Alleviated cerebral infarction in male mice lacking all nitric oxide synthase isoforms after middle cerebral artery occlusion.

PURPOSE: The role of the nitric oxide synthases (NOSs) system in cerebral infarction has been examined in pharmacological studies with non-selective NOSs inhibitors. However, due to the non-specificity of the non-selective NOSs inhibitors, its role remains to be fully elucidated. We addressed this issue in mice in which neuronal, inducible, and endothelial NOS isoforms were completely disrupted. METHODS AND RESULTS: We newly generated mice lacking all three NOSs by crossbreeding each single NOS-/- mouse. In the male, cerebral infarct size at 24 h after middle cerebral artery occlusion (MCAO) was significantly smaller in the triple n/i/eNOSs-/- genotype as compared with wild-type genotype. Neurological deficit score and mortality rate were also significantly lower in the triple n/i/eNOSs-/- than in the WT genotype. In contrast, in the female, there was no significant difference in the cerebral infarct size in the two genotypes. In the male triple n/i/eNOSs-/- genotype, orchiectomy significantly increased the cerebral infarct size, and in the orchiectomized male triple n/i/eNOSs-/- genotype, treatment with testosterone significantly reduced it. Cyclopaedic and quantitative comparisons of mRNA expression levels in cerebral infarct lesions between the male wild-type and triple n/i/eNOSs-/- genotypes at 1 h after MCAO revealed significant involvements of decreased oxidative stress and mitigated mitochondrial dysfunction in the alleviated cerebral infarction in the male triple n/i/eNOSs-/- genotype. CONCLUSIONS: These results provide the first evidence that the NOSs system exerts a deleterious effect against acute ischemic brain injury in the male.

Oral allergy induction through skin exposure to previously tolerated food antigens in murine models.

Food allergies (FAs) are caused by a failure of the immune system to regulate oral tolerance (OT). The use of soap containing hydrolyzed wheat overrides acquired OT to wheat through skin exposure. However, in mouse models, the experimental OT is robust, suggesting that acquired OT to allergens prevents the development of FAs. We aimed to analyze the mechanisms and developed a mouse model of FA that overrides acquired OT via skin exposure. Three murine FA models (intraperitoneal [IP], epicutaneous [EC], and intradermal [ID]) were compared to evaluate if allergies to ovalbumin (OVA) that had been previously tolerated orally could be induced. In the ID model, OT was overridden, and allergic reactions of severe anaphylaxis were developed. To analyze this effect in the ID model, we measured the migration of dendritic cells (DCs) into lymph nodes. The induction of OT promoted the migration of CD103+ dermal DCs; moreover, repeated percutaneous doses of OVA for sensitization gradually increased the migration of CD11b+ dermal DCs. The difference in the proportion of regulatory T cells between ID-sensitized groups at the first ID injection disappeared at the tenth injection. Although OT was robust in the IP model, ID sensitization was found to override OT.

Food allergy is linked to season of birth, sun exposure, and vitamin D deficiency.

The season of birth and ultraviolet B exposure have been related to the occurrence of food allergy. The levels of vitamin D produced from skin by ultraviolet B exposure might reflect this relationship. Vitamin D is known to induce antimicrobial peptides, protect intestinal flora, enhance the gut epithelial barrier, suppress mast cell activation and IgE synthesis from B cells, and increase the number of tolerogenic dendritic cells and IL-10-producing regulatory T cells. Vitamin D deficiency has been shown to exacerbate sensitization and allergic symptoms in a murine model of food allergy. However, in clinical situations, contradictory observations have been reported regarding the relationship between food allergy and vitamin D deficiency/supplementation. In this review, we have explored the links between food allergy and vitamin D levels. One explanation for the discrepant findings is confounding factors such as race, age, residency, skin color, and epigenetic changes that contribute to vitamin D levels. In addition, the season of birth influences the development of atopic dermatitis, which could lead to food sensitization. Finally, ultraviolet radiation could lead to regulatory T cell expansion and immunosuppression, irrespective of vitamin D status. Based on our current understanding, we believe that correction of vitamin D deficiency by supplementation, appropriate skin care, and sufficient ultraviolet radiation exposure could alter the prognosis of food allergy. To identify potential treatment strategies for food allergy, it is essential to gain a better understanding of the appropriate levels of vitamin D and ultraviolet radiation exposure.

Immune suppression of food allergy by maternal IgG in murine models.

BACKGROUND: Most of the patients develop food allergy early in life. The factors related to parental immune condition might be one of the conceivable causes. METHODS: We reported murine models of food allergy and oral OVA tolerance. To investigate the influence of parental immune condition on infant food allergy, female and male mice with food allergy or oral tolerance were mated with each other. RESULTS: Food allergy was suppressed by decreased IgE production in the offspring of mice with food allergy. On the contrary, anaphylaxis for OVA was induced in the offspring of mice with oral tolerance. The suppression of food allergy being dependent on a maternal factor was revealed in the offspring after cross-mating mice with food allergy and oral tolerance. Because OVA-specific IgG, presumed to be from the allergic mother, was detected in the serum of naïve infants from mothers allergic to food, we assumed that the suppression was dependent on a specific IgG. The serum IgG purified by a G-protein column was administered before OVA sensitization in the food allergy model, and OVA-specific IgE production was found to be diminished in the administered mice. However, OVA-specific monoclonal IgG1 and IgG2a administration could not suppress food allergy. Because we detected OVA-IgG immune complex in the serum of mothers allergic to food, it might be a cause of maternal immune suppression. CONCLUSIONS: We demonstrated that maternal specific IgG conjugated food antigen is an important factor related to the development of food allergy and acquiring tolerance.

Eppikajutsuto Protects against Food Allergy Induced by Ovalbumin in a Murine Model.

BACKGROUND: Currently, there are no efficient medications available for the prevention and treatment of food allergy (FA). Herbal medicines, including traditional Japanese Kampo medicines (TJKMs), are promising therapeutic drugs. METHODS: We screened 18 TJKMs for treatment of FA symptoms in a mouse FA model induced by ovalbumin (OVA). BALB/c mice were sensitized intraperitoneally by an OVA/aluminum hydroxide gel mixture followed by 4 booster doses of oral OVA and FA symptom induction by 50 mg of OVA. TJKMs were orally administered for 28 days from the day of sensitization to the day before FA symptom induction. Evaluated FA symptoms included a decrease in body temperature and allergic diarrhea. Allergic sensitization was determined by plasma OVA-specific IgE levels. Cytokine mRNA levels in mesenteric lymph nodes, plasma mouse mast cell protease-1, and the number of mast cells in the small and large intestines were analyzed. Additionally, the therapeutic effect of the TJKM eppikajutsuto (EJT) on mast cell degranulation was determined in active anaphylaxis and passive cutaneous anaphylaxis models. RESULTS: EJT effectively prevented FA symptoms. Although OVA-specific IgE levels and the intestinal mast cell numbers were not different between the EJT-treated and untreated FA mice, plasma mMcpt1 and IL-4 levels were lower in EJT-treated FA mice than untreated FA mice. EJT could alleviate symptoms in both active and passive anaphylaxis models. CONCLUSION: EJT prevented OVA-induced FA symptoms in a mouse model, suggesting that EJT might exert its therapeutic activity via IL-4 suppression and the inhibition of mucosal mast cell degranulation.

Oligomerization-function relationship of EGFR on living cells detected by the coiled-coil labeling and FRET microscopy.

The epidermal growth factor receptor (EGFR) is a well-studied receptor tyrosine kinase and an important anticancer therapeutic target. The activity of EGFR autophosphorylation and transphosphorylation, which induces several cell signaling pathways, has been suggested to be related to its oligomeric state. However, the oligomeric states of EGFRs induced by EGF binding and the receptor-ligand stoichiometry required for its activation are still controversial. In the present study, we performed Förster resonance energy transfer (FRET) measurements by combining the coiled-coil tag-probe labeling method and spectral imaging to quantitatively analyze EGFR oligomerization on living CHO-K1 cell membranes at physiological expression levels. In the absence of its ligands, EGFRs mainly existed as monomers with a small fraction of predimers (~10%), whereas ~70% of the EGFRs formed dimers after being stimulated with the ligand EGF. Ligand-induced dimerization was not significantly affected by the perturbation of membrane components (cholesterol or monosialoganglioside GM3). We also investigated both dose and time dependences of EGF-dependent EGFR dimerization and autophosphorylation. The formation of dimers occurred within 20s of the ligand stimulation and preceded its autophosphorylation, which reached a plateau 90 s after the stimulation. The EGF concentration needed to evoke half-maximum dimerization (~1 nM) was lower than that for half-maximum autophosphorylation (~8 nM), which suggested the presence of an inactive dimer binding a single EGF molecule.

Collagen gel contraction assay using human bronchial smooth muscle cells and its application for evaluation of inhibitory effect of formoterol.

Collagen gel contraction assay is a method for evaluating contraction of cells embedded in collagen gel matrices through measuring the gel size. In the present study, we established a protocol for collagen gel contraction assay using human bronchial smooth muscle cells obtained commercially, and applied it for evaluation of inhibitory effect of formoterol on histamine-induced contraction. Human bronchial smooth muscle cells were embedded in collagen gel in wells of 24-well plates, and gel contraction against histamine or acetylcholine was observed. Gel size was measured at an interval of 10 min for 60 min from the addition of a stimulant. Both acetylcholine and histamine caused gel contraction in a concentration-dependent manner and the contraction by histamine was apparently potent than that by acetylcholine. Formoterol at concentrations of 10(-10)-10(-7) M inhibited collagen gel contraction caused by histamine concentration-dependently. Pre-treatment with fluticasone at a concentration of 10(-8) M apparently potentiated the inhibitory effect of formoterol at 10(-10) and 10(-8) M on collagen gel contraction by histamine. Prolonged pre-treatment with 10(-8) M formoterol abolished the inhibitory effect of 10(-8) M formoterol. Furthermore, 4 h simultaneous pre-treatment with 10(-8) M formoterol and fluticasone partially but significantly recovered the inhibitory effect of 10(-8) M formoterol. Present results indicate that the collagen gel contraction assay using human bronchial smooth muscle cells is useful for evaluating the effects of bronchodilating drugs, and that fluticasone potentiates the inhibitory effect of formoterol on histamine-induced collagen gel contraction.

Treatment of the chronic itch of atopic dermatitis using standard drugs and kampo medicines.

Atopic dermatitis is a common skin disease accompanied by intense itching. Relapsing eczema is caused by immune imbalances and skin-barrier disruption. The immunopathy and barrier dysfunction are closely related to the onset of itching and subsequent scratching, and intractable dermatitis is amplified by the itch-scratch cycle. The standard therapy for atopic dermatitis is topical corticosteroids and immunosuppressants to lessen the inflammation, along with moisturizing agents to improve the physiologic skin dysfunction. Corticosteroids are the primary treatment for the inflammation in atopic dermatitis. Some clinical trials demonstrated a tendency for the alleviation of pruritus with long-term treatment. Tacrolimus results in instant burning and itching in the short term, but they resolve a few days after the beginning of use and then are relieved. Substance P is a neuropeptide released from sensory nerve fibers and a neurotransmitter of pain and itching. Basic experimental reports indicated that the antipruritic effect of tacrolimus is probably dependent on depleting substance P, followed by transient induction. Oral administration of antihistamines and antiallergics is used as adjunctive pharmacotherapy for pruritus. It is known that second-generation antihistamines are less sedative or nonsedative drugs compared with the first generation, and the drugs have additional efficacy in blocking some chemical mediators. Japanese traditional Kampo medicines are also used for the treatment of atopic dermatitis. This paper discusses the efficacy of representative Kampo medicines in the treatment of inflammation and itching based on murine atopic dermatitis models. Information on the mechanism of action of Kampo medicines will result in more choice of pharmacotherapeutic agents for complex diseases such as atopic dermatitis.

Transient receptor potential vanilloid 1 - a polymodal nociceptive receptor - plays a crucial role in formaldehyde-induced skin inflammation in mice.

Formaldehyde (FA) is irritating to the skin and is the main cause of sick building syndrome. However, the cutaneous reaction induced by long-term FA exposure has not been fully investigated. In our previous study, we demonstrated that repeated painting of 2% - 10% FA on mouse ears caused marked ear swelling and increased mRNA expression of transient receptor potential vanilloid 1 (TRPV1) and neurotrophins in the ear. TRPV1 is reported to be involved in neurogenic inflammation; therefore, in the present study, we investigated the role of TRPV1 in FA-induced skin inflammation using TRPV1 gene-knockout mice. Mice were painted with 5% FA once a week for 5 weeks, and ear swelling and mRNA expression were investigated. Ear swelling and increased expression of neurotrophins mRNA by FA provocation in wild-type mice were attenuated by disruption of the TRPV1 gene. Furthermore, painting with a threshold dose of capsaicin, which does not induce ear swelling in intact mice, caused marked ear swelling after painting the ear 5 times with FA, indicating that inflamed tissues after FA application are hypersensitive to various ligands of TRPV1 in mice. These results demonstrated that neurogenic inflammation via TRPV1 and neurotrophins could be involved in FA-induced dermatitis.

Lactobacillus acidophilus strain L-92 induces CD4(+)CD25(+)Foxp3(+) regulatory T cells and suppresses allergic contact dermatitis.

The anti-allergic mechanism of heat-killed Lactobacillus acidophilus strain L-92 has not been fully investigated. Recent studies have reported that CD4(+)CD25(+)Foxp3(+) (forkhead box P3) T regulatory (Treg) cells play important roles in controlling allergic diseases. Hence, we examined the effect of orally administered L-92 on CD4(+)CD25(+)Foxp3(+) cell populations. BALB/c mice were supplemented daily with L-92 by gavage for 5 weeks. 2,4-Dinitrofluorobenzene (DNFB) was used to induce allergic contact dermatitis (ACD) in mice. Fluorescent-activated cell sorter (FACS) analysis was used to determine CD4(+)CD25(+)Foxp3(+) T cell populations in spleen and cervical lymph nodes (CLN). Interleukin-10 (IL-10), transforming growth factor-ß (TGF-ß), and Foxp3 mRNA expressions in mouse ear skin were investigated by real-time reverse transcription-polymerase chain reaction (RT-PCR). The percentage of CD4(+)CD25(+)Foxp3(+) T cell populations were significantly increased in both spleen and CLN of L-92-fed group than vehicle and control. In addition, L-92 produced higher levels of Foxp3, IL-10 and TGF-ß compared to control mice. These results suggest that L-92 can up-regulate the number of Treg cells to suppress the progression of DNFB-induced contact dermatitis in mice.

Characterization of skin inflammation induced by repeated exposure of toluene, xylene, and formaldehyde in mice.

Volatile organic compounds (VOCs) are considered the main cause of sick building syndrome and they are likely to irritate the skin, eyes, and mucous membrane; however, the toxic threshold and the mechanisms of cutaneous reaction induced by long-time VOC exposure have not been clarified. In the present study, we investigated the effect of repeated painting of VOCs onto mouse skin. Various concentrations of toluene, xylene, and formaldehyde (FA) were applied once a week for 5 weeks. While FA solution (2-10%) induced remarkable ear swelling and caused evident infiltration of inflammatory cells, high concentrations of toluene and xylene (50 or 100%) evoked mild ear swelling and marginal inflammatory cell invasion. In addition, FA exposure markedly increased the expression of interleukin-4 (IL-4), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and transient receptor potential vanilloid-1 (TRPV-1) mRNAs in the ears and IL-4 and NT-3 mRNAs in the cervical lymph nodes. Furthermore, capsazepine, a TRPV-1 antagonist, significantly suppressed ear swelling caused by repeated painting of 5% FA. These findings demonstrate that FA has more potent irritancy against skin than toluene or xylene and suggest that the Th2 response, neurotrophins and TRPV-1 play important roles in FA-induced skin inflammation.

[A case of parahiatal hernia with vomiting, and a review of the literature].

A parahiatal hernia, that occurs from muscular diaphragmatic defects causing separation from the esophageal hiatus, is rare. We treated a 68-year-old Japanese woman with the symptom of vomiting. Based on imaging studies (upper gastrointestinal studies, gastroscopy, contrast-enhanced computed tomography) we diagnosed parahiatal hernia. On laparoscopic surgery, the hernial orifice was separated from the esophageal hiatus and the crus of the diaphragm was between the hiatus and the orifice. We closed the hernial orifice with mesh. Parahiatal hernia is rare and is difficult to diagnose preoperatively. We present a case and the clinical discuss the characteristics and management of this rare disease.

Spontaneous pulmonary emphysema in mice lacking all three nitric oxide synthase isoforms.

The roles of endogenous nitric oxide (NO) derived from the entire NO synthases (NOSs) system have yet to be fully elucidated. We addressed this issue in mice in which all three NOS isoforms were deleted. Under basal conditions, the triple n/i/eNOSs-/- mice displayed significantly longer mean alveolar linear intercept length, increased alveolar destructive index, reduced lung elastic fiber content, lower lung field computed tomographic value, and greater end-expiratory lung volume as compared with wild-type (WT) mice. None of single NOS-/- or double NOSs-/- genotypes showed such features. These findings were observed in the triple n/i/eNOSs-/- mice as early as 4 weeks after birth. Cyclopaedic and quantitative comparisons of mRNA expression levels between the lungs of WT and triple n/i/eNOSs-/- mice by cap analysis of gene expression (CAGE) revealed that mRNA expression levels of three Wnt ligands and ten Wnt/ß-catenin signaling components were significantly reduced in the lungs of triple n/i/eNOSs-/- mice. These results provide the first direct evidence that complete disruption of all three NOS genes results in spontaneous pulmonary emphysema in juvenile mice in vivo possibly through down-regulation of the Wnt/ß-catenin signaling pathway, demonstrating a novel preventive role of the endogenous NO/NOS system in the occurrence of pulmonary emphysema.

Effect of diesel exhaust particles on house dust mite-induced airway eosinophilic inflammation and remodeling in mice.

Recent research has focused on the effects of ambient particulate pollution and much evidence has indicated that particulate pollution is associated with the onset of asthma and allergy; however, the effect of diesel exhaust particles (DEP) on the development of allergen-induced airway remodeling has not been fully investigated in vivo. In the present study, we examined the effects of DEP on Dermatophagoides farinae allergens (Der f)-induced asthma-like phenotypes in mice. Mice were administered i.t. 8 times with Der f. DEP were injected i.t. with Der f 4 times throughout the experiment or twice at the sensitization period. In both cases, DEP aggravated Der f-induced increases in airway responsiveness to acetylcholine, the number of eosinophils and neutrophils in the bronchoalveolar lavage fluid (BALF), serum Der f-specific IgG1 levels, Th2 cytokines and transforming growth factor-beta1 levels in BALF, and amount of hydroxyproline in the right lungs. Furthermore, goblet cell hyperplasia and subepithelial fibrosis were also markedly aggravated. These findings indicate that DEP can potentiate airway remodeling induced by repeated allergen challenge as well as Th2-drived airway hyperresponsiveness, eosinophilic inflammation, and IgG1 production and that DEP can exhibit adjuvant activity for airway remodeling, probably due to the enhancement of allergen sensitization and/or of Th2 polarizing pathways.

Staphylococcal Phage in Combination with Staphylococcus Epidermidis as a Potential Treatment for Staphylococcus Aureus-Associated Atopic Dermatitis and Suppressor of Phage-Resistant Mutants.

Atopic dermatitis is accompanied by the abnormal overgrowth of Staphylococcus aureus, a common cause of skin infections and an opportunistic pathogen. Although administration of antibiotics is effective against S. aureus, the resulting reduction in healthy microbiota and the emergence of drug-resistant bacteria are of concern. We propose that phage therapy can be an effective strategy to treat atopic dermatitis without perturbing the microbiota structure. In this study, we examined whether the S. aureus phage SaGU1 could be a tool to counteract the atopic exacerbation induced by S. aureus using an atopic mouse model. Administration of SaGU1 to the back skin of mice reduced both S. aureus counts and the disease exacerbation caused by S. aureus. Furthermore, the S. aureus-mediated exacerbation of atopic dermatitis with respect to IgE plasma concentration and histopathological findings was ameliorated by the application of SaGU1. We also found that Staphylococcus epidermidis, a typical epidermal symbiont in healthy skin, significantly attenuated the emergence of SaGU1-resistant S. aureus under co-culture with S. aureus and S. epidermidis in liquid culture infection experiments. Our results suggest that phage therapy using SaGU1 could be a promising clinical treatment for atopic dermatitis.

Convenient preparation of an antigenic oligosaccharide from white kidney bean powder: A useful plant oligosaccharide for synthesis of immunoactive glycopolymer.

Plant glycoproteins, especially allergenic glycoproteins such as pollen allergens, often carry antigenic N-glycans with α1-3 fucose and/or ß1-2 xylose residue(s) on the trimannosyl core structure. We previously reported that one of such antigenic free-form N-glycans, Man3Xyl1Fuc1GlcNAc2 (M3FX) suppressed IL-4 production from Th2 cells of pollinosis patients. For the molecular-level analysis of this immunoactivity, an effective and convenient procedure for large scale preparation of the immunoactive free-form N-glycan and a synthesis of glycopolymers bearing multivalent M3FX has been required. During the preparation of prebiotic oligosaccharides from several edible beans, we found that the free-form M3FX accumulates in relatively large amounts in white kidney beans. In this report, we describe a new procedure for preparation of M3FX from white kidney bean powders by a combination of ion-exchange method, gel-filtration, and hydrophilic partitioning. The high-purity of M3FX prepared by this procedure was confirmed by MS-analysis and 1H-NMR, suggesting that the free-form M3FX can be used for the synthesis of neoglycopolymer. Using this new procedure, the immunoactive oligosaccharide can be prepared without the chemical method such as hydrazinolysis and other purification steps required to exclude other type of N-glycans.

Pharmacological characterization of a chronic pruritus model induced by multiple application of 2,4,6-trinitrochlorobenzene in NC mice.

Female NC/Jic mice were sensitized and challenged repeatedly at 48 h intervals for 10 and 30 days by painting 1% 2,4,6-trinitrochlorobenzene (TNCB) on both ears. Mice challenged with TNCB for 30 days developed an inflammatory dermatitis with high immunoglobulin E (IgE) titer. Histological analysis with acidic Toluidine Blue staining revealed that dermal mast cells markedly differentiated and intensely degranulated, consistent with a dramatic increase in scratching behavior. A significant increase in total scratching events could be observed in mice treated with TNCB for a short period of 10 days. Extending the term of TNCB application to 30 days, the IgE titer and number of mast cells elevated significantly, and thus various drugs were evaluated pharmacologically by using the mice treated with TNCB for 30 days. Terfenadine and cyproheptadine attenuated the chronic scratching behavior. Tacrolimus and dexamethasone were less effective and cromolyn showed no effect. In addition, terfenadine and tacrolimus suppressed the degranulation of mast cells. The present chronic scratching model could be suitable to evaluate drugs effective for suppression of mast cell differentiation and degranulation by irritation, and may represent a promising tool to develop new drugs for inflammatory pruritus associated with, for example, atopic dermatitis.

Histamine-releasing factor enhances food allergy.

Food allergy occurs due to IgE- and mast cell-dependent intestinal inflammation. Previously, we showed that histamine-releasing factor (HRF), a multifunctional protein secreted during allergy, interacts with a subset of IgE molecules and that the HRF dimer activates mast cells in an HRF-reactive IgE-dependent manner. In this study, we investigated whether HRF plays any role in food allergy. Specifically, we determined that prophylactic and therapeutic administration of HRF inhibitors that block HRF-IgE interactions reduces the incidence of diarrhea and mastocytosis in a murine model of food allergy. Food allergy-associated intestinal inflammation was accompanied by increased secretion of the HRF dimer into the intestine in response to proinflammatory, Th2, and epithelial-derived cytokines and HRF-reactive IgE levels at the elicitation phase. Consistent with these data, patients with egg allergy had higher blood levels of HRF-reactive IgE compared with individuals that were not hypersensitive. Successful oral immunotherapy in egg-allergy patients and food-allergic mice reduced HRF-reactive IgE levels, thereby suggesting a pathological role for HRF in food allergy. Together, these results suggest that antigen and HRF dimer amplify intestinal inflammation by synergistically activating mast cells and indicate that HRF has potential as a therapeutic target in food allergy.