Shigemi Kondo

Granulysin in human serum as a marker of cell‐mediated immunity

Granulysin is a cytolytic granule protein of natural killer (NK) cells and cytotoxic T lymphocytes (CTL) with a broad range of antimicrobial and tumoricidal activities. Two molecular forms of granulysin, the 15‐kDa precursor and 9‐kDa mature form, are produced in these cells. In this study, we developed monoclonal antibodies against granulysin and found that the 15‐kDa granulysin is spontaneously secreted by peripheral blood NK and T cells via a non‐granule exocytotic pathway. When NK cells killed the target cells, the released granulysin levels in culture supernatants significantly increased through the granule exocytosis. The granulysin protein was found in the sera of healthy individuals at an average concentration of 3.7±3.2 ng/ml (age 0–99 years, n=244). The serum levels of granulysin were transiently highly elevated among patients with acute viral infections. In addition, the serum granulysin levels in patients with severe immunodeficiency treated bycell therapy fluctuated proportionately to the improvement of other immunological parameters. Our results suggest that granulysin is well associated with diverse activities of NK cells and CTL in physiological and pathological settings and could be a useful novel serum marker to evaluate the overall status of host cellular immunity.

Effect of the continuous intake of probiotic-fermented milk containing Lactobacillus casei strain Shirota on fever in a mass outbreak of norovirus gastroenteritis and the faecal microflora in a health service facility for the aged.

For conducting effective risk management in long-stay elderly people at a health service facility, we performed an open case-controlled study to evaluate the effect of the intake of probiotic-fermented milk containing Lactobacillus casei strain Shirota (LcS-fermented milk) on norovirus gastroenteritis occurring in the winter season during the intake period. A total of seventy-seven elderly people (mean age 84 years) were enrolled in the study. During a 1-month period, there was no significant difference in the incidence of norovirus gastroenteritis between the LcS-fermented milk-administered (n 39) and the non-administered (n 38) groups; however, the mean duration of fever of >37°C after the onset of gastroenteritis was 1·5 (SD 1·7) d in the former and 2·9 (SD 2·3) d in the latter group, showing a significant shortening in the former group (P < 0·05). RT-quantitative PCR analysis targeting ribosomal RNA showed both Bifidobacterium and Lactobacillus to be significantly dominant, whereas Enterobacteriaceae decreased in faecal samples from the administered group (n 10, mean age 83 years), with a significant increase in faecal acetic acid concentration. Continuous intake of LcS-fermented milk could positively contribute to the alleviation of fever caused by norovirus gastroenteritis by correcting the imbalance of the intestinal microflora peculiar to the elderly, although such consumption could not protect them from the disease.

A Novel Serum Protein That Is Selectively Produced by Cytotoxic Lymphocytes

Cytotoxic lymphocytes such as CTL and NK cells play principal roles in the host defense mechanisms. Monitoring these effector cells in vivo is helpful to understand the immune responses in disorders such as cancer and infectious diseases. In this study, we identified a novel secretory protein, killer-specific secretory protein of 37 kDa (Ksp37), as a Th1-specific protein by a subtractive cloning method between human Th1 and Th2 cells. In peripheral blood leukocytes, Ksp37 expression was limited to Th1-type CD4+ T cells, effector CD8+ T cells, γδ T cells, and CD16+ NK cells. Most of these Ksp37-expressing cells coexpressed perforin, indicating that Ksp37 is selectively and commonly expressed in the lymphocytes that have cytotoxic potential. Ksp37 was released at constant rate from both unstimulated and stimulated PBMCs in vitro and also detected in normal human sera. In healthy individuals, serum Ksp37 levels were significantly higher in children (mean ± SD; 984 ± 365 ng/ml for age 0–9) than in adults (441 ± 135 ng/ml for age 20–99), consistent with reported differences in the absolute counts of blood T and NK cells between children and adults. In patients with infectious mononucleosis, transient elevation of serum Ksp37 levels was observed during the early acute phase of primary EBV infection. These results suggest that Ksp37 may be involved in an essential process of cytotoxic lymphocyte-mediated immunity and that Ksp37 may also have clinical value as a new type of serum indicator for monitoring cytotoxic lymphocytes in vivo.

Heterogeneously vancomycin-intermediate Staphylococcus aureus (hVISA) emerged before the clinical introduction of vancomycin in Japan: a retrospective study

Vancomycin-intermediate Staphylococcus aureus (VISA) and its precursor, heterogeneous VISA (hVISA), are increasingly the cause of vancomycin treatment failure. Prolonged glycopeptide treatment causes the emergence of these pathogens. However, we recently reported that hVISA can be generated by methicillin-resistant S. aureus (MRSA) exposure to imipenem (Katayama et al., Antimicrob Agents Chemother. 53:3190–6). We report here a retrospective prevalence study of VISA and hVISA on 750 MRSA clinical strains isolated from 31 Japanese national university hospitals in 1990, the year before the introduction of injectable vancomycin into clinical use in Japan in 1991. No VISA strain was identified, but population analysis identified 38 hVISA strains (5.1%) from 19 hospitals. We also determined the nucleotide sequences of vraSR, walRK,clpP, and rpoB genes whose mutations are known to be associated with vancomycin resistance. When compared with vancomycin-susceptible MRSA strain N315, six of the 38 hVISA strains possessed nonsynonymous mutations in vraSR, seven in walRK, and two in rpoB genes, Thirteen of 38 (34.2%) hVISA strains possessed at least one of these mutations. Results were consistent with our hypothesis that hVISA was present in Japanese hospitals before the clinical introduction of vancomycin.

Seasonal variation in liver function tests: a time-series analysis of outpatient data

Background Long-term physiological variations, such as seasonal variations, affect the screening efficiency at medical checkups. This study examined the seasonal variation in liver function tests using recently described data-mining methods. Methods The ‘latent reference values’ of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyltransferase (γGT), cholinesterase (ChE) and total bilirubin (T-Bil) were extracted from a seven-year database of outpatients (aged 20–79 yr; comprising approximately 1,270,000 test results). After calculating the monthly means for each variable, the time-series data were separated into trend and seasonal components using a local regression model (Loess method). Then, a cosine function model (cosinor method) was applied to the seasonal component to determine the periodicity and fluctuation range. A two-year outpatient database (215,000 results) from another hospital was also analysed to confirm the reproducibility of these methods. Results The serum levels of test results tended to increase in the winter. The increase in AST and ALT was about 6% in men and women, and was greater than that in ChE, ALP (in men and women) and γGT (in men). In contrast, T-Bil increased by 3.6% (men) and 5.0% (women) in the summer. The total protein and albumin concentrations did not change significantly. AST and ALT showed similar seasonal variation in both institutions in the comparative analysis. Conclusions The liver function tests were observed to show seasonal variations. These seasonal variations should therefore be taken into consideration when establishing either reference intervals or cut-off values, which are especially important regarding aminotransferases.

Association between antimicrobial consumption and clinical isolates of methicillin-resistant Staphylococcus aureus: a 14-year study

The objective of this study was to determine the relationship between clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and antimicrobial consumption in hospitalized patients over a 14-year period. The study was retrospectively conducted between January 1995 and December 2008 at Juntendo University Hospital, Tokyo, Japan, a 1,020-bed tertiary-care teaching hospital. The incidence of MRSA isolates was examined using clinical specimens presented to the microbiology laboratory in the hospital. Antimicrobial consumption through intravenous injection was calculated in terms of the number of defined daily doses per 100 bed-days. The correlation between the incidence of MRSA isolates and antimicrobial consumption was determined employing a multiple stepwise regression analysis. A total of 109,946 bacterial isolates were consecutively collected over the 14-year period, and, of these, 13,872 (64% of S. aureus strains excluding coagulase-negative staphylococci) were MRSA strains. The longitudinal observation showed that the number and rate of MRSA isolates marginally decreased. The rate of MRSA isolates among S. aureus strains in 1995 was 68.5%, whereas that in 2008 was 53.8%. Consumption of cephalosporins decreased. Among carbapenems, the rate of imipenem (IPM) consumption decreased, whereas that of meropenem increased. A multiple stepwise regression analysis revealed that the antimicrobial consumption of cefmetazole, cefotiam, and IPM was positively correlated with the incidence of MRSA isolates. The use of β-lactam antimicrobials may contribute to the development of MRSA strains.

Analysis of Staphylococcal cassette chromosome mec in BD GeneOhm MRSA assay-negative strains.

ABSTRACT The BD GeneOhm MRSA assay could identify methicillin-resistant Staphylococcus aureus (MRSA) strains at a high ratio (97.8%). Analysis of 11 assay-negative MRSA strains suggested that insertion of non-mec staphylococcal cassette chromosome elements (SCCs) downstream of orfX, and carriage of SCCmecs with a left extremity that cannot be detected by the kit, might lead to their being given an incorrect negative status.