ß-Glucans in food modify colonic microflora by inducing antimicrobial protein, calprotectin, in a Dectin-1-induced-IL-17F-dependent manner.

Dectin-1 (gene symbol: Clec7a) is a receptor for ß-glucans that play an important role for the host defense against fungi. Recently, we showed that Clec7a-/- mice are resistant against dextran sodium sulfate (DSS)-induced colitis because of regulatory T-cell population expansion in the colon. The regulatory T-cell expansion is caused by expansion of commensal Lactobacillus murinus whose growth is suppressed by an antimicrobial protein, calprotectin S100A8/A9. In this report, we showed that S100A8 was mainly produced by mouse colonic epithelial cells. S100A8 was not induced directly by Dectin-1 but by Dectin-1-induced cytokines, especially interleukin-17F (IL-17F), that were produced by several types of innate immune cells including CD11c+/CD11b+ myeloid cells in colonic lamina propria. S100A8/A9 heterodimer preferentially suppressed the growth of L. murinus that was increased in both Clec7a-/- and Il17f-/- mice. Furthermore, similar expansion of L. murinus and DSS-colitis resistance were observed in mice fed with ß-glucan-free food. These observations suggest that food-derived ß-glucans control the specific commensal microbiota via the Dectin-1-IL-17F-calprotectin axis to maintain the intestinal homeostasis.

Human papilloma virus detection in oropharyngeal cancer with gargle samples.

Human papilloma virus detection in oropharyngeal cancer with gargle samples. OBJECTIVE: human papilloma virus (HPV) is a major risk factor for oropharyngeal squamous cell carcinoma (OPSCC) and knowledge of a patient's HPV status is clinically important in terms of treatment and prognosis. The practicality of using oral gargle samples to reliably detect HPV in patients with OPSCC remains unclear. Therefore, we evaluated the feasibility of HPV detection in gargle samples of OPSCC patients using an HPV-dedicated nucleic acid amplification test (cobas 4800 HPV Test; Roche Diagnostics K.K.). METHODOLOGY: 15 patients with histologically proven OPSCC were evaluated from May 2014 to March 2015. Swab sam- ples served as positive controls and were tested using both the Hybrid Capture II HPV Test (HC-II; Digene Corporation) and the cobas 4800 HPV Test. Oral gargle samples were tested using the cobas 4800 HPV Test. Five of the 15 patients were confirmed to be HPV-positive by a combination of p16 immunohistochemistry, HPV-DNA in situ hybridization and nucleic acid amplification. RESULTS: the sensitivity and specificity of the gargling method were 60% and 100%, respectively. No false-positives were obtained. Detection of HPV in two very small tumours rising from the base of the tongue was difficult and these cases were overlooked as HPV-negative. CONCLUSIONS: use of the gargling method to determine HPV positivity in OPSCC patients appears feasible, except in patients with very small tumours. Real-time polymerase chain reaction using gargle samples may have greater clinical efficacy than the swabbing method.

The role of TNFalpha and IL-17 in the development of excess IL-1 signaling-induced inflammatory diseases in IL-1 receptor antagonist-deficient mice.

IL-1 receptor antagonist (IL-1Ra)-deficient mice spontaneously develop several inflammatory diseases, resembling rheumatoid arthritis, aortitis, and psoriasis in humans. As adoptive T cell transplantation could induce arthritis and aortitis in recipient mice, it was suggested that an autoimmune process is involved in the development of diseases. In contrast, as dermatitis developed in scid/scid-IL-IRa-deficient mice and could not be induced by T cell transfer, a T cell-independent mechanism was suggested. The expression of proinflammatory cytokines was augmented at the inflammatory sites. The development of arthritis and aortitis was significantly suppressed by the deficiency of TNFalpha or IL-17. The development of dermatitis was also inhibited by the deficiency of TNFalpha. These observations suggest that TNFalpha and IL-17 play a crucial role in the development of autoimmunity downstream of IL-1 signaling, and excess IL-1 signaling-induced TNFalpha also induces skin inflammation in a T cell-independent manner.

IgG reactive to CTL-directed epitopes of self-antigens is either lacking or unbalanced in atopic dermatitis patients.

We previously demonstrated that CTL-directed epitopes derived from non-mutated self-antigens elicit a type-I allergy in the majority of healthy donors (HD) as did the presence of IgE and IgG reactive to these peptides in the sera of the donors. We investigated in this study whether Igs reactive to eight types of CTL-directed peptides were elevated in the sera of 40 patients with atopic dermatitis (AD). Total IgE levels in the sera of AD patients were significantly higher than those of HD, however, no significant differences between the AD patients and the HD were observed in either the serum levels or the positive rates of IgE reactive to seven of the eight peptides. Total IgG levels were not different from each other, however, IgG reactive to the two peptides with no sequence similarity to other species and one peptide that had similarity to DNA helicase II of enterobacteria were not detectable in the sera of the AD patients. Although IgG reactive to the remaining five peptides, which had sequence similarity to other species, were detectable in both the AD patients and the HD, ratios of peptide-specific IgG1/IgG2 were mostly lower in the AD patients than in the HD. These results indicate that IgG reactive to CTL-directed epitopes of self-antigens is either lacking or unbalanced in AD patients. This information may provide new insight into the immune-mechanisms of elevated auto-reactivity of AD patients.

Evaluation of Kampo medicines used to treat rheumatoid arthritis in collagen-induced arthritic and pX transgenic mice.

Abstract To evaluate the usefulness of Kampo medicines (traditional herbal medicines) used clinically for the treatment of rheumatoid arthritis (RA), we selected eight of them and examined their effects on collagen-induced arthritic and pX transgenic mice. Among these, Dai-bofu-to, Kanzo-bushi-to, and Makyo-yokkan-to significantly reduced the severity of arthritis in collagen-induced arthritis (CIA) mice. The onset of arthritis was delayed by three Kampo medicines, but only the effect of Makyo-yokkan-to was statistically significant. In addition, three Kampo medicines suppressed the arthropathy of pX transgenic mice, which had developed spontaneously. The onset of arthritis was delayed by 10.7, 8.3, and 15.4 days following treatment with Dai-bofu-to, Kanzo-bushi-to, and Makyo-yokkan-to, respectively. A study of the underlying mechanism showed that Kanzo-bushi-to decreased serum antitype II collagen antibody levels, suggesting that Kanzo-bushi-to possesses immunomodulating activity. This study shows that some Kampo medicines are effective in an induced or spontaneously developed arthritis animal model of human RA.

Crystallization and preliminary X-ray analysis of catalase-peroxidase from the halophilic archaeon Haloarcula marismortui.

Catalase-peroxidases are bifunctional enzymes found in many microorganisms. Crystals of catalase-peroxidase from the halophilic archaeon Haloarcula marismortui were obtained using the hanging-drop vapour-diffusion method. The rhombic plate-shaped crystals were grown from purified protein solution using (NH(4))(2)SO(4) as precipitant at 293 K. The crystal belongs to the monoclinic system, space group C2, and diffracted beyond 2.0 A resolution.

Crystallization and preliminary X-ray studies of V(1)-ATPase of Thermus thermophilus HB8 complexed with Mg-ADP.

Crystals have been grown of the V(1)-ATPase sector of the V-type ATP synthase complex (V(0)V(1)) from the thermophilic eubacterium Thermus thermophilus HB8. These crystals are grown by the vapor diffusion method in the presence of 5 mM Mg-ADP, from solutions containing 100 mM sodium acetate and 2 M sodium formate, pH 5.5. The crystals diffracted X rays beyond 3.4 A in resolution on a synchrotron radiation source. The crystals belong to the trigonal space group P3, with unit cell dimensions of a = b = 89.0 A, c = 179.2 A, and gamma = 120 degrees. The unit cell presumably contains one molecule of V(1)-ATPase and the V(m) value is calculated as 3.0 A(3)/Da.

Presence of Streptococcus infection in extra-oropharyngeal head and neck squamous cell carcinoma and its implication in carcinogenesis.

To investigate the involvement of S. anginosus infection in head and neck cancer in the extra-oropharyngeal cavity, we analyzed 3 DNA samples prepared from squamous cell carcinoma of the external auditory canal and one from squamous cell carcinoma of the skin using polymerase chain reaction (PCR) analysis and Southern blot analysis to detect the DNA sequence of S. anginosus. We also examined these four specimens by Gram's stain to detect the streptococcal bacterial bodies. By PCR analysis, the DNA sequence of S. anginosus was found in 4 out of 4 (100%) DNA samples obtained from these tumors. By Southern blot analysis, positive bands were detected in one out of the 3 (33%) samples from the tumor taken from the external auditory canal. We detected streptococcal bacterial bodies in one of the three specimens from the tumor obtained from cancer of the external auditory canal and in the one specimen from the skin cancer by the method of Gram's stain. Contrary to our expectations, these bacterial bodies were located in the middle of the tumor. Since S. anginosus is thought to exist in the mouth as a normal flora and to be located mainly in the gingiva and dental plaque, these data strongly indicate that S. anginosus infection is implicated in the carcinogenesis of head and neck squamous cell carcinoma.

A clinical study of oral tongue cancer.

Thirty-nine previously untreated patients with squamous cell carcinoma of the oral tongue treated with curative intent in our hospital from 1993 through 1998 are reviewed. Of these patients, those in the early stage (stages I and II) constituted 64%. The over all 5-year survival rate of all the patients was 60%. The 5-year survival rate of the patients with early stage cancers was unsatisfactory (stage I: 73%; II: 56%). This was thought to be related to the absence of elective neck dissection and the administration of chemotherapy in the patients with early stage cancer. We concluded that elective neck dissection for levels I, II and III is the first choice of treatment strategy for patients with stage II cancer. Our data indicate that chemotherapy in patients with early stage cancer was not beneficial and might have increased the risk of late lymph node metastasis in the clinically NO patients without neck dissection. There were 9 patients younger than 40 years of age and their survival rate at 5 years was 80%, which was better than that of the older patients. The treatment strategy for patients younger than 40 years of age was similar to that of older patients.

Streptococcus anginosus in head and neck squamous cell carcinoma: implication in carcinogenesis.

It has been suggested that Helicobacter pylori (H. pylori) infection might be associated with not only gastric ulcers but also gastric malignancies. Recently, it was reported that the Streptococcus anginosus (S. anginosus) DNA sequence was found in DNA samples extracted from esophageal cancers. Because smoking and alcohol abuse are regarded as risk factors for both esophgeal cancer and head and neck cancer, infection of S. anginosus might be associated with carcinogenesis of head and neck cancer. To investgate the involvement of S. anginosus infection in head and neck cancer, we analyzed 217 DNA samples prepared from head and neck squamous cell carcinomas. We performed PCR analysis with S. anginosus-16S ribosomal DNA-specific primers, and Southern blot analysis. For detection of S. anginosus in the oral and pharyngeal cavities, we used oropharyngeal bacteriological culture and PCR analysis of gingival smears of the patients. By PCR analysis, the S. anginosus DNA sequence was found in 217 out of 217 (100%) DNA samples obtained from head and neck cancers. By Southern blot analysis, positive bands were detected in 41 out of 125 (33%) samples. We could find no S. anginosus colony in oropharyngeal bacteriological culture dishes of 53 patients with and without head and neck cancer. On the other hand, we found the S. anginosus DNA fragment in 8 out of 8 DNA samples obtained from gingival smears by PCR analysis. These data indicate that the upper aerodigestive environment of the patients permitting S. anginosus infection was implicated in the carcinogenesis of head and neck squamous cell carcinoma.

Lipopolysaccharide-induced HIV-1 expression in transgenic mice is mediated by tumor necrosis factor-alpha and interleukin-1, but not by interferon-gamma nor interleukin-6.

BACKGROUND: As serum HIV-1 load correlates well with the prognosis of the disease, it is suggested that the viral load is one of the major determinants of the disease progression of AIDS. Accordingly, HIV-1 activation mechanisms were extensively studied in vitro, and involvement of cytokines including tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, IL-6 and interferon (IFN)-gamma has been suggested in this process. However, so far the roles of these cytokines in the HIV-1 expression in vivo have not been well elucidated because of the lack of appropriate animal disease models. OBJECTIVE: To elucidate the roles of cytokines in HIV-1 activation in vivo. DESIGN AND METHODS: Transgenic mice carrying a defective HIV-1 genome were used as a model for HIV-1 carriers. In order to examine the possible involvement of cytokines in HIV-1 expression, TNF-alpha-, IL-1-, IL-6- and IFN-gamma-deficient HIV-1 transgenic mice, were produced and HIV-1 expression was analyzed after activation with bacterial lipopolysaccharides (LPS). RESULTS: HIV-1 expression in the transgenic mouse spleen was activated 10- to 20-fold by LPS, and the serum p24 Gag protein levels reached 400 pg/ml, which is nearly equal to the levels that occur in AIDS patients. However, this augmentation was suppressed by 60% in TNF-alpha-deficient mice and by 40% in IL-1alpha/beta-deficient mice. In contrast, no suppression was observed in either IL-6-, IFN-gamma-, IL-1alpha, or IL-1beta-deficient mice. CONCLUSIONS: Results suggest that TNF-alpha and IL-1 play important roles in HIV-1 gene activation and selective suppression of these cytokines could improve clinical prognosis and potentially slow progression of the disease.

Enhancement in the perfection of orthorhombic lysozyme crystals grown in a high magnetic field (10 T).

Orthorhombic crystals of hen egg-white (HEW) lysozyme were grown in a homogeneous and static magnetic field of 10 T. All crystals grown at 10 T were oriented such that their crystallographic c axes were parallel to the magnetic field direction and showed a narrower average full-width at half-maximum (FWHM) of the rocking curve than those grown at 0 T. Rocking-width measurements were made at the BL-10A station at the Photon Factory, Tsukuba, Japan, using a high-resolution vertical-type four-circle diffractometer. Crystal perfection was evaluated using the FWHM of the rocking curve; the effects of the magnetic field on the quality of the crystals were examined by comparison of the FWHM of seven crystals grown at 10 and 0 T. The FWHMs of the reflections along the a, b and c axes decreased by 23.5, 35.3 and 27.8%, respectively, and those of other general reflections decreased by 17.4-42.2% in the crystals grown at high magnetic field. These results clearly showed that a magnetic field of 10 T improved the crystal perfection of the orthorhombic lysozyme crystals. As a result, the maximum resolution of X-ray diffraction increased from 1.3 A at 0 T to 1.13 A at 10 T. The magnetic field also affected the dimensions of the unit cell, increments being 0.2% for the a and c axes and 0.1% for the b axis, respectively. These facts suggest that the application of a high magnetic field during crystallization might result in remarkable enhancements in the diffraction power of protein crystals having magnetic anisotropy.

Columnar epidermal necrosis: a unique manifestation of transfusion-associated cutaneous graft-vs-host disease.

BACKGROUND: In 1978, the first case of columnar epidermal necrosis was reported in a 6-year-old boy. There were scaly, partially vesicular or crusty, erythematous lesions mainly involving the extremities that histopathologically showed peculiar features of focal, total epidermal necrosis accompanied by a lichenoid tissue reaction. He developed the skin eruption after receiving a blood transfusion from his mother when he showed debility induced by vaccination with an alternated live measles virus vaccine. The lesions rapidly regressed after sun exposure. To our knowledge, there has been no report of a similar case despite such unique features. OBSERVATION: We encountered a similar case of columnar epidermal necrosis in a 15-year-old Japanese girl with chronic graft-vs-host disease; the lesions occurred 3 months after the transfusion of peripheral blood stem cells from her HLA antigen-matched brother. However, there was no exacerbation of liver dysfunction, diarrhea, or bone marrow aplasia. The peculiar cutaneous lesions responded well to topical phototherapy. CONCLUSION: These 2 patients shared a similarity in their lesions and circumstances under which the blood transfusion was performed to a debilitated patient from a close family member. We believe that focal epidermal necrosis observed in patients with this condition represents a variant of blood transfusion-associated lichenoid graft-vs-host disease that occurs uniquely in a skin-targeted fashion.

Development of chronic inflammatory arthropathy resembling rheumatoid arthritis in interleukin 1 receptor antagonist-deficient mice.

Interleukin (IL)-1 is a proinflammatory cytokine that plays important roles in inflammation, host defense, and the neuro-immuno-endocrine network. IL-1 receptor antagonist (ra) is an endogenous inhibitor of IL-1 and is supposed to regulate IL-1 activity. However, its pathophysiological roles in a body remain largely unknown. To elucidate the roles of IL-1ra, IL-1ra-deficient mice were produced by gene targeting, and pathology was analyzed on different genetic backgrounds. We found that all of the mice on a BALB/cA background, but not those on a C57BL/6J background, spontaneously developed chronic inflammatory polyarthropathy. Histopathology showed marked synovial and periarticular inflammation, with articular erosion caused by invasion of granulation tissues closely resembling that of rheumatoid arthritis in humans. Moreover, elevated levels of antibodies against immunoglobulins, type II collagen, and double-stranded DNA were detected in these mice, suggesting development of autoimmunity. Proinflammatory cytokines such as IL-1beta, IL-6, and tumor necrosis factor alpha were overexpressed in the joints, indicating regulatory roles of IL-1ra in the cytokine network. We thus show that IL-1ra gene deficiency causes autoimmunity and joint-specific inflammation and suggest that IL-1ra is important in maintaining homeostasis of the immune system. Possible involvement of IL-1ra gene deficiency in RA will be discussed.

[An adult case of asymptomatic congenital tracheal stenosis].

We report an adult case of asymptomatic congenital tracheal stenosis. A 42-year-old woman was admitted to our hospital, whose chief complaint was tongue pain. Clinical examination revealed a tumor 35 x 20 mm in diameter along the middle, right edge of the tongue, and histopathological examination determined a diagnosis of squamous cell carcinoma of the tongue. She had no history of dyspnea, stridor nor recurrent pneumonia, and enjoyed playing table tennis in her daily life. Physical examination, electrocardiogram, spirogram and laboratory data showed no abnormal signs although chest roentgenogram revealed a narrowed trachea 6 mm in diameter, however, the tracheal stenosis was not noted at the time. On the 10th hospital day, radical operation for cancer of the tongue including right hemiglossectomy and supraomohyoid neck dissection, were performed. Oral intubation was not successful, and a tracheostomy was conducted. The trachea was incised, and revealed that complete tracheal rings existed without posterior membraneous tissue. The operation time was limited because of high airway pressure for a narrow tracheal tube (5 mm in diameter). The main postoperative complication was one crust formation in the tracheal lumen that resulted in CO2 narcosis due to check valve obstruction of the trachea on the 4th postoperative day. A nebulizer provided continuous moisture and was effective in preventing recurrence of the crust attachment to the tracheal membrane thus, the tracheostoma was closed by the 17th postoperative day. This case indicates that among healthy people there are extremely rare patients with congenital tracheal stenosis who survive their postnatal and infantile periods, and who experience no trouble in their daily lives. As physicians, we must be aware of this disease in adult patients in our clinics.

Bone marrow-derived cells are responsible for the development of autoimmune arthritis in human T cell leukemia virus type I-transgenic mice and those of normal mice can suppress the disease.

Previously, we reported that human T cell leukemia virus type I env-pX region-introduced transgenic (pX-Tg) mice developed an inflammatory polyarthropathy associated with a development of autoimmunity. To elucidate roles of autoimmunity in the development of arthritis, the immune cells were reciprocally replaced between pX-Tg mice and non-transgenic (Tg) mice. When bone marrow (BM) cells and spleen cells from pX-Tg mice were transferred into irradiated non-Tg mice, arthritis developed in these mice. In contrast, arthritis in pX-Tg mice was completely suppressed by non-Tg BM and spleen cells. Similar results were obtained with BM cells only. After the transplantation, T cells, B cells, and macrophages were replaced completely, whereas cells in the joints were replaced partially. In those mice, serum Ig and rheumatoid factor levels correlated with the disease development, and inflammatory cytokine expression was elevated in the arthritic joints. Furthermore, involvement of T cells in the joint lesion was suggested, because the incidence was greatly reduced in athymic nu/nu mice although small proportion of the mice still developed arthritis. These observations suggest that BM stem cells are abnormal, causing autoimmunity in pX-Tg mice, and this autoimmunity plays an important, but not absolute, role in the development of arthritis in this Tg mouse.

[Loss of heterozygosity of 3p21 and 9p21 in head and neck squamous cell carcinomas and its prognostic implication].

To examine whether genetic factors influence the prognosis of cancer patients, several microsatellite markers were used to determine the allelic loss of certain areas of the genome. Three microsatellite markers, D3S1067, IFNA and D9S171 were used to study the loss of heterozygosity (LOH) of 3p21 and 9p21 in 93 head and neck squamous cell carcinomas. Of 57 informative cases, LOH was detected in 27 of 57 (47%) DNA samples obtained from cancer specimens when at least one marker was used. The frequency of LOH was not correlated with the clinical factors. However, the frequency of LOH was significantly higher in the recurrent cases than in the non-recurrent cases, and patients with 3p21 and/or 9p21 LOH tended to survive for a shorter period of time. These results suggested that the allelic loss at 3p21 and/or 9p21 could be correlated with the prognosis of the patients, and that it was a novel prognostic factor independent of other clinical factors concerning head and neck cancers. LOH at 3p21 and/or 9p21 may help to identify head and neck cancer patients with a poor prognosis, who need an intensive postoperative follow-up protocol, or who are suitable for novel investigational therapeutic approaches.

[Superselective intra-arterial infusion chemotherapy of high-dose cisplatin for advanced paranasal sinus carcinomas].

Eighteen patients with advanced paranasal sinus carcinomas were treated by "two-route" intra-arterial chemotherapy using cis-diamminedichloroplatinum (CDDP) and sodium thiosulfate (STS) to preserve the hard palate and the eye. In these patients, 100 mg/m2 of CDDP was administered weekly through each feeding artery of the tumor superselectively at 5 mg/min. During infusion of CDDP, STS at a two-hundred fold dose of CDDP was injected through a catheter placed in the brachiocephic vein introduced via the subclavian vein. The complete and partial response rates were 14/18 (78%) and 4/18 (22%), respectively. None of the nine patients following operation showed residual tumors histologically. The peak of the mean total plasma platinum concentration was 5.5 micrograms/ml, and this concentration was rapidly reduced to 1.5 micrograms/ml in 5 hours. The peak of the plasma protein unbound platinum was 3.9 micrograms/ml. This concentration rapidly decreased to almost zero within 5 hours after IA infusion. The mean tumor platinum content achieved by superselective IA infusion was as high as 6.0 micrograms/g tumor, and this decreased rapidly to 2.4 micrograms/g tumor on the 5th day after the 1st intra-arterial infusion. All patients were free from chemotoxicity such as renal, hematological dysfunctions, or gastrointestinal symptoms. Each chemotherapy treatment could be done weekly on schedule. All but one patient was alive for 5-40 months. This new method of chemotherapy appears very effective for advanced paranasal sinus carcinomas.

The human T cell leukemia virus type I-tax gene is responsible for the development of both inflammatory polyarthropathy resembling rheumatoid arthritis and noninflammatory ankylotic arthropathy in transgenic mice.

We previously reported that inflammatory arthropathy resembling rheumatoid arthritis (RA) develops among transgenic mice carrying the long terminal repeat (LTR)-env-pX-LTR region of human T cell leukemia virus type I (LTR-pX-Tg mice). Because four genes are encoded in this region, we produced transgenic mice that only express the tax gene to examine its role in the development of arthritis. Transgenic mice were produced by constructing DNAs that express the tax gene alone under the control of either its own LTR or CD4 enhancer/promoter and by microinjecting them into C3H/HeN-fertilized ova. We produced seven transgenic mice carrying the LTR-tax gene and nine mice carrying the CD4-tax and found that one of the LTR-tax-Tg mice and five of CD4-tax-Tg mice developed RA-like inflammatory arthropathy similar to LTR-pX-Tg mice, indicating that the tax gene is arthritogenic. On the other hand, the other two LTR-tax-Tg mice had ankylotic changes caused by new bone formation without inflammation. In these ankylotic mice, tax mRNA, inflammatory cytokine mRNA, and autoantibody levels except for TGF-beta1 level were lower than those in LTR-pX- or CD4-tax-Tg mice. These results show that Tax is responsible for the development of inflammatory arthropathy resembling RA and that this protein also causes ankylotic arthropathy.