Intranasal administration of ceramide liposome suppresses allergic rhinitis by targeting CD300f in murine models.

Allergic rhinitis (AR) is caused by type I hypersensitivity reaction in the nasal tissues. The interaction between CD300f and its ligand ceramide suppresses immunoglobulin E (IgE)-mediated mast cell activation. However, whether CD300f inhibits the development of allergic rhinitis (AR) remains elusive. We aimed to investigate the roles of CD300f in the development of AR and the effectiveness of intranasal administration of ceramide liposomes on AR in murine models. We used ragweed pollen-induced AR models in mice. Notably, CD300f deficiency did not significantly influence the ragweed-specific IgE production, but increased the frequency of mast cell-dependent sneezing as well as the numbers of degranulated mast cells and eosinophils in the nasal tissues in our models. Similar results were also obtained for MCPT5-exprssing mast cell-specific loss of CD300f. Importantly, intranasal administration of ceramide liposomes reduced the frequency of sneezing as well as the numbers of degranulated mast cells and eosinophils in the nasal tissues in AR models. Thus, CD300f-ceramide interaction, predominantly in mast cells, alleviates the symptoms and progression of AR. Therefore, intranasal administration of ceramide liposomes may be a promising therapeutic approach against AR by targeting CD300f.

Murine model identifies tropomyosin as IgE cross-reactive protein between house dust mite and coho salmon that possibly contributes to the development of salmon allergy.

Background: Recently, we have developed a method to identify IgE cross-reactive allergens. However, the mechanism by which IgE cross-reactive allergens cause food allergy is not yet fully understood how. In this study, we aimed to understand the underlying pathogenesis by identifying food allergens that cross-react with house dust mite allergens in a murine model. Material and methods: Allergenic protein microarray analysis was conducted using serum from mice intraperitoneally injected with Dermatophagoides pteronyssinus (Der p) extract plus alum or alum alone as controls. Der p, Dermatophagoides farinae (Der f), coho salmon extract-sensitized and control mice were analyzed. Serum levels of IgE against Der p, Der f, coho salmon extract, protein fractions of coho salmon extract separated by ammonium sulfate precipitation and anion exchange chromatography, and recombinant coho salmon tropomyosin or actin were measured by an enzyme-linked immunosorbent assay. A murine model of cutaneous anaphylaxis or oral allergy syndrome (OAS) was established in Der p extract-sensitized mice stimulated with coho salmon extract, tropomyosin, or actin. Results: Protein microarray analysis showed that coho salmon-derived proteins were highly bound to serum IgE in Der p extract-sensitized mice. Serum IgE from Der p or Der f extract-sensitized mice was bound to coho salmon extract, whereas serum IgE from coho salmon extract-sensitized mice was bound to Der p or Der f extract. Analysis of the murine model showed that cutaneous anaphylaxis and oral allergic reaction were evident in Der p extract-sensitized mice stimulated by coho salmon extract. Serum IgE from Der p or Der f extract-sensitized mice was bound strongly to protein fractions separated by anion exchange chromatography of coho salmon proteins precipitated with 50% ammonium sulfate, which massively contained the approximately 38 kDa protein. We found that serum IgE from Der p extract-sensitized mice was bound to recombinant coho salmon tropomyosin. Der p extract-sensitized mice exhibited cutaneous anaphylaxis in response to coho salmon tropomyosin. Conclusion: Our results showed IgE cross-reactivity of tropomyosin between Dermatophagoides and coho salmon which illustrates salmon allergy following sensitization with the house dust mite Dermatophagoides. Our method for identifying IgE cross-reactive allergens will help understand the underlying mechanisms of food allergies.

Staphylococcus aureus δ-toxin present on skin promotes the development of food allergy in a murine model.

Background: Patients with food allergy often suffer from atopic dermatitis, in which Staphylococcus aureus colonization is frequently observed. Staphylococcus aureus δ-toxin activates mast cells and promotes T helper 2 type skin inflammation in the tape-stripped murine skin. However, the physiological effects of δ-toxin present on the steady-state skin remain unknown. We aimed to investigate whether δ-toxin present on the steady-state skin impacts the development of food allergy. Material and methods: The non-tape-stripped skins of wild-type, KitW-sh/W-sh, or ST2-deficient mice were treated with ovalbumin (OVA) with or without δ-toxin before intragastric administration of OVA. The frequency of diarrhea, numbers of jejunum or skin mast cells, and serum levels of OVA-specific IgE were measured. Conventional dendritic cell 2 (cDC2) in skin and lymph nodes (LN) were analyzed. The cytokine levels in the skin tissues or culture supernatants of δ-toxin-stimulated murine keratinocytes were measured. Anti-IL-1α antibody-pretreated mice were analyzed. Results: Stimulation with δ-toxin induced the release of IL-1α, but not IL-33, in murine keratinocytes. Epicutaneous treatment with OVA and δ-toxin induced the local production of IL-1α. This treatment induced the translocation of OVA-loaded cDC2 from skin to draining LN and OVA-specific IgE production, independently of mast cells and ST2. This resulted in OVA-administered food allergic responses. In these models, pretreatment with anti-IL-1α antibody inhibited the cDC2 activation and OVA-specific IgE production, thereby dampening food allergic responses. Conclusion: Even without tape stripping, δ-toxin present on skin enhances epicutaneous sensitization to food allergen in an IL-1α-dependent manner, thereby promoting the development of food allergy.

Changes in Physical and Oral Function after a Long-Term Care Prevention Program in Community-Dwelling Japanese Older Adults: A 12-Month Follow-Up Study.

BACKGROUND: The aim of this prospective cohort study was to clarify changes in physical and oral function in older adults after completing a 3-month health program combining physical and oral exercise, oral health instruction, and nutritional guidance. METHODS: Subjects were 34 women aged at least 70 years (mean age 79.2 years) in Bungotakada City, Oita Prefecture, Japan. Physical and oral function was investigated on the first day (baseline), at the end of the program, and 6 and 12 months after completing the health program. Physical function was measured using handgrip strength test, timed up and go (TUG) test and one-leg standing time test. Oral diadochokinesis test and repetitive saliva swallowing test (RSST) were employed to assess oral function. RESULTS: TUG scores were significantly lower at 6 and 12 months than at baseline in participants aged ≥70 and <80 years. The repetition rate of the monosyllables /pa/, /ta/, and /ka/ was improved at the end of program in participants aged ≥70 and <80 years and increased to more than 6 times/second at 12 months. CONCLUSIONS: Our 3-month health program maintained improvements in oral and physical function in older women 1 year after completing the program.

Budesonide Foam for Ulcerative Colitis Patients Experiencing Inadequate Response to Biological Therapy.

BACKGROUND In recent years, a plethora of therapeutic agents for ulcerative colitis (UC), especially novel biologics (Bio), have become available. Although it is now possible to use biological drugs, there should be no need for frequently changing medications. To avoid first-pass metabolism in the liver, thus reducing systemic bioavailability, budesonide foam has been applied as a topical steroid. We therefore evaluated whether budesonide foam has therapeutic value in UC patients who responded inadequately to Bio or to tacrolimus. MATERIAL AND METHODS We enrolled 10 patients who were experiencing an inadequate response to Bio (n=7) or to tacrolimus (n=3) at Juntendo University. We used Lichtiger's index to assess UC activity and clinical response. RESULTS Of the study patients, 4 were receiving adalimumab, 3 golimumab, and 3 tacrolimus. The average Lichtiger's index before budesonide administration was 7.1 (range 13-3), which improved to 3.4 (range 7-0) after budesonide therapy (p=0.01). Notably, 4 of the 6 cases with a Lichtiger's index >4 before budesonide administration achieved improvement of ≥3 points or remission. CONCLUSIONS Although the number of patients was small, budesonide foam had significant efficacy when added to the treatment of patients having an inadequate response to Bio or to tacrolimus. These results suggest that in cases responding poorly to Bio, adding budesonide foam as combination therapy can achieve a clinical remission.

The phytosphingosine-CD300b interaction promotes zymosan-induced, nitric oxide-dependent neutrophil recruitment.

Zymosan is a glucan that is a component of the yeast cell wall. Here, we determined the mechanisms underlying the zymosan-induced accumulation of neutrophils in mice. Loss of the receptor CD300b reduced the number of neutrophils recruited to dorsal air pouches in response to zymosan, but not in response to lipopolysaccharide (LPS), a bacterial membrane component recognized by Toll-like receptor 4 (TLR4). An inhibitor of nitric oxide (NO) synthesis reduced the number of neutrophils in the zymosan-treated air pouches of wild-type mice to an amount comparable to that in CD300b-/- mice. Treatment with clodronate liposomes decreased the number of NO-producing, CD300b+ inflammatory dendritic cells (DCs) in wild-type mice, thus decreasing NO production and neutrophil recruitment. Similarly, CD300b deficiency decreased the NO-dependent recruitment of neutrophils to zymosan-treated joint cavities, thus ameliorating subsequent arthritis. We identified phytosphingosine, a lipid component of zymosan, as a potential ligand of CD300b. Phytosphingosine stimulated NO production in inflammatory DCs and promoted neutrophil recruitment in a CD300b-dependent manner. Together, these results suggest that the phytosphingosine-CD300b interaction promotes zymosan-dependent neutrophil accumulation by inducing NO production by inflammatory DCs and that CD300b may contribute to antifungal immunity.

Improvement of physical and oral function in community-dwelling older people after a 3-month long-term care prevention program including physical exercise, oral health instruction, and nutritional guidance.

Objective: There is limited evidence about the most appropriate way to improve physical and oral function in older people. The aim of this study was to clarify the effectiveness of a long-term care prevention program combining physical exercise, oral health instruction, and nutritional guidance among community-dwelling older people. Materials and methods: We included 43 older people aged at least 65 years (seven men and 23 women; mean age 75.3 years) in Bungotakada City, Japan. The 3-month program involved a weekly intervention. Physical and oral function was investigated on the first day of the program (i.e., baseline) and at the end of the program (i.e., after 3 months). Physical function was examined using measures such as handgrip strength, timed up-and-go test, and one-leg standing time with eyes open. An oral diadochokinesis test was used to assess oral function. Results: Mean maximum handgrip strength increased significantly in older people aged ≤74 years (younger participants) and those aged ≥75 years (older participants) after 3 months compared with the baseline. The timed up-and-go test duration was significantly lower in older participants after the program. Repetition of the monosyllables pa and ka and the repetitive saliva swallowing test was significantly improved in both groups. The dysphagia risk assessment for the community-dwelling elderly score was significantly lower in younger participants after 3 months. Conclusion: Participation in a 3-month program combining physical exercise, oral health instruction, and nutritional guidance may contribute to improvement or maintenance of oral and physical function in older people.

Leukocyte mono-immunoglobulin-like receptor 8 (LMIR8)/CLM-6 is an FcRγ-coupled receptor selectively expressed in mouse tissue plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) produce large amounts of type-I interferon (IFN) in response to viral infection or self nucleic acids. Leukocyte mono-immunoglobulin-like receptor 8 (LMIR8), also called CMRF-35-like molecule-6 (CLM-6), is a putative activating receptor among mouse LMIR/CLM/CD300 members; however, the expression and function of LMIR8 remain unclear. Here, we characterize mouse LMIR8 as a pDC receptor. Analysis of Flag-tagged LMIR8-transduced bone marrow (BM)-derived mast cells demonstrated that LMIR8 can transmit an activating signal by interacting with immunoreceptor tyrosine-based activating motif (ITAM)-containing FcRγ. Flow cytometric analysis using a specific antibody for LMIR8 showed that LMIR8 expression was restricted to mouse pDCs residing in BM, spleen, or lymph node. FcRγ deficiency dampened surface expression of LMIR8 in mouse pDCs. Notably, LMIR8 was detected only in pDCs, irrespective of TLR9 stimulation, suggesting that LMIR8 is a suitable marker for pDCs in mouse tissues; LMIR8 is weakly expressed in Flt3 ligand-induced BM-derived pDCs (BMpDCs). Crosslinking of transduced LMIR8 in BMpDCs with anti-LMIR8 antibody did not induce IFN-α production, but rather suppressed TLR9-mediated production of IFN-α. Taken together, these observations indicate that LMIR8 is an FcRγ-coupled receptor selectively expressed in mouse tissue pDCs, which might suppress pDC activation through the recognition of its ligands.

The CD300e molecule in mice is an immune-activating receptor.

CD300 molecules (CD300s) belong to paired activating and inhibitory receptor families, which mediate immune responses. Human CD300e (hCD300e) is expressed in monocytes and myeloid dendritic cells and transmits an immune-activating signal by interacting with DNAX-activating protein 12 (DAP12). However, the CD300e ortholog in mice (mCD300e) is poorly characterized. Here, we found that mCD300e is also an immune-activating receptor. We found that mCD300e engagement triggers cytokine production in mCD300e-transduced bone marrow-derived mast cells (BMMCs). Loss of DAP12 and another signaling protein, FcRγ, did not affect surface expression of transduced mCD300e, but abrogated mCD300e-mediated cytokine production in the BMMCs. Co-immunoprecipitation experiments revealed that mCD300e physically interacts with both FcRγ and DAP12, suggesting that mCD300e delivers an activating signal via these two proteins. Binding and reporter assays with the mCD300e extracellular domain identified sphingomyelin as a ligand of both mCD300e and hCD300e. Notably, the binding of sphingomyelin to mCD300e stimulated cytokine production in the transduced BMMCs in an FcRγ- and DAP12-dependent manner. Flow cytometric analysis with an mCD300e-specific Ab disclosed that mCD300e expression is highly restricted to CD115+Ly-6Clow/int peripheral blood monocytes, corresponding to CD14dim/+CD16+ human nonclassical and intermediate monocytes. Loss of FcRγ or DAP12 lowered the surface expression of endogenous mCD300e in the CD115+Ly-6Clow/int monocytes. Stimulation with sphingomyelin failed to activate the CD115+Ly-6Clow/int mouse monocytes, but induced hCD300e-mediated cytokine production in the CD14dimCD16+ human monocytes. Taken together, these observations indicate that mCD300e recognizes sphingomyelin and thereby regulates nonclassical and intermediate monocyte functions through FcRγ and DAP12.

Disrupting ceramide-CD300f interaction prevents septic peritonitis by stimulating neutrophil recruitment.

Sepsis is a serious clinical problem. Negative regulation of innate immunity is associated with sepsis progression, but the underlying mechanisms remains unclear. Here we show that the receptor CD300f promotes disease progression in sepsis. CD300f -/- mice were protected from death after cecal ligation and puncture (CLP), a murine model of septic peritonitis. CD300f was highly expressed in mast cells and recruited neutrophils in the peritoneal cavity. Analysis of mice (e.g., mast cell-deficient mice) receiving transplants of wild-type or CD300f -/- mast cells or neutrophils indicated that CD300f deficiency did not influence intrinsic migratory abilities of neutrophils, but enhanced neutrophil chemoattractant production (from mast cells and neutrophils) in the peritoneal cavity of CLP-operated mice, leading to robust accumulation of neutrophils which efficiently eliminated Escherichia coli. Ceramide-CD300f interaction suppressed the release of neutrophil chemoattractants from Escherichia coli-stimulated mast cells and neutrophils. Administration of the reagents that disrupted the ceramide-CD300f interaction prevented CLP-induced sepsis by stimulating neutrophil recruitment, whereas that of ceramide-containing vesicles aggravated sepsis. Extracellular concentrations of ceramides increased in the peritoneal cavity after CLP, suggesting a possible role of extracellular ceramides, CD300f ligands, in the negative-feedback suppression of innate immune responses. Thus, CD300f is an attractive target for the treatment of sepsis.

Endoscopic submucosal dissection with a combination of small-caliber-tip transparent hood and flex knife for superficial esophageal neoplasia. Is it safe for elderly patients?

BACKGROUND: Safety and efficacy of endoscopic submucosal dissection (ESD) for esophageal neoplasias have not been adequately investigated in elderly patients. This study was designed to evaluate the safety and efficacy of ESD for esophageal neoplasias in elderly patients. METHODS: Fifty-three superficial esophageal neoplasias treated with ESD using a combination of small-caliber-tip transparent hood and flex knife from May 2006 to June 2009 were divided into elderly group (aged 70 years or older: 25 lesions in 23 patients) and nonelderly group (younger than aged 70 years: 28 lesions in 25 patients). Therapeutic efficacy, complications, and follow-up results were evaluated retrospectively. RESULTS: The history of cerebral infarction or cardiopulmonary disease and the usage of antiplatelet agents or anticoagulants were significantly higher in elderly group (p 0.0050 and p 0.0013, respectively). Median procedural times in the elderly group and the nonelderly group were 93 ± 53 (range, 42-235) min and 95 ± 55 (range, 40-230) min (p 0.73), respectively. Median sizes of the neoplasias and the resected specimens were 14 ± 11 (range, 5-45) mm and 15 ± 17 (range, 5-83) mm (p 0.56), and 35 ± 12 (range, 18-60) mm and 38 ± 17 (range, 18-90) mm (p 0.38), respectively. En bloc resection rate was 100% in each group. Body temperature and white blood cell counts of the next day after ESD were significantly higher in the nonelderly group than in the elderly group (p 0.0087 and p 0.0043, respectively). There were no complications, such as postoperative bleeding or perforation, in each group. The median follow-up period of 23 ± 10 (range, 4-35) months in the elderly group revealed no local or distant metastasis. CONCLUSIONS: ESD with a combination of small-caliber-tip transparent hood and flex knife is a safe and effective treatment for superficial esophageal neoplasia in elderly and nonelderly patients.

Endoscopic resection after endoscopic hemostasis for hemorrhagic gastric cancer.

A 68 years old man was referred to our hospital with symptoms of hematemesis and melena. An emergent gastroscopy showed a gastric ulcerative lesion with an exposed vessel (Forrest IIa) protruding from its base, which was located at the posterior wall of the upper portion of the gastric body. Endoscopic hemostasis was performed with endoclips and antiulcer treatment was done. Although the ulcerative lesion was healed two months after endoscopic hemostasis, the histopathological examination of the biopsy specimens revealed well differentiated adenocarcinoma (0-IIc). The tumor could be resected en-bloc by endoscopic submucosal dissection (ESD) without any complications such as perforation or postoperative bleeding. The resected specimen showed that the resected tumor was well differentiated intramucosal adenocarcinoma (13 x 10 mm) with a clear lateral margin. There was no recurrence during 12 months follow-up after ESD treatment. Follow-up endoscopy with biopsies should be performed for accurate diagnosis of gastric ulcerative lesions and ESD after endoscopic hemostasis with endoclips was an effective method for early gastric cancer presenting with massive hemorrhage in our case.

Endoscopic submucosal dissection and preoperative assessment with endoscopic ultrasonography for the treatment of rectal carcinoid tumors.

BACKGROUND: Rectal carcinoid tumors 10 mm in diameter or smaller located within the submucosal layer can be cured by local excision including endoscopic treatment. But complete resection of these tumors with endoscopic polypectomy is difficult. This study aimed to evaluate the usefulness of endoscopic submucosal dissection (ESD) and endoscopic ultrasonography (EUS) for the treatment of rectal carcinoid tumors. METHODS: In this study, 22 rectal carcinoid tumors in 21 patients were evaluated with EUS and treated using ESD from January 2004 to December 2008. RESULTS: The mean size of the resected tumors was 6.1 mm (range, 2.0-10 mm) on histopathologic evaluations. When the sizes of the tumors shown by EUS and histopathologic evaluation were compared, the mean values were not significantly different. All the tumors were located within the submucosal layer, and the accuracy of the preoperative depth determination with EUS was 100% (22/22). The mean duration of the ESD procedure was 37 min (range, 20-71 min). The overall rate of en bloc resection with ESD was 100% (22/22). Although postoperative bleeding occurred in two cases (9%), both cases were successfully managed by endoscopic hemostasis. No perforation or recurrence was observed during the mean follow-up period of 30 months (range, 7-66 months). CONCLUSIONS: Endoscopic submucosal dissection and preoperative assessment with EUS are effective for treating rectal carcinoid tumors and enabling en bloc resection.

Endoscopic submucosal dissection with a combination of small-caliber-tip transparent hood and flex knife is a safe and effective treatment for superficial esophageal neoplasias.

BACKGROUND: Superficial esophageal neoplasias resected in piecemeal manner with endoscopic mucosal resection (EMR) sometimes recur locally, and additional treatments need to be developed. Efficacy and safety of endoscopic submucosal dissection (ESD) for esophageal neoplasias are not sufficiently demonstrated, so we conducted a retrospective study to evaluate the efficacy and safety of ESD for superficial esophageal neoplasias. METHODS: Thirty-seven superficial esophageal neoplasias consisted of 34 squamous cell neoplasias and 3 columnar neoplasias in 35 patients were treated with ESD from May 2006 to July 2008. Patients were regularly followed up with endoscopy every 6 months, and with echoendoscope and computed tomography every year. Therapeutic efficacy, complications, and follow-up results were evaluated. RESULTS: The mean size of the resected neoplasias and that of the resected specimens were 22 mm (range 10-83 mm) and 41 mm (range 18-90 mm), respectively. The mean duration of the ESD procedures was 117 min (range 40-235 min). The overall rates of en bloc resection and of free margin resection were 100% (37/37) and 95% (35/37), respectively. The mean follow-up period of 19 months (range 7-32 months) revealed no local or distant recurrence. There were no complications such as bleeding or perforation in any case. In all nine cases, the strictures were successfully managed with prophylactic endoscopic balloon dilation. CONCLUSIONS: ESD with a combination of small-caliber-tip transparent hood and flex knife is a safe endoscopic treatment for superficial esophageal neoplasias and enables large en bloc resection.