Effectiveness of patient and staff cohorting to reduce the risk of vancomycin-resistant enterococcus (VRE) acquisition: a retrospective cohort study during a VRE outbreak in Japan.

BACKGROUND: Patient and staff cohorting is part of a bundle approach in the response to multi-drug-resistant organisms, but its effectiveness is not fully clarified. This study compared the risks of acquiring vancomycin-resistant Enterococcus faecium (VREfm) at a hospital during a VREfm outbreak based on contact characteristics in order to better understand the effectiveness of cohorting. METHODS: Exposure came from contact with patients with VREfm (infectors), including existing patients with VREfm and patients who acquired VREfm during the study period. Contact was defined as length of contact time, degree of sharing space, and care by the same nurses as those caring for infectors between January and March 2018. The outcome was VREfm acquisition as determined through monthly stool or rectal screening cultures. Incidence rates were calculated based on contact patterns, and incidence rate ratios (IRRs) were compared. FINDINGS: Among 272 inpatients (4038 patient-days), 43 patients acquired VREfm with the same or similar pulsotype. Incidence rates were 8.45 per 1000 patient-days when susceptible inpatients were on the same ward as an infector but cared for by different nurses (reference), 16.96 when susceptible inpatients were on the same ward as an infector and cared for by the same nurses [IRR 2.01, 95% confidence interval (CI) 0.62-10.28], and 52.91 when susceptible inpatients shared a room with an infector (IRR 6.26, 95% CI 1.61-35.40). CONCLUSION: Compared with susceptible inpatients in a different room from infectors and not being cared for by the same nurses, the risk of VREfm acquisition could be six times higher for susceptible inpatients who are in the same room as infectors, and could be double for susceptible inpatients cared for by the same nurses as infectors.

Movement strategies during car transfers in individuals with tetraplegia: a preliminary study.

STUDY DESIGN: Three-dimensional kinematic analysis of car transfer (CT) movement in four adult males with C6 tetraplegia. OBJECTIVES: The aim of the present study was to assess the normal transfer technique movement from a wheelchair to a car (that is, CT) in subjects with tetraplegia. A better understanding of CT movement is invaluable knowledge for spinal cord injury rehabilitation. This type of knowledge will improve rehabilitation programs so that patients with tetraplegia will have greater societal participation. SETTING: School of Comprehensive Rehabilitation, Osaka Prefecture University, Osaka, Japan. METHODS: Four adult males with C6 tetraplegia, an impairment grade of A according to the American Spinal Injury Association guidelines, took part in the study. The subjects used their own wheelchair and car in our assessments of their CT movement technique. Movements were assessed using a three-dimensional video analysis system with six digital video cameras. CT data, which included lateral displacement of the head and buttocks, and angular displacement of neck flexion and trunk forward inclination, were collected and correlation coefficients were calculated. RESULTS: All four subjects demonstrated negative correlations in lateral displacements greater than 0.70. As for correlation coefficients of angular displacement, two subjects demonstrated negative correlations (r = -0.98 and r = -0.77) and one subject demonstrated a positive correlation (r = 0.75). The neck flexion and trunk forward inclination strategy was different among the four subjects. CONCLUSIONS: Each subject with C6 tetraplegia demonstrated different strategies during CT movement.

Deficient biosynthesis of N-acetylglucosaminyl-phosphatidylinositol, the first intermediate of glycosyl phosphatidylinositol anchor biosynthesis, in cell lines established from patients with paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a hemolytic disorder caused by a deficiency of biosynthesis of the glycosyl phosphatidylinositol (GPI) anchor, but the biochemical defect is not completely understood. In the present study, we have analyzed affected cell lines established recently from two Japanese patients with PNH. Two lines of evidence indicate that these cells do not synthesize N-acetylglucosaminyl-phosphatidylinositol, the first intermediate in the GPI anchor biosynthesis. First, somatic cell hybridization analysis using Thy-1-deficient murine thymoma cell lines with known biochemical defects as fusion partners showed that the PNH cell lines belong to complementation class A, which is known not to synthesize N-acetylglucosaminyl-phosphatidylinositol. Second, analysis of in vitro glycolipid biosynthesis demonstrated that cell lysates of these PNH cell lines in fact did not support biosynthesis of N-acetylglucosaminyl-phosphatidylinositol. Thus, we have characterized for the first time the exact biochemical defect leading to PNH.

CD49d and CD49e induce cell adhesion-mediated drug resistance through the nuclear factor-κB pathway in Burkitt lymphoma.

Burkitt lymphoma (BL) is a highly aggressive form of non-Hodgkin's B-cell lymphoma. Currently, multi-agent chemotherapy regimens are being used to significantly improve cure rates and achieve complete remissions in BL patients. However, drug resistance can often occur within 6 months in BL patients, contributing to poor prognosis. Mounting evidence suggests that cell adhesion-mediated drug resistance (CAM-DR), caused by the interaction between the bone marrow microenvironment and tumour cells may play an important role in drug resistance to chemotherapy. However, the molecular mechanism underlying CAM-DR in BL has not been identified yet. In this study, we investigated the molecular mechanism responsible for CAM-DR in BL cells. We also examined the therapeutic targets of CAM-DR in BL cells and found CD49d and CD49e to be the important adhesion molecules involved. However, CD49a, CD49b, CD11a, CD29, CD18, and CD61 were not found to be associated with CAM-DR in BL cells. Furthermore, we clarified that CD49d- and CD49e-mediated CAM-DR could be attributed to an increase in the expression of B cell leukemia-xL (Bcl-xL) and survivin proteins, and a decrease in the expression of Bcl-2 associated X (Bax), Bcl-2 interacting mediator (Bim) and p53 upregulated modulator of apoptosis (PUMA) proteins via nuclear factor kappaB (NF-κB) activation. In addition, bortezomib was found to overcome CAM-DR in BL cells by inhibiting NF-κB. Thus, bortezomib may have potential clinical applications in the treatment of CD49d- and CD49e-mediated CAM-DR in BL patients.

Distinct human prolactin (hPRL) and growth hormone (hGH) behavior under bacteriophage lambda PL promoter control: temperature plays a major role in protein yields.

When producing recombinant protein for therapy, it is desirable not only to obtain substantial amounts of the protein, but also to make sure that potential contaminants such as inducing agents are not present in the final product. To prevent this, one can use expression systems in which the promoter (lambdaP(L)) is activated by a temperature shift that denatures a repressor (e.g., cIts). In this manner, hGH was successfully expressed and secreted in Escherichia coli periplasm, with specific yields well above 1 microg ml(-1) A(600)(-1), after a temperature shift from 30 to 42 degrees C. However, attempts to express a related hormone, human prolactin, employing the same protocol were unsuccessful, providing 0.03 microg ml(-1) A(600)(-1) at the most. A process is described in which this labile protein is obtained from a cIts(-) strain under optimized temperature condition (37 degrees C). The highest periplasmic secretions of prolactin ever reported were thus obtained: 0.92+/-0.10 microg ml(-1) A(600)(-1) at an optical density of approximately 3 A(600) units in shake flask cultures and approximately 1 microg ml(-1) A(600)(-1), at an OD of 35 A(600) units, via a rapid and flexible batch feed process in laboratory bioreactor. Purified hPRL was monomeric, correctly processed (Mr=22,906), properly folded and bioactive (51.5+/-24.1 IU mg(-1)).

The ROR nuclear orphan receptor subfamily: critical regulators of multiple biological processes.

The nuclear receptor superfamily, a group of structurally related, ligand-dependent transcription factors, includes a large number of orphan receptors for which no ligand has yet been identified. These proteins function as key regulators of many physiological processes that occur during embryonic development and in the adult. The retinoid-related orphan receptors (RORs) alpha, beta, and gamma comprise one nuclear orphan receptor gene subfamily. RORs exhibit a modular structure that is characteristic for nuclear receptors; the DNA-binding domain is highly conserved and the ligand-binding domain is moderately conserved among RORs. By a combination of alternative promoter usage and exon splicing, each ROR gene generates several isoforms that differ only in their amino terminus. RORs bind as monomers to specific ROR response elements (ROREs) consisting of the consensus core motif AGGTCA preceded by a 5-bp A/T-rich sequence. RORE-dependent transcriptional activation by RORs is cell type-specific and mediated through interactions with nuclear cofactors. RORs have been shown to interact with certain corepressors as well as coactivators, suggesting that RORs are not constitutively active but that their activity is under some regulatory control. RORs likely can assume at least two different conformations: a repressive state, which allows interaction with corepressor complexes, and an active state, which promotes binding of coactivator complexes. Whether the transition between these two states is regulated by ligand binding and/or by phosphorylation remains to be determined. Ca2+/calmodulin-dependent kinase IV (CaMKIV) can dramatically enhance ROR-mediated transcriptional activation. This stimulation involves CaMKIV-mediated phosphorylation not of RORs, but likely of specific nuclear cofactors that interact with RORs. RORalpha is widely expressed. In the cerebellum, its expression is limited to the Purkinje cells. RORalpha-/- mice and the natural RORalpha-deficient staggerer mice exhibit severe cerebellar ataxia due to a defect in Purkinje cell development. In addition, these mice have thin long bones, suggesting a role for RORalpha in bone metabolism, and develop severe atherosclerosis when placed on a high-fat diet. Expression of RORbeta is very restricted. RORbeta is highly expressed in different parts of the neurophotoendocrine system, the pineal gland, the retina, and suprachiasmatic nuclei, suggesting a role in the control of circadian rhythm. This is supported by observations showing alterations in circadian behavior in RORbeta-/- mice. RORgamma, which is most highly expressed in the thymus, plays an important role in thymopoiesis. Thymocytes from RORgamma-/- mice undergo accelerated apoptosis. The induction of apoptosis is, at least in part, due to a down-regulation of the expression of the antiapoptotic gene Bcl-XL. In addition to the thynic phenotype, RORgamma-/- mice lack lymph nodes, indicating that RORgamma is essential for lymph node organogenesis. Overexpression of RORgamma has been shown to inhibit T cell receptor-mediated apoptosis in T cell hybridomas and to repress the induction of Fas-ligand and interleukin 2. These studies demonstrate that RORs play critical roles in the regulation of a variety of physiological processes. Further characterization of the mechanisms of action of RORs will not only lead to the identification of ROR target genes and provide additional insight into their normal physiological functions, but will also determine their roles in disease.

Affinity chromatography of angiotensin I-converting enzyme from rabbit lung using hippurylhistidylleucyl-OH.

Approximately 50-fold purification of angiotensin I-converting enzyme (Peptidyldipeptide hydrolase, EC 3.4.15.1) from rabbit lung was achieved by affinity chromatography using the synthetic substrate Hippuryl-His-Leu-OH. The specific activity of the enzyme was increased from 0.044 units/mg protein to 1.911 units/mg protein for Hippuryl-His-Leu-OH and from 0.33 nmol/min per mg protein to 13.8 nmol/min per mg protein for angiotensin I.

Solubilization of angiotensin I-coverting enzyme from rabbit lung using trypsin treatment.

The solubilization of angiotensin I-converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) from rabbit lung was carried out using trypsin treatment. A good recovery of 76% was obtained. The enzyme from solubilized fraction was purified using colums of Sephadex G-200, hydroxyapatite and DEAE-cellulose. The purified enzyme was shown to convert angiotensin I to angiotensin II and also to inactivate bradykinin. The specific activity of the enzyme was 24.3 units/mg protein for hippurylhistidylleucyl hydroxide and 0.182 mumol/min per mg protein for angiotensin I. The enzymic activity obtained after trypsin treatment for 5 h could be divided into two components: (i) an enzyme of molecular weight 300 000 (peak II) and (ii) an enzyme of molecular weight 145 000 (peak III), by Sephadex G-200 gel filtration. The molecular weight of the denatured enzyme was found to be 155 000 by disc gel electrophoresis in the presence of sodium dodecyl sulfate. Km values of peak II and peak III fraction for Hippuryl-His Leu-OH were 2.6 mM.

Purification of angiotensin I-converting enzyme from human lung.

Angiotensin I-converting enzyme (peptidyl dipeptide hydrolase, EC 3.4.15.1) was solubilized from the membrane fraction of human lung using trypsin treatment and purfied using columns of DE 52-cellulose, hydroxyapatite and Sephadex G-200. The purified enzyme was shown to convert angiotensin I to angiotensin II and also to inactivate bradykinin. The specific activity of the enzyme was 9.5 units/mg protein for Hippuryl-His-Leu-OH and 0.665 mumol/min per mg protein for angiotensin I. The enzymic activity obtained after trypsin treatment (1 mg/200 mg protein) for 2 h could be divided into three components: (i) an enzyme of molecular weight 290 000 (peak I), (ii) an enzyme of molecular weight 180 000 (peak II) and (iii) an enzyme of molecular weight 98 000 (peak III), by columns of DE 52-cellulose and Sephadex G-200. Km values of peak I, II and III fraction for Hippuryl-His-Leu-OH were identical at 1.1 mM. pH optimum of the enzyme was 8.3 for Hippuryl-His-Leu-OH.

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on leukemic cells from chronic B cell leukemia.

It is not clear whether cells from various chronic B cell leukemias including B-chronic lymphocytic leukemia (CLL), CLL in prolymphocytoid transformation (CLL-PLT), B-prolymphocytic leukemia (PLL), and hairy cell leukemia (HCL) simply represent different stages of a single B cell differentiation pathway. Furthermore, it is not known whether cells from any given B cell leukemia are characterized by the same population during the differentiation process. Differentiation of various B cell leukemic cells was induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), and the resulting changes in their morphology, cytoplasmic immunoglobulin (clg), and cytochemistry were evaluated. With respect to peculiar morphological change, i.e. extending long thin processes (spreading) and the appearance of clg, each sample showed different responses. According to these two indices samples were classified into three groups; spread+ clg- samples (one case of CLL-PLT, all HCL), spread+ clg+ samples (one of CLL, one of CLL-PLT), and spread- clg+ samples (a majority of CLL, one of CLL-PLT, and all PLL). Unexpectedly, both CLL and CLL-PLT consisted of heterogenous populations as to the reactivity to TPA. In the process of TPA-induced differentiation in CLL cells, features similar to those of HCL cells were not found. Since three different TPA-induced response patterns were observed in each chronic B cell leukemia type, it was not possible to sequentially assign each of these leukemias along a single B cell differentiation pathway. In order to explain this result, we introduced the hypothesis that these groups might be divided into different lineages in B cell differentiation. Since TPA-induced spreading cells were present in the B cell fraction of normal peripheral blood mononuclear cells, this morphological change should not be associated with malignant transformation.

Expression of decay-accelerating factor on hematopoietic progenitors and their progeny cells grown in cultures with fractionated bone marrow cells from normal individuals and patients with paroxysmal nocturnal hemoglobinuria.

Decay-accelerating factor (DAF), a complement-regulating glycoprotein, has been shown to be expressed on hematopoietic progenitors and their progeny cells in normal individuals but not on abnormal cells in patients with paroxysmal nocturnal hemoglobinuria (PNH). Fluorescence histograms of bone marrow cells showed the heterogeneity of DAF expression on their cell membranes, suggesting mixed hematopoietic cell populations in PNH. We have previously shown that DAF is a maturational protein expressed on normal hematopoietic cells. In order to elucidate the relationship between DAF expression and cell maturity in PNH, we fractionated bone marrow cells according to amount of DAF and cultured these cells in methylcellulose for clonal assay of erythroid burst-forming units (BFU-E) and granulocyte-macrophage colony-forming units (CFU-GM), followed by reanalysis of DAF expression on their progeny. The matured cells from the bursts/colonies in cultures with DAF-negative PNH marrow cells had no or little DAF, but those grown from DAF-positive PNH progenitors showed nearly as much of this factor as those grown from normal progenitors. These results clearly indicate that there are at least two distinct populations of hematopoietic progenitors with respect to the membrane expression of DAF and that abnormalities occur at the level of the stem cell in PNH.

The Challenge of e-Health Presence on a Petroleum Platform: Using Telemedicine to Make Operation of Pre-Salt Wells a Reality.

Telemedicine can be defined as the use of electronic media for the transmission of clinical data and information from one location to another using information technology and telecommunication in order to provide immediate clinical health care at long distances. This new approach can involve specialized medical service centers in the oil production at great distances from the offshore installations in Brazil. The importance of the right health diagnosis, taken at the proper time, will make a serious difference in the facilities, which will be located around 300 km offshore. This paper presents an overview of telemedicine and its different applications, comparing them according to level of maturity and applicability. Important results from a case study in a fixed oil platform are analyzed. At the end of this work, the strategy of telemedicine implementation in a Brazilian petroleum operator is discussed.

Cholesterol sulfate activates transcription of transglutaminase 1 gene in normal human keratinocytes.

Cholesterol sulfate and transglutaminase 1 are essential for the process of keratinization. Cholesterol sulfate is formed during keratinization and activates the eta isoform of protein kinase C. Transglutaminase 1 is a key enzyme for formation of the cornified envelope in terminally differentiated keratinocytes. In this study, we demonstrated that cholesterol sulfate acts as a transcriptional activator of the transglutaminase 1 gene in normal human keratinocytes. Growth of normal human keratinocytes was inhibited by cholesterol sulfate, but not by its parental cholesterol. Treatment of normal human keratinocytes with cholesterol sulfate induced activity of transglutaminase 1 in a dose- and time-dependent manner. Activation of transcription of transglutaminase 1 by cholesterol sulfate was demonstrated by northern blotting analysis, whereas that by cholesterol was not. In order to identify a cholesterol sulfate responsive region in the transglutaminase 1 gene, plasmids were constructed containing a luciferase reporter gene ligated to deletion fragments of the 5' upstream region of the tranglutaminase 1 gene and were transfected into normal human keratinocytes. Transfected cells were treated with cholesterol sulfate, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate and a high concentration of Ca2+. Our results indicate that the responsive element(s) for cholesterol sulfate and phorbol ester is located upstream of the human transglutaminase 1 gene at a position(s) between -819 and -549, whereas the responsive element for Ca2+ is located at a position between -79 and -49.

Minimal model analyzing response of glycine receptors expressed in Xenopus oocyte: inhibition by a lipid hydroperoxide.

Glycine receptor (GlyR) was expressed in Xenopus oocytes by injecting rat brain mRNA. Glycine (Gly)-elicited responses in the oocyte were measured by the voltage-clamping method. The following measurements were made to establish the relationship between Gly concentration and the current: 1) Gly-induced membrane current before desensitization, 2) Gly-induced membrane current after desensitization equilibrium, 3) fraction of the active form of the receptor after desensitization equilibrium, 4) rate of recovery of the desensitized receptors upon removal of Gly. These results were analyzed on the basis of the minimal model proposed for nicotinic acetylcholine and gamma-aminobutyric acid A receptor. The equilibrium and rate constants of the model were evaluated for GlyR. The effects of procaine and 13-L-hydroperoxylinoleic acid (LOOH) on GlyR were examined electrophysiologically. LOOH noncompetitively inhibited the receptor with the inhibition constant of 27 microM, while 1 mM procaine, a local anesthetic, did not inhibit GlyR at all.

Proteolytic cleavage of an activated subcomponent of the first component of rabbit complement, C1s.

When rabbit C1 purified by affinity chromatography on IgG-Sepharose 6B was chromatographed on DEAE-cellulose in the presence of ethylenediaminetetraacetate, C1s was isolated as two forms, C1s(I) and C1s(II), having different molecular weights. On the other hand, incubation of the C1 with soybean trypsin inhibitor before the chromatography resulted in the isolation of C1s(I) alone, indicating that, during the purification, C1s(II) was derived from C1s(I) by proteolytic cleavage of C1s(I) by a contaminating protease, probably plasmin [EC 3.4.21.7]. In fact, C1s(I) was completely converted to C1s(II) or a C1s(II)-like fragment by highly purified plasmin. Analysis of the polypeptide chain structures revealed that C1s(I), which consisted of H and L chains with molecular weights of 70,000 and 36,000, respectively, was converted to C1s(II) by cleavage of the H chain, since C1s(II) consisted of two chains each with a molecular weight of 37,000. This conversion proceeded without any alteration in C1 esterase activity, but was accompanied by loss of the ability to form C1r-C1s complex.

Clinical study on cardiac sarcoidosis.

Forty-one patients with sarcoidosis were investigated for cardiac involvement. The results are summarized as follows: The focal left ventricular perfusion defect was found on the TMPS's of 5 out of 41 patients with sarcoidosis. The defect disappeared in four of these five patients over a 3-year period, but remained in the patient with fatal myocardial sarcoidosis. A decreased left ventricular ejection fraction was found in 14 out of 41 patients with sarcoidosis, and in 3 out of 5 patients with the left ventricular perfusion defect. Echocardiographic examination did not reveal left ventricular wall dysfunction at the site of the left ventricular perfusion defect, except in one patient with fatal myocardial sarcoidosis. Holter ECG's showed such abnormalities as CRBBB, heart blocks, PVC, and PSC in 13 out of 32 patients with sarcoidosis. SACE activity was elevated in all three patients with the left ventricular perfusion defect that were examined, and it could be correlated with changes in the left ventricular perfusion defect in one of them.

Effects of captopril (SQ 14, 225) on the renin-angiotensin-aldosterone system in normal rats.

High doses of captopril (SQ 14, 225) (120-160 mg/kg/day) were administered orally to normal rats, and the effects on the renin-angiotensin-aldosterone system were observed. Plasma angiotensin converting enzyme (ACE) activity was elevated significantly on the 3rd, 7th and 30th days of captopril administration. ACE activity in the lung and the kidney was significantly decreased on the 1st day then gradually increased, becoming significantly higher than that of controls by the 30th day. Plasma renin activity (PRA) was significantly elevated on the 1st day and remained at a high level until the 30th day. Renal renin content was found to be significantly lower on the 1st and 3rd days. Plasma aldosterone concentration was not affected by captopril treatment, whereas serum potassium concentration was found to be significantly lower on the 1st, 3rd and 30th days. It is suggested that besides its inhibitory action on ACE, captopril has a direct or indirect stimulating action on ACE production as well as on renin release.

Angiotensin I-converting enzyme in human urine.

It was demonstrated that angiotensin I-converting enzyme was excreted in human urine. The mean activity of the enzyme in normal urine was found to be 0.38 +/- 0.04 (S.E.M.) units/day (n = 18) and the enzymic activity correlated well with the concentration of the excreted sodium (r = 0.76, p less than 0.005). Urinary angiotensin I-converting enzyme was partially purified. Three different molecular weights of enzyme (greater than 400 000, 290 000 and 140 000) were demonstrated by Sephadex G-200 gel filtration. The enzymic properties of these three enzymes were identical with those of angiotensin I-converting enzyme from human lung with regard to inhibitory effects (bradykinin potentiator c and Arg-Pro-Pro), Cl- dependency, pH optimum and KM value.

A semi-automated measurement of urinary catecholamines using high-speed ion-exchange column chromatography.

Semi-automated measurement of urinary catecholamines, specific for dopa, noradrenaline, adrenaline and dopamine and other metabolites has been developed. Steps included alumina extraction, separation by high-speed ion-exchange column chromatography and fluorescent development by trihydroxyindole (THI) method without interference by other fluorescent substances. Recovery, reproducibility and sensitivity were satisfactory. alpha-Methyldopa, a metabolite of alpha-methyldopa, and isoproterenol were separated completely without interfering with the measurement of noradrenaline, adrenaline and dopamine.

Ni(II)-based immobilized metal ion affinity chromatography of recombinant human prolactin from periplasmic Escherichia coli extracts.

A novel, two-step preparative technique is described for the purification of authentic recombinant human prolactin (rhPRL) secreted into the periplasm of transformed Escherichia coli cells. The first step is based on immobilized metal ion affinity chromatography of periplasmic extract, using Ni(II) as a relatively specific ligand for hPRL in this system. It gives superior resolution and yield than established ion-exchange chromatography. Size-exclusion chromatography is used for further purification to >99.5% purity. The methodology is reproducible, leading to 77% recovery. Identity and purity of the rhPRL were demonstrated using sodium dodecylsulphate-polyacrylamide electrophoresis, isoelectric focusing, mass spectrometry (matrix-assisted laser desorption ionization time-of-flight), radioimmunoassay, RP-HPLC and high-performance size-exclusion chromatography. In the Nb2 bioassay, the hormone showed a bioactivity of 40.9 IU/mg.