Appropriateness of surgical antimicrobial prophylaxis in Japanese university hospitals.

BACKGROUND: Surgical antimicrobial prophylaxis (SAP) is one of the major purposes of antimicrobial use. AIM: To determine the adherence to the Japanese SAP guidelines in Japanese university hospitals. METHODS: This was a retrospective cohort study including 15 general hospitals and one dental university hospital. Up to three cases of 18 designated surgeries were evaluated regarding adherence to Japanese SAP guidelines: selection of antibiotics, timing of administration, re-dosing intervals, and duration of SAP. When all items were appropriate, surgery was defined as 'appropriate'. FINDINGS: In total, 688 cases (22-45 cases per surgery) were included. The overall appropriateness was 46.8% (322/688), and the appropriateness of each surgery ranged from 8.0% (2/25, cardiac implantable electronic device implantation) to 92.1% (35/38, distal gastrectomy). The appropriateness of each item was as follows: pre/intraoperative selections, 78.5% (540/688); timing of administrations, 96.0% (630/656); re-dosing intervals, 91.6% (601/656); postoperative selection, 78.9% (543/688); and duration of SAP, 61.4% (423/688). The overall appropriateness of hospitals ranged from 17.6% (9/51) to 73.3% (33/45). The common reasons for inappropriateness were the longer duration (38.5%, 265/688) and choice of antibiotics with a non-optimal antimicrobial spectrum before/during, and after surgery (19.0%, 131/688 and 16.9%, 116/688, respectively), compared to the guideline. CONCLUSIONS: Adherence to the guidelines differed greatly between the surgeries and hospitals. Large-scale multi-centre surveillance of SAP in Japanese hospitals is necessary to identify inappropriate surgeries, factors related to the appropriateness, and incidences of surgical site infections.

Bayesian reconstruction of a vancomycin-resistant Enterococcus transmission route using epidemiologic data and genomic variants from whole genome sequencing.

BACKGROUND: Outbreaks of vancomycin-resistant enterococcus (VRE) are a serious problem in hospitals. Inferring the transmission route is an important factor to institute appropriate infection control measures; however, the methodology has not been fully established. AIM: To reconstruct and evaluate the transmission model using sequence variants extracted from whole genome sequencing (WGS) data and epidemiological information from patients involved in a VRE outbreak. METHODS: During a VRE outbreak in our hospital, 23 samples were collected from patients and environmental surfaces and analysed using WGS. By combining genome alignment information with patient epidemiological data, the VRE transmission route was reconstructed using a Bayesian approach. With the transmission model, evaluation and further analyses were performed to identify risk factors that contributed to the outbreak. FINDINGS: All VREs were identified as Enterococcus faecium belonging to sequence type 17, which consisted of two VRE genotypes: vanA (N = 8, including one environmental sample) and vanB (N = 15). The reconstruction model using the Bayesian approach showed the transmission direction with posterior probability and revealed transmission through an environmental surface. In addition, some cases acting as VRE spreaders were identified, which can interfere with appropriate infection control. Vancomycin administration was identified as a significant risk factor for spreaders. CONCLUSION: A Bayesian approach for transmission route reconstruction using epidemiologic data and genomic variants from WGS can be applied in actual VRE outbreaks. This may contribute to the design and implementation of effective infection control measures.

Effects of heme degradation products on reactivation of latent HIV-1.

Human immunodeficiency virus (HIV-1) infection can be currently controlled by combined antiretroviral therapy, but a sterilizing cure is not achievable as this therapy does not target persistent HIV-1 in latent reservoirs. Therefore, different latency reversal agents are intensively explored in various models. We have previously observed that heme arginate, a drug approved for human use, reveals a strong synergism with PKC inducers in reactivation of the latent provirus. Heme is physiologically decomposed by heme oxygenases into 3 degradation products: iron (Fe2+), carbon monoxide (CO) and biliverdin which is further converted to bilirubin by biliverdin reductase. In this paper, we have studied the effects of individual heme-degradation products on latent HIV-1 reactivation in ACH-2 cells harboring integrated HIV-1 provirus and in H12 clone of Jurkat cells harboring HIV-minivirus expressing EGFP. We employed addition of ascorbate to generate Fe2+, resulting in increased expression of both HIV-1 p24 Ag and EGFP in PMA-stimulated ACH-2 and H12 cells, respectively, as characterized on RNA and protein levels. On the other hand, addition of a CO-donor or bilirubin decreased the p24 expression. The reactivation of latent HIV-1 by iron or heme arginate was inhibited by antioxidant N-acetyl cysteine, or by an iron chelator desferrioxamine, suggesting that the effects were mediated by iron- or heme-induced redox stress. Finally, we demonstrated the stimulatory effects of heme arginate and PMA on HIV-1 expression in peripheral blood mononuclear cells of HIV-infected patients cultured ex vivo. These results may constitute a new direction in the latent HIV-1 reactivation and therapy.

Iron Overload Causes Alterations of E-Cadherin in the Liver.

Iron overload causes tissue damage in the liver, but its initial effects at the molecular and cellular level are not well understood. Epithelial cadherin (E-cad) is a major adhesion protein in adherens junctions and is associated with several signal transduction pathways. Dysfunction of E-cad causes instability of adherens junctions, which leads to cell invasion, cell migration, and carcinogenesis. We found in liver samples from iron-overloaded mice that the apparent molecular mass of E-cad was reduced from 125 to 115 kDa in sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing conditions and immunoblotting, and that the cellular expression of E-cad was decreased in immunohistochemistry. The mRNA level of E-cad, however, did not change significantly, suggesting that the alterations are posttranslational. Interestingly, incubation of control liver extracts with Fe2+ alone also produced the same mobility shift. Neither an oxidant nor an antioxidant influenced this shift in vitro, suggesting that reactive oxygen species, which are generated by iron and known to cause damage to macromolecules, are not involved. Treatment of the 115 kDa E-cad with deferoxamine, an iron chelator, thus removing Fe2+, shifted the molecular mass back to 125 kDa, demonstrating that the shift is reversible. The observation also implies that the alteration that causes the mobility shift is not due to transcriptional control, deglycosylation, and proteolysis. This reversible mobility shift of E-cad has not been previously known. The alteration of E-cad that causes the mobility shift might be an initial step to liver diseases by iron overload.

Decreased hemojuvelin protein levels in mask mice lacking matriptase-2-dependent proteolytic activity.

Matriptase-2, a membrane protein encoded by the Tmprss6 gene, is a negative regulator of hepcidin expression. Although matriptase-2 has been proposed to cleave membrane hemojuvelin, we have recently found decreased hemojuvelin protein levels in Tmprss6 -/- mice. The purpose of this study was to confirm this observation by determining hemojuvelin protein levels in another strain of mice with disrupted Tmprss6 gene, and to determine the effect of matriptase-2 deficiency on the expression of other membrane proteins participating in the bone morphogenetic protein signal transduction. Mask mice, which lack the proteolytic domain of matriptase-2, displayed decreased liver hemojuvelin protein content, while Id1 mRNA level, an indicator of hemojuvelin-dependent signal transduction, was increased. Protein levels of bone morphogenetic protein receptors Alk3 and Acvr2a were unchanged, and transferrin receptor 2 and neogenin protein levels were slightly decreased. The results confirm that the loss of matriptase-2 increases bone morphogenetic protein-dependent signaling, while paradoxically decreasing liver hemojuvelin protein content. The regulation of transferrin receptor 2 protein levels by transferrin saturation was not affected in mask mice. How the loss of matriptase-2 proteolytic activity leads to decreased hemojuvelin protein levels is at present unclear.

Inhibition of arginase ameliorates experimental ulcerative colitis in mice.

Nitric oxide (NO) is produced from the conversion of L-arginine by NO synthase (NOS) and regulates a variety of processes in the gastrointestinal tract. Considering the increased activity of arginase in colitis tissue, it is speculated that arginase could inhibit NO synthesis by competing for the same L-arginine substrate, resulting in the exacerbation of colitis. We examined the role of arginase and its relationship to NO metabolism in dextran sulfate sodium (DSS)-induced colitis. Experimental colitis was induced in mice by administration of 2.5% DSS in drinking water for 8 days. Treatment for arginase inhibition was done by once daily intraperitoneal injection of N(ω)-hydroxy-nor- arginine (nor-NOHA). On day 8, we evaluated clinical parameters (body weight, disease activity index, and colon length), histological features, the activity and expression of arginase, L-arginine content, the expression of NO synthase (NOS), and the concentration of NO end-product (NOx: nitrite + nitrate). Administration of nor-NOHA improved the worsened clinical parameters and histological features in DSS-induced colitis. Treatment with nor-NOHA attenuated the increased activity of arginase, upregulation of arginase Ι at both mRNA and protein levels, and decreased the content of L-arginine in colonic tissue in the DSS-treated mice. Conversely, despite the decreased expression of NOS2 mRNA, the decreased concentration of NOx in colonic tissues was restored to almost normal levels. The consumption of L-arginine by arginase could lead to decreased production of NO from NOS, contributing to the pathogenesis of the colonic inflammation; thus, arginase inhibition might be effective for improving colitis.

Termination of the vertebral veins: Evaluation by multidetector row computed tomography.

The purpose of this study was to evaluate the topographic anatomy of the vertebral vein (VV) in the lower neck and thoracic inlet using CT scans. Enhanced CT scans using 32-MDCT were obtained for 199 consecutive patients. Reconstructed images with 1-mm section thickness/intervals were evaluated by two radiologists examining the drainage point, number, and route of VVs using frame forwarding and the rewind function on the DICOM viewer. The VV was classified into four types as follows: Type A (80.6%), a VV that descended ventral to the subclavian artery (SA) and drained into the upper portion of the brachiocephalic vein (BCV); Type B (5.8%), a VV that descended dorsal to the SA and drained into the upper portion or the lower portion of the BCV; Type C (8.3%), a doubled VVs that crossed both sides of the SA and drained into the upper portion of the BCV and formed a common trunk; Type D (5.3%), a VV ventral to the SA that drained into the upper portion of the BCV and another VV dorsal to the SA drained into the upper portion or the lower portion of the BCV. Some variations were observed in regard to the drainage point, number, and route of the VVs. Classification of the VV may be useful for interpreting chest CT scans and in better understanding the embryologic development of the vertebral vein.

Hepcidin downregulation by repeated bleeding is not mediated by soluble hemojuvelin.

Hepcidin is a key regulator of iron homeostasis, while hemojuvelin is an important component of the hepcidin regulation pathway. It has been recently proposed that soluble hemojuvelin, produced from hemojuvelin by the protease furin, decreases hepcidin expression. The aim of the presented study was to examine the downregulation of hepcidin by chronic bleeding in hemojuvelin-mutant mice. Male mice with targeted disruption of the hemojuvelin gene (Hjv-/- mice) and wild-type littermates were maintained on an iron-deficient diet and subjected to weekly phlebotomies for 7 weeks. Gene expression was examined by real-time PCR. In wild type mice, repeated bleeding decreased hepcidin mRNA by two orders of magnitude. In Hjv-/- mice, basal hepcidin expression was low; however, repeated bleeding also decreased hepcidin mRNA content by an order of magnitude. Phlebotomies reduced hepatic iron overload in Hjv-/- mice by 80 %. Liver and muscle furin mRNA content was not significantly changed. No effect on hepatic Tmprss6 mRNA content was observed. Results from the study indicate that soluble hemojuvelin is not the sole factor responsible for hepcidin downregulation. In addition, the presented data suggest that, under in vivo conditions, tissue hypoxia does not transcriptionally regulate the activity of furin or TMPRSS6 proteases.

Analysis of gap junction formation in rat hepatocytes by intravenous injection of an anti-connexin32 monoclonal antibody (HAM8).

HAM8 monoclonal antibody was used to examine the mechanism of connexin32 (Cx32) formation in the rat model in vivo by immunofluorescence and immunoelectron microscopy. After a single intravenous injection of HAM8 IgG, a number of HAM8 signals were observed at the sinusoidal face, between adjacent hepatocytes as well as in the cytoplasm stained with only 2nd fluorescent antibody. Moreover, the in vivo localization of the HAM8 antigen appeared to change with time. At 5 min after the antibody injection, Cx32 signals from liver sections were clearly detected at the sinusoidal face. Fifteen minutes later, numerous linear and dotted fluorescent signals were observed between hepatocytes; in addition, much punctate staining was found at the sinusoidal face and in hepatocytes. These findings were identified by immunoelectron microscopy. Interestingly, 1 hr later, much punctate staining considered to be similar to those seen in the normal rat liver tissues was observed between adjacent hepatocytes, suggesting that a great deal of Cx32 combined with HAM8 have been assembled into identifiable gap junction plaques. Five hours later, intercellular and intracellular Cx32 signals were infrequently detected. When staining was performed with HAM8 and 2nd antibody, however, numerous Cx32 signals were again observed between neighboring hepatocytes, as punctate staining appearing in a pattern approximately the same as that seen in the normal liver tissue. Based on these results, we assumed that a precursor gap was present during Cx32 formation, and discussed the pathways of Cx32 formation and the degradation of Cx32 as well as that of HAM8.

Isolation and characterization of a human alternative complement pathway-inhibiting protein from larval hemolymph of the silkworm, Bombyx mori.

An alternative complement pathway-inhibiting protein (ACPIP), which inhibits the activation of the alternative complement pathway (ACP) of the human serum, was isolated from larval hemolymph of the silkworm, Bombyx mori, by using ammonium sulfate fractionation and column chromatographies to homogeneity. About 400microg of ACPIP was routinely obtained from 20ml hemolymph. The purified ACPIP preparation consisted of two distinct polypeptides (34 and 32kDa) on SDS-PAGE. The amino acid compositions of the two polypeptides were nearly identical; 21% of the amino acid residues were acidic. The amino terminal amino acid sequences up to 20 residues in these two polypeptides were also identical. Addition of the ACPIP to human serum resulted in a dose-dependent inhibition of the hemolysis of intact rabbit erythrocytes via the ACP, whereas in no inhibition of hemolysis of sensitized-sheep erythrocytes (EA) via the classical pathway.

Ultrastructural and functional changes of the proximal tubular epithelial cells in the renal cortex from spontaneously diabetic KKAy mice.

To investigate the morphological and functional changes of epithelial cells of proximal tubules in the early stage of diabetic nephropathy, we examined the ultrastructural changes of proximal convoluted tubular cells in spontaneously diabetic KKAy mice. KKAy mice already showed significant elevation of urinary albumin excretion at 16 weeks of age. Examination revealed that lysosomes were increased in number and conspicuous in the epithelial cells of the proximal convoluted tubules from KKAy mice at 40 weeks of age when compared to control C57BL mice. Furthermore, endogenous IgG was detected in the lysosomes of proximal tubular cells from KKAy mice. These findings suggested that reabsorption activity was elevated in the proximal tubules of KKAy mice at 40 weeks of age.

Vascular cell adhesion molecule-1 expression in the renal interstitium of diabetic KKAy mice.

To investigate the mechanism of interstitial inflammation in diabetic nephropathy, we used spontaneously diabetic KKAy mice. Twelve KKAy mice were divided into two groups; six mice were fed standard mouse chow ad libitum and six mice were placed on a diet (i.e. they received the same amount of chow as six control C57BL mice). Diabetic KKAy mice developed hypercholesterolemia and albuminuria. Animals were killed at 16 weeks of age and renal tissues were immunostained for vascular cell adhesion molecule-1 (VCAM-1). In diabetic KKAy mice, the renal interstitium was infiltrated by monocytes, lymphocytes, plasma cells, and other cells. The walls of venules near the infiltrating cells were more intensely stained for VCAM-1 when compared with other sites. In contrast, the VCAM-1 staining of arterioles and peritubular capillaries was not significantly increased. There was weak VCAM-1 staining of the infiltrating cells, including lymphocytes, monocytes, and other cells. Electron microscopy demonstrated immunolabeling for VCAM-1 on the cell surface and in the cytoplasm of both infiltrating cells and vascular endothelial cells. In KKAy mice placed on a diet, there was less staining for VCAM-1 and cellular infiltration was also decreased. Thus, increased expression of VCAM-1 by the endothelial cells of venules and VCAM-1 expression by infiltrating cells were demonstrated in the interstitium of kidneys from diabetic mice. These results suggest that increased expression of VCAM-1 by endothelial cells and infiltrating cells contributes to interstitial inflammation in diabetic nephropathy.

Successful nerve regeneration and persistence of donor cells after a limited course of immunosuppression in rat peripheral nerve allografts.

BACKGROUND: The origin of Schwann cells and effect of a limited course of immunosuppression using cyclosporine (CsA) were examined in rat peripheral nerve allotransplants. METHODS: Phenotypes of Schwann cells in groups without, with continuing, and with limited (12 weeks) CsA treatment were examined immunohistochemically in allogeneically and syngeneically transplanted animals from 4 to 36 weeks after transplantation. RESULTS: In the group receiving no CsA, little nerve regeneration was obtained; donor Schwann cells were rejected and replaced by recipient cells. In continuing and limited-course CsA groups, successful nerve regeneration was achieved at postoperative week 36, as was also observed in the syngeneic group. Schwann cells in the continuing CsA group remained donor-derived. In the limited-course CsA group, graft rejection and loss of function occurred after the withdrawal of CsA, and donor Schwann cells were replaced by recipient cells in the part of the graft where rejection had been complete. However, many donor Schwann cells remained at week 36, when the rejection response subsided. CONCLUSION: Possible clinical use of a limited course of immunosuppression was supported by this demonstration of long term persistence of donor Schwann cells.

Mutations of connexin32 in Charcot-Marie-Tooth disease type X interfere with cell-to-cell communication but not cell proliferation and myelin-specific gene expression.

Connexin32 (Cx32) is a gap junction protein and its mutations are responsible for X-linked Charcot-Marie-Tooth disease. We examined the functional abnormality of C6 glioma cells transfected with mutant (C53S and P172R) Cx32 genes. Nontransfected C6 did not express Cx32. Northern and Western blot analyses showed Cx32 mRNA and protein in cells with the wild-type gene as well as with the mutant Cx32 genes. An immunocytochemical study of cells with the wild-type gene showed the immunoreactive spots in the cell membrane. In cells with C53S or P172R mutant gene, however, the immunoreactivity was found in the cytoplasm. The scrape-loading method produced effective dye transfer in cells with the wild-type gene but not in those with mutant genes. A cell proliferation assay showed no differences in nontransfected cells, cells with the wild-type gene and those with the mutant genes. Messenger RNA expression for proteolipid protein did not change. These findings suggest that Cx32 gene mutation results in loss of cell-to-cell communication because of failure to incorporate Cx32 protein in the cell membrane. The mutations do not, however, interfere with cell proliferation or myelin-specific gene expression, at least myelin proteolipid protein expression in C6 glioma cells.

Expression of ICAM-1 in implanted primary and metastatic squamous cell carcinomas in rats.

FF6 tumor cells are derived from a spontaneous rat squamous cell carcinoma (SCC) which originally arose in the facial skin of a DA rat. In this study, FF6 tumor cells were implanted into rat oral mucosa to establish an ex vivo metastatic model. We analyzed the expression of intercellular cell adhesion molecule-1 (ICAM-1) in the implanted primary and metastatic FF6 tumors by immuno-staining with a monoclonal antibody (mAb) against ICAM-1. The implanted primary FF6 cells showed strong expression of ICAM-1, whereas the tumor cells of metastatic lesions showed weak or negative expression of ICAM-1. By immunostaining with mAb OX6, a number of MHC class II-positive macrophages were detected in tumor mesenchyme and surrounding the metastatic foci. These results suggested that the local immune reaction in the lymph node influenced the expression of ICAM-1 on tumor cells, and that MHC class II-positive macrophages may play a role in transplanted tumor growth and metastases.

[Effects of beta-adrenergic agonists and phosphodiesterase inhibitors on the outflow facility of the eye].

Changes in the outflow facility of perfused bovine eyes and in the shape of cells in cultured trabecular meshwork (TM) were studied after exposure to adrenergic drugs. Dobutamine, a selective beta 1 agonist caused confluent TM cells to change from their usual polygonal shape to a characteristic stellate shape. Salbutamol, a selective beta 2 agonist showed no effect. The phosphodiesterase (PDE) inhibitors, isobutylmethylxanthine (IBMX), theophylline, and caffeine were also very effective in producing this shape change. Epinephrine, isoproterenol, dobutamine or salbutamol did not increase the outflow facility, at temperatures of 22 degrees C or 36 degrees C, whereas theophylline, caffeine and IBMX increased the facility in a dose-dependent manner. The high concentrations of beta-adrenergic agents required to produce even a small change in outflow facility and the cell shape argue against the involvement of adrenergic receptor mediation; on the other hand, the enhancement of the effects of epinephrine by PDE inhibitors and a similar effect produced by cAMP suggest that the changes in the cell shape are produced by beta-receptor activation. The beta-adrenergic agents were ineffective on changing outflow facility but the PDE inhibitors were remarkably effective in producing both shape change and increasing facility. PDE inhibition has much greater influence on and around the outflow channels than pure beta-adrenergic agonists, at least in enucleated bovine eyes.

Analysis of allogenic lymphocytes in rat thymus following sublethal irradiation.

The effects of allogeneic lymphocytes on the rat thymus following sublethal irradiation were investigated using immunofluorescence. The recovery of thymus weight following irradiation was delayed in rats 6 days after receiving lymphocytes compared to controls. Allogeneic cells forming colonies were detected by immunofluorescence in both the cortex and medulla of the host thymus, most frequently on day 15 when an appropriate number (3 x 10(6)) was injected. The allogeneic cells detected in the host thymus, presumably T lymphocytes, appeared to disturb thymic reconstitution following irradiation. However, double-immunofluorescence staining revealed that allogeneic cells did not affect the thymic stromal microenvironment. Allogeneic cells may have subsequently affected thymic tissue via cytokines. It is important to investigate not only the character of allogeneic cells in the host thymus but also the interactions of donor allogeneic cells, host immature lymphocytes and thymic epithelial cells because of the possibility that these allogeneic cells in the host thymus could prevent the rejection of allogeneic transplants.

Morphological and flow cytofluorometrical analyses of regenerated rat thymus after irradiation.

Reconstituted rat thymuses were studied by immunohistochemistry, transmission electron microscopy (TEM) and flow cytofluorometry on days 0, 1, 2, 3, 5, and 7 after whole-body sublethal irradiation (6 Gy). One day after irradiation, numerous apoptotic cells were seen in the cortical thymus; the percentage of the sub-G1 peak representing apoptotic cells was 8.9% in the DNA content histogram of cytofluorometry. On day 3, the thymic structure had been destroyed and no distinction was drawn between the cortex and medulla. In this stage, few thymocytes but many macrophages were present, and the percentage of the sub-G1 peak reached a peak at 13.0%. Bromodeoxyuridine (BrdU) incorporated cells gradually increased after irradiation, and immunohistochemically numerous apoptotic cells were found primarily in the cortex on day 7. These thymocytes showed some levels of electron density of the nucleus as revealed by TEM. The percentage of S phase cells did not change markedly (20-30%) based on one-color DNA content histograms, but the percentage of early S and S phase cells was extremely high on day 7 (70%). These data indicate that a part of DNA synthetic cells may result in apoptosis. The combination of immunohistochemistry, TEM and flow cytofluorometry to analyze DNA content and BrdU incorporation proved a useful tool for investigating the reconstituted thymus.

Recognition of a special membrane antigen of squamous cell carcinoma in rats with a monoclonal antibody UB23.

This article describes the recognition of a special membrane antigen of the rat squamous cell carcinoma (SCC) by a monoclonal antibody (mAb), UB23, and the characterization of the UB23 antigen expression in the implanted primary and metastatic SCC in rat models. The mAb UB23 was raised against the FF6 tumor, a well-differentiated rat SCC, and it recognized the 120- to 130-kD cell surface antigen in FF6 tumor cells. The UB23 antigen was found in frequently observed small 'basal' cells but not in keratinocytes, and an increased expression was seen in the cells at the interface with peritumoral stroma in both the implanted primary FF6 tumors and metastases. These results indicated that the UB23 antigen is closely related with the cell differentiation and invasion of FF6 cells, and could be useful for analyzing the mechanism of differentiation, invasion and metastasis of SCC in animal models.