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.

Free and protein-bound plasma estradiol-17 beta during the menstrual cycle.

Methods are described for the measurement of the estradiol-binding capacity of TeBG and of the free, TeBG-bound, and non-specifically protein-bound fractions of plasma estradiol. Each determination used undiluted plasma at 37 C, and a total volume of less than 2.0 ml of plasma was required to complete all the assays. The measurement of the per cent of free estradiol was affected by changes in plasma dilution. The measurement of the other fractions of estradiol was not influenced by changes in either the dilution or the volume of plasma. The distribution of plasma estradiol was determined daily throughout 5 individual menstrual cycles. The per cent of free, the per cent of TeBG-bound, and the TeBG binding capacity of estradiol remained constant throughout the cycle with mean values of 2.21 +/- 0.04% (SE), 38.4 +/- 0.7%, and 16.6 +/- 0.43 ng/ml, respectively. The mean association constant of TeBG for estradiol was 6.58 +/- 0.25 x 10(7)M-1. The concentration of the free and non-specifically protein-bound fractions of estradiol paralleled the total plasma concentration of estradiol. The results show that biologic events related to normal cyclic changes of plasma estradiol may be attributed to fluctuations in the free estradiol and to estradiol which is bound with low affinity to non-specific plasma proteins.

Effects of chronic sulpiride-induced hyperprolactinemia on menstrual cycles of normal women.

We investigated the influence of chronic (27-65 days) sulpiride-induced hyperprolactinemia on the menstrual cycles of four normal women. The hyperprolactinemia (206.4 ng/mL, the average of the mean values of each subject obtained by sulpiride treatment) suppressed the LH surge and the secretion of plasma estradiol-17 beta and progesterone to their basal levels. The results suggest that the endocrine changes in normal women with sulpiride-induced hyperprolactinemia are similar to those in women with spontaneous hyperprolactinemia. Sulpiride-induced hyperprolactinemia may be useful as a model for studying spontaneous hyperprolactinemia.

Estrogen-androgen balance in anovulation.

The balance of estradiol (E2) and testosterone (T) in 15 anovulatory patients was evaluated by measuring the daily plasma concentration of E2 and T, and their free and protein-bound fractions for a 3- to 4-week period. Similar daily plasma E2 and T data were obtained from five normal ovulatory cycles as a control group. The daily concentration of the free, non-testosterone-estradiol-binding globulin (TeBG)-bound (index), and total E2 fluctuated in a wider range than that of the T in the ovulatory as well as in the anovulatory cycles. The percentage of free (%F) and TeBG-bound (%TeBG) fractions of both E2 and T were relatively constant. The concentration of the free, index, and total E2 and T showed a parallel pattern even in anovulatory cycles. An increased %F fraction associated with a decreased %TeBG fraction of E2 and T was observed in anovulatory patients who were hypo- or normoestrogenic; however, an opposite shifting of these two fractions was observed in anovulatory patients who were severely hypoestrogenic. In a hyperestrogen-normoandrogenic state, there was a significant increase in the binding of E2 and T. The daily binding capacity of plasma TeBG revealed a greater fluctuation than the binding fractions, and it decreased in anovulatory patients, especially in the hyperandrogenic state. E2:T ratio of concentration showed a curve-linear relationship to %F, %TeBG, and binding capacity of sex steroids.

An enzymologic study of corpora lutea in early pregnant rats treated with abortifacient agents.

Luteal metabolism was investigated in corpora lutea of early pregnant rats treated with four abortifacient agents. In corpora lutea of rats treated with prostaglandin F2alpha or of rats 1 day postpartum, glucose-6-phosphate dehydrogenase (G6PDH) activity increased 140 to 170% and 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) was activated to significantly high levels, whereas malic enzyme activity decreased to 29% of control values. In aminoglutethimide-treated rats, the activities of G6PDH and malic enzyme decreased, while 20alpha-HSD activity was maintained at a very low level. With the increased dose, complete abortion was observed. In corpora lutea of these aborted rats, 20alpha-HSD was activated moderately and G6PDH values were slightly higher than control values, whereas malic enzyme activity fell to lower levels. All rats treated with clomiphene citrate aborted within 63 hours after the last injection. The activities of G6PDH, malic enzyme, and ATP citrate lyase in these corpora lutea decreased to 66, 68, and 72% of control levels, respectively; 20alpha-HSD activity was maintained at a very low level, and activities of 3beta-hydroxysteroid dehydrogenase, 6-phosphogluconate dehydrogenase, and pyruvate kinase were not appreciably altered. These findings indicated that, at the beginning of luteolysis and fetal resorption, the activities of steroidogenic enzymes decreased and 20alpha-HSD was not yet activated. Therefore, we could gauge the early changes of luteolysis by measuring the activities of G6PDH, MALIC ENZYME, AND ATP citrate lyase as well as 20alpha-HSD.

PIP: An enzymologic study of corpora lutea in early pregnant rats treated with abortifacient agents is presented. Prostaglandin F2 alpha treatmen t (500 mcg twice daily 3 or 4 consecutive times) revealed an increase in corpora lutea glucose-6-phosphate dehydrogenase (G6PDH) activity of 110-140% and a moderate increase in 20 alpha-hydroxysteroid dehydrogenas e (20 alpha-HSD), whereas malic enzyme decreased to 27% of control values. Aminoglutethimide treatment (10-20 mg twice daily 6 or 7 consecutive times) revealed decreased G6PDH and malic enzyme activities while 20 alpha-HSD activity was maintained at a very low level. Corpora lutea of these aborted rats revealed moderately active 20 alpha-HSD valu es and slightly higher than control values for G6PDH, whereas malic enzy me activity fell to lower levels. Clomiphene citrate treatment (.5 ml of 3 mg/ml or .5 ml plus .5 ml of 10 mg/ml progesterone) caused abortion within 63 hours postinjection. G6PDH, malic enzyme, and adenosine triphosphate (ATP) citrate lyase activities in these corpora lutea decreased to 66, 68, and 72% of control levels, respectively, while 20 alpha-HSD activity was maintained at a very low level. Activities of 3beta hydroxysteroid dehydrogenase, 6-phosphogluconate dehydrogenase and pyruvate kinase were not appreciably altered. These results indicate that at the beginning of luteolysis and fetal resorption the activities of steroidogenic enzymes decreased and 20 alpha-HSD was not yet activated. Therefore, G6PDH, malic enzyme, and ATP citrate lyase activities could be measured to gauge early changes of luteolysis.

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.

The use of an enzyme-linked immunosorbent assay for estimation of the immune status of chickens artificially immunized against coccidiosis.

Nine thousand commercial breeder chicks (Chankee) reared in a floor pen were exposed to restricted numbers of Eimeria tenella and E. necatrix oocysts to confer immunity. Antibody induction in these chicks was examined by the enzyme-linked immunosorbent assay (ELISA) with antigen prepared from E. tenella oocysts. The oocyst excretion pattern demonstrated recycled infections which continued in these chicks for greater than or equal to 22 days after exposure. Antibody levels in their sera, as determined by the mean absorbence values in ELISA, increased gradually up to 38 days post-inoculation. Mean absorbence values of sera from control chicks remained at a low level. When infected and control chicks were challenged with the two species of coccidia, the test chicks were protected against both species. The antibody level did not change for 8 days in the challenge groups, while in the control chicks, absorbence in ELISA rose significantly and the mean absorbence value was higher than that in immunized chicks. Some factors which influence the results of ELISA are considered and the applicability of this method to measuring immunity against coccidiosis in chickens is discussed.

[Congenital large field color vision defects].

To investigate changes in the way of seeing colors due to expansion of the visual angle in patients with congenital color vision defects, an anomaloscope which can alter the visual angle to 2 degrees, 6 degrees, 10 degrees, 15 degrees and 20 degrees was prepared using three colored light emission diodes, and 28 patients with congenital color vision defects were examined. The results showed that the patients could be categorized into two groups: one with no change in the equation range and one in which the equation range was contracted. Contraction of the equation range was marked when the visual angle was 10 degrees or more.

[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.

[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.

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 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.

Increased expression of Fc epsilon R2/CD23 on peripheral blood B lymphocytes and serum IgE levels in Kawasaki disease.

We analyzed the expression of Fc epsilon R2/CD23 on the peripheral blood B lymphocytes of 10 patients with Kawasaki disease (KD) using a fluorescence-activated cell sorter. The absolute count of CD23-positive B lymphocytes and the ratio of CD23-positive B lymphocytes to B lymphocytes were high during the acute stage of KD in comparison to those during the convalescent stage and in control subjects. In addition, both the increased number of CD23-positive B lymphocytes and the increased serum IgE levels appeared during the latter part of the acute stage. These results suggest that the increased number of CD23-positive B lymphocytes during the latter part of the acute stage in KD reflect the activation of B lymphocytes and have an important role in IgE-mediated immunity.