Validation of a novel method to identify healthcare-associated infections.

Despite its potential for use in large-scale analyses, previous attempts to utilise administrative data to identify healthcare-associated infections (HAI) have been shown to be unsuccessful. In this study, we validate the accuracy of a novel method of HAI identification based on antibiotic utilisation patterns derived from administrative data. We contemporaneously and independently identified HAIs using both chart review analysis and our method from four Japanese hospitals (N=584). The accuracy of our method was quantified using sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) relative to chart review analysis. We also analysed the inter-rater agreement between both identification methods using Cohen's kappa coefficient. Our method showed a sensitivity of 0.93 (95% CI: 0.87-0.96), specificity of 0.91 (0.89-0.94), PPV of 0.75 (0.68-0.81) and NPV of 0.98 (0.96-0.99). A kappa coefficient of 0.78 indicated a relatively high level of agreement between the two methods. Our results show that our method has sufficient validity for identification of HAIs in large groups of patients, though the relatively lower PPV may imply limited utilisation in the pinpointing of individual infections. Our method may have applications in large-scale HAI identification, risk-adjusted multicentre studies involving cost of illness, or even as the starting point of future cost-effectiveness analyses of HAI control measures.

Preparation of gallium oxynitride powder and its nanofibers by the nitridation of a gallium oxide precursor doped with nickel or cobalt obtained via the citrate route.

Acicular crystals were grown in gallium oxynitride powder prepared by ammonia nitridation of amorphous gallium oxide precursors containing less than 5 at% of either Ni or Co, via the citrate route. The crystals were several tens of nanometres wide, several micrometres long, and grown in the temperature range 750 to 850 degrees C in a flow of ammonia of less than 200 mL min(-1). The crystal structure of the gallium oxynitride was a highly disordered 2H wurtzite-type with some 3C zinc blende-type stacking faults. The crystals grew in their basal plane changing their aspect ratio with the supplying method of small amounts of Ni or Co and an amount of residual carbon. The acicular crystals were grown by the catalytic behavior of Ni or Co to enhance one-dimensional growth in the hexagonal c-plane.

A transmembrane trap method for efficient cloning of genes encoding proteins possessing transmembrane domain.

To facilitate searching for genes encoding cell membrane proteins, we developed a method for isolating cDNAs that contain sequences for hydrophobic transmembrane runs. This cloning strategy, termed the "transmembrane (TM) trap method," utilizes a vector that directs the cell surface expression of mouse CD4 fusion protein when an insert encoding hydrophobic transmembrane sequences is cloned in-frame with correct orientation. We applied this novel method to isolation of cytokine receptor cDNAs. Our strategy enabled efficient isolation of relatively rare species encoding receptors such as IL-2Rgamma, IL-3Rbeta, IL-4Ralpha, IL-5Ralpha, and IL-6Ralpha. This method also could be used to isolate cDNAs for intracellular molecules with a transmembrane region, e.g., bcl-2. These results indicate that the TM trap method provides an efficient cloning strategy for identification of various families of genes encoding proteins with one or more transmembrane regions.

Increased cell surface expression of C-terminal truncated erythropoietin receptors in polycythemia.

Primary familial and congenital polycythemia (PFCP) is a disorder characterized by an increased number of erythrocytes despite normal blood oxygen pressure and a normal serum erythropoietin (EPO) level. Recent studies revealed that erythroid progenitor cells from certain individuals with PFCP express various forms of EPO receptor (EPOR) truncated at the terminal carboxyl site (EPOR-TTC(PFCP)). EPOR-TTC(PFCP) can transmit EPO-mediated proliferative signals more efficiently than can full-length EPOR (EPOR-F), at least partly because of defective recruitment of SHP-1 phosphatase to these receptors. In agreement with previous studies, Ba/F3 transfectants expressing EPOR-TTC(PFCP) showed higher proliferative responses to EPO. In those transfectants, we found that EPOR-TTC(PFCP) was expressed more abundantly on the cell surface than was EPOR-F. This tendency was confirmed by a transient-expression experiment using COS7 cells. Since expression levels of EPOR protein were not significantly different among these transfectants, differences in cell surface expression were likely dependent on post-translational mechanism(s). In addition to defective recruitment of SHP-1 to EPOR-TTC(PFCP), more efficient transport and expression on the cell surface appear to serve as mechanisms responsible for increased EPO-responsiveness of erythroid progenitor cells in PFCP.

Molecular cloning and chromosomal mapping of a novel five-span transmembrane protein gene, M83.

In an attempt to identify novel transmembrane molecules expressed on hematopoietic cells, we identified a novel transmembrane protein gene, M83. Cloning of the full-length cDNAs of human and mouse M83 revealed that M83 encodes a type I transmembrane protein with a region containing five hydrophobic segments within the C-terminal part of the protein, suggesting that M83 is a five-span transmembrane molecule. The M83 protein was expressed on the cell surface as a glycosylated protein with a molecular mass of 84 kDa. The M83 gene was localized to human chromosome 16p13.3, mouse chromosome 17B1, and rat chromosome 10q12.3 distal. In human, M83 mRNA was highly expressed in placenta, pancreas, and lymphohematopoietic tissues including peripheral blood, spleen, and bone marrow. Among hematopoietic cells, it was highly expressed in resting T lymphocytes and was downregulated by cell activation, suggestive of its biological role related to the T cell resting status.

Characterization of the mouse interleukin-13 receptor alpha1 gene.

Interleukin (IL)-13 is a pleiotropic immune regulatory cytokine that shares structural and biological characteristics with IL-4. The receptor for IL-13 is comprised of the IL-4 receptor alpha (IL-4Ralpha) subunit and a low-affinity IL-13-binding subunit, IL-13Ralpha1. An additional receptor, IL-13Ralpha2, binds to IL-13 with high affinity, but lacks the cytoplasmic domain for signaling. In this study, we isolated the mouse IL-13Ralpha1 gene (Il13ra1) of approximately 56 kb that spans the entire coding region. The mouse Il13ra1 gene is composed of 11 exons, and shows striking similarity in genomic structure to the previously reported class I cytokine receptor genes. Motifs characteristic of the cytokine receptor family are similarly organized on the genome, including conserved cysteines, a WSxWS motif, and Box1, indicating closely related genetic evolution of the cytokine receptor superfamily. Alternative mRNA splicings were demonstrated to generate variant transcripts that encode soluble IL-13Ralpha1. The mouse Ill13ra1 gene was mapped to the proximal region of the mouse X chromosome, and was closely linked to the DXPas3 locus by interspecific backcross analysis. Il13ra1 mRNA was co-expressed with I14ra mRNA in mouse myeloid and natural killer cells on which IL-13 has been known to act, whereas the Il13ra2 mRNA was not detected in these cells, indicating that IL-13Ralpha1 is the major component of the IL-13 receptor complex in lymphohematopoietic cells.

Impaired erythropoiesis in transgenic mice overexpressing a truncated erythropoietin receptor.

Erythropoietin (EPO), one of the pivotal regulators of erythrocyte production, transmits signals through the EPO receptor (EPOR). We have previously reported that human bone marrow (BM) cells express two dominant forms of the EPOR, one full-length and one truncated (EPOR-F and EPOR-T). Experiments with a cell line have shown that the EPOR-T acts as a dominant-negative regulator of EPOR-F-mediated signals. Its role in erythropoiesis in vivo, however, has yet to be clarified. Here we show the presence in mouse BM of a truncated form of the EPOR that is essentially the same as EPOR-T in humans. To investigate its role in vivo, we generated transgenic mice overexpressing mouse EPOR-T (EPOR-T-Tg mice). As a result, two independent EPOR-T-Tg lines were established. One line revealed mild anemia, but another line did not. When anemia was induced experimentally in these mice, however, both lines showed apparently poor recovery resulting in higher mortality than wild-type control mice. The impaired erythropoiesis found in these mice thus strongly suggests the EPOR-T's role as a negative regulator of erythropoiesis in vivo.

Nephropathy and hypertension as manifestations in a 13-y-old girl with primary antiphospholipid syndrome.

Severe renal hypertension due to both unilateral renal arterial occlusion and renal thrombotic microangiopathy developed in a 13-y-old girl as a manifestation of primary antiphospholipid antibody syndrome. The combination of the intravenous high-dose urokinase therapy and oral anticoagulation therapy, comprising aspirin, warfarin and dipyridamole, was significantly effective in improving her renal function and preventing thrombotic events during an 18-month follow-up period.

Decrease in the concentrations of transforming growth factor-beta 1 in the sera of patients with Kawasaki disease.

Kawasaki disease (KD) is one of the most important forms of vasculitis, and is characterized by the initiation of a proinflammatory cytokine cascade. To further characterize the immunological profile of KD, we measured the serum levels of transforming growth factor-beta 1 (TGF-beta 1) as a regulatory cytokine. We determined the concentration of TGF-beta 1 in the sera of the patients with KD, anaphylactoid purpura (AP), and scarlet fever, using a sandwich enzyme linked immunosorbent assay. The serum levels of TGF-beta 1 were decreased in patients with KD, but not in patients with AP or scarlet fever during the acute stage. We found an inverse correlation between TGF-beta 1 and soluble tumor necrosis factor (TNF) receptor levels in KD patients during the acute and subacute stage. Decreased levels of TGF-beta 1, in particular to suppress TNF alpha (TNF-alpha) production, is an important part of the regulatory system of increased TNF-alpha production which cause vasculitis.

A multi-well version of in situ hybridization on whole mount embryos of Caenorhabditis elegans.

We report an efficient procedure for in situ hybridization with a multi-well format on Caenorhabditis elegans embryos for large scale screening of gene expression patterns in this organism. Each hybridization well contains embryos at various stages throughout embryogenesis. The validity of the method was confirmed through results with control genes whose expression patterns have been reported; glp-1 in very early embryos, myo-2 in pharyngeal muscle and unc-54 in body wall muscle. Several collagen genes and a pepsinogen gene were also examined to establish a set of lineage-specific markers. As a pilot project, we examined approximately 100 unique cDNA species classified by our cDNA project, finding that approximately 10% of the cDNA groups were expressed in specific cells and at specific stages.

Susceptibility of mice to bacterial and fungal infections after intragastric administration of ebselen.

The seleno-organic compound ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) has anti-inflammatory activity and exhibits glutathione peroxidase-like activity in-vitro. Ebselen inhibited candidacidal activity over the same range of concentrations as it inhibited the production of microbicidal H2O2 by human neutrophils and macrophage-like cells. Therefore, the long-term administration of ebselen might be expected to induce an immunocompromised state in the host. To examine such a possibility, mice (5-weeks-old ddY, male) were given daily intragastric doses of 0, 10 or 100 mg/kg-1 ebselen for 21 days and then infected intraperitoneally with Candida albicans (10(8) cells/mouse), Pseudomonas aeruginosa (1.5 x 10(7) cells/mouse) or methicillin-resistant Staphylococcus aureus (5 x 10(8) cells/mouse). Ebselen at none of the tested doses affected the increase in body weight of mice during administration of the drug. No evidence was obtained that mice became more susceptible to the various microorganisms after the administration of ebselen at any tested dose.

Soluble CD23 antigen in Kawasaki disease and other acute febrile illnesses.

Kawasaki disease (KD) is an acute febrile illness of early childhood. Although the epidemiology of KD suggests an infectious agent, the cause still remains unknown. Intense immune activation during the acute disease has been well documented. Quantitative determination of soluble CD23 in serum can serve as an index of macrophage/monocyte or B-cell activation. To further characterize the immunological profile in KD, we investigated whether soluble CD23 levels in serum increase during the acute disease. In addition, we compared soluble CD23 levels in 33 patients with acute KD with levels in ten patients each with measles, rubella, infectious mononucleosis, and scarlet fever to determine if marked elevations in soluble CD23 were unique to acute KD. Patients with KD, rubella and infectious mononucleosis, but not patients with measles or scarlet fever, had increased soluble CD23 levels in serum during the acute stage, as compared to age-matched control subjects (P < 0.01). These data suggest infection with Epstein-Barr virus and rubella and acute KD are all characterized by B-cell and macrophage/monocyte activation.

Pentoxifylline and intravenous gamma globulin combination therapy for acute Kawasaki disease.

We compared the efficacy of oral administration of pentoxifylline (PTX) and intravenous infusions of gamma globulin (IVGG) combination therapy with that of IVGG in reducing the frequency of coronary-artery lesions (CAL) in children with Kawasaki disease (KD), in a randomized trial. All patients with KD received acetylsalicylic acid (30 mg/kg per day), until the 30th day, after the onset of fever, followed by daily acetylsalicylic acid at a dose of 3-5 mg/kg per day there-after, and intravenous IVGG, 200 mg/kg per day, for 5 consecutive days. In addition, patients randomly assigned to PTX and IVGG combination therapy groups received oral PTX at a dosage of 10 mg/kg per day (low-dose) or 20 mg/kg per day (high-dose), in three divided doses until the 30th day. Patients with KD were all free from CAL prior to treatment. We assessed the presence of CAL by two-dimensional echocardiography which was also done prior to treatment and then twice a week after hospital admission. We detected CAL in 3 of 18 patients (16.7%) in the IVGG therapy group, as compared with 2 of 18 patients (11.1%) in the low-dose PTX and IVGG combination therapy group. There were no significant difference between the two groups. In the next study, we detected CAL in 3 of 21 patients (14.3%) in the IVGG therapy group, as compared with none of 22 patients (0%) in the high-dose PTX and IVGG combination therapy group (chi 2 = 6.4, P < 0.02). No adverse side-effects were observed in 79 patients with KD.(ABSTRACT TRUNCATED AT 250 WORDS)

[Serum soluble CD2, CD4 and CD8 levels in infectious mononucleosis].

Acute infectious mononucleosis (IM) is a lymphoproliferative disease caused by the Epstein-Barr virus (EBV) infection. It has been reported that soluble T cell antigens are released from cells in response to T cell activation. In the present study, we investigated whether soluble antigen levels of CD2, CD4 and CD8 in serum increase during acute IM. Soluble CD2, CD4 and CD8 levels in serum were measured by a sandwich enzyme immunoassay. In addition, peripheral blood T cell subsets were analyzed by single and two color flow-cytometric analyses in IM. Patients with IM had increased levels of soluble CD2, CD4 and CD8 in serum samples obtained during acute stages. We found a positive correlation between serum levels of soluble CD8 and absolute counts of HLA-DR+CD8+T cells during acute IM. In addition, the correlation between soluble CD8 levels and serum GOT or GPT levels was shown to be positive during acute IM. Our findings suggest that the soluble antigen levels of CD2, CD4 and CD8, in particular CD8, in serum are an important immunologic parameter for determining the activation of T cells during acute IM.

Soluble ICAM-1 levels in serum during acute infectious mononucleosis.

Intercellular adhesion molecule-1 (ICAM-1) has been reported as a T-cell, B-cell or macrophage/monocyte activation antigen. We investigated whether soluble ICAM-1 levels in serum increased during the acute stage in 11 patients with infectious mononucleosis (IM). Serum ICAM-1 levels were measured by a double determinant immunoassay using 2 monoclonal antibodies in the FAST system. Serum ICAM-1 levels in patients with IM increased during the acute stage. There was a positive correlation between serum ICAM-1 levels and absolute counts of peripheral blood mononuclear cells. Our results suggest that the elevated serum ICAM-1 levels are based on the increased counts of activated peripheral blood mononuclear cells and that ICAM-1 is a part of the regulatory system of immune reactions during acute IM.

[Serum soluble CD4 and CD8 levels in Kawasaki disease].

The levels of soluble CD4 (sCD4) and sCD8 in serum correlate with T cell subset activation and may be important in monitoring and characterizing disease processes in immunological diseases. We compared acute Kawasaki disease (KD) with anaphylactoid purpura (AP) and measles, in terms of serum sCD4 and sCD8 levels. The levels of serum sCD4 and sCD8 were measured by a sandwich enzyme immunoassay. In addition, peripheral blood T-cell subsets were analysed by single and two-colour flow-cytometric analyses in KD patients. The levels of serum sCD4 and sCD8 were significantly elevated in patients during the acute stages of KD and measles, but not in AP. Peripheral blood CD4+, CD8+ and also HLA-DR+T cell counts did not increase during the acute stage of KD. Our results suggest that there is a low level of activation of peripheral blood T cells during acute KD, or that infiltrating T cells in some local tissues of KD patients contribute to the elevated levels of serum sCD4 and sCD8.

Immunological abnormalities in Kawasaki disease with coronary artery lesions.

A review of our previous immunological studies on Kawasaki disease (KD) was undertaken. The results showed that peripheral blood macrophages/monocytes, T-cells and B-cells become activated during acute KD in terms of numerical changes in immunocompetent cells, expression of activated antigens on the cell surfaces and cytokine production. Also, during acute KD with coronary artery lesions (CALs) the numbers of macrophages/monocytes are increased. In addition, both the increased levels of tumor necrosis factor-alpha and shed intercellular adhesion molecule-1 in serum are more evident in KD patients with CALs than in those without. Our results further suggest that the main characteristics of the pathogenesis of KD are increased numbers of peripheral blood macrophages/monocytes with the secretion of monokines by these activated cells, and the expression of adhesion molecules on immunocompetent cells. These immune responses develop more vigorously in KD patients with CALs.

Serum soluble CD4 and CD8 levels in Kawasaki disease.

The levels of soluble CD4 (sCD4) and sCD8 in serum correlate with the T cell subset activation and may be important in monitoring and characterizing disease processes during immunological diseases. We compared acute Kawasaki disease (KD) with anaphylactoid purpura (AP) and acute febrile viral infections, such as measles and infectious mononucleosis (IM), in terms of serum sCD4 and sCD8 levels. The levels of serum sCD4 and sCD8 were measured by a sandwich enzyme immunoassay. In addition, peripheral blood mononuclear cell subsets were analysed by single and two-colour flow-cytometric analyses in KD and IM patients. The levels of serum sCD4 and sCD8 were significantly elevated in patients during acute stages of KD, measles and IM, but not AP. Peripheral blood CD4+, CD8+ and also HLA-DR+ T cells count did not increase during the acute stage of KD; however, peripheral blood CD8+ and HLA-DR+ T cell counts were increased during the acute stage of IM. Our results suggest that there is a low level of activation of peripheral blood T cells during acute KD, or that infiltrated T cells in some local tissues of KD patients contribute to the elevated levels of serum sCD4 and sCD8.