chemotherapy may result in ovarian atrophy, a depletion of the primordial follicle pool, diminished ovarian weight, cortical and stromal fibrosis. Imatinib mesylate is an anticancer agent that inhibits competitively several receptor tyrosine kinases (RTKs). RTKs play important roles in cell metabolism, proliferation, and apoptosis. In clinic, imatinib mesylate is also known as an anti-fibrotic medicine. In the present study, the impact of imatinib on the ovarian tissue was investigated by assessing ovarian tissue fibrosis in postnatal rat administered with or without imatinib for three days. Fibrosis in the ovarian tissue was determined by histology (Picrosirius and Masson's trichrome staining) and the protein expression of vimentin and alpha-smooth muscle actin (alpha-SMA). Furthermore, mRNA expression of Forkhead box transcription factor O1 and O3 (FOXO1 and FOXO3), which are markers of cell proliferation was quantified. A short-term exposure to imatinib showed to increase tissue fibrosis in ovaries. This was observed by Masson's trichrome staining. Exposure to imatinib led also to a down-regulation of vimentin protein expression and up-regulation mRNA expression of FOXO3. This may indicate a role of FOXO3 in ovarian tissue fibrosis in postnatal rat ovaries.

Extracellular ATP is a signaling molecule that often serves as a danger signal to alert the immune system of tissue damage. This molecule activates P2 nucleotide receptors, that include the ionotropic P2X receptors and metabotropic P2Y receptors. Recently, it has been reported that ATP accumulates in the airways of both asthmatic patients and sensitized mice after allergen challenge. The role and function of ATP in the pathogenesis of chronic obstructive pulmonary diseases (COPD) are not well understood. In this study we investigated the effect of cigarette smoke on purinergic receptors and ATP release by neutrophils. Neutrophils and their mediators are key players in the pathogenesis of lung emphysema. Here we demonstrated that in an in vivo model of cigarette smoke-induced lung emphysema, the amount of ATP was increased in the bronchoalveolar lavage fluid. Moreover, activation of neutrophils with cigarette smoke extract induced ATP release. Treatment of neutrophils with apyrase (catalyses the hydrolysis of ATP to yield AMP) and suramin (P2-receptor antagonist) abrogated the release of CXCL8 and elastase induced by cigarette smoke extract and exogenous ATP. These observations indicate that activation of purinergic signaling by cigarette smoke may take part in the pathogenesis of lung emphysema.

Disintegration of the colonic epithelial barrier is considered a key event in the initiation and progression of inflammatory bowel and celiac disease. As the primary etiology of these diseases remains unknown, we hypothesized that the trichothecene deoxynivalenol (DON), a fungal metabolite found in grain-based human diets, might be one of the triggers resulting in an impairment of the intestinal tight junction network preceding an inflammatory response. Using horizontal impedance measurements, we demonstrate that DON disintegrates a human Caco-2 cell monolayer within

Objective Proline–glycine–proline (PGP) has been shown to have chemotactic effects on neutrophils via CXCR2 in several lung diseases. PGP is derived from collagen by the combined action of matrix metalloproteinase (MMP) 8 and/or MMP9 and prolyl endopeptidase (PE). We investigated the role of PGP in inflammatory bowel disease (IBD). Design In intestinal tissue from patients with IBD and mice with dextran sodium sulfate (DSS)-induced colitis, MMP8, MMP9 and PE were evaluated by ELISA, immunoblot and immunohistochemistry. Peripheral blood polymorphonuclear cell (PMN) supernatants were also analysed accordingly and incubated with collagen to assess PGP generation ex vivo. PGP levels were measured by mass spectrometry, and PGP neutralisation was achieved with a PGP antagonist and PGP antibodies. Results In the intestine of patients with IBD, MMP8 and MMP9 levels were elevated, while PE was expressed at similar levels to control tissue. PGP levels were increased in intestinal tissue of patients with IBD. Similar results were obtained in intestine from DSS-treated mice. PMN supernatants from patients with IBD were far more capable of generating PGP from collagen ex vivo than healthy controls. Furthermore, PGP neutralisation during DSS-induced colitis led to a significant reduction in neutrophil infiltration in the intestine. Conclusions The proteolytic cascade that generates PGP from collagen, as well as the tripeptide itself, is present in the intestine of patients with IBD and mice with DSS-induced colitis. PGP neutralisation in DSS-treated mice showed the importance of PGP-guided neutrophilic infiltration in the intestine and indicates a vicious circle in neutrophilic inflammation in IBD.

Rationale: Neutrophils are key players in chronic obstructive pulmonary disease (COPD), and increased numbers of neutrophils are present in sputum and lung tissue of patients with COPD. Interestingly, immunoglobulin free light chains (IgLC) are able to prolong the life of neutrophils; therefore, IgLC may contribute to the chronic state of inflammation.Objectives: In this study, the relation between IgLC and COPD has been investigated.Methods: We investigated the presence of IgLC in different murine lung emphysema models. IgLC levels in serum from mice and patients with COPD were examined by Western blot analysis and ELISA, respectively. IgLC levels in lung tissue were determined by immunohistochemistry. Fluorescence-activated cell sorter and immunofluorescent analysis were used to detect binding between IgLC and human neutrophils. Interleukin-8 (CXCL8) release by neutrophils after IgLC incubation was measured by ELISA. The effect of F991, an IgLC antagonist, was examined on the neutrophil influx in murine lungs after 5 days of smoke exposure.Measurements and Main Results: Increased levels of IgLC in serum of cigarette smoke-exposed and cigarette smoke extract-treated mice compared with control mice were observed. Patients with COPD showed increased serum IgLC and expression of IgLC in lung tissue compared with healthy volunteers. Interestingly, IgLC bound to neutrophils and activated neutrophils to release CXCL8. F991 inhibited the IgLC binding to neutrophils and reduced the smoke-induced neutrophil influx in murine lungs after smoke exposure.Conclusions: This study describes for the first time an association between neutrophils and IgLC in the pathophysiology of COPD, which could open new avenues to targeted treatment of this chronic disease.

Background Cigarette smoking induces peripheral inflammatory responses in all smokers and is the major risk factor for neutrophilic lung disease such as chronic obstructive pulmonary disease. The aim of this study was to investigate the effect of cigarette smoke on neutrophil migration and on β2-integrin activation and function in neutrophilic transmigration through endothelium. Methods and results Utilizing freshly isolated human PMNs, the effect of cigarette smoke on migration and β2-integrin activation and function in neutrophilic transmigration was studied. In this report, we demonstrated that cigarette smoke extract (CSE) dose dependently induced migration of neutrophils in vitro. Moreover, CSE promoted neutrophil adherence to fibrinogen. Using functional blocking antibodies against CD11b and CD18, it was demonstrated that Mac-1 (CD11b/CD18) is responsible for the cigarette smoke-induced firm adhesion of neutrophils to fibrinogen. Furthermore, neutrophils transmigrated through endothelium by cigarette smoke due to the activation of β2-integrins, since pre-incubation of neutrophils with functional blocking antibodies against CD11b and CD18 attenuated this transmigration. Conclusion This is the first study to describe that cigarette smoke extract induces a direct migratory effect on neutrophils and that CSE is an activator of β2-integrins on the cell surface. Blocking this activation of β2-integrins might be an important target in cigarette smoke induced neutrophilic diseases.

Neutrophils are innate immune cells in chronic inflammatory diseases including chronic obstructive pulmonary disease (COPD) and can be attracted to the site of inflammation via the collagen breakdown product N-acetyl Proline-Glycine-Proline (N-Ac-PGP). To elucidate whether CXCR2 is involved in N-Ac-PGP-induced neutrophil migration and activation, studies using specific antagonists were performed in vivo. N-Ac-PGP and keratinocyte cell-derived chemokine (KC; CXCL1) were administered in C57Bl/6 mice via oropharyngeal aspiration. Intraperitoneal applications of CXCR2 antagonist SB225002 or SB332235 were administered 1h prior and 1h after oropharyngeal aspiration. Six hours after oropharyngeal aspiration mice were sacrificed. Neutrophil counts and CXCL1 levels were determined in bronchoalveolar lavage fluid, myleoperoxidase (MPO) levels were measured in lung tissue homogenates and an immunohistological staining for neutrophils was performed on lung tissue. N-Ac-PGP and CXCL1 induced a neutrophil influx in the bronchoalveolar lavage fluid and lung tissue, which was also reflected by increased MPO levels in lung tissue. The N-Ac-PGP- and CXCL1-induced neutrophil influx and the increased pulmonary tissue MPO levels were inhibited by the CXCR2 antagonists SB225002 and SB332235. Moreover, N-Ac-PGP administration enhanced the CXCL1 levels in bronchoalveolar lavage fluid, which could not be attenuated by both CXCR2 antagonists. In conclusion, neutrophil migration induced by N-Ac-PGP is mediated via direct CXCR2 interaction. The N-Ac-PGP-induced release of CXCL1 is independent of CXCR2. Related to the maximal effect of CXCL1, N-Ac-PGP is more potent at inducing neutrophil migration in the pulmonary tissue than into the bronchoalveolar lavage fluid, or N-ac-PGP may be more potent at inducing MPO levels in the lung tissue.

There is increasing evidence that the neutrophil chemoattractant proline-glycine-proline (PGP), derived from the breakdown of the extracellular matrix, plays an important role in neutrophil recruitment to the lung. PGP formation is a multistep process involving neutrophils, metalloproteinases (MMPs), and prolyl endopeptidase (PE). This cascade of events is now investigated in the development of lung emphysema. A/J mice were whole body exposed to cigarette smoke for 20 wk. After 20 wk or 8 wk after smoking cessation, animals were killed, and bronchoalveolar lavage fluid and lung tissue were collected to analyze the neutrophilic airway inflammation, the MMP-8 and MMP-9 levels, the PE activity, and the PGP levels. Lung tissue degradation was assessed by measuring the mean linear intercept. Additionally, we investigated the effect of the peptide l-arginine-threonine-arginine (RTR), which binds to PGP sequences, on the smoke-induced neutrophil influx in the lung after 5 days of smoke exposure. Neutrophilic airway inflammation was induced by cigarette smoke exposure. MMP-8 and MMP-9 levels, PE activity, and PGP levels were elevated in the lungs of cigarette smoke-exposed mice. PE was highly expressed in epithelial and inflammatory cells (macrophages and neutrophils) in lung tissue of cigarette smoke-exposed mice. After smoking cessation, the neutrophil influx, the MMP-8 and MMP-9 levels, the PE activity, and the PGP levels were decreased or reduced to normal levels. Moreover, RTR inhibited the smoke-induced neutrophil influx in the lung after 5 days' smoke exposure. In the present murine model of cigarette smoke-induced lung emphysema, it is demonstrated for the first time that all relevant components (neutrophils, MMP-8, MMP-9, PE) involved in PGP formation from collagen are upregulated in the airways. Together with MMPs, PE may play an important role in the formation of PGP and thus in the pathophysiology of lung emphysema.

Emphysema is characterized by enlargement of the alveoli, which is the most important parameter to assess the presence and severity of this disease. Alveolar enlargement is primarily defined on morphological criteria; therefore, characterization of this disease with morphological parameters is a prerequisite to study the pathogenesis. For this purpose, different methods of lung fixation were evaluated in a murine model of LPS-induced lung emphysema. Five different methods of lung fixation were evaluated: intratracheal instillation of fixatives, in situ fixation, fixed-volume fixation, vascular whole body perfusion, and vacuum inflation. In addition, the effects of three different fixatives (10% formalin, Carnoy's, and agarose/10% formalin solution) and two embedding methods (paraffin and plastic) were investigated on the murine lung morphology. Mice received intranasal administration of LPS to induce alveolar wall destruction. Quantification of air space enlargement was determined by mean linear intercept analysis, and the histological sections were analyzed for the most optimal fixation method. Additionally, routine immunohistological staining was performed on lung tissue of PBS-treated mice. Intratracheal instillation of formalin or agarose/formalin solution, in situ fixation, and fixed-volume fixation provided a normal lung architecture, in contrast to the lungs fixed via whole body perfusion and vacuum inflation. Formalin-fixed lungs resulted in the most optimal lung morphology for lung emphysema analysis when embedded in paraffin, while for Carnoy's fixed lungs, plastic embedding was preferred. The histological findings, the mean linear intercept measurement, and the immunohistochemistry data demonstrated that fixation by intratracheal instillation of 10% formalin or in situ fixation with 10% formalin are the two most optimal methods to fix lungs for alveolar enlargement analysis to study lung emphysema.

Background Tobacco smoking irritates and damages the respiratory tract and contributes to a higher risk of developing lung emphysema. At present, smoking cessation is the only effective treatment for reducing the progression of lung emphysema, however, there is hardly anything known about the effects of smoking cessation on cytokine and chemokine levels in the airways. To the best of our knowledge, this is the first reported in vivo study in which cytokine profiles were determined after cessation of cigarette smoke exposure. Methods The severity of airway remodeling and inflammation was studied by analyzing alveolar enlargement, heart hypertrophy, inflammatory cells in the bronchoalveolar lavage fluid (BALF) and lung tissue and by determining the cytokine and chemokine profiles in the BALF of A/J mice exposed to cigarette smoke for 20 weeks and 8 weeks after smoking cessation. Results The alveolar enlargement and right ventricle heart hypertrophy found in smoke-exposed mice remained unchanged after smoking cessation. Although the neutrophilic inflammation in the BALF of cigarette smoke-exposed animals was reduced after smoking cessation, a sustained inflammation in the lung tissue was observed. The elevated cytokine (IL-1α and TNF-α) and chemokine (CCL2 and CCL3) levels in the BALF of smoke-exposed mice returned to basal levels after smoking cessation, while the increased IL-12 levels did not return to its basal level. The cigarette smoke-enhanced VEGF levels did not significantly change after smoking cessation. Moreover, IL-10 levels were reduced in the BALF of smoke-exposed mice and these levels were still significantly decreased after smoking cessation compared to the control animals. Conclusion The inflammatory changes in the airways caused by cigarette smoke exposure were only partially reversed after smoking cessation. Although smoking cessation should be the first step in reducing the progression of lung emphysema, additional medication could be provided to tackle the sustained airway inflammation.