Shintaro Yanagimoto

Clostridium difficile flagellin stimulates toll-like receptor 5, and toxin B promotes flagellin-induced chemokine production via TLR5

AIMS Clostridium difficile is an important pathogen in nosocomial infections. Although C. difficile toxins are considered to be major virulence factors, pathogenesis of C. difficile associated diseases remains to be determined. In this study, we investigated whether C. difficile flagellin is involved in the pathogenesis of C. difficile-associated diseases. MAIN METHODS C. difficile flagellin was extracted from bacterial body by using a combination of ultracentrifugation and low speed centrifugation. Extracted C. difficile flagellin was added to HEK293T cells transiently transfected with pUNO-mcs (empty vector) or pUNO-hTLR5, and NF-kappaB activation was compared by a dual-luciferase assay. The amount of C. difficile flagellin-induced inflammatory mediators such as interleukin-8 and CCL20 was measured by ELISA assay in the culture media of intestinal epithelial cell lines, HT29 cells and Caco-2 cells. Flagellin induced phosphorylation of p38 mitogen-activated protein kinase was examined by Western blotting analysis in Caco-2 cells. The amount of C. difficile flagellin-induced inflammatory mediators in the presence, or absence of C. difficile toxin B was also measured by ELISA assay. KEY FINDINGS C. difficile flagellin induced activation of NF-kappaB in HEK293T cells via toll-like receptor 5. C. difficile flagellin also induced activation of p38 mitogen-activated protein kinase, and promoted the production of interleukin-8 and CCL20 in intestinal epithelial cells via toll-like receptor 5. Pretreatment with toxin B enhanced flagellin-induced cytokine productions. SIGNIFICANCE Our results indicate that toxin B promotes flagellin-induced activation of intestinal epithelial cells, and that C. difficile flagellin may play a role in the occurrence of C. difficile-associated diseases.

Bacterial flagellin inhibits T cell receptor‐mediated activation of T cells by inducing suppressor of cytokine signalling‐1 (SOCS‐1)

Flagellin, the structural component of bacterial flagella, is secreted by pathogenic and commensal bacteria, and is recognized by Toll‐like receptor (TLR) 5. Flagellin is a common bacterial antigen present on most motile bacteria and is speculated to contribute to the activation of CD4+ T cells in the intestine. However, molecular mechanisms by which flagellin regulate T cell activation remains to be determined. Using Jurkat T cells or human primary T cell, we showed that flagellin stimulation induced tyrosine phosphorylation of TLR5 and activation of both mitogen‐activated protein kinases and nuclear factor κB. In addition, stimulation by flagellin did not induce nuclear factor of activated T cells (NFAT) activation, while stimulation via the T cell receptor (TCR) leads to activation of NFAT. However, TCR‐mediated NFAT activation and tyrosine phosphorylation of zeta‐associated protein 70 (Zap‐70) were inhibited in cells pre‐stimulated by flagellin. Furthermore, flagellin stimulation induced suppressor of cytokine signalling‐1 (SOCS‐1), which formed a complex with Zap‐70 after stimulation via TCR, and inhibition of SOCS‐1 expression by SOCS‐1‐specific small interfering RNA reinstated TCR‐mediated activation of NFAT in cells pre‐stimulated with flagellin. These results collectively indicate that bacterial flagellin inhibits TCR‐mediated activation of T cells by inducing SOCS‐1.

Raf1 plays a pivotal role in lipopolysaccharide-induced activation of dendritic cells.

Activation of extracellular-regulated kinases 1/2 (ERK) is involved in lipopolysaccharide (LPS)-induced cellular responses such as the increased production of proinflammatory cytokines. However, mitogen-activated protein kinases (MAPKs) such as p38 are also activated by LPS and have been postulated to be important in the control of these end points. Therefore, establishing the relative contribution of MAPKs in each cell type is important, as is elucidating the molecular mechanisms by which these MAPKs are activated in LPS-induced signaling cascades. We demonstrated in DC2.4 dendritic cells that ERK regulates tyrosine phosphorylation of phosphatidyl-inositol-3-kinase (PI3-K) and the production of TNF-alpha. We also demonstrated that Raf1 is phosphorylated and involved in the production of TNF-alpha and tyrosine phosphorylation of PI3-K via ERK. Raf1 also regulates the activation of NF-kappaB. We propose that Raf1 plays a pivotal role in LPS-induced activation of the dendritic cells.

MYD88 (L265P) mutation is associated with an unfavourable outcome of primary central nervous system lymphoma.

Primary central nervous system lymphomas (PCNSL) are rare forms of diffuse large B cell lymphoma (DLBCL) that arise within the brain or the eyes. The current molecular classification of DLBCL distinguishes two main subtypes: activated B-cell-like (ABC)-lymphoma and germinal centre B-cell-like (GCB)-lymphoma (Zhang et al, 2013). PCNSL typically show an immunophenotype resembling that of ABC-DLBCL (Zhang et al, 2013). Somatic mutations in the myeloid differentiation primary response gene 88. (MYD88), CD79b molecule, immunoglobulin-associated beta (CD79B) and caspase recruitment domain family, member 11 (CARD11) genes were recently shown to control cell survival of ABC-DLBCL by promoting the nuclear factor (NF)jB signalling pathway (Zhang et al, 2013). The mutation frequencies of MYD88, CD79B and CARD11 are 15–30%, 17–23% and 8–18% in ABC-DLBCL (Zhang et al, 2013) and are reported to be 40–79%, 30~-44% and 11–30% in PCNSL, Indicating that they are strongly over-represented in PCNSL (Braggio et al, 2015; Yamada et al, 2015). In addition, several other gene mutations were more prevalent in PCNSL than in DLBCL, not otherwise specified; “pim-1 oncogene” (PIM1: 20–44%), “transducin (beta)-like 1 X-linked receptor 1” (TBL1XR1: 7–23%), “BTG family, member 2” (BTG2: 22– 30%), “PR domain containing 1, with ZNF domain” (PRDM1: 7–20%) and “tumor necrosis factor, alpha-induced protein 3” (TNFAIP3: 11–20%) (Braggio et al, 2015). Nonetheless, the clinical relevance of these mutations remains unclear, although several specific clinical parameters may predict the treatment outcome of PCNSL patients. These include altered mental activity and elevated creatinine clearance (CrCl) (Taoka et al, 2010); age >50 years and Karnofsky performance score <70 (Memorial Sloan-Kettering Cancer Center prognostic score; Abrey et al, 2006); age >75 years, increased serum lactate dehydrogenase level, cerebrospinal fluid protein concentration and lymphoma involvement of deep brain structures (International Extranodal Lymphoma Study Group prognostic scoring system; Abrey et al, 2006), were identified as prognostic factors. However, recent advances in genetic knowledge are not incorporated in any of these proposals. Hence, we conducted this study to elucidate the impact of gene mutations on clinical outcome of PCNSL. We analysed 42 human immunodeficiency virus (HIV)negative PCNSL patients who were treated with a modified version of the European Organization for Research and Treatment of Cancer protocol (Taoka et al, 2010) at the University of Tsukuba Hospital between March 2005 and May 2015. This protocol consisted of intermediate-dose methotrexate (MTX), lomustine, procarbazine, methylprednisolone and intrathecal chemotherapy with MTX and cytarabine (Table SI) (Taoka et al, 2010). All PCNSL patients in this study had DLBCL. GCB and ABC phenotypes were assessed by immunohistochemistory using anti-CD10, MUM1 and BCL6 antibodies. Targeted deep sequencing was performed by using Ion Ampliseq technology (Methods S1, Table SII). Clinical parameters were collected from the clinical record (Methods S1). Overall survival (OS) and progression-free survival (PFS) were estimated using the KaplanMeier method and compared by the Peto-Peto generalized Wilcoxon test. All data were analysed with EZR (http://www. jichi.ac.jp/saitama-sct/SaitamaHP.files/statmedEN.html). Multivariate analysis was performed with Cox regression model. Significance was set at P ≤ 0 05. The major clinical characteristics of the 42 patients are summarized in Table SIII. At least one mutation was detected in 38 out of 42 cases (90 4%). Notably, we found recurrent mutations in MYD88 (67%), CD79B (43%), PIM1 (69%), TBL1XR1 (24%), BTG2 (29%), PRDM1 (24%) and TNFAIP3 (12%) (Table SIV, Fig. S1) at frequencies similar to those previously reported (Braggio et al, 2015; Yamada et al, 2015). For the entire cohort, the median follow-up time was 26 months (range, 1–105 months), with 3-years OS and PFS of 46% and 26%, respectively, comparable to the survival rate of our previous cohort treated with the same protocol (Taoka et al, 2010). Univariate analysis showed that age >75 years (P = 0 0105) and altered mental activity (P = 0 0202) (Table SV) were significantly associated with inferior OS. In addition, MYD88 L265P mutation (P = 0 127, Fig. 1A), CrCl >90 ml/min (P = 0 124) and deep brain involvement (P = 0 0649) showed a tendency to be associated with inferior OS, although statistically non-significant (Table SV). When adjusted in a multivariate Cox regression analysis, MYD88 L265P mutation remained as a significant risk factor for death (hazard ratio (HR), 2 903; 95% confidence interval (CI), 1 013–8 323, P = 0 0474), together with altered mental activity (HR, 4 729; 95% CI, 1 522–14 7, P = 0 0072) (Table I). Regarding PFS, CrCl >90 ml/min (P = 0 0286) and altered mental activity (P = 0 0473) were significant factors for inferior PFS (Table SV). MYD88 L265P mutation (P = 0 0872, Fig. 1B) was not significantly associated, but showed a tendency to be

Detection of the G17V RHOA mutation in angioimmunoblastic T-cell lymphoma and related lymphomas using quantitative allele-specific PCR.

Angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) are subtypes of T-cell lymphoma. Due to low tumor cell content and substantial reactive cell infiltration, these lymphomas are sometimes mistaken for other types of lymphomas or even non-neoplastic diseases. In addition, a significant proportion of PTCL-NOS cases reportedly exhibit features of AITL (AITL-like PTCL-NOS). Thus disagreement is common in distinguishing between AITL and PTCL-NOS. Using whole-exome and subsequent targeted sequencing, we recently identified G17V RHOA mutations in 60–70% of AITL and AITL-like PTCL-NOS cases but not in other hematologic cancers, including other T-cell malignancies. Here, we establish a sensitive detection method for the G17V RHOA mutation using a quantitative allele-specific polymerase chain reaction (qAS-PCR) assay. Mutated allele frequencies deduced from this approach were highly correlated with those determined by deep sequencing. This method could serve as a novel diagnostic tool for 60–70% of AITL and AITL-like PTCL-NOS.

A Single Amino Acid of Toll-like Receptor 4 That Is Pivotal for Its Signal Transduction and Subcellular Localization

Toll-like receptor 4 (TLR4) is essential for recognizing a Gram-negative bacterial component, lipopolysaccharide (LPS). A single amino acid mutation at position 712 of murine TLR4 leads to hyporesponsiveness to LPS. In this study we determined that an amino acid, a leucine at position 815 of human TLR4, is also pivotal for LPS responsiveness and subcellular distribution. By replacing the leucine with alanine, the mutant TLR4 lost responsiveness to LPS and did not localize on the plasma membrane. In addition, it does not coprecipitate with myeloid differentiation-2, an accessory protein that is necessary for TLR4 to recognize LPS. These results suggest that the leucine at position 815 is required for the normal maturation of TLR4 and for formation of the TLR4·MD-2 complex.

Discrimination of eating habits with a wearable bone conduction sound recorder system

Continuous monitoring of eating habits could be useful in preventing lifestyle diseases such as the metabolic syndrome. Conventional methods consist in self-reporting and mastication frequency calculation from myoelectric potential of the masseter muscle, both resulting in a significant burden for the user. We developed a non-invasive wearable sensing system that can record eating habits over a long period of time in daily life. Our original sensing system is composed by a bone conduction microphone placed in the ears, from which sound data are collected to a portable IC recorder. Applying frequency spectrum analysis on collected sound data, we could not only count the mastication number during eating, but also accurately differentiate eating, drinking, and speaking activities, which can be used to evaluate the regularity of meals. Moreover, using clustering of sound spectra, we found it is possible to classify types of foods eaten regarding their texture.

Scedosporium prolificans Endocarditis: Case Report and Literature Review.

Scedosporium prolificans, a hyaline filamentous fungus, is widely distributed in the environment and is currently an emerging human pathogen, especially among immunocompromised patients. However, S. prolificans endocarditis is rare. We herein report a case of S. prolificans endocarditis in a 64-year-old patient with breast cancer in complete remission for 30 years after chemotherapy and radiation treatment who was not cured. Susceptibility testing showed resistance to all antifungal drugs, except echinocandin. A review of the literature revealed 10 cases of S. prolificans endocarditis; of these, only one involved an immunocompetent host with no risk factors and only two patients survived. In order to improve the mortality rate, it is necessary to establish rapid diagnostic methods and efficient therapeutic approaches.

A pathologically proven case of adult-onset HIV-related lymphocytic interstitial pneumonia with acute exacerbation treated with steroid and antiretroviral therapy

Lymphocytic interstitial pneumonia (LIP) is a rare opportunistic illness in human immunodeficiency virus (HIV)-infected adults, although it is relatively common among HIV-infected children. Most adult cases have been reported in African and Afro-Caribbean patients and few cases have been reported from Asia. Acute exacerbation of HIV-related LIP has not been well described. Here we report a pathologically proven case of acute exacerbation of adult-onset HIV-related LIP. The patient was an African immigrant living in Japan who presented with chronic dyspnea and diffuse bilateral pulmonary infiltrates. His clinical, radiological, and pathological findings were consistent with those of LIP. After a diagnostic surgical lung biopsy, his hypoxemia and pulmonary infiltrates exacerbated rapidly over a few days, although his condition had not progressed during the previous year. LIP may be an important differential diagnosis among adult patients in Asian countries, especially patients of non-Asian ethnicity.

A Comparative Study of Oral Health Status between International and Japanese University Student Patients in Japan

Background: The number of international students enrolled in universities in Japan is increasing. To provide better oral care services for international students, we have to understand their oral environment and dental health behaviors. However, few studies have investigated the oral health status of international university students. The object of the present study was to clarify the current oral status of international university students. Methods: The subjects were students who visited the dental department at the University of Tokyo’s Health Services Center between April 2012 and March 2013. Our medical records were reviewed with regard to the following items: attributes (nationality, gender, and age); chief complaint (reason for visit); history of dental treatment; mean number of decayed (D), missing (M) or filled (F) teeth as a single (DMFT) index; degree of calculus deposition; gingival condition; and oral hygiene status. Results: The records of 554 university students (138 international and 416 non-international students) were analyzed; 88.4% of the 138 international students were from Asian countries (n = 122), of which 47.1% were from China and 10.9% from Korea, followed by North America (5.8%), Europe (4.3%), and Africa (1.5%). Although no significant differences were found regarding the history of dental treatment between international and non-international students (49.3% and 48.8%, respectively), international students had a significantly higher dental caries morbidity rate (60.1%) than non-international students (49.0%). The international students showed a significantly higher DMFT value compared with the non-international students: 5.0 and 4.0 per individual, respectively. Severe calculus deposition was observed in international students compared with non-international students (51.9% and 31.7%, respectively). Conclusions: The international university students had poorer oral health status than the non-international students, even though the result might include many uncertainties and possible biases.