Methicillin-Resistant Staphylococcus epidermidis Is Part of the Skin Flora on the Hands of Both Healthy Individuals and Hospital Workers.

Staphylococcus epidermidis, a major skin flora on hands, acts as a reservoir of various antimicrobial resistance determinants including staphylococcal cassette chromosome mec (SCCmec) and contributes to multidrug resistance for S. aureus. The aim of this study was understanding the characteristics of commensal S. epidermidis on the hands of hospital workers and healthy individuals. A total of 23 hospital workers (physicians, nurses, and hospital pharmacists), 13 community pharmacists, and 24 healthy individuals (students) were studied. Commensal bacteria on hands were recovered using a glove-juice method. For methicillin-resistant S. epidermidis (MRSE), we performed SCCmec typing, pulsed-field gel electrophoresis (PFGE), and determined the antimicrobial susceptibility. The detection rates of MRSE in community pharmacists (92.3%) and students (87.5%) were higher than those in hospital workers (66.7 to 81.8%). SCCmec type IV strains were predominant in both hospital workers and students. PFGE analysis strongly suggested that the MRSE of hospital workers and students were normal inhabitants of each subject. The antimicrobial resistance rates and levels in MRSE of hospital workers were higher than those of students. Our findings showed that MRSE was frequently colonized on the hands of healthy individuals as well as hospital workers.

[Disinfection efficacy of hand hygiene based on chlorhexidine gluconate content and usage of alcohol-based hand-rubbing solution].

Recently, the procedure for surgical hand hygiene has been switching to a two-stage method and hand-rubbing method from the traditional hand-scrubbing method. Both the two-stage and hand-rubbing methods use alcohol-based hand-rubbing after hand washing. The former requires 5 min of antiseptic hand washing, and the latter 1 min of nonantiseptic hand washing. For a prolonged bactericidal effect in terms of surgical hand hygiene, chlorhexidine gluconate (CHG) has been noted due to its residual activity. However, no detailed study comparing the disinfection efficacy and prolonged effects according to different contents of CHG and the usage of alcohol-based hand-rubbing has been conducted. The glove juice method is able to evaluate disinfection efficacy and prolonged effects of the disinfectants more accurately because it can collect not only transitory bacteria but also normal inhabitants on hands. In the present study, we examined the disinfection efficacy and prolonged effects on alcohol-based hand-rubbing containing CHG by six hand-rubbing methods and three two-stage methods using the glove juice method. In both methods, 3 mL (one pump dispenser push volume) alcohol-based hand-rubbing solution containing 1% (w/v) CHG showed the highest disinfection efficacy and prolonged effects, and no significant difference was found between the hand-rubbing and two-stage methods. In the two methods of hand hygiene, the hand-rubbing method was able to save time and cost. Therefore, the data strongly suggest that the hand-rubbing method using a one pump dispenser push volume of alcohol-based hand-rubbing solution containing 1% (w/v) CHG is suitable for surgical hand hygiene.

Release of arachidonic acid induced by tumor necrosis factor-alpha in the presence of caspase inhibition: evidence for a cytosolic phospholipase A2alpha-independent pathway.

Stimulation of L929 cells with tumor necrosis factor-alpha (TNFalpha) caused cell death accompanied by a release of arachidonic acid (AA). Although the inhibition of caspases has been shown to cause necrosis in TNFalpha-treated L929 cells, its role in the TNFalpha-induced release of AA has not been elucidated. The release of AA is tightly regulated by phospholipase A(2) (PLA(2)). To find out the mechanisms underlying the TNFalpha-induced release of AA, we investigated the relationship between TNFalpha stimulation and PLA(2) regulation with and without zVAD, an inhibitor of caspases. In the present study, we found that treatment with TNFalpha and zVAD stimulated release of AA and cell death in C12 cells (a variant of L929 cells lacking alpha type of cytosolic PLA(2) (cPLA(2)alpha)). Stimulation with TNFalpha/zVAD also caused the release of AA from L929-cPLA(2)alpha-siRNA cells. Treatment with pyrrophenone (a selective inhibitor of cPLA(2)alpha) completely inhibited the TNFalpha-induced release of AA, but only partially inhibited the TNFalpha/zVAD-induced response in L929 cells. The TNFalpha/zVAD-induced release of AA from C12 and L929-cPLA(2)alpha-siRNA cells was pyrrophenone-insensitive, but inhibited by treatment with butylated hydroxyanisole (BHA, an antioxidant). Treatment with dithiothreitol, which inactivates secretory PLA(2) activity, decreased the amount of AA released by TNFalpha/zVAD. TNFalpha/zVAD appears to stimulate release of AA from C12 cells in a cPLA(2)alpha-independent, BHA-sensitive manner. The possible roles of secretory PLA(2) and reactive oxygen species from different pools in the release of AA and cell death were discussed.

Expression of cytosolic phospholipase A2 alpha in murine C12 cells, a variant of L929 cells, induces arachidonic acid release in response to phorbol myristate acetate and Ca2+ ionophores, but not to tumor necrosis factor-alpha.

Tumor necrosis factor-alpha (TNFalpha)-induced cell death is regulated through the release of arachidonic acid (AA) by group IVA cytosolic phospholipase A2 (cPLA2alpha) in the murine fibroblast cell line L929. However, the signaling pathway by which TNFalpha activates cPLA2alpha remained to be solved. We examined AA release in L929 cells, in a variant of L929 (C12 cells) lacking cPLA2alpha, and in C12 cells transfected with cPLA2alpha expression vectors. In transient and stable clones of C12 cells expressing cPLA2alpha, Ca2+ ionophore A23187 and phorbol myristate acetate (PMA) stimulated AA release within 90 min, although no response to TNFalpha was observed within 6 h. These results suggest that C12 cells may lack the components necessary for TNFalpha-induced AA release, in addition to cPLA2alpha. PMA is known to stimulate AA release via phosphorylation of Ser505 in cPLA2alpha by activating extracellular signal-regulated kinases (ERK1/2). However, PMA-induced AA release from C12 cells expressing mutant cPLA2alpha S505A (mutation of Ser505 to Ala), which is not phosphorylated by ERK1/2, was similar to that from L929 cells and C12 cells expressing wild-type cPLA2alpha. The role of Ser505 phosphorylation in AA release induced by PMA is also discussed.

Involvement of protein kinase C-related anti-apoptosis signaling in radiation-induced apoptosis in murine thymic lymphoma(3SBH5) cells.

Protein kinase C (PKC; also known as PRKC) is known to be an important participant in radiation-induced apoptosis. However, its role is not fully clarified. Using 3SBH5 cells, which are radiation-sensitive thymic lymphoma cells, the involvement and functions of PKC were assessed in radiation- induced apoptosis. PMA (phorbol 12-myristate 13-acetate), a PKC activator, inhibited the radiation-induced apoptosis in 3SBH5 cells. On the other hand, chelerythrine, a PKC inhibitor, potentiated apoptosis. In addition, Gö6976, a classical PKC (cPKC) inhibitor, which specifically inhibits PKC (alpha and betaI), also promoted apoptosis. Interestingly, post-treatment (20 min after irradiation) with Gö6976 had no effect on the radiation-induced apoptosis. These results suggest that cPKC is activated early after irradiation for anti-apoptosis signaling and contributes to the balance between cell survival and death. Indeed, an increase of cPKC activity involving PKC (alpha, betaI and betaII) was observed in the cytosolic fraction 3 min after irradiation with 0.5 Gy. However, no translocation of cPKC was observed in the cells after irradiation. Our findings indicate that activation of cPKC (alpha or beta) soon after irradiation is critical to the understanding of the regulation of radiation-induced apoptosis in radiation-sensitive cells.

Effects of 4-hydroxy-2-nonenal, a marker of oxidative stress, on the cyclooxygenase-2 of human placenta in chorioamnionitis.

Chorioamnionitis (CAM) is one of the causes of preterm labour. A recent study has indicated that NADPH oxidase, a reactive oxygen species (ROS)-producing enzyme, is activated in CAM. CAM is thought to be closely associated with oxidative stress. We have hypothesized that oxidative stress in CAM may induce preterm labour. The purpose of this study is to examine the effect of 4-hydroxy-2-nonenal (HNE), which is a marker of oxidative stress, on human placenta during preterm labour. We initially examined the HNE-modified proteins in human placentas by immunoblotting and immunohistochemistry using anti-HNE antibody. To examine the effect of HNE on human placenta, we stimulated human placental tissue with HNE. The expressions of cyclooxygenase-2 (COX-2) mRNA and protein were observed by RT-PCR and western blot analysis respectively. Furthermore, we measured the peroxidase activity of COX-2 by COX activity assay kit. Prostaglandin E(2) (PGE(2)) in the supernatants of placental tissue was also determined by enzyme-linked immunosorbent assay. Immunoblotting and immunohistochemistry showed that the levels of HNE-modified proteins were increased in the placentas with CAM, compared to the normal placenta. HNE induced the expression of COX-2 mRNA, protein and activity in the placental tissue culture stimulated with HNE. In addition, PGE(2) was also released into the medium in a time-dependent fashion. These findings suggest that HNE-modified proteins, which were increased in the placenta with CAM, play an important role in preterm labour.

Changes of lipocalin-type prostaglandin D synthase level during pregnancy.

AIM: Prostaglandin D (PGD), synthesized by lipocalin-type prostaglandin D synthase (L-PGDS), has marked effects on a number of biological processes, including the prevention of platelet aggregation and the relaxation of vascular smooth muscle. The aim of the study presented here was to examine the significance of L-PGDS in human pregnancy. METHODS: We measured the concentration of plasma L-PGDS in pregnant and non-pregnant women, and the concentration of L-PGDS in the umbilical cord blood, amniotic fluid and urine of newborns by enzyme-linked immunoabsorbent assay. To determine the localization of L-PGDS, we performed immunohistochemical analysis. To evaluate the usefulness of diagnosis of rupture of membranes (ROM), we determined the concentration of L-PGDS in cervicovaginal secretions. RESULTS: Pregnant women and non-pregnant women had similar L-PGDS concentrations (0.57 +/- 0.13 microg/mL vs 0.53 +/- 0.07 microg/mL). Umbilical cord blood, amniotic fluid and newborn urine contained higher L-PGDS concentrations (1.87 +/- 0.73 microg/mL, 2.62 +/- 0.86 microg/mL, 6.31 +/- 4.62 microg/mL, respectively) than maternal blood. The concentration of L-PGDS in amniotic fluid from 19 weeks onward was significantly greater than that at 15-18 weeks (3.201 +/- 0.384 microg/mL, n = 6 vs 1.735 +/- 0.477 microg/mL, n = 4; P < 0.05). Immunohistochemistry revealed that the amniotic cells of the placenta expressed L-PGDS. The sources of L-PGDS in amniotic fluid are fetus urine and amniotic cells. The concentration of L-PGDS in cervicovaginal secretions with rupture of membrane (ROM) were significantly higher than those without ROM. CONCLUSION: The measurement of L-PGDS in cervicovaginal fluid was useful in the detection of ROM during pregnancy.

Detection of oxidative stress in seminal plasma and fractionated sperm from subfertile male patients.

OBJECTIVES: Oxidative stress in the reproductive system is thought to affect the fertilizing ability of sperm. Since 8-hydroxydeoxyguanosine (8-OHdG) and lipid peroxides are widely used as markers to quantify oxidative stress, we compared 8-OHdG and lipid peroxide concentrations in seminal plasma and spermatozoa from subfertile and fertile men. STUDY DESIGN: Semen obtained from 37 men of subfertile couples (21 men with normozoospermia and 16 with asthenozoospermia) and from eight fertile volunteers were examined. Seminal plasma and spermatozoa were fractionated by four-step discontinuous Percoll gradient centrifugation. 8-OHdG in seminal plasma was measured by ELISA, and lipid peroxides in seminal plasma and spermatozoa were determined using a thiobarbituric acid (TBA) assay. RESULTS: The concentrations of 8-OHdG and lipid peroxides in the seminal plasma of the subfertile group were significantly higher than those of the fertile group. There were no significant differences in these values between patients with normozoospermia and asthenozoospermia. In all four fractions obtained by Percoll gradient fractionation, the lipid peroxide levels in spermatozoa recovered from subfertile males were significantly higher than those of fertile controls. CONCLUSIONS: Seminal plasma and spermatozoa from subfertile males showed elevated levels of oxidative stress that were detectable in ejaculated semen specimens by ELISA or TBA assay. Even the spermatozoa fraction considered to be mature and normal showed elevated oxidative stress in the subfertile group. Our results confirm the importance of oxidative stress in male reproductive function, and could be applied for the selection of patients for antioxidant therapy.

The expression of secretory leukocyte protease inhibitor (SLPI) in the fallopian tube: SLPI protects the acrosome reaction of sperm from inhibitory effects of elastase.

BACKGROUND: Secretory leukocyte protease inhibitor (SLPI) is a protein found in various fluids, including parotid secretions, cervical mucus, seminal plasma and ascites, and is a potent inhibitor of human leukocyte elastase activity. The objective of the study was 2-fold, to evaluate (i) the presence of SLPI in the Fallopian tube, and (ii) the effect of SLPI on the acrosome reaction of sperm. METHODS AND RESULTS: Western blot analysis revealed that SLPI protein was detected as a 12 kDa band in the isthmus, ampulla and infundibulum of the Fallopian tube. Immunohistochemistry using an anti-SLPI polyclonal antibody revealed positive staining of epithelial cells in the Fallopian tube. RT-PCR demonstrated that SLPI transcripts were expressed in the Fallopian tube. To determine the function of SLPI in the Fallopian tube, the effects of SLPI and elastase on the sperm acrosome reaction were examined. SLPI prevented the reduction of the acrosome reaction by elastase in a dose-dependent manner. CONCLUSION: The present findings suggest that SLPI in the Fallopian tube contributes to sperm-oocyte interaction.

Cervical fluid oncofetal fibronectin as a predictor of early ectopic pregnancy. Is it affected by blood contamination?

OBJECTIVE: To evaluate (1) the diagnostic usefulness of oncofetal fibronectin (fFN) in the cervical fluid in unruptured ectopic pregnancy, and (2) the contribution of maternal blood contamination of the specimen to the test results. STUDY DESIGN: A total of 111 cases in the first trimester of pregnancy, including 12 cases of ectopic pregnancy, 26 of spontaneous abortion and 73 of viable intrauterine pregnancy, were studied. fFN was determined with a commercial enzyme-linked immunosorbent assay kit with a threshold of 50 ng/mL in cervical fluid. To determine the effect of blood contamination on the fFN assay, the positive rate of the fFN test was examined when maternal blood in the first trimester was mixed with the dilution buffer. RESULTS: Eighty-three percent (10/12), 38% (10/26) and 12% (9/73) of cases with ectopic pregnancy, miscarriage and normal pregnancy, respectively, were positive for fFN. The positive rate in ectopic pregnancy was significantly higher than in normal pregnancy and spontaneous abortion. The test was positive for fFN in 8% with 0.5% of blood contamination, 67% with 1% contamination and 100% with 10% contamination.

Expression of platelet-derived endothelial cell growth factor/thymidine phosphorylase in cervical intraepithelial neoplasia.

Angiogenesis contributes to the growth and secondary spreading of solid tumors. Platelet-derived endothelial cell growth factor (PD-ECGF)/thymidine phosphorylase (TP) has been identified as such an angiogenic factor. In this study, the expression of PD-ECGF/TP and VEGF was evaluated by immunohistochemical staining of tumor specimens from 40 patients with cervical intraepithelial neoplasia (10 with moderate dysplasia; 10 with severe dysplasia; 10 with carcinoma in situ; 10 with invasive carcinoma). The microvessel density was assessed by immunostaining for factor VIII-related antigen in the most highly neovascularized area. In both the nucleus and cytoplasm, the intensity of PD-ECGF/TP expression in carcinoma in situ and invasive carcinoma was significantly stronger than that in moderate dysplasia. However, the intensity of VEGF expression was not significantly different in the various specimens. The microvessel density in mild dysplasia was significantly different from that in carcinoma in situ (p<0.05), and that in invasive carcinoma (p<0.05). There was no significant relationship between the microvessel density and the expression of PD-ECGF/TP or that of VEGF. These results show that the expression of PD-ECGF/TP appears to be involved in the promotion of angiogenesis in cervical intraepithelial neoplasia.

Detection of fractalkine in human seminal plasma and its role in infertile patients.

BACKGROUND: Fractalkine is a relatively newly discovered CX(3)C chemokine, which is a chemoattractant for T cells, monocytes and natural killer cells. Several reports have demonstrated the association between chemokine levels in seminal plasma and semen quality. The fractalkine levels in ejaculates from normal donors and infertile male patients with or without asthenozoospermia, were examined and correlated with sperm motility and morphology. METHODS AND RESULTS: Western blot analysis showed fractalkine protein to be present in the seminal plasma. Fractalkine titres in the seminal plasma of infertile men with asthenozoospermia (0.64 +/- 0.04 microg/ml; n = 58) were lower than those in patients without asthenozoospermia (0.94 +/- 0.10 microg/ml; n = 22, P < 0.01) and fertile donors (1.04 +/- 0.07 microg/ml; n = 10, P < 0.001). There was no significant difference between fractalkine levels in patients with and without leukospermia. No significant correlation was found between fractalkine and interleukin-8 levels in seminal plasma. Sperm motility was positively correlated (R(2) = 0.14, P < 0.001) with fractalkine concentration. The existence of CX(3)CR-positive leukocytes in semen was confirmed using specific primers for CX(3)CR. CONCLUSIONS: These results suggest that fractalkine is a chemokine associated with sperm motility and the migration of CX(3)CR-positive leukocytes into semen.

Effectiveness of hormonal tourniquet by vasopressin during myomectomy through vasopressin V1a receptor ubiquitously expressed in myometrium.

To elucidate the mechanism by which myometrial vasopressin injection promotes hemostasis during myomectomy, we examined the expression of vasopressin V1a receptor transcripts in the myometrium. Vasopressin V1a receptor expression was ubiquitous, and the transcripts were detected in the myometrium not only of cycling and pregnant patients, but also of postmenopausal or GnRH agonist-treated patients. Based on these observations, we applied intraoperative myometrial vasopressin injection during myomectomy in a non-randomized study in a total of 84 patients. Vasopressin injection significantly reduced the intraoperative blood loss and postoperative hemoglobin fall in patients without and with GnRH agonist pretreatment. No serious complications occurred on account of the vasopressin injection. We conclude that intraoperative vasopressin injection is effective as a hormonal tourniquet even in GnRH agonist-pretreated myomectomy.