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Introduction: The lack of effective anti-inflammatory therapies for pneumonia represents a challenge for identifying new alternatives. Non-digestible galacto-oligosaccharides (GOS) are attractive candidates due to their anti-inflammatory and immunomodulatory effects both locally and systemically. Objectives: The anti-inflammatory properties of GOS were investigated in calves with lung infections and in calf primary bronchial epithelial cells (PBECs) and human lung epithelial cells (A549). To delineate the mechanism, the potential capacity of GOS to inhibit the NLR family pyrin domain containing 3 (NLRP3) inflammasome has been investigated. Methods: GOS were administrated orally to calves with naturally occurring lung infections during early life or used as pretreatments in cell cultures exposed to M. haemolytica, lipopolysaccharides (LPS), leukotoxin or ATP. The cell composition, cytokine/chemokine concentrations, and M. haemolytica-LPS lgG levels in broncho-alveolar lavage fluid (BALF) and blood were investigated, while the M. haemolytica positivity in BALF and bronchial mucosa was detected in vivo. Key markers of NLRP3 inflammasome activation were measured in vivo and in vitro. Results: GOS reduced M. haemolytica positivity and M. haemolytica-LPS lgG levels in calves with lung infections. Regulation of immune function and suppression of inflammatory response by GOS is related to the inhibition of NLRP3 inflammasome as observed in bronchial mucosal tissue of infected calves. The M. haemolytica-induced IL-1β production in PBECs was lowered by GOS, which was associated with NLRP3 inflammasome inhibition caused by the decreased reactive oxygen species and ATP production. GOS inhibited leukotoxin-induced ATP production in PBECs. The LPS- and ATP-induced NLRP3 inflammasome activation in PBECs and A549 cells was suppressed by GOS. Conclusion: GOS exert anti-inflammatory properties by inhibiting the NLRP3 inflammasome activation in vitro and in vivo, suggesting a potential role for GOS in the prevention of lung infections.

Clostridioides difficile infection is expected to become the most common healthcare-associated infection worldwide. C. difficile-induced pathogenicity is significantly attributed to its enterotoxin, TcdA, which primarily targets Rho-GTPases involved in regulating cytoskeletal and tight junction (TJ) dynamics, thus leading to cytoskeleton breakdown and ultimately increased intestinal permeability. This study investigated whether two non-digestible oligosaccharides (NDOs), alginate (AOS) and chitosan (COS) oligosaccharides, possess antipathogenic and barrier-protective properties against C. difficile bacteria and TcdA toxin, respectively. Both NDOs significantly reduced C. difficile growth, while cell cytotoxicity assays demonstrated that neither COS nor AOS significantly attenuated the TcdA-induced cell death 24 h post-exposure. The challenge of Caco-2 monolayers with increasing TcdA concentrations increased paracellular permeability, as measured by TEER and LY flux assays. In this experimental setup, COS completely abolished, and AOS mitigated, the deleterious effects of TcdA on the monolayer’s integrity. These events were not accompanied by alterations in ZO-1 and occludin protein levels; however, immunofluorescence microscopy revealed that both AOS and COS prevented the TcdA-induced occludin mislocalization. Finally, both NDOs accelerated TJ reassembly upon a calcium-switch assay. Overall, this study established the antipathogenic and barrier-protective capacity of AOS and COS against C. difficile and its toxin, TcdA, while revealing their ability to promote TJ reassembly in Caco-2 cells.

One cluster of the extrapulmonary manifestations in chronic obstructive pulmonary disease (COPD) is related to the brain, which includes anxiety, depression and cognitive impairment. Brain-related comorbidities are related to worsening of symptoms and increased mortality in COPD patients. In this study, a murine model of COPD was used to examine the effects of emphysema and repetitive pulmonary inflammatory events on systemic inflammatory outcomes and brain function. In addition, the effect of a dietary intervention on brain-related parameters was assessed. Adult male C57Bl/6J mice were exposed to elastase or vehicle intratracheally (i.t.) once a week on three consecutive weeks. Two weeks after the final administration, mice were i.t. exposed to lipopolysaccharide (LPS) or vehicle for three times with a 10 day interval. A dietary intervention enriched with omega-3 PUFAs, prebiotic fibers, tryptophan and vitamin D was administered from the first LPS exposure onward. Behavior and cognitive function, the degree of emphysema and both pulmonary and systemic inflammation as well as blood-brain barrier (BBB) integrity and neuroinflammation in the brain were assessed. A lower score in the cognitive test was observed in elastase-exposed mice. Mice exposed to elastase plus LPS showed less locomotion in the behavior test. The enriched diet seemed to reduce anxiety-like behavior over time and cognitive impairments associated with the presented COPD model, without affecting locomotion. In addition, the enriched diet restored the disbalance in splenic T-helper 1 (Th1) and Th2 cells. There was a trend toward recovering elastase plus LPS-induced decreased expression of occludin in brain microvessels, a measure of BBB integrity, as well as improving expression levels of kynurenine pathway markers in the brain by the enriched diet. The findings of this study demonstrate brain-associated comorbidities – including cognitive and behavioral impairments – in this murine model for COPD. Although no changes in lung parameters were observed, exposure to the specific enriched diet in this model appeared to improve systemic immune disbalance, BBB integrity and derailed kynurenine pathway which may lead to reduction of anxiety-like behavior and improved cognition.

Abstract Hypoxia and hyperthermia, which can be induced by high environmental temperature or strenuous exercise, are two common stressors that affect intestinal epithelial integrity and lead to multiple clinical symptoms. In this study, we developed an in-vitro intestinal monolayer model using two human colonic epithelial cell lines, Caco-2 and HT-29, co-cultured in Transwell inserts, and investigated the effects of heat treatment and/or hypoxia on the epithelial barrier function. The monolayer with a ratio of 9:1 (Caco-2:HT-29) showed high trans-epithelial electrical resistance (TEER), low Lucifer Yellow permeability and high mucin production. Hyperthermia and/or hypoxia exposure (2 h) triggered heat shock and oxidative stress responses. HSP-70 and HSF-1 protein levels were up-regulated by hyperthermia, which were further enhanced when hyperthermia was combined with hypoxia. Increased HIF-1α protein expression and Nrf2 nuclear translocation was only caused by hypoxia. Hyperthermia and/or hypoxia exposure disrupted the established monolayer by increasing paracellular permeability, decreasing ZO-1, claudin-3 and occludin protein/mRNA expression, while enhancing E-cadherin protein expression. Tight junction protein distribution in the monolayer was also modulated by the hyperthermia and/or hypoxia exposure. In addition, transcription levels of mucin genes, MUC-2 and MUC-5AC, were increased after 2 h of hyperthermia and/or hypoxia exposure. In conclusion, this Caco-2/HT-29 cell model is valid and effective for studying detrimental effects of hyperthermia and/or hypoxia on intestinal barrier function and related heat shock and oxidative stress pathways and can be used to investigate possible interventions to reverse hyperthermia and/or hypoxia-induced intestinal epithelial injury.

The worldwide increase in the incidence of antibiotic resistance of the atypical bacterium Mycoplasma pneumoniae (MP) challenges the treatment of MP infections, especially in children. Therefore, alternative strategies for the treatment of MP infections are warranted. Galacto- and fructo-oligosaccharides (GOS and FOS) are a specific group of complex carbohydrates that were recently shown to possess direct anti-pathogenic properties. In this study, we assessed whether GOS and FOS exert anti-microbial and anti-infective effects against MP and, especially, macrolide-resistant MP (MRMP) in vitro. The MIC values of GOS for MP and MRMP were 4%. In contrast, the MIC values of FOS for both MP and MRMP were 16%. A time-kill kinetic assay showed that FOS possess bacteriostatic properties, while for GOS, a bactericidal effect against MP and MRMP was observed after 24 h at a concentration of 4x MIC. In co-cultures with human alveolar A549 epithelial cells, GOS killed adherent MP and MRMP and also concentration-dependently inhibited their adherence to A549 cells. Further, GOS suppressed (MR)MP-induced IL-6 and IL-8 in A549 cells. None of the aforementioned parameters were affected when FOS were added to these co-cultures. In conclusion, the anti-infective and anti-microbial properties of GOS could provide an alternative treatment against MRMP and MP infections.

Deoxynivalenol (DON), a highly prevalent contaminant of grain-based products, is known to induce reproductive- and immunotoxicities. Considering the importance of immune development in early life, the present study investigated the effects of perinatal DON exposure on allergy development and vaccine responsiveness in the offspring. Pregnant mice received control or DON-contaminated diets (12.5 mg/kg diet) during pregnancy and lactation. After weaning, female offspring were sensitized to ovalbumin (OVA) by oral administration of OVA with cholera toxin (CT). Male offspring were injected with Influvac vaccine. OVA-specific acute allergic skin response (ASR) in females and vaccine-specific delayed-type hypersensitivity (DTH) in males were measured upon intradermal antigen challenge. Immune cell populations in spleen and antigen-specific plasma immunoglobulins were analyzed. In female CT+OVA-sensitized offspring of DON-exposed mothers ASR and OVA-specific plasma immunoglobulins were significantly higher, compared to the female offspring of control mothers. In vaccinated male offspring of DON-exposed mothers DTH and vaccine-specific antibody levels were significantly lower, compared to the male offspring of control mothers. In both models a significant reduction in regulatory T cells, Tbet+ Th1 cells and Th1-related cytokine production of the offspring of DON-exposed mothers was observed. In conclusion, early life dietary exposure to DON can adversely influence immune development in the offspring. Consequently, the immune system of the offspring may be skewed towards an imbalanced state, resulting in an increased allergic immune response to food allergens and a decreased immune response to vaccination against influenza virus in these models.

Increased exposure to household air pollution and ambient air pollution has become one of the world’s major environmental health threats. In developing and developed countries, environmental cigarette smoke (CS) exposure is one of the main sources of household air pollution (HAP). Moreover, results from different epidemiological and experimental studies indicate that there is a strong association between HAP, specifically CS exposure, and the development of allergic diseases that often persists into later life. Here, we investigated the impact of prenatal and postnatal CS exposure on offspring susceptibility to the development of allergic airway responses by using a preclinical mouse model. Pregnant BALB/c mice were exposed to either CS or air during pregnancy and lactation and in order to induce allergic asthma the offspring were sensitized and challenged with house dust mite (HDM). Decreased lung function parameters, like dynamic compliance and pleural pressure, were observed in PBS-treated offspring born to CS-exposed mothers compared to offspring from air-exposed mothers. Maternal CS exposure significantly increased the HDM-induced airway eosinophilia and neutrophilia in the offspring. Prenatal and postnatal CS exposure increased the frequency of Th2 cells in the lungs of HDM-treated offspring compared to offspring born to air-exposed mothers. Offspring born to CS-exposed mothers showed increased levels of IL-4, IL-5 and IL-13 in bronchoalveolar lavage fluid compared to offspring from air-exposed mothers. Ex-vivo restimulation of lung cells isolated from HDM-treated offspring born to CS-exposed mothers also resulted in increased IL-4 production. Finally, serum immunoglobulins levels of HDM-specific IgE and HDM-specific IgG1 were significantly increased upon a HDM challenge in offspring born to CS-exposed mothers compared to offspring from air-exposed mothers. In summary, our results reveal a biological plausibility for the epidemiological studies indicating that prenatal and postnatal CS exposure increases the susceptibility of offspring to allergic immune responses.

The bacterial pathogens Streptococcus agalactiae (GBS) and Staphylococcus aureus (S. aureus) cause serious infections in humans and animals. The emergence of antibiotic-resistant isolates and bacterial biofilm formation entails the urge of novel treatment strategies. Recently, there is a profound scientific interest in the capabilities of non-digestible oligosaccharides as antimicrobial and anti-biofilm agents as well as adjuvants in antibiotic combination therapies. In this study, we investigated the potential of alginate oligosaccharides (AOS) and chitosan oligosaccharides (COS) as alternative for, or in combination with antibiotic treatment. AOS (2–16%) significantly decreased GBS V growth by determining the minimum inhibitory concentration. Both AOS (8 and 16%) and COS (2–16%) were able to prevent biofilm formation by S. aureus wood 46. A checkerboard biofilm formation assay demonstrated a synergistic effect of COS and clindamycin on the S. aureus biofilm formation, while AOS (2 and 4%) were found to sensitize GBS V to trimethoprim. In conclusion, AOS and COS affect the growth of GBS V and S. aureus wood 46 and can function as anti-biofilm agents. The promising effects of AOS and COS in combination with different antibiotics may offer new opportunities to combat antimicrobial resistance.

Chronic obstructive pulmonary disease (COPD), often caused by smoking, is a chronic lung disease with systemic manifestations including metabolic comorbidities. This study investigates adaptive and pathological alterations in adipose and skeletal muscle tissue following cigarette smoke exposure using in vivo and in vitro models. Mice were exposed to cigarette smoke or air for 72 days and the pre-adipose cell line 3T3-L1 was utilized as an in vitro model. Cigarette smoke exposure decreased body weight, and the proportional loss in fat mass was more pronounced than the lean mass loss. Cigarette smoke exposure reduced adipocyte size and increased adipocyte numbers. Adipose macrophage numbers and associated cytokine levels, including interleukin-1β, interleukine-6 and tumor necrosis factor-α were elevated in smoke-exposed mice. Muscle strength and protein synthesis signaling were decreased after smoke exposure; however, muscle mass was not changed. In vitro studies demonstrated that lipolysis and fatty acid oxidation were upregulated in cigarette smoke-exposed pre-adipocytes. In conclusion, cigarette smoke exposure induces a loss of whole-body fat mass and adipose atrophy, which is likely due to enhanced lipolysis.

Cigarette smoke exposure during pregnancy and lactation is associated with adverse pregnancy outcomes. Here, we investigated the effects of maternal smoke exposure on pregnancy and offspring immunity and explored whether, epidermal growth factor (EGF), an important growth-promoting factor in human colostrum and milk, might be a possible missing link in maternal smoke exposure and changes in infants’ immune responses. Pregnant BALB/c mice were exposed to either cigarette smoke or air during gestation and lactation, and effects on pulmonary inflammation in dams and immune responses in offspring were examined. Maternal smoke exposure increased airway hyperresponsiveness and accumulation of inflammatory cells in the lungs of pregnant dams compared to non-pregnant dams. The E-cadherin protein expression was reduced in mammary glands of cigarette smoke-exposed pregnant dams. EGF levels were higher in mammary glands and serum of smoke-exposed pregnant dams compared to air-exposed pregnant dams. Offspring from cigarette smoke-exposed dams exhibited elevated levels of IL-17A, MCP-1, IL-22, and IL-13 in anti-CD3 stimulated spleen cell culture supernatants. EGF levels were also increased in serum of offspring from smoke-exposed dams. A positive correlation was observed between serum EGF levels and neutrophil numbers in bronchoalveolar lavage fluid of the dams. Interestingly, IL-17A, MCP-1, IL-22, IL13, and IFN-γ levels in anti-CD3 stimulated spleen cell culture supernatants of male pups also showed a positive correlation with EGF serum levels. In summary, our results reveal that maternal smoke exposure predisposes dams to exacerbated airway inflammation and offspring to exacerbated immune responses and both phenomena are associated with elevated EGF concentrations.

There is a strong correlation between dysregulation of the gastrointestinal microbiota and development of allergic diseases. The most prevalent therapies for relieving asthma symptoms are associated with serious side effects, and therefore novel approaches are needed. Our objective was to elucidate whether oral administration of Lactobacillus rhamnosus GG (LGG) as a probiotic or turmeric powder (TP) as a prebiotic or both as a synbiotic mitigate allergic inflammation including lung function, airway inflammatory cell infiltration, Th2 cytokines/chemokine in a murine model of house dust mite (HDM)-induced asthma. BALB/c mice were intranasally sensitized and challenged with HDM received TP (20 mg/Kg mouse), or/and LGG (105 or 107 cfu/ml), or both orally. Interestingly, the synbiotic intervention (HDM-TP-LGG E7) specifically suppress the developement of airway hyperresponsiveness in response to methacholine. Besides, our synbiotic, TP, and LGG strongly down-regulated eosinophilia, IL-5, CCL17, IL-13. In terms of T cell response, CD4+ Th2 cells and CD4+ Th17 population were reduced in the splenocytes of the treatment groups compared to control. The synbiotic group not only elevated CD25+Foxp3+Treg frequency compared to asthmatic group, but also increased T reg cells compared to the probiotic group. The synbiotic also indicated the superior effect in suppressing Th2 cells compared to probiotic. Although, TP and LGG alone displayed suppressive effects, this study showed that the combination therapy consisting of TP and LGG (synbiotic) is more effective in some of the parameters than either of the treatments alone. This novel synbiotic, might be considered as a potential food-based drug for translational medicine and can possibly be used along with corticosteroid treatment.

Enterotoxin-producing bacteria (EPB) have developed multiple mechanisms to disrupt gut homeostasis, and provoke various pathologies. A major part of bacterial cytotoxicity is attributed to the secretion of virulence factors, including enterotoxins. Depending on their structure and mode of action, enterotoxins intrude the intestinal epithelium causing long-term consequences such as hemorrhagic colitis. Multiple non-digestible oligosaccharides (NDOs), and short chain fatty acids (SCFA), as their metabolites produced by the gut microbiota, interact with enteropathogens and their toxins, which may result in the inhibition of the bacterial pathogenicity. NDOs characterized by diverse structural characteristics, block the pathogenicity of EPB either directly, by inhibiting bacterial adherence and growth, or biofilm formation or indirectly, by promoting gut microbiota. Apart from these abilities, NDOs and SCFA can interact with enterotoxins and reduce their cytotoxicity. These anti-virulent effects mostly rely on their ability to mimic the structure of toxin receptors and thus inhibiting toxin adherence to host cells. This review focuses on the strategies of EPB and related enterotoxins to impair host cell immunity, discusses the anti-pathogenic properties of NDOs and SCFA on EPB functions and provides insight into the potential use of NDOs and SCFA as effective agents to fight against enterotoxins.

Background: Dietary non-digestible galacto-oligosaccharides (GOS) suppress allergic responses in mice sensitized and challenged with house dust mite (HDM). Budesonide is the standard therapy for allergic asthma in humans but is not always completely effective.Aim: To compare the efficacy of budesonide or different doses of GOS alone or with a combination therapy of budesonide and GOS on HDM-allergic responses in mice.Methods:BALB/c mice were sensitized and challenged with HDM, while fed a control diet or a diet supplemented with 1 or 2.5 w/w% GOS, and either or not oropharyngeally instilled with budesonide. Systemic and local inflammatory markers, such as mucosal mast cell protease-1 (mMCP-1) in serum, pulmonary CCL17, CCL22, and IL-33 concentrations and inflammatory cell influx in the bronchoalveolar lavage fluid (BALF) were determined.Results: Budesonide or GOS alone suppressed the number of eosinophils in the BALF of HDM allergic mice whereas budesonide either or not combined with GOS lowered both eosinophil and lymphocyte numbers in the BALF of HDM-allergic mice. Both 1 w/w% and 2.5 w/w% GOS and/or budesonide suppressed serum mMCP-1 concentrations. However, budesonide nor GOS alone was capable of reducing Th2 driving chemokines CCL17, CCL22 and IL-33 protein levels in supernatants of lung homogenates of HDM allergic mice, whereas the combination therapy did. Moreover, IL-13 concentrations were only significantly suppressed in mice treated with budesonide while fed GOS. A similar tendency was observed for the frequency of GATA3+CD4+ Th2 and CD4+RORγt+ Th17 cells in the lungs of the allergic mice.Conclusion: Dietary intervention using GOS may be a novel way to further improve the efficacy of anti-inflammatory drug therapy in allergic asthma by lowering Th2 driving mediators and mast cell degranulation.

During recent years the impact of microbial communities on the health of their host (being plants, fish, and terrestrial animals including humans) has received increasing attention. The microbiota provides the host with nutrients, induces host immune development and metabolism, and protects the host against invading pathogens (1–6). Through millions of years of co-evolution bacteria and hosts have developed intimate relationships. Microbial colonization shapes the host immune system that in turn can shape the microbial composition (7–9). However, with the large scale use of antibiotics in agriculture and human medicine over the last decades an increase of diseases associated with so-called dysbiosis has emerged. Dysbiosis refers to either a disturbed microbial composition (outgrowth of possible pathogenic species) or a disturbed interaction between bacteria and the host (10). Instead of using more antibiotics to treat dysbiosis there is a need to develop alternative strategies to combat disturbed microbial control. To this end, we can learn from nature itself. For example, the plant root (or “rhizosphere”) microbiome of sugar beet contains several bacterial species that suppress the fungal root pathogen Rhizoctonia solani, an economically important fungal pathogen of this crop (11). Likewise, commensal bacteria present on healthy human skin produce antimicrobial molecules that selectively kill skin pathogen Staphylococcus aureus. Interestingly, patients with atopic dermatitis (inflammation of the skin) lacked antimicrobial peptide secreting commensal skin bacteria (12). In this review, we will give an overview of microbial manipulation in fish, plants, and terrestrial animals including humans to uncover conserved mechanisms and learn how we might restore microbial balance increasing the resilience of the host species.

Thermal stress can evoke a stress response and enhance the synthesis of heat shock proteins, while gut barrier dysfunction is considered as an important adverse effect of thermal stress. Considering the previously described effects of galacto-oligosaccharides, nowadays mainly used in infant formulas, we hypothesized that galacto-oligosaccharides may protect the intestinal barrier against heat stress. Human epithelial colorectal adenocarcinoma cells were pre-treated with galacto-oligosaccharides prior to thermal stress exposure (40–42 °C) for 24 h. Pre-treatment of galacto-oligosaccharides prevented the heat stress-induced up-regulation of heat shock proteins and reduced the heat-induced stress response as observed by a decrease in haem oxygenase-1. Galacto-oligosaccharides partly prevented the heat-induced effects on monolayer integrity as measured by transepithelial electrical resistance, paracellular permeability and E-cadherin expression. In addition to their prebiotic effect, galacto-oligosaccharides may have beneficial potency to protect the intestinal epithelial barrier against heat stress and may be an attractive dietary application for people who are at high risk of developing heat stress.

Deoxynivalenol (DON) is one of the major mycotoxins produced by Fusarium fungi, and exposure to this mycotoxin requires an assessment of the potential adverse effects, even at low toxin levels. The aim of this study was to investigate the effects of a short-term, low-dose DON exposure on various gut health parameters in pigs. Piglets received a commercial feed or the same feed contaminated with DON (0.9 mg/kg feed) for 10 days, and two hours after a DON bolus (0.28 mg/kg BW), weight gain was determined and samples of different segments of the intestine were collected. Even the selected low dose of DON in the diet negatively affected weight gain and induced histomorphological alterations in the duodenum and jejunum. The mRNA expression of different tight junction (TJ) proteins, especially occludin, of inflammatory markers, like interleukin-1 beta and interleukin-10 and the oxidative stress marker heme-oxigenase1, were affected along the intestine by low levels of DON in the diet. Taken together, our results indicate that even after low-level exposure to DON, which has been generally considered as acceptable in animal feeds, clinically-relevant changes are measurable in markers of gut health and integrity.

High ambient temperatures negatively affect the human well-being as well as animal welfare and production. The gastrointestinal tract is predominantly responsive to heat stress. The currently available information about the multifaceted response to heat stress within different parts of the intestine is limited, especially in avian species. Hence, this study aims to evaluate the heat stress-induced sequence of events in the intestines of chickens. Furthermore, the gut health-promoting effect of dietary galacto-oligosaccharides (GOS) was investigated in these heat stress-exposed chickens. Chickens were fed a control diet or diet supplemented with 1% or 2.5% GOS (6 days) prior to and during a temperature challenge for 5 days (38-39°C, 8h per day). The parameters measured in different parts of the intestines included the genes (qPCR) HSF1, HSF3, HSP70, HSP90, E-cadherin, claudin-1, claudin-5, ZO-1, occludin, TLR-2, TLR-4, IL-6, IL-8, HO-1, HIF-1α) and their associated proteins HSP70, HSP90 and pan-cadherin (western blots). In addition, IL-6 and IL-8 plasma concentrations were measured by ELISA. In the jejunum, HSF3, HSP70, HSP90, E-cadherin, claudin-5, ZO-1, TLR-4, IL-6 and IL-8 mRNA expression and HSP70 protein expression were increased after heat stress exposure and a more pronounced increase in gene expression was observed in ileum after heat stress exposure, and in addition HSF1, claudin-1 and HIF-1α mRNA levels were upregulated. Furthermore, the IL-8 plasma levels were decreased in chickens exposed to heat stress. Interestingly, the heat stress-related effects in the jejunum were prevented in chickens fed a GOS diet, while dietary GOS did not alter these effects in ileum. In conclusion, our results demonstrate the differences in susceptibility to heat stress along the intestine, where the most obvious modification in gene expression is observed in ileum, while dietary GOS only prevent the heat stress-related changes in jejunum.

A novel neutrophil chemoattractant derived from collagen, proline-glycine-proline (PGP), has been recently characterized in chronic obstructive pulmonary disease (COPD). This peptide is derived via the proteolytic activity of matrix metalloproteases (MMP's)-8/9 and PE, enzymes produced by neutrophils and present in COPD serum and sputum. Valproic acid (VPA) is an inhibitor of PE and could possibly have an effect on the severity of chronic inflammation. Here the interaction site of VPA to PE and the resulting effect on the secondary structure of PE is investigated. Also, the potential inhibition of PGP-generation by VPA was examined in vitro and in vivo to improve our understanding of the biological role of VPA. UV-visible, fluorescence spectroscopy, CD and NMR were used to determine kinetic information and structural interactions between VPA and PE. In vitro, PGP generation was significantly inhibited by VPA. In vivo, VPA significantly reduced cigarette-smoke induced neutrophil influx. Investigating the molecular interaction between VPA and PE showed that VPA modified the secondary structure of PE, making substrate binding at the catalytic side of PE impossible. Revealing the molecular interaction VPA to PE may lead to a better understanding of the involvement of PE and PGP in inflammatory conditions. In addition, the model of VPA interaction with PE suggests that PE inhibitors have a great potential to serve as therapeutics in inflammatory disorders.

Cigarette smoking induces inflammatory responses in all smokers and is the major risk factor for lung disease such as chronic obstructive pulmonary disease (COPD). In this progressive disease, chronic inflammation in the lung contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated Proline-Glycine-Proline (N-ac-PGP). The generation of this tripeptide is mediated by a multistep pathway involving matrix metalloproteases (MMPs) 8 and 9 and prolyl endopeptidase (PE). Here we investigated whether cigarette smoke extract (CSE) stimulates human PMNs to breakdown whole matrix collagen leading to the generation of the chemotactic collagen fragment N-ac-PGP.Incubating PMNs with CSE led to the release of chemo-attractant CXCL8 and proteases MMP8 and MMP9. PMNs constitutively expressed PE activity as well as PE protein. Incubating CSE-primed PMNs with collagen resulted in collagen breakdown and in N-ac-PGP generation. Incubation of PMNs with the tripeptide N-ac-PGP resulted in the release of CXCL8, MMP8 and MMP9. Moreover, we tested whether PMNs from COPD patients are different from PMNs from healthy donors. Here we show that the intracellular basal PE activity of PMNs from COPD patients increased 25-fold compared to PMNs from healthy donors. Immunohistological staining of human lung tissue for PE showed that besides neutrophils, macrophages and epithelial cells express PE.This study indicates that neutrophils activated by cigarette smoke extract can breakdown collagen into N-ac-PGP and that this collagen fragment itself can activate neutrophils, which may lead in vivo to a self-propagating cycle of neutrophil infiltration, chronic inflammation and lung emphysema. MMP-, PE- or PGP-inhibitors can serve as an attractive therapeutic target and may open new avenues towards effective treatment of COPD.

Considering the diverse toxic effects of the Fusarium toxin deoxynivalenol (DON), its common occurrence in wheat-based products, and its stability during processing, DON constitutes an increasing health concern for humans and animals. In addition to the parent compound DON, human and animal exposure encompasses the acetylated fungal metabolites 3-acetyl-deoxynivalenol (3ADON) and 15-acetyl-deoxynivalenol (15ADON) as well as the plant-derived DON-glucoside (DON3G) and the bacterial product de-epoxy-DON (DOM-1). In the current study we used the well-established Caco-2 cell model to compare the effects of these naturally occurring forms of DON on cell viability and markers of barrier integrity, as well as on the release of the pro-inflammatory chemokine chemokine CXC motif ligand (CXCL8). Results show that 3ADON is less potent in inducing adverse effects on barrier integrity when compared to DON, whereas 15ADON appears to be slightly more potent than DON. In contrast, DON3G and DOM-1 exerted no measurable adverse effects on the intestinal barrier. It was also demonstrated that galacto-oligosaccharides (GOS) are able to protect epithelial cells against DON and its acetylated forms, which suggests that GOS are beneficial food additives in the protection of vulnerable segments of the human population against adverse effects of DON and its derivatives.