Impaired intestinal tolerance in the absence of a functional complement system.

BACKGROUND: Cells of the innate immune system regulate both adaptive immune responses and the maintenance of tolerance, especially in the gut. However, relatively little is known about the effects of complement on lymphocyte homeostasis. OBJECTIVE: This study explored complement C3 deficiency in mice and human subjects for its effect on intestinal tolerance. METHODS: C3-deficient mice and control C57BL/6 mice were fed ovalbumin (OVA) by means of gavage, and subsequent response to immunization with OVA in Freund's adjuvant was monitored. Serum antibodies against commensal microbes were measured, and the activation status of peripheral blood lymphocytes bearing mucosal homing markers was determined from 2 rare cases of C3-deficient patients. RESULTS: We show in C3-deficient mice and human patients that intestinal tolerance fails in the absence of functional complement. In contrast to wild-type control animals, in which oral tolerance was induced, intragastric administration of OVA did not result in a significantly decreased response to subsequent subcutaneous OVA challenge in C3-deficient mice. In the jejunum of C3-deficient mice the cytokine ratio between IL-10 and IFN-γ or IL-17 levels was decreased, indicating a shift in favor of proinflammatory cytokines. In 2 C3-deficient children the frequency of gut-homing T cells expressing activation markers was increased, and the patients had increased serum IgG levels against gut commensal microbes. The data also suggest that the impaired oral tolerance was at least partly caused by the absence of signaling through C3-binding complement regulators in T cells. CONCLUSIONS: Taken together, our results identify complement as an important and nonredundant regulator of intestinal tolerance.

In Search of Clinical Markers: Indicators of Exposure in Dampness and Mold Hypersensitivity Syndrome (DMHS).

Potential markers were sought to diagnose mold hypersensitivity. Indoor air condensed water and human macrophage THP-1 test were applied to evaluate the buildings. Basophil activation tests (BAT) were conducted and mold-specific immunoglobulins (IgE, IgG, IgA, and IgD) were measured in study subjects' serum and feces. Exposed subjects reported markedly more symptoms from occupational air than controls. Basophils from exposed subjects died/lost activity at 225 times lower concentrations of toxic extracts from the target building than recommended in the common BAT protocol. Fecal IgG and IgD levels against Acrostalagmus luteoalbus and Aspergillus versicolor produced receiver operating curves (ROC) of 0.928 and 0.916, respectively, when plotted against the inflammation marker MRP8/14. Assaying serum immunoglobulin concentrations against the toxic Chaetomium globosum (MTAV35) from another building, a test control, did not differentiate study individuals. However, if liver metabolism produced the same core molecule from other Chaetomium globosum strains, this would explain the increased response in fecal immunoglobulins in the exposed. The altered immunoglobulin values in the samples of exposed when compared to controls revealed the route of mold exposure. The toxicity of indoor air condensed water samples, BAT and serology confirmed the severity of symptoms in the target building's employees, supporting earlier findings of toxicity in this building.

Toxic Indoor Air Is a Potential Risk of Causing Immuno Suppression and Morbidity-A Pilot Study.

We aimed to establish an etiology-based connection between the symptoms experienced by the occupants of a workplace and the presence in the building of toxic dampness microbiota. The occupants (5/6) underwent a medical examination and urine samples (2/6) were analyzed by LC-MS/MS for mycotoxins at two time-points. The magnitude of inhaled water was estimated. Building-derived bacteria and fungi were identified and assessed for toxicity. Separate cytotoxicity tests using human THP-1 macrophages were performed from the office's indoor air water condensates. Office-derived indoor water samples (n = 4/4) were toxic to human THP-1 macrophages. Penicillium, Acremonium sensu lato, Aspergillus ochraceus group and Aspergillus section Aspergillus grew from the building material samples. These colonies were toxic in boar sperm tests (n = 11/32); four were toxic to BHK-21 cells. Mycophenolic acid, which is a potential immunosuppressant, was detected in the initial and follow-up urine samples of (2/2) office workers who did not take immunosuppressive drugs. Their urinary mycotoxin profiles differed from household and unrelated controls. Our study suggests that the presence of mycotoxins in indoor air is linked to the morbidity of the occupants. The cytotoxicity test of the indoor air condensate is a promising tool for risk assessment in moisture-damaged buildings.

Perceived food hypersensitivity: a review of 10 years of interdisciplinary research at a reference center.

Perceived food hypersensitivity is a prevalent, but poorly understood condition. In this review article, we summarize narratively recent literature including results of our 10 years' interdisciplinary research program dealing with such patients. The patients (more than 400) included in our studies were all adults referred to a university hospital because of gastrointestinal complaints self-attributed to food hypersensitivity. Despite extensive examinations, food allergy was seldom diagnosed. The majority of the patients fulfilled the diagnostic criteria for irritable bowel syndrome. In addition, most suffered from several extra-intestinal health complaints and had considerably impaired quality of life. However, psychological factors could explain only approximately 10% of the variance in the patients' symptom severity and 90% of the variance thus remained unexplained. Intolerance to low-digestible carbohydrates was a common problem and abdominal symptoms were replicated by carbohydrate ingestion. A considerable number of patients showed evidence of immune activation by analyses of B-cell activating factor, dendritic cells and "IgE-armed" mast cells. Multiple factors such as immune activation, disturbed intestinal fermentation, enteric dysmotility, post-infectious changes and "local" allergy in the gut as well as psychological disturbances may play a role in the pathophysiology of perceived food hypersensitivity. Hence, our results support the view that management of these patients should be interdisciplinary.

Association of toxic indoor air with multi-organ symptoms in pupils attending a moisture-damaged school in Finland.

BACKGROUND: There is an on-going debate on how best to test toxic indoor air. Toxicological methods based on condensed water samples and cell culture technique are newly introduced research tools which were tested in this study. METHODS: Pupils (n=47) from a water-damaged and (n=56) healthy schools were interviewed using a questionnaire. Indoor air was collected with a novel condensed water sampling technique and human THP-1 macrophages were exposed to the condensate. The cytotoxicity of cotton wool swab samples was tested using human BJ fibroblasts. Conventional microbiological culture methods were also performed. RESULTS: Gastrointestinal problems (GI) were reported by 51% from the study cohort but only 4% of the control cohort, relative risk RR=14.30. For any neurological or neuropsychological symptoms, the RR was 63.04, muscular-skeletal pain RR=58.28, headache RR=31.00, respiratory symptoms RR=22.64, fatigue RR=21.45, sub febrility RR=15.49, ear infections RR=7.74, skin rash RR=5.96, all being statistically significant (P<0.001). All indoor air (n=7) and cotton wool samples (n=2) taken from the water-damaged classroom or in proximity of the problematic classrooms were toxic in cell culture assays. Low numbers of moisture-damage indicators were recovered from wall, passive air, and swab samples, namely Aspergillus ochraceus species group, Aspergillus, Eurotium species group, Fusarium, Tritirachium, Scopulariopsis genus group and Aspergillus versicolores species group. CONCLUSIONS: Indoor air toxicity and dampness-related microbiota recovered from the classrooms were associated with multi-organ morbidity of the school occupants. These results corroborated our previous reports from two adult cohorts i.e. evidence of causality. These new toxicological methods based on condensed water and cell culturing techniques seem to be superior to conventional microbiological methods in correlating with clinical symptoms.

Food allergy alters jejunal circular muscle contractility and induces local inflammatory cytokine expression in a mouse model.

BACKGROUND: We hypothesized that food allergy causes a state of non-specific jejunal dysmotility. This was tested in a mouse model. METHODS: Balb/c mice were epicutaneously sensitized with ovalbumin and challenged with 10 intragastric ovalbumin administrations every second day. Smooth muscle contractility of isolated circular jejunal sections was studied in organ bath with increasing concentrations of carbamylcholine chloride (carbachol). Smooth muscle layer thickness and mast cell protease-1 (MMCP-1) positive cell density were assayed histologically. Serum MMCP-1 and immunoglobulins were quantified by ELISA, and mRNA expressions of IFN-gamma, IL-4, IL-6 and TGFbeta-1 from jejunal and ileal tissue segments were analyzed with quantitative real-time PCR. RESULTS: Ovalbumin-specific serum IgE correlated with jejunal MMCP-1+ cell density. In the allergic mice, higher concentrations of carbachol were required to reach submaximal muscular stimulation, particularly in preparations derived from mice with diarrhoea. Decreased sensitivity to carbachol was associated with increased expression of IL-4 and IL-6 mRNA in jejunum. Smooth muscle layer thickness, as well as mRNA of IFN-gamma and TGF-beta1 remained unchanged. CONCLUSION: In this mouse model of food allergy, we demonstrated a decreased response to a muscarinic agonist, and increased levels of proinflammatory IL-6 and Th2-related IL-4, but not Th1-related IFN-gamma mRNAs in jejunum. IgE levels in serum correlated with the number of jejunal MMCP-1+ cells, and predicted diarrhoea. Overall, these changes may reflect a protective mechanism of the gut in food allergy.

Hemin and Cobalt Protoporphyrin Inhibit NLRP3 Inflammasome Activation by Enhancing Autophagy: A Novel Mechanism of Inflammasome Regulation.

Inflammasomes are intracellular protein platforms, which, upon activation, produce the highly proinflammatory cytokines interleukin (IL)-1ß and IL-18. Heme, hemin and their degradation products possess significant immunomodulatory functions. Here, we studied whether hemin regulates inflammasome function in macrophages. Both hemin and its derivative, cobalt protoporphyrin (CoPP), significantly reduced IL-1ß secretion by cultured human primary macrophages, the human monocytic leukemia cell line and also mouse bone marrow-derived and peritoneal macrophages. Intraperitoneal administration of CoPP to mice prior to urate crystal-induced peritonitis alleviated IL-1ß secretion to the peritoneal cavity. In cultured macrophages, hemin and CoPP inhibited NLRP3 inflammasome assembly by reducing the amount of intracellular apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC). The reduction of ASC was associated with enhanced autophagosome formation and autophagic flux. Inhibition of autophagy prevented the CoPP-induced depletion of ASC, implying that the depletion was caused by increased autophagy. Our data indicate that hemin functions as an endogenous negative regulator of the NLRP3 inflammasome. The inhibition is mediated via enhanced autophagy that results in increased degradation of ASC. This regulatory mechanism may provide a novel approach for the treatment of inflammasome-related diseases.

Effect of simvastatin on development of obliterative airway disease: an experimental study.

BACKGROUND: Obliterative bronchiolitis after lung transplantation is characterized by airway inflammation leading to obliteration of small airways. Statins are known to have lipid-independent immunomodulatory properties. We investigated the effect of simvastatin treatment on innate and adaptive immune responses and the development of obliterative airway disease (OAD). METHODS: In fully MHC-mismatched rat tracheal allograft recipients, we used simvastatin at different doses (0.1 to 20 mg/kg/day orally) to assess its effect on OAD development. No immunosuppressive treatment was administered. Histologic, immunohistochemical and real-time RT-PCR analyses were performed 3, 10 and 30 days after transplantation. RESULTS: Simvastatin treatment with doses ranging from 0.5 to 20 mg/kg/day significantly enhanced early epithelial recovery and reduced the development of OAD. No dose response was observed. Simvastatin treatment markedly reduced IL-23 mRNA and lymphocyte chemokine CCL20 production, and the infiltration of CD4(+) and CD8(+) T cells into allografts already at 3 days. At 10 days, simvastatin significantly attenuated the production of pro-inflammatory cytokines, IL-1ß, TNF-α, MCP-1 and IP-10, and Th17-polarizing cytokines, IL-6 and IL-17e, and inhibited allograft infiltration by inflammatory cells. The protective effects of simvastatin on inflammation and OAD were partially mediated through nitric oxide synthase. CONCLUSIONS: Simvastatin treatment inhibited adaptive T-cell alloimmune activation as depicted by reduced expression of lymphocyte chemokine and pro-inflammatory cytokine mRNA and reduced allograft infiltration by inflammatory cells. Importantly, simvastatin inhibits the development of OAD and this effect is partially mediated by increased nitric oxide activity. These results suggest a role for simvastatin in the prevention of obliterative bronchiolitis.

A functional complement system is required for normal T helper cell differentiation.

Complement is a fundamental part of the innate immune system, and also modulates B cell responses. Its effects on T cells, however, are less well studied. Here we have studied antigen-specific T cell responses in C3-knockout (C3-KO) C57BL/6 mice. The animals were immunized with ovalbumin (OVA) in complete Freund's adjuvant, which favors T helper 1 (Th1)-type responses. Splenic lymphocytes from C3-KO mice proliferated less in response to OVA stimulation than splenocytes from control wild type (WT) mice. The response in the C3-KO mice was also qualitatively different. The expression of Th1 lineage determining transcription factor T-bet was decreased in OVA-stimulated splenocytes, and the induction of Th1-associated IgG subclasses impaired. In WT mice T cell proliferation in response to OVA was positively correlated with antigen-specific IgG2a and IgG3 levels. In C3-KO mice the proliferative response correlated with antigen-specific IgE levels, consistent with Th2 deviation. The expression of Th1-inducing cytokines IL-12 and IFN-γ was also decreased in the collecting lymph nodes in the C3-KO mice after immunization. Our results show that the complement system and its component C3 participate in the regulation of T cell responses, and that complement function is required for normal T helper cell differentiation.

Murine model of food allergy after epicutaneous sensitization: role of mucosal mast cell protease-1.

OBJECTIVE: Studies of the pathological mechanisms of food allergy have been impeded by the lack of relevant animal models. The purpose of this study was to develop a physiological model of food allergy that was not dependent on immunostimulatory adjuvants. MATERIAL AND METHODS: Balb/c mice were epicutaneously sensitized four times at varying intervals over a 22-day period, and challenged orally from day 40, 6 times every 1-3 days with either saline or ovalbumin. RESULTS: After sensitization (day 35) but before the oral challenges, the ovalbumin-sensitized groups showed increased specific IgE and IgG1 production when compared with the sham-sensitized groups. Mucosal mast cell protease-1 (MMCP-1) was undetectable in serum before the intragastric challenge. MMCP-1 concentrations were increased after the first ovalbumin dose, solely in the ovalbumin-sensitized and -challenged group. After the challenge period, the mean serum MMCP-1 concentration increased from an undetectable level in controls to an over 44-fold level in the ovalbumin-sensitized and -challenged mice. In this group, MMCP-1-positive cells were present in the small intestine and expressions of IFN-gamma and CXCL-9 mRNA were decreased in the ileum, suggesting an impaired Th-1-type response. Within one hour of the last ovalbumin challenge, 5 out of 6 mice developed diarrhea in the ovalbumin-sensitized and -challenged group, but there was no diarrhea in the other groups. CONCLUSIONS: A murine model of food allergy based on sensitization via epicutaneous exposure to allergen without immunostimulatory adjuvants was developed. Effective production of MMCP-1 together with specific IgE and IgG1 suggests a breakdown in oral tolerance to the allergen. Intragastric challenges were accompanied by mast cell-dependent immunopathological changes and diarrhea.

Napins, 2S albumins, are major allergens in oilseed rape and turnip rape.

BACKGROUND: Children with IgE-mediated allergy to foods frequently react to seeds of oilseed rape (Brassica napus ssp. oleifera) and turnip rape (Brassica rapa ssp. oleifera) in skin prick tests (SPTs). Sensitization pathways are not known. OBJECTIVE: We identified possible major allergens in oilseed rape and turnip rape using sera from 72 atopic children (mean age, 3.3 years) with positive SPT responses to oilseed rape and turnip rape. METHODS: Allergens from oilseed rape and turnip rape seed extracts were purified by using gel filtration and cation exchange chromatography and characterized by means of reversed-phase chromatography, N-terminal amino acid sequencing, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. IgE binding of sera from 72 children with positive SPT reactions to oilseed rape and turnip rape and 72 age- and sex-matched atopic control subjects with negative SPT responses were analyzed by means of IgE ELISA and immunoblotting. In vivo reactivity of the purified allergens was tested with SPTs in 6 children. RESULTS: In IgE immunoblotting and IgE ELISA major reactivity was to a group of homologous, approximately 9.5- to 14.5-kd proteins. These allergens were identified as 2S albumins, also known as napins, by means of N-terminal amino acid sequencing. In ELISA approximately 80% of the patients had IgE to purified napins from both plants. In SPTs purified napins caused positive reactions in all 6 children tested. CONCLUSIONS: This study shows that 2S albumins in oilseed rape and turnip rape are new potential food allergens. Further studies are needed to clarify the routes of exposure and mechanisms of sensitization.

Enzymic degradation of a beta-lactam antibiotic, ampicillin, in the gut: a novel treatment modality.

Antibiotics can cause severe alterations in the gut microflora and promote diarrhoea and overgrowth of pathogenic bacteria. The present study investigated the potency of targeted recombinant beta-lactamase (TRBL) to degrade a beta-lactam antibiotic in the jejunum of fistula-operated beagles. We used different peroral doses of purified beta-lactamase (PenP) of Bacillus licheniformis in enteric-coated pellets together with intravenous ampicillin. Serum and jejunal samples were collected for ampicillin and beta-lactamase analysis. A dose-response effect of TRBL on ampicillin concentrations in the jejunal samples could be observed. The highest doses applied decreased the jejunal ampicillin concentrations to undetectable levels. In the serum samples, the ampicillin concentrations were not affected by the beta-lactamase dose used. Our results indicate that it may be possible to evolve a targeted treatment to degrade beta-lactam antibiotics intestinally and, thus, decrease antibiotic-induced adverse effects on the gut microflora.