Yoshihisa Itoh

Alterations in penicillin binding protein gene of Streptococcus pneumoniae and their correlation with susceptibility patterns

Penicillin binding protein (pbp) gene alterations of 328 clinical isolates of Streptococcus pneumoniae were examined for a correlation with their antibiotic-resistance. The frequency of penicillin G (PEN-G) resistance was determined to clarify susceptibility to several antibiotics, namely PEN-G, ampicillin, sulbactam/ampicillin, cefozopram, panipenem (PAPM), clarithromycin (CLR), azithromycin (AZM) and levofloxacin (LVX). Oligonucleotide primers for three pbp genes (pbp1a, pbp2x and pbp2b) were used to detect mutations in pbp. Of the strains, 25.9% were classified as Pen-Gs, 68.0% as Pen-Gir and 6.1% as Pen-Gr. The polymerase chain reaction product for wild-type pbp1a was found in 185 isolates, that for wild-type pbp2x was found in 66 isolates and that for wild-type pbp2b was found in 213 isolates. None of these three genes was detectable in 100 isolates while all of them were detected in 64 isolates (1aw/2xw/2bw). Of those 64 isolates with 1aw/2xw/2bw, the minimum inhibitory concentration (MIC) of PEN-G was < or =0.06 mg/l for 54 isolates and 0.12 mg/l for 10 isolates. Of the 272 strains for which the MIC of PAPM was < or =0.03 mg/l, there were 85 Pen-Gs, 184 Pen-Gir and three Pen-Gr isolates. Three strains for which the MIC of LVX was > or =4.0 mg/l included one Pen-Gs and two Pen-Gir isolates. The MICs of CLR correlated significantly with those of AZM. The MIC of CLR was > or =1 mg/l for 216 isolates, and the MIC of AZM was > or =1 mg/l for 244 of them. These data suggested that PAPM may be effective against S. pneumoniae infection, although acquisition of resistance should be considered. LVX also seemed to be effective against S. pneumoniae.

Evaluation of adsorption of urine cystatin C to the polymer materials on the microplate by an antigen capture enzyme-linked immunosorbent assay.

BACKGROUNDnCystatin C is a low molecular weight protein of 13 kDa with an isoelectric point of 9.3. Its adsorption on the urine sampling containers may cause the underestimation of cystatin C levels. We newly developed an antigen capture enzyme-linked immunosorbent assay (ELISA) of sandwich method for measurement of adsorbed level.nnnMETHODSnWe used a polystyrene microplates with 3 different polymers. These include high hydrophobic, low hydrophobic, and hydrophilic materials. Using the same microplate, the absorbed protein was measured by an antigen Capture ELISA, and calibration was conducted by an ordinary ELISA.nnnRESULTSnIn normal urine the concentrations of absorbed cystatin C levels to the 3 materials at day 1 were 0.50, 0.32-0.84 microg/l (median, interquartile range), 0.28, 0.21-0.37 microg/l, and <0.08, <0.08-0.09 microg/l in high hydrophobic, low hydrophobic, and high hydrophilic material, respectively. The absorption rate was 6%, 3%, and 1%, respectively. The adsorption is dependent on urine pH. It changes reciprocally with urine protein concentration. In pathologic urine, the absolute absorption level was <0.08 microg/l on the median, and the adsorption ratio (absorption level/urine level) was much less than 0.5% of that in normal urine.nnnCONCLUSIONnIn the clinical setting, the absorption of cystatin C to sample containers is negligible since the rate of adsorption is low both in normal and pathologic urine. The material with high hydrophilic surface processing may be used for other proteins when interaction of the proteins with surface material affects the value to clinical decision.

Recovery of susceptibility to penicillin G in clinical isolates of Streptococcus pneumoniae despite increased accumulation of pbp gene alterations

Two hundred consecutive clinical isolates of Streptococcus pneumoniae isolated in 2005 and 2006 were analysed for susceptibility to various antimicrobials, pbp gene alterations and macrolide resistance gene expression (2007 analysis) and the results were compared with previous data (2003 analysis). The average minimum inhibitory concentration (MIC) of penicillin G in isolates with 1a(m)/2x(m)/2b(m) decreased from 1.135+/-0.503 mg/L in the 2003 analysis to 0.872+/-0.540 mg/L in the 2007 analysis (P=0.0046). The prevalence of isolates with 1a(m)/2x(m)/2b(m) increased from 30.5% to 32.3%, but the difference was not statistically significant (P=0.6979). The prevalence of isolates with a clarithromycin MIC > or = 1.0mg/L increased from 65.9% to 80.0% (P=0.0005). Isolates expressing ermB increased from 46.6% to 62.6% (P=0.0004). We conclude that the decrease in penicillin resistance of S. pneumoniae does not correlate with a decrease in pbp mutations; on the contrary, the prevalence of isolates with pbp mutations increased. A decrease in penicillin resistance in S. pneumoniae with pbp mutations appears to explain the present results regarding the recovery of penicillin susceptibility. Our results suggest that the spread of mutated pbp genes among S. pneumoniae itself is not responsible for acquisition of the penicillin-resistant phenotype. Use of beta-lactams, especially oral cephalosporins, appears to be responsible for the acquisition of penicillin resistance.

Urinary protein 1/Clara cell 16 concentrations and lung functions in male subjects with pneumoconiosis

Background: Protein 1 (P1)/Clara cell 16 kDa protein (CC16, previously named CC10), a potentially immunosuppressive protein secreted by non-ciliated cells of the tracheobronchial epithelium, has been found to be a new useful lung-specific biomarker in several pathological lung conditions. Particularly, urinary P1 (uP1) may reflect the altered lung functions in pneumoconiosis. Methods: We investigated the relationship between uP1 values and lung functions in 31 non-smoking pneumoconiotic males (mean age 73 years) with a history of dust exposure work in shipbuilding. The protein was measured using an originally prepared enzyme-linked immunosorbent assay system. The forced expiratory volume in 1 s % (FEV1.0%) and % vital capacity (%VC) were tested with a spirometer. Results: The mean values of uP1 were 4.62 ± 4.82 (mean ± standard deviation) ng/mol creatinine. A univariable correlation test showed a significant positive correlation between uP1 and %VC (r = 0.356, P = 0.049). Also, a multiple regression analysis, when adjusted for age, disease duration, FEV1.0% and %VC, showed a significant correlation of uP1 with %VC (β = 0.467, P = 0.030). Conclusion: The results suggest that a decreased uP1, corroborated by a decreased %VC, may be the result of damage to secretory cells. Measurement of uP1 may become a possible index of fibrotic changes in pneumoconiosis.

Expression of uteroglobin in normal and carcinogenic endometrium and influence of hormone replacement therapy.

Uteroglobin, first reported in 1968 as a steroid secreted in rabbit uterine fluid during early pregnancy, is a progesterone‐regulated and progesterone‐binding protein. There is evidence that indicates that uteroglobin is inversely correlated to neoplastic growth but its role to endometrial carcinogenesis is not known. Therefore we analyzed the expression of uteroglobin in 13 normal endometrium, 19 hyperplasia and 21 endometrial carcinoma samples and the relation to estrogen receptor‐α (ER‐α) and progesterone receptor (PR) by immunohistochemistry and Western blotting. We also analyzed the expression of uteroglobin in 15 menopausal women who received hormone replacement therapy (HRT). The expression of uteroglobin was higher during the secretory phase than in the proliferative phase; however, it was detected in endometrial hyperplasia as weakly as in the proliferative phase and decreased according to the loss of differentiation in endometrial carcinoma. The results were basically in accord with those for PR; however, the expression of uteroglobin was weak, though PR was most detected in endometrial hyperplasia. In menopausal endometrium, the group treated with estrogen plus progesterone exhibited higher expression of uteroglobin than the group treated only with estrogen. The evidence suggests that uteroglobin expression is regulated by progesterone in the normal endometrium but that the regulation by PR is lost in endometrial hyperplasia and carcinoma according to acquirement of tumorigenesis and that estrogen plus progesterone therapy reduces the risk for endometrial carcinoma by restoring uteroglobin.

Call for the use of a common equation for glomerular filtration rate estimation in East and South-East Asia.

BACKGROUNDnEstimated glomerular filtration rate (eGFR) is currently calculated using various equations and serum creatinine (Scr) value measured by different assays. Differences among these eGFRs deserve further study.nnnMETHODSnVolunteers from eight Asian regions (n=3283; age 20-65 years, 1454 men, 1829 women) were recruited. The Chronic Kidney Disease Epidemiology Collaboration equation (EPI), Modification of Diet in Renal Disease Study equation (MDRD) for Japanese (MDRDJap) and MDRD for Chinese (MDRDChi) were selected. Jaffe and enzymatic assays were used to measure Scr. Six eGFRs were obtained for each volunteer: EPI equation using Scr value of enzymatic assay (EPI/E) and Jaffe assay (EPI/J); MDRDJap equation using Scr value of the two assays (MDRDJap/E, MDRDJap/J); and MDRDChi equation using Scr value of the two assays (MDRDChi/E, MDRDChi/J).nnnRESULTSnNeither Scr nor eGFR showed significant regional difference. We compared eGFR calculated using the same equation but with different assays. The medians (2.5%, 97.5%) of eGFR difference were 2.0 (-7, 14) mL/min/1.73 m(2) for EPI, 3.0 (-12.0, 18.0) mL/min/1.73 m(2) for MDRDJap, and 5.0 (-18, 30) mL/min/1.73 m(2) for MDRDChi. We also compared eGFR calculated using different equations but with the same assay. The medians (2.5%, 97.5%) of eGFR difference were 11 (-6, 56) mL/min/1.73 m(2) between MDRDChi/E and EPI/E; 26 (9, 35) mL/min/1.73 m(2) between EPI/E and MDRDJap/E; and 39 (22, 65) mL/min/1.73 m(2) between MDRDChi/E and MDRDJap/E, respectively.nnnCONCLUSIONSneGFR difference caused by using different equations is much larger than that caused by using different Scr assays. A common equation for GFR estimation is encouraged for use in Asians.

Questões atuais relativas à dosagem e à descrição da excreção urinária de albumina

ANTECEDENTES: A excrecao urinaria de albumina indica lesao nos rins e e reconhecida como fator de risco para a progressao das doencas renal e cardiovascular. A dosagem da albumina urinaria chama a atencao sobre a necessidade clinica de relatos de resultados precisos e claramente descritos. O National Kidney Disease Education Program e a Federacao Internacional de Quimica Clinica e Medicina Laboratorial (IFCC) reuniram-se para avaliar o estado atual das questoes pre-analiticas, analiticas e pos-analiticas que afetam as dosagens da albumina na urina e para identificar as areas que necessitam de melhorias. CONTEUDO: A quimica da albumina na urina nao e completamente compreendida. Diretrizes atuais recomendam a utilizacao da relacao albumina/creatinina (RAC) como substituta para a coleta de amostras cronometradas de urina, frequentemente inadequadas. Os resultados da RAC sao afetados pela preparacao do paciente, pela hora do dia da coleta das amostras e nao e padronizada. Foram relatadas consideraveis diferencas intermetodos para a dosagem tanto de albumina quanto de creatinina, mas a verdade e desconhecida, porque nao existem procedimentos de referencia para a dosagem de albumina e nao ha materiais de referencia para qualquer um desses analitos na urina. Os intervalos de referencia recomendados para a RAC nao consideram as grandes diferencas intergrupos na excrecao da creatinina (por exemplo, relacionadas com diferencas em idade, sexo e etnia), nem o aumento continuo no risco relacionado com a excrecao de albumina. DISCUSSAO: Necessidades clinicas foram identificadas para a padronizacao de (a) metodos de coleta da urina, (b) dosagens de albumina e de creatinina na urina com base em um sistema de referencia completo, (c) relatorios dos resultados dos testes e (d) intervalos de referencia para a RAC.

The current status of urinary proteins as diagnostic and prognostic markers

Numerous proteins appear in urine under different pathophysiological conditions. Individual proteins are measured by immunoassays, while mass spectrometry is being introduced to evaluate proteins and peptides. The protein in urine is more or less unstable and heterogeneous due to degradation or modification by urine proteases, resulting in altered immunochemical reactions. A quality assurance system for each analyte by each is a key, by setting proper pre-analytical conditions and establishing reference measurement systems, especially by qualified reference material. Indication for urine protein measurement is to seek pathogenesis, localisation of the affected organ or tissue, evaluation of prognosis or response to therapy in systemic, renal and genitourinary diseases. Albumin, a marker for glomerular dysfunction, is essential not only for diabetic nephropathy, but a reliable risk and prognostic marker for cardiovascular diseases. Measurement of very low levels is underway for early detection and prevention of related diseases. α1-microglobulin is a well-established reliable tubular marker. Among post-renal markers IL-8 is a good inflammatory marker especially for urinary tract infection (UTI). The pre-renal marker may specifically detect systemic diseases; a specific antibody against microorganisms is in use by point of care testing (POCT). Clinical usefulness is being expanded by discovery of new proteins and peptides that may be incorporated in the expert system.

Epitope analysis of human immunoglobulin D with a mouse monoclonal anti-Fab δ chain antibody

Abstract Background: The clinical significance of IgD measurements in serum is limited as the frequency of abnormal concentrations is rare. Methods: We prepared a mouse monoclonal antibody and a rabbit polyclonal antibody against Fab δ and Fc δ chain and compared epitope recognition by the monoclonal antibody against Fab δ (anti-Fab δ mono) with that by other antibodies. Results: Anti-Fab δ mono specifically reacted with purified IgD and Fab δ of myelomatous origin, but not with other isotypes and light chains. In 19 of 22 myeloma sera, the monoclonal antibody recognized intact IgD and/or its fragments when analyzed by immunoblotting. Of these there were only four cases in which possible Fab δ fragments were identified. The other three sera showed no reactivity with the antibody and the IgD value was low on a chemiluminescence enzyme immunoassay. Conclusions: These findings indicate the presence of at least two immunochemically different IgD molecules in the sera. No positive reaction with any synthetic peptide was observed for the antibody on δ-chain ranging from N-terminus of JH, C δ 1 to a hinge region. We suggest that the epitope recognized by the antibody is related to the variable region or a conformational structure on the Fd δ region of IgD.

Study on an improved isolation method of human α1-antichymotrypsin and the measurement using radioimmunoassay in various human body fluids

α1-antichymotrypsin (α1AC) is known to specifically inhibit the activity of chymotrypsin-like proteases, and considered to be one of the acute phase reactants. In our previous study, the α1AC level was increased in gastric juice from the patients with gastric cancer, when compared to those in benign gastrointestinal disorders or normal controls, α1AC was purified from serum using the method of salting out and gel chromatography which was modified from the method of Travis, et al. The moleculer weight of the purified α1AC was 58, 000 daltons by SDS-polyacrylamide gel electrophoresis, which was completely identical to the report by Lame and Hayem. The new highly specific and sensitive radioimmunoassay (RIA) was established for α1AC. The RIA was prepared using the purified α1AC and anti-α1AC antibody commarcially available. The α1AC level was first determined in some human body fluids, such as gastric juice, bile, ascitic, pleural, cerebrospinal and synovial fluids.