Tropospheric winds from northeastern China carry the etiologic agent of Kawasaki disease from its source to Japan.

Evidence indicates that the densely cultivated region of northeastern China acts as a source for the wind-borne agent of Kawasaki disease (KD). KD is an acute, coronary artery vasculitis of young children, and still a medical mystery after more than 40 y. We used residence times from simulations with the flexible particle dispersion model to pinpoint the source region for KD. Simulations were generated from locations spanning Japan from days with either high or low KD incidence. The postepidemic interval (1987-2010) and the extreme epidemics (1979, 1982, and 1986) pointed to the same source region. Results suggest a very short incubation period (<24 h) from exposure, thus making an infectious agent unlikely. Sampling campaigns over Japan during the KD season detected major differences in the microbiota of the tropospheric aerosols compared with ground aerosols, with the unexpected finding of the Candida species as the dominant fungus from aloft samples (54% of all fungal strains). These results, consistent with the Candida animal model for KD, provide support for the concept and feasibility of a windborne pathogen. A fungal toxin could be pursued as a possible etiologic agent of KD, consistent with an agricultural source, a short incubation time and synchronized outbreaks. Our study suggests that the causative agent of KD is a preformed toxin or environmental agent rather than an organism requiring replication. We propose a new paradigm whereby an idiosyncratic immune response, influenced by host genetics triggered by an environmental exposure carried on winds, results in the clinical syndrome known as acute KD.

Extracellular microRNA-21 and microRNA-26a increase in body fluids from rats with antigen induced pulmonary inflammation and children with recurrent wheezing.

BACKGROUND: This study aims to find out whether extracellular miRNAs is implicated in recurrent childhood wheezing with asthmatic risk. METHODS: One hundred and forty children of Chinese Han population were recruited for this study. Plasma and intracellular miRNAs from children with recurrent wheezing and rats with antigen induced pulmonary inflammation (AIPI) were detected by using reverse transcription-quantitative PCR. Differential leukocytes in blood were automatically counted. Total IgE was detected by enzyme-linked immunosorbent assay. Clinical implication in diagnosis was evaluated using receiver operating characteristic curves. RESULTS: The increase of plasma miR-21 and miR-26a was screened out from 11 candidate miRNAs and validated in wheezing children. The level of expression for both miRNAs were comparable in different age and gender. Plasma miR-21 was more preferable to miR-26a and total IgE for diagnosis. Plasma miR-21 and miR-26a levels were not significantly correlated with various leukocyte counts or miRNA expression in blood cells. In acute and chronic AIPI rats, miR-21 levels increased in both plasma and lavaged lung compared with control. Moreover, circulating miR-21 and miR-26a levels were highly positively correlated with infiltrated cell counts in bronchoalveolar lavage fluid of AIPI rats. CONCLUSIONS: Circulating miR-21 and miR-26a increase in wheezing children and AIPI rats. This not only manifests their strong clinical implication in recurrent childhood wheezing with asthma risk, but also provides novel insights into the role of extracellular miRNAs during development of airway inflammation and recurrent wheezing.

IgE/antigen-mediated enhancement of IgE production is a mechanism underlying the exacerbation of airway inflammation and remodelling in mice.

IgE is known to enhance some antibody responses to specific antigens, but whether this contributes to allergic asthma remains unclear. We have previously found that repeated antigen challenges in mice sensitized with antigen-specific IgE monoclonal antibody (mAb) exacerbated airway inflammation and remodelling accompanied by increased levels of endogenous antigen-specific IgE and IgG1. Here, we investigated whether IgE/antigen-mediated enhancement of endogenous IgE production contributes to the exacerbation of airway inflammation and remodelling. BALB/c mice passively sensitized with ovalbumin (OVA) -specific IgE mAb were challenged with OVA intratracheally seven times; anti-IgE mAb was intraperitoneally administered 1 day before the fourth challenge. Treatment with anti-IgE mAb inhibited the increased level of endogenous OVA-specific IgE in serum, but not OVA-specific IgG1, and a biphasic increase in airway resistance at the fourth challenge. Furthermore, a biphasic increase in airway resistance, airway hyper-responsiveness to methacholine, OVA-specific IgE and IgG1 production, and infiltrations by neutrophils and eosinophils in the lungs at the seventh challenge were suppressed by treatment; airway remodelling, such as goblet cell hyperplasia and sub-epithelial fibrosis, was also reduced. In addition, the production of interleukin-17A, interleukin-33 and CXCL1 in the lungs related to these IgE-mediated responses was decreased by treatment. Collectively, we found that the mechanism leading to the exacerbation of allergic asthma is closely related to IgE/antigen-mediated enhancement of IgE production, suggesting that this may create a vicious circle leading to the chronic status in asthmatic patients having levels of antigen-specific IgE ready to form complexes with antigen.

Antigen-specific regulation of IgE antibodies by non-antigen-specific γδ T cells.

We re-examined the observation that γδ T cells, when transferred from mice tolerized to an inhaled conventional Ag, suppress the allergic IgE response to this Ag specifically. Using OVA and hen egg lysozyme in crisscross fashion, we confirmed the Ag-specific IgE-regulatory effect of the γδ T cells. Although only Vγ4(+) γδ T cells are regulators, the Ag specificity does not stem from specificity of their γδ TCRs. Instead, the Vγ4(+) γδ T cells failed to respond to either Ag, but rapidly acquired Ag-specific regulatory function in vivo following i.v. injection of non-T cells derived from the spleen of Ag-tolerized mice. This correlated with their in vivo Ag acquisition from i.v. injected Ag-loaded splenic non-T cells, and in vivo transfer of membrane label provided evidence for direct contact between the injected splenic non-T cells and the Vγ4(+) γδ T cells. Together, our data suggest that Ag itself, when acquired by γδ T cells, directs the specificity of their IgE suppression.

Upregulated protein arginine methyltransferase 1 by IL-4 increases eotaxin-1 expression in airway epithelial cells and participates in antigen-induced pulmonary inflammation in rats.

Protein arginine methyltransferases (PRMTs), catalyzing methylation of both histones and other cellular proteins, have emerged as key regulators of various cellular processes. This study aimed to identify key PRMTs involved in Ag-induced pulmonary inflammation (AIPI), a rat model for asthma, and to explore the role of PRMT1 in the IL-4-induced eosinophil infiltration process. E3 rats were i.p. sensitized with OVA/alum and intranasally challenged with OVA to induce AIPI. The expressions of PRMT1-6, eotaxin-1, and CCR3 in lungs were screened by real-time quantitative PCR. Arginine methyltransferase inhibitor 1 (AMI-1, a pan-PRMT inhibitor) and small interfering RNA-PRMT1 were used to interrupt the function of PRMT1 in A549 cells. In addition, AMI-1 was administrated intranasally to AIPI rats to observe the effects on inflammatory parameters. The results showed that PRMT1 expression was mainly expressed in bronchus and alveolus epithelium and significantly upregulated in lungs from AIPI rats. The inhibition of PRMTs by AMI-1 and the knockdown of PRMT1 expression were able to downregulate the expressions of eotaxin-1 and CCR3 with the IL-4 stimulation in the epithelial cells. Furthermore, AMI-1 administration to AIPI rats can also ameliorate pulmonary inflammation, reduce IL-4 production and humoral immune response, and abrogate eosinophil infiltration into the lungs. In summary, PRMT1 expression is upregulated in AIPI rat lungs and can be stimulated by IL-4. Intervention of PRMT1 activity can abrogate IL-4-dependent eotaxin-1 production to influence the pulmonary inflammation with eosinophil infiltration. The findings may provide experimental evidence that PRMT1 plays an important role in asthma pathogenesis.

Allogeneic mesenchymal stem cells prevent allergic airway inflammation by inducing murine regulatory T cells.

BACKGROUND: Adult bone marrow-derived mesenchymal stem cells (MSC) possess potent immune modulatory effects which support their possible use as a therapy for immune-mediated disease. MSC induce regulatory T cells (T(reg)) in vitro although the in vivo relevance of this is not clear. OBJECTIVE: This study addressed the hypothesis that adult bone marrow derived-MSC would prevent the pathology associated with allergen-driven airway inflammation, and sought to define the effector mechanism. METHODS: The influence of allogeneic MSC was examined in a model system where T(reg) induction is essential to prevent pathology. This was tested using a combination of a model of ovalbumin-driven inflammation with allogeneic MSC cell therapy. RESULTS: Systemic administration of allogeneic MSC protected the airways from allergen-induced pathology, reducing airway inflammation and allergen-specific IgE. MSC were not globally suppressive but induced CD4(+) FoxP3(+) T cells and modulated cell-mediated responses at a local and systemic level, decreasing IL-4 but increasing IL-10 in bronchial fluid and from allergen re-stimulated splenocytes. Moderate dose cyclophosphamide protocols were used to differentially ablate T(reg) responses; under these conditions the major beneficial effect of MSC therapy was lost, suggesting induction of T(reg) as the key mechanism of action by MSC in this model. In spite of the elimination of T(reg) , a significant reduction in airway eosinophilia persisted in those treated with MSC. CONCLUSION: These data demonstrate that MSC induce T(reg) in vivo and reduce allergen-driven pathology. Multiple T(reg) dependent and independent mechanisms of therapeutic action are employed by MSC.

Inflammatory role of ASC in antigen-induced arthritis is independent of caspase-1, NALP-3, and IPAF.

Because IL-1beta plays an important role in inflammation in human and murine arthritis, we investigated the contribution of the inflammasome components ASC, NALP-3, IPAF, and caspase-1 to inflammatory arthritis. We first studied the phenotype of ASC-deficient and wild-type mice during Ag-induced arthritis (AIA). ASC(-/-) mice showed reduced severity of AIA, decreased levels of synovial IL-1beta, and diminished serum amyloid A levels. In contrast, mice deficient in NALP-3, IPAF, or caspase-1 did not show any alteration of joint inflammation, thus indicating that ASC associated effects on AIA are independent of the classical NALP-3 or IPAF inflammasomes. Because ASC is a ubiquitous cytoplasmic protein that has been implicated in multiple cellular processes, we explored other pathways through which ASC may modulate inflammation. Ag-specific proliferation of lymph node and spleen cells from ASC-deficient mice was significantly decreased in vitro, as was the production of IFN-gamma, whereas IL-10 production was enhanced. TCR ligation by anti-CD3 Abs in the presence or absence of anti-CD28 Abs induced a reduction in T cell proliferation in ASC(-/-) T cells compared with wild-type ones. In vivo lymph node cell proliferation was also significantly decreased in ASC(-/-) mice, but no effects on apoptosis were observed either in vitro or in vivo in these mice. In conclusion, these results strongly suggest that ASC modulates joint inflammation in AIA through its effects on cell-mediated immune responses but not via its implication in inflammasome formation.

Compositional optimization and safety assessment of a hydrogel patch as a transcutaneous immunization device.

The development of a simple, easy-to-use, and non-invasive vaccination system is in high demand. For transcutaneous immunization (TCI), we previously developed a hydrogel patch formulation that accelerates the penetration of an antigen (Ag) through the stratum corneum (SC) and effectively elicits Ag-specific immune responses. The present studies were performed to optimize the composition and assess the safety of the patch formulation. A hydrogel patch with a water content ratio of 5% more effectively induced an immune response compared to patches with a different composition, suggesting that the moisture content of the hydrogel patch formulation has optimal ratio for SC hydration to promote Ag penetration through the SC. TCI using a hydrogel patch induced few local and systemic adverse reactions. Based on these results, we are now advancing translational research to evaluate the safety and efficacy of our novel TCI system in humans.

Biological and Inflammatory Effects of Antigen 5 from Polybia paulista (Hymenoptera, Vespidae) Venom in Mouse Intraperitoneal Macrophages.

The social wasp Polybia paulista (Hymenoptera, Vespidae) is highly aggressive, being responsible for many medical occurrences. One of the most allergenic components of this venom is Antigen 5 (Poly p 5). The possible modulation of the in vitro immune response induced by antigen 5 from P. paulista venom, expressed recombinantly (rPoly p 5), on BALB/c mice peritoneal macrophages, activated or not with LPS, was assessed. Here, we analyzed cell viability changes, expression of the phosphorylated form of p65 NF-κB subunit, nitric oxide (NO), proinflammatory cytokines production, and co-stimulatory molecules (CD80, CD86). The results suggest that rPoly p 5 does not affect NO production nor the expression of co-stimulatory molecules in mouse peritoneal macrophages. On the other hand, rPoly p 5 induced an increase in IL-1ß production in non-activated macrophages and a reduction in the production of TNF-α and MCP-1 cytokines in activated macrophages. rPoly p 5 decreased the in vitro production of the phosphorylated p65 NF-κB subunit in non-activated macrophages. These findings suggest an essential role of this allergen in the polarization of functional M2 macrophage phenotypes, when analyzed in previously activated macrophages. Further investigations, mainly in in vivo studies, should be conducted to elucidate Polybia paulista Ag5 biological role in the macrophage functional profile modulation.

Reduced incidence and severity of antigen-induced autoimmune diseases in mice lacking interferon regulatory factor-1.

Interferon regulatory factor-1 (IRF-1) is a transcription factor that regulates interferon-induced genes and type I interferons. Recently, studies of IRF-l-deficient mice have revealed that IRF-I regulates the induction of molecules that play important roles in inflammation, such as inducible nitric oxide synthase (iNOS) and interleukin-l beta-converting enzyme (ICE). To study the role of IRF-1 in autoimmunity, we investigated type II collagen-induced arthritis (CIA), and experimental allergic encephalomyelitis (EAE), in mice lacking IRF-1. The incidence and severity of CIA were significantly decreased in IRF-1-/- mice compared with IRF-l +/- mice, as was the production of interferon (IFN)-gamma in lymph node cells. Both IRF-l+/- and IRF-1-/- mice exhibited mild and transient disease after adoptive transfer of a type II collagen (CII)-specific T cell line together with sera from arthritic mice, but the IRF-1-/- mice were less severely affected than the IRF-1+/- mice. In addition, the incidence of EAE in IRF-1-/- mice was decreased as compared with IRF-1 +/- mice. Reverse transcription polymerase chain reaction showed that IRF-1 mRNA was constitutively expressed in the spinal cords of IRF-1+/- mice, and was upregulated in mice with clinical EAE. Expression of iNOS was also detected in inflamed spinal cords. These results suggest that IRF-I plays a key role in promoting inflammation and autoimmunity in CIA and EAE animal models.

Interleukin 12 inhibits antigen-induced airway hyperresponsiveness, inflammation, and Th2 cytokine expression in mice.

Allergic asthma is characterized by airway hyperresponsiveness and pulmonary eosinophilia, and may be mediated by T helper (Th) lymphocytes expressing a Th2 cytokine pattern. Interleukin (IL) 12 suppresses the expression of Th2 cytokines and their associated responses, including eosinophilia, serum immunoglobulin E, and mucosal mastocytosis. We have previously shown in a murine model that antigen-induced increases in airway hyperresponsiveness and pulmonary eosinophilia are CD4+ T cell dependent. We used this model to determine the ability of IL-12 to prevent antigen-induced increases in airway hyperresponsiveness, bronchoalveolar lavage (BAL) eosinophils, and lung Th2 cytokine expression. Sensitized A/J mice developed airway hyperresponsiveness and increased numbers of BAL eosinophils and other inflammatory cells after single or repeated intratracheal challenges with sheep red blood cell antigen. Pulmonary mRNA and protein levels of the Th2 cytokines IL-4 and IL-5 were increased after antigen challenge. Administration of IL-12 (1 microgram/d x 5 d) at the time of a single antigen challenge abolished the airway hyperresponsiveness and pulmonary eosinophilia and promoted an increase in interferon (IFN) gamma and decreases in IL-4 and IL-5 expression. The effects of IL-12 were partially dependent on IFN-gamma, because concurrent treatment with IL-12 and anti-IFN-gamma monoclonal antibody partially reversed the inhibition of airway hyperresponsiveness and eosinophilia by IL-12. Treatment of mice with IL-12 at the time of a second antigen challenge also prevented airway hyperresponsiveness and significantly reduced numbers of BAL inflammatory cells, reflecting the ability of IL-12 to inhibit responses associated with ongoing antigen-induced pulmonary inflammation. These data show that antigen-induced airway hyperresponsiveness and inflammation can be blocked by IL-12, which suppresses Th2 cytokine expression. Local administration of IL-12 may provide a novel immunotherapy for the treatment of pulmonary allergic disorders such as atopic asthma.

Pre-conceptional exposure to multiwalled carbon nanotubes suppresses antibody production in mouse offspring.

Prenatal particle exposure has been shown to increase allergic responses in offspring. Carbon nanotubes (CNTs) possess immunomodulatory properties, but it is unknown whether maternal exposure to CNTs interferes with offspring immune development. Here, C57Bl/6J female mice were intratracheally instilled with 67 of µg multiwalled CNTs on the day prior to mating. After weaning, tolerance and allergy responses were assessed in the offspring. Offspring of CNT-exposed (CNT offspring) and of sham-exposed dams (CTRL offspring) were intranasally exposed to ovalbumin (OVA) once weekly for 5 weeks to induce airway mucosal tolerance. Subsequent OVA sensitization and aerosol inhalation caused low or no OVA-specific IgE production and no inflammation. However, the CNT offspring presented with significantly lower OVA-specific IgG1 levels than CTRL offspring. In other groups of 5-week-old offspring, low-dose sensitization with OVA and subsequent OVA aerosol inhalation led to significantly lower OVA-specific IgG1 production in CNT compared to CTRL offspring. OVA-specific IgE and airway inflammation were non-significantly reduced in CNT offspring. The immunomodulatory effects of pre-gestational exposure to multiwalled CNTs were unexpected, but very consistent. The observations of suppressed antigen-specific IgG1 production may be of importance for infection or vaccination responses and warrant further investigation.

Generation of Highly Efficient Equine-Derived Antibodies for Post-Exposure Treatment of Ricin Intoxications by Vaccination with Monomerized Ricin.

Ricin, a highly lethal toxin derived from the seeds of Ricinus communis (castor beans) is considered a potential biological threat agent due to its high availability, ease of production, and to the lack of any approved medical countermeasure against ricin exposures. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this work was to generate anti-ricin antitoxin that confers high level post-exposure protection against ricin challenge. Due to safety issues regarding the usage of ricin holotoxin as an antigen, we generated an inactivated toxin that would reduce health risks for both the immunizer and the immunized animal. To this end, a monomerized ricin antigen was constructed by reducing highly purified ricin to its monomeric constituents. Preliminary immunizing experiments in rabbits indicated that this monomerized antigen is as effective as the native toxin in terms of neutralizing antibody elicitation and protection of mice against lethal ricin challenges. Characterization of the monomerized antigen demonstrated that the irreversibly detached A and B subunits retain catalytic and lectin activity, respectively, implying that the monomerization process did not significantly affect their overall structure. Toxicity studies revealed that the monomerized ricin displayed a 250-fold decreased activity in a cell culture-based functionality test, while clinical signs were undetectable in mice injected with this antigen. Immunization of a horse with the monomerized toxin was highly effective in elicitation of high titers of neutralizing antibodies. Due to the increased potential of IgG-derived adverse events, anti-ricin F(ab')2 antitoxin was produced. The F(ab')2-based antitoxin conferred high protection to intranasally ricin-intoxicated mice; ~60% and ~34% survival, when administered 24 and 48 h post exposure to a lethal dose, respectively. In line with the enhanced protection, anti-inflammatory and anti-edematous effects were measured in the antitoxin treated mice, in comparison to mice that were intoxicated but not treated. Accordingly, this anti-ricin preparation is an excellent candidate for post exposure treatment of ricin intoxications.

Natural killer cells play an essential role in resolution of antigen-induced inflammation in mice.

This study examined whether NK cells are important for resolution of antigen-induced inflammation. C57BL/6 mice were immunized twice with methylated BSA (mBSA) and inflammation induced by intraperitoneal injection of mBSA. Mice were injected intravenously with anti-asialo GM1 (αASGM1) or a control antibody 24h prior to peritonitis induction and peritoneal exudate collected at different time points. Expression of surface molecules and apoptosis on peritoneal cells was determined by flow cytometry and concentration of chemokines, cytokines, soluble cytokine receptors and lipid mediators by ELISA and LC-MS/MS. Apoptosis in parathymic lymph nodes and spleens was determined by TUNEL staining. Mice administered αASGM1 had lower peritoneal NK cell numbers and a higher number of peritoneal neutrophils 12h after induction of inflammation than control mice. The number of neutrophils was still high in the αASGM1 treated mice when their number had returned to baseline levels in the control mice, 48h after induction of inflammation. Peritoneal concentrations of the neutrophil regulators G-CSF and IL-12p40 were higher at 12h in the αASGM1 treated mice than in the control mice, whereas concentrations of lipid mediators implicated in resolution of inflammation, i.e. LXA4 and PGE2, were lower. Reduced apoptosis was detected in peritoneal neutrophils as well as in draining lymph nodes and spleens from the αASGM1 treated mice compared with that in the control mice. In addition, αASGM1 treated mice had lower number of peritoneal NK cells expressing NKp46 and NKG2D, receptors implicated in NK cell-induced neutrophil apoptosis. Furthermore, αASGM1 treatment completely blocked the increase in CD27+ NK cells that occurred in control mice following induction of inflammation, but CD27+ NK cells have been suggested to have a regulatory role. These results indicate a crucial role for NK cells in resolution of antigen-induced inflammation and suggest their importance in tempering neutrophil recruitment and maintaining neutrophil apoptosis.

B and T lymphocyte attenuator inhibits antigen-induced eosinophil recruitment into the airways.

BACKGROUND: Signaling through CD28 family co-receptors regulates activation of CD4(+) T cells positively and negatively. It has been shown that stimulatory co-receptors such as CD28 and ICOS play critical roles in the induction of allergic airway inflammation. However, the role of B and T lymphocyte attenuator (BTLA), an inhibitory co-receptor expressed preferentially in Th1 cells, in the regulation of allergic airway inflammation remains to be determined. METHODS: We examined antigen-induced eosinophil recruitment and cytokine production in the airways in antigen-sensitized BTLA-deficient (BTLA-/-) mice. We also examined antigen-induced cytokine production and cell proliferation of splenic T cells in antigen-sensitized BTLA-/- mice. RESULTS: Antigen-induced eosinophil recruitment and IL-5 production in the airways was enhanced in antigen-sensitized BTLA-/- mice. On the other hand, antigen-induced Th1 and Th2 cytokine production as well as T cell proliferation of splenocytes was normal in BTLA-/-mice. CONCLUSION: BTLA inhibits antigen-induced eosinophil recruitment into the airways by preventing IL-5 production from Th2 cells.

Investigation of the immunogenicity of diclofenac and diclofenac metabolites.

Oral administration of the non-steroidal anti-inflammatory drug diclofenac (DCF) is associated with a high incidence of adverse drug reactions, some of which are thought to be mediated by the immune system. It has been proposed that metabolic activation of DCF and covalent binding to protein generates an antigenic determinant that stimulates immune cells; however, the nature of the metabolite remains ill-defined. The aim of this study was to synthesize and evaluate the antigenic potential of DCF metabolites in the mouse. DCF and DCF metabolites were administered via subcutaneous injection over a 5-day period to BALB/C strain mice to induce immune activation. Proliferation was measured by the addition of [(3)H] thymidine to ex vivo isolated draining auricular lymph node cells. Results were compared with those provoked by exposure to 2,4-dinitrochlorobenzene. Lymph node activation was observed following treatment with 2,4-dinitrochlorobenzene, 5-hydroxy DCF quinoneimine and 4'-hydroxy DCF quinoneimine, but not DCF acyl glucuronide or DCF itself. Interestingly, lymph node cells from 5-hydroxy DCF treated mice were also found to proliferate, when compared with cells from vehicle-treated mice, while 4'-hydroxy DCF did not stimulate lymph node cell activation. The reactivity of 5-hydroxy DCF quinoneimine was confirmed by synthesis and characterization of an N-acetyl cysteine adduct. These data show that formation of 5-hydroxy DCF and subsequent autoxidation provides an antigenic determinant for immune cell activation in the mouse.

Protective effect of beta-glucan extracted from Saccharomyces cerevisiae, against DNA damage and cytotoxicity in wild-type (k1) and repair-deficient (xrs5) CHO cells.

A large number of functional foods, including those that contain beta-glucan, have been shown to prevent the development of cancer and other chronic diseases. The aim of the present study was to elucidate its mechanism of action, as well as to understand its effects as an antigenotoxic, anticlastogenic agent, and to determine its capacity to preserve cell viability. The investigation was carried out in the CHO-k1 and CHO-xrs5 cell lines. The cytokinesis-blocked micronucleus assay indicated that the different doses of beta-glucan examined (5, 10, 20 and 40 microg/ml) did not show clastogenic effects. In the CHO-k1 cell line, a chemopreventive effect could be observed in all the protocols tested: pre-treatment (% reduction of 35.0-57.3), simultaneous treatment (simple--5 reduction of 19.7-55.6 and with pre-incubation--of 42.7-56.4) and post-treatment (% reduction of 17.9-37.6). This finding indicates mechanisms of action involving desmutagenesis and bioantimutagenesis, albeit the latter having a lesser role. However, in the repair-deficient CHO-xrs5 cells, beta-glucan did not show a protective effect with post-treatment (% reduction of 2.96), thus supporting the involvement of bioantimutagenesis. The comet assay in CHO-k1 cells demonstrated that beta-glucan has neither a genotoxic nor an antigenotoxic effect. Cell viability tests indicated that beta-glucan preserves cell viability in both cell lines, preventing apoptotic events. These findings suggest that beta-glucan, when present in foods, could provide them with nutraceutical characteristics and act as a dietary supplement, or that beta-glucan could be used in new drug development.

Design and evaluation of immunotoxicity studies.

The evaluation of potential adverse effects of pharmaceuticals on the immune system is part of the standard drug development procedures and needs to be available prior to the start of phase III clinical trials. Although the histopathological assessment of lymphoid organs/tissues is considered fundamental for the identification and characterization of immunotoxic reactions, additional investigations are now recommended by the European guidelines for repeated-dose toxicity testing of medicinal products in order to achieve an accurate assessment of immune system functionality with regard to immunomodulation. In the present paper, we describe and discuss a study design which permits the investigation of the immune function in a 4-week study in rats following immunization by subcutaneous administration of the T-dependent antigen Keyhole Limpet Hemocynin (KLH). This includes assessment of hematology parameters, titration of KLH-specific antibodies in serum, lymphocyte immunophenotyping in blood, thymus, spleen and lymph nodes, macroscopic pathology and histopathological evaluation of the lymphatic organs/tissues and of the injection sites.

ß1-Adrenergic and M2 Muscarinic Autoantibodies and Thyroid Hormone Facilitate Induction of Atrial Fibrillation in Male Rabbits.

Activating autoantibodies to the ß1-adrenergic and M2 muscarinic receptors are present in a very high percentage of patients with Graves' disease and atrial fibrillation (AF). The objective of this study was to develop a reproducible animal model and thereby to examine the impact of these endocrine-like autoantibodies alone and with thyroid hormone on induction of thyroid-associated atrial tachyarrhythmias. Five New Zealand white rabbits were coimmunized with peptides from the second extracellular loops of the ß1-adrenergic and M2 muscarinic receptors to produce both sympathomimetic and parasympathomimetic antibodies. A catheter-based electrophysiological study was performed on anesthetized rabbits before and after immunization and subsequent treatment with thyroid hormone. Antibody expression facilitated the induction of sustained sinus, junctional and atrial tachycardias, but not AF. Addition of excessive thyroid hormone resulted in induced sustained AF in all animals. AF induction was blocked acutely by the neutralization of these antibodies with immunogenic peptides despite continued hyperthyroidism. The measured atrial effective refractory period as one parameter of AF propensity shortened significantly after immunization and was acutely reversed by peptide neutralization. No further decrease in the effective refractory period was observed after the addition of thyroid hormone, suggesting other cardiac effects of thyroid hormone may contribute to its role in AF induction. This study demonstrates autonomic autoantibodies and thyroid hormone potentiate the vulnerability of the heart to AF, which can be reversed by decoy peptide therapy. These data help fulfill Witebsky's postulates for an increased autoimmune/endocrine basis for Graves' hyperthyroidism and AF.

Effects of bemiparin on airway responses to antigen in sensitized Brown-Norway rats.

Heparins have demonstrated activity in asthma. The effects of bemiparin, a low molecular weight heparin, were examined on antigen-induced responses in sensitized Brown-Norway rats. Inhaled bemiparin (1 mg/ml) reduced the acute bronchospasm produced by aerosol antigen, prevented airway hyperresponsiveness to 5-hydroxytryptamine postantigen exposure, and reduced the eosinophil count (from 0.205+/-0.062 to 0.054+/-0.016 x 10(6) cells/ml in antigen and antigen+bemiparin groups, respectively; P<0.05), eosinophil peroxidase activity, and proteins in the bronchoalveolar lavage fluid (BALF), as well as the transiently augmented mucin Muc5ac expression. Hyperresponsiveness to adenosine was not affected by bemiparin. In similar experiments, inhaled fondaparinux (1 mg/ml) did not affect the antigen-induced responses, while a low-anticoagulant low molecular weight heparin was effective. In conclusion, bemiparin showed beneficial effects in experimental asthma, probably unrelated to its anticoagulant activity, which extends the previous positive findings obtained with other heparins.