Takatoshi Kitazawa

Cryptococcus gattii Genotype VGIIa Infection in Man, Japan, 2007

We report a patient in Japan infected with Cryptococcus gattii genotype VGIIa who had no recent history of travel to disease-endemic areas. This strain was identical to the Vancouver Island outbreak strain R265. Our results suggest that this virulent strain has spread to regions outside North America.

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.

Clinical efficacy of peramivir in adult patients with seasonal influenza during the winter of 2012 in Japan

Peramivir is an intravenously administered neuraminidase inhibitor for influenza. The clinical efficacy of peramivir remains unclear because it is used in a limited number of countries. We compared the clinical efficacy of peramivir with that of oseltamivir in influenza patients during the 2012–2013 season.

Characteristics of Serum Endocan Levels in Infection

Objectives Endocan is a newly recognized biomarker of sepsis. However, there have been no studies of the trends in endocan levels during infection and their associations with other clinical factors. The aim of this study was to assess the time course of endocan levels and the associations of endocan with clinical factors during infection by comparison with other biomarkers. Methods Serum samples and blood cultures were obtained from patients who were diagnosed with infection from June 2013 to March 2014. Serum endocan, C-reactive protein (CRP), and procalcitonin (PCT) levels during four periods during infection were measured (day 0, day 1-2, day 3-5, and day 6-10). Results A total of 78 patients were enrolled in this study. The median endocan level decreased by only 23% during infection, whereas both serum CRP and PCT levels decreased by more than 80%. Endocan levels were correlated to neither CRP levels nor PCT levels in each period. Endocan levels at day 0 in patients with bacteremia were higher than those without bacteremia (1.09 ng/mL vs 0.82 ng/mL, P=0.002), but neither CRP levels nor PCT levels at day 0 were different between the two groups. Areas under the receiver operator characteristic (ROC) curves of endocan, CRP, and PCT at day 0 were 0.662, 0.343, and 0.563, respectively. Positive blood cultures tended to be related to high endocan levels, but not significantly (odds ratio: 4.24, 95% CI: 0.99-10.34, P=0.05). Conclusions In bacteremic cases, serum endocan levels in bacteremia tended to be higher than in non-bacteremic cases. Although endocan level was not identified as a prognostic factor of bacteremia, further prospective study concerning the relationship between serum endocan level and bacteremia would be needed.

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.

Lopinavir inhibits insulin signaling by promoting protein tyrosine phosphatase 1B expression

Treatment with antiretroviral therapy, including protease inhibitors (PIs), may result in metabolic side-effects, for example insulin resistance. The aim of the present study was to investigate the mechanism of the dysregulation of insulin signaling by two PIs, lopinavir and darunavir, by analyzing changes in the expression or activity of proteins associated with insulin signaling. 3T3-L1 preadipocytes were pretreated with lopinavir or darunavir for 48 h and then stimulated with insulin for 30 min. The cell lysates were subjected to western blotting with anti-phospho-insulin receptor substrate (IRS) 1, anti-IRS1, anti-suppressor of cytokine signaling (SOCS) 1, anti-SOCS3 and anti-protein tyrosine phosphatase (PTP) 1B antibodies and to immunoprecipitation with anti-IRS1 antibody. Translocation of glucose transporter 4 (GLUT4) following treatment with lopinavir or darunavir was observed using immunofluorescence. While GLUT4 was recruited to the cellular membrane in control adipocytes following insulin stimulation, it was diffusely distributed in the cytosol in lopinavir-treated adipocytes. In darunavir-treated adipocytes, GLUT4 was mainly recruited to the cellular membrane, but some GLUT4 remained in the cytosol. After insulin stimulation, IRS1 was tyrosine-phosphorylated to a greater extent in control adipocytes compared with darunavir-treated adipocytes. Tyrosine phosphorylation of IRS1 was inhibited in lopinavir-treated adipocytes. The expression of PTP1B was upregulated in adipocytes pretreated with the PIs, particularly lopinavir, compared with those pretreated with a vehicle control. The degree of regulation in insulin signaling differs between lopinavir and darunavir. One mechanism by which lopinavir regulates insulin signaling is by the promotion of PTP1B expression.

Infective endocarditis due to Enterobacter cloacae resistant to third- and fourth-generation cephalosporins

We report the case of using a long-term combination of meropenem and amikacin to treat infective endocarditis caused by Enterobacter cloacae resistant to third- and fourth-generation cephalosporins. Multi-drug resistant Gram-negative bacilli, such as the E. cloacae in our study, may become possible pathogens of infective endocarditis. Our experience with this case indicates that long-term use of a combination of β-lactam and aminoglycosides might represent a suitable management option for future infective endocarditis cases due to non-Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, Kingella spp. (HACEK group) Gram-negative bacilli such as ours.

Increase of 25-hydroxyvitamin D levels after initiation of combination antiretroviral therapy

Although vitamin D deficiency in HIV patients reported worldwide, the mechanisms and the effect of combination antiretroviral therapy (cART) on vitamin D levels are unclear. Patients were 50 male Japanese with HIV who visited Teikyo University Hospital, Tokyo, Japan. Patients were divided into those receiving cART (cART-experienced group, n = 30) and those who had not received cART (cART-naïve group, n = 20). Patients in the cART-experienced group had received treatment with cART for more than one year and those in the cART-naïve group were just about to start cART at study entry. Patients underwent measurement of serum 25-hydroxyvitamin D (25(OH)D) levels and assessment of clinical factors twice at one year intervals. At study entry, 23 (76.7%) in the cART-experienced group and 19 (95.0%) in the cART-naïve group had vitamin D insufficiency or deficiency. Mean 25(OH)D values were significantly higher in the cART-experienced group (25.2 ng/ml vs. 19.3 ng/ml, p = 0.01). However, levels of 25(OH)D at one year increased more in the cART-naïve group (-1.1 ng/ml vs. 5.0 ng/ml, p = 0.01), with mean 25(OH)D values in the cART-naïve group increasing to match those in the cART-experienced group. HIV infected patients who initiated cART showed increases in vitamin D levels in one year.

Prevalence and risk factors for loss of bone mineral density in male Japanese patients with HIV.

To the Editors: As the mortality of HIV-infected individuals was improved by combination antiretroviral therapy (cART), opportunistic infections were replaced by long-term complications including loss of bone mineral density (BMD). It was first reported in 1999 and has emerged in the last decade and has probably placed HIV-infected individuals at higher risk of bone fractures. The causes are considered to be multifactorial and include HIV infection itself, body mass index (BMI), estimated glomerular filtration rate (eGFR), testosterone level, and hepatitis coinfection. Some articles link the use of protease inhibitors and the tenofovir (TDF) with lower BMD. Most of the articles associated with BMD loss in HIV-infected patients have been reported from North America, Europe, and Oceania. Accordingly, the BMD research on HIV-infected Asians is underrepresented. In this work, we focused on male Japanese HIV-infected patients and studied the prevalence and severity of BMD loss among them and attempted to determine clinical factors related to BMD loss. Forty male Japanese HIV-infected individuals aged from 21 to 70 years who visited the Teikyo University Hospital were enrolled in this study. This study was approved by the ethical committee of the Teikyo University School of Medicine, and the patients gave written informed consent. The patients underwent BMD analyses from March 2010 to February 2012 with the same dual energy X-ray absorptiometry scan (Discovery SI, Hologic Inc, Bedford, MA). Thirty-nine patients were analyzed in both the lumbar spines and bilateral femoral necks, whereas 1 patient was limited to the bilateral femoral necks. The BMD data of the lumbar spines and the smaller BMD value of the femoral necks were employed to calculate T-scores that were calculated as comparison with young normal reference value expressed as SD units. Osteopenia was defined as a T-score of between −1 and −2.5SD, and osteoporosis was defined as that of