Clinical characteristics and molecular analysis of USA300 and ST 764 methicillin-resistant Staphylococcus aureus isolates from outpatients in Japan by PCR-Based open reading frame typing.

INTRODUCTION: USA300 is the most common community-acquired methicillin-resistant Staphylococcus aureus (MRSA) strain. Sequence type (ST) 764 MRSA is a new local variant of the ST 5 lineage. The objective of this study was to determine the clinical characteristics of USA300 and ST 764 infections among outpatients in Japan. METHODS: We obtained MRSA isolates from 132 outpatients who visited our hospital from January 2016 to December 2017 and compared USA300 infection group to ST 764 infection group. Molecular analysis, including that of various toxins and other virulence factors, of the MRSA isolates were performed. In particular, we investigated the relationships among PCR-based open reading frame typing (POT) scores, MRSA clones, and virulence factors. RESULTS: Twenty-seven USA300 isolates (20.5%) and 16 ST 764 isolates (12.1%) were identified. Although USA300 and ST 764 had lower rates of risk factors, their infection rates were higher. USA300-infected patients had higher rates of deep skin and soft tissue infections compared with the non-USA300 CA-MRSA-infected patients. Notably, the USA300 and ST 764 isolates had unique POT scores. CONCLUSIONS: Our results indicated that USA300 MRSA was spreading in an area 120 km west of Tokyo, Japan. We observed multiple cases of ST 764 MRSA infection, raising concerns about the antimicrobial resistance of ST 764, as it limits the choices of antibiotics to treat infection. The POT score can predict the presence of toxins and virulence factors, as well as the clone identity of MRSA with high accuracy.

The Circulation of Type F Clostridium perfringens among Humans, Sewage, and Ruditapes philippinarum (Asari Clams).

Clostridium perfringens is an important pathogen that is responsible for gastroenteritis; the causative agent for the symptoms is C. perfringens enterotoxin (CPE), which is mainly produced by type F C. perfringens. Since shellfishes may gather C. perfringens in the water environment, this study estimated the potential circulation of type F C. perfringens among humans, sewage, and Ruditapes philippinarum (asari clams) as a result of sewage pollution. A comparison of the characteristics among the isolates from 86 sewage influents, 36 effluents, 76 asari clams, and 37 humans was conducted. Serotyping, cpe genotyping, and toxin genotyping showed that C. perfringens with a plasmid IS1151 sequence downstream of cpe was predominant among sewage influents, effluents, humans, and asari clams. Multilocus sequence typing suggested that some isolates from a human, sewage influents, effluents, and asari clams were linked to each other. These results demonstrated that asari clams are the necessary infection sources of C. perfringens responsible for carriers and foodborne diseases, and that these pathogens from humans infected by asari clams can pollute the water environment. It is useful to assess bacteria such as C. perfringens isolates from sewage to estimate the trend of those from the community.

Characterization of Salmonella Isolates from Wastewater Treatment Plant Influents to Estimate Unreported Cases and Infection Sources of Salmonellosis.

Salmonella enterica is a major cause of gastroenteritis usually caused by animal-based contaminated foods. Since the current passive surveillance is not sufficient to detect all infections and infection sources, we determined the prevalence of Salmonella isolated from sewage influent of wastewater treatment plants (WWTPs) and compared the characteristics of human and food isolates to identify the infection sources. Sewage influent samples were collected monthly from two WWTPs located in the Yamanashi Prefecture, Japan, for three years. Serotypes, antimicrobial resistances, isolation periods, isolated areas, and pulsed-field gel electrophoresis patterns of six isolates belonging to five serotypes were consistent with those of the isolates from patients. Real-time PCR for Salmonella indicated that sewage influents reflect cases of patients infected with Salmonella, including unreported cases. Serovars Schwarzengrund and Anatum were predominant in sewage, but not in humans, and their characteristics were closely related or identical to those isolated from poultry heart and liver, respectively. These results suggest that sewage influent contains Salmonella isolates from humans and that some originated from unreported human cases infected by poultry-associated products. Therefore, it is necessary to take countermeasures against Salmonella infection based on the unreported cases, which would be disclosed by analysis of sewage influent.

Prediction of skeletal-related events in patients with non-small cell lung cancer.

PURPOSE: Advanced lung cancer frequently causes bone metastasis which can be associated with skeletal-related events (SREs) that may cause significant deterioration of the patient's quality of life (QoL). The Spinal Instability Neoplastic Score (SINS) can be used to assist in standardizing evaluations of neoplastic spinal instability between spinal and non-spine surgeons. This research investigated the association between SREs and SINS for patients with non-small cell lung cancer (NSCLC). METHODS: Between 2009 and 2013, 47 patients with NSCLC who were diagnosed with bone metastasis were classified using SINS into either a stable group (SINS, 0-6 points) or unstable group (SINS, 7-18 points). The primary endpoint was time from diagnosis of metastasis to SREs. Secondary endpoints included tumor type and epidermal growth factor receptor (EGFR) mutational status. SREs were defined as spinal compression, pathologic fracture, spinal surgery, and hypercalcemia. RESULTS: Patients included 37 cases of adenocarcinoma and 10 cases of squamous cell carcinoma. Mean follow-up time was 10.2 ± 13.7 months. SRE incidence was 15.0 % (3/20) in the stable group versus 44.4 % (12/27) in the unstable group (p = 0.048). A Cox regression model revealed that an EGFR-positive mutational status (hazard ratio [HR] = 0.15, 95 % CI, 0.030.71; p = 0.017) or good spinal stability (HR = 0.49; 0.08-0.99; p = 0.049) were favorable prognostic factors. CONCLUSION: The incidence of SREs was significantly lower in NSCLC patients with better spinal stability as determined by SINS, which was a good prediction tool for SREs from bone metastasis. The lower incidence of SREs in EGFR-positive patients suggests tumor biology should be considered when predicting SREs.

Transplanting neural progenitors into a complete transection model of spinal cord injury.

Neural progenitor cell (NPC) transplantation is a promising therapeutic strategy for spinal cord injury (SCI) because of the potential for cell replacement and restoration of connectivity. Our previous studies have shown that transplants of NPC, composed of neuron- and glia-restricted progenitors derived from the embryonic spinal cord, survived well in partial lesion models and generated graft-derived neurons, which could be used to form a functional relay. We have now examined the properties of a similar NPC transplant using a complete transection model in juvenile and adult rats. We found poor survival of grafted cells despite using a variety of lesion methods, matrices, and delays of transplantation. If, instead of cultured progenitor cells, the transplants were composed of segmental or dissociated segments of fetal spinal cord (FSC) derived from similar-staged embryos, grafted cells survived and integrated well with host tissue in juvenile and adult rats. FSC transplants differentiated into neurons and glial cells, including astrocytes and oligodendrocytes. Graft-derived neurons expressed glutaminergic and GABAergic markers. Grafted cells also migrated and extended processes into host tissue. Analysis of axon growth from the host spinal cord showed serotonin-positive fibers and biotinylated dextran amine-traced propriospinal axons growing into the transplants. These results suggest that in treating severe SCI, such as complete transection, NPC grafting faces major challenges related to cell survival and formation of a functional relay. Lessons learned from the efficacy of FSC transplants could be used to develop a therapeutic strategy based on neural progenitor cells for severe SCI.

The Sp1 transcription factor is essential for the expression of gliostatin/thymidine phosphorylase in rheumatoid fibroblast-like synoviocytes.

INTRODUCTION: Gliostatin/thymidine phosphorylase (GLS/TP) has angiogenic and arthritogenic activities, and aberrant GLS production has been observed in the active synovial membranes of rheumatoid arthritis (RA) patients. The human GLS gene promoter contains at least seven consensus binding sites for the DNA binding protein Sp1. Here we examined whether Sp1 is necessary for GLS production in RA. We also studied the effects of the Sp1 inhibitor mithramycin on GLS production in RA fibroblast-like synoviocytes (FLSs). METHODS: FLSs from RA patients were treated with specific inhibitors. The gene and protein expression of GLS were studied using the quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and an enzyme immunoassay. Intracellular signalling pathway activation was determined by western blotting analysis, a luciferase assay, a chromatin immunoprecipitation (ChIP) assay and a small interfering RNA (siRNA) transfection. RESULTS: The luciferase and ChIP assays showed that Sp1 binding sites in the GLS promoter were essential for GLS messenger RNA (mRNA) expression. GLS production was suppressed in FLSs by siRNA against Sp1 transfection. Mithramycin decreased GLS promoter activity, mRNA and protein expression in FLSs. Tumour necrosis factor-α (TNF-α) significantly increased GLS expression in RA FLSs; this effect was reduced by pre-treatment with cycloheximide and mithramycin. CONCLUSIONS: Pretreatment of mithramycin and Sp1 silencing resulted in a significant suppression of GLS production in TNF-α-stimulated FLSs compared to controls. GLS gene expression enhanced by TNF-α was partly mediated through Sp1. As physiological concentrations of mithramycin can regulate GLS production in RA, mithramycin is a promising candidate for anti-rheumatic therapy.

Phenotypic analysis of astrocytes derived from glial restricted precursors and their impact on axon regeneration.

Although astrocytes are involved in the production of an inhibitory glial scar following injury, they are also capable of providing neuroprotection and supporting axonal growth. There is growing appreciation for a diverse and dynamic population of astrocytes, specified by a variety of glial precursors, whose function is regulated regionally and temporally. Consequently, the therapeutic application of glial precursors and astrocytes by effective transplantation protocols requires a better understanding of their phenotypic and functional properties and effective protocols for their preparation. We present a systematic analysis of astrocyte differentiation using multiple preparations of glial-restricted precursors (GRP), evaluating their morphological and phenotypic properties following treatment with fetal bovine serum (FBS), bone morphogenetic protein 4 (BMP-4), or ciliary neurotrophic factor (CNTF) in comparison to controls treated with basic fibroblast growth factor (bFGF), which maintains undifferentiated GRP. We found that treatments with FBS or BMP-4 generated similar profiles of highly differentiated astrocytes that were A2B5-/GFAP+. Treatment with FBS generated the most mature astrocytes, with a distinct and near-homogeneous morphology of fibroblast-like flat cells, whereas BMP-4 derived astrocytes had a stellate, but heterogeneous morphology. Treatment with CNTF induced differentiation of GRP to an intermediate state of GFAP+cells that maintained immature markers and had relatively long processes. Furthermore, astrocytes generated by BMP-4 or CNTF showed considerable experimental plasticity, and their morphology and phenotypes could be reversed with complementary treatments along a wide range of mature-immature states. Importantly, when GRP or GRP treated with BMP-4 or CNTF were transplanted acutely into a dorsal column lesion of the spinal cord, cells from all 3 groups survived and generated permissive astrocytes that supported axon growth and regeneration of host sensory axons into, but not out of the lesion. Our study underscores the dynamic nature of astrocytes prepared from GRP and their permissive properties, and suggest that future therapeutic applications in restoring connectivity following CNS injury are likely to require a combination of treatments.

FK506 inhibition of gliostatin/thymidine phosphorylase production induced by tumor necrosis factor-α in rheumatoid fibroblast-like synoviocytes.

Gliostatin/thymidine phosphorylase (GLS/TP) is known to have angiogenic and arthritogenic activities. The purpose of this study was to determine the inhibitory effects of FK506 (tacrolimus) on GLS production in rheumatoid arthritis (RA). We investigated the modulation of serum GLS by FK506 therapy and the effect of FK506 on the production of GLS in fibroblast-like synoviocytes (FLSs). Serum samples were collected from 11 RA patients with active disease at baseline and after 12 weeks of FK506 treatment. Serum concentrations of GLS and matrix metalloproteinase (MMP)-3 were measured by ELISA and found to be down-regulated in responders evaluated with a disease activity score. Patient FLSs were cultured and stimulated by tumor necrosis factor (TNF)-α with or without FK506. The expression levels of GLS were determined using reverse transcription-polymerase chain reaction (RT-PCR) and enzyme immunoassay and shown to be significantly increased. GLS levels in TNF-α-stimulated FLSs were reduced by FK506 treatment. Our data show a novel mechanism for the action of physiological concentrations of FK506 in RA that regulates the production of GLS in FLSs.

TNF-alpha-induced aquaporin 9 in synoviocytes from patients with OA and RA.

OBJECTIVES: To determine whether aquaporins (AQPs) are expressed in the synovial tissues of patients with OA and RA, and to examine the patterns of expression in patients with and without hydrarthrosis. METHODS: AQPs were detected in synovial tissue samples from patients with OA and RA using RT-PCR and immunohistochemistry. Fibroblast-like synoviocytes (FLSs) from patients with OA and RA were cultured and stimulated with TNF-alpha. The expression of AQPs in FLSs was examined using RT-PCR and western blot analyses and the function of aquaglyceroporins was examined by a glycerol uptake assay. RESULTS: AQP1, -3 and -9 mRNAs were expressed in synovial tissues from patients with OA and RA. AQP1, -3 and -9 proteins were also detected by immunohistochemistry. AQP9 mRNA was expressed more strongly in the synovial tissues of OA patients with hydrarthrosis than those without. AQP9 mRNA and protein expression were strongly induced with TNF-alpha treatment in FLSs, whereas the expression of AQP1 and -3 mRNAs was not induced with TNF-alpha treatment. AQP9 as an aquaglyceroporin was induced by TNF-alpha. CONCLUSIONS: AQP9 mRNA was detected in synovial tissues from OA and RA patients with hydrarthrosis. AQP9 expression was strongly induced in FLSs with TNF-alpha. Although the functions of AQP1, -3 and -9 in synovial tissues remain to be elucidated, it suggested that AQP9 might be related to the pathogenesis of hydrarthrosis and inflammatory synovitis.

[Two cases of group A rotavirus outbreak among adults].

Two cases of group infections with the group A rotavirus (ARV) occurred among adult residents at welfare facilities. Case 1 occurred at a care facility for the elderly between March and April of 2000, where 14 patients aged 68 to 93 (average: 85) of 93 residents developed cardinal symptoms of diarrhea, vomiting and fever. ARV was found in 7 stool samples and their G-serotype was G2. Case 2 occurred at a facility for the disabled in April 2006, where 15 patients aged 28 to 64 (average: 48) of 51 residents developed cardinal symptoms of diarrhea, vomiting and fever. ARV was found in 11 stool samples and their G-serotype was G1. The ARV route of infection was not specified in either case. Since G2 was identified among sporadic diarrheas in the general public during the 1999/2000 season when Case 1 occurred, and similarly for G1 during the 2005/2006 season for Case 2, there may be an associations between group-infection cases among adults and sporadic diarrhea in the general public. Of the three types of antigen detection kit and RT-PCR used to detect ARV for this study, RT-PCR showed the highest detection. RT-PCR was considered to be the most effective in detecting the virus for group infection cases.

[Evaluation of Norwalk virus detection kit by enzyme immunoassay].

We briefly examined detection kit using the EIA method for Norwalk virus, and compared the results of the tests using the EIA method with those using RT-PCR method. In reproducibility, an amount of variation was observed in data obtained from positive controls and in lower values. The sensitivity obtained from the EIA method was about 300 times lower than that obtained from the RT-PCR method. Results accordance ratio between EIA method and RT-PCR method was 70%. This results discrepancy was presumably caused by a difference of sensitivity and specificity between these two methods. In conclusion, this detection kit using the EIA method is easily manually operated so that this kit can be considered as an effective and simple detection tool for Norwalk virus.