, 2005). For the ‘SFG’ set, a mean cycle threshold (Ct) value below 35 indicates the sample is

positive, and a Ct value above 35 indicates the sample is positive if another set is positive and/or a sequence is obtained and/or serology is positive. Thus, samples are run in duplicate using sets targeting two different genes. From January 2009 to December 2009, the set ‘RAF-plasmid’ was used to detect R. africae; its target gene is located on a plasmid of the species. Following recent R. africae genome sequencing, it was reported that this plasmid might be unstable. check details To avoid false-negative results, we designed a new primer and probe set targeting a non-plasmidic gene. Consequently, the set ‘RAF’ was used to detect R. africae in clinical samples from January 2010 to December 2010. We retrospectively collected data for the molecular diagnosis

of rickettsioses from January 2009 to December 2010 to assess the usefulness of this strategy. Except for the ‘SFG’ set, which had been previously described (Socolovsch et al., 2010), the sets were found to be specific for the corresponding rickettsial species both in silico and in vitro, when tested against a panel of 30 rickettsial strains (Fig. 1a). Sensitivity was also evaluated using 10-fold serial dilutions (Fig. 1b). A total of 643 clinical specimens corresponding to 465 different patients were received at the FNRC from January 2009 to December 2010. Among these, find more 204 originated from locally hospitalized patients, 218 from other French hospitals and 43 from international hospitals. Forty-five positive qPCRs

were obtained: 31/150 cutaneous biopsies, 8/42 cutaneous swab specimens, 2/223 total blood samples and 4/94 serum samples. The first molecular screening of SFG Rickettsia using the set labelled ‘SFG’ was positive for 44 samples; the 45th sample was positive using the set labelled ‘TG’, which detects TG Rickettsia. Among 45 positive results, 11 were obtained from locally hospitalized Orotic acid patients, 32 from other French hospitals and two from international hospitals. A final diagnosis of R. africae was obtained for 15 samples (13 cutaneous biopsies, two eschar swabs) corresponding to 15 different patients with a diagnosis of ATBF; five samples were positive for the sets ‘SFG’ and ‘RAF-plasmid’, and 10 samples were positive for the sets ‘SFG’ and ‘RAF’. A final diagnosis of R. conorii was obtained for nine samples corresponding to nine different patients with a diagnosis of MSF; eight samples (cutaneous biopsies) were positive for the sets ‘SFG’ and ‘RCO’. One remaining sample (serum) was positive for the set ‘SFG’ and negative for ‘RCO’; a final diagnosis of R. conorii was obtained using conventional PCR followed by sequencing. A final diagnosis of R. honei was obtained for one sample (serum) corresponding to a patient whose final diagnosis was FISF (Murphy et al., 2011); it was positive for the set ‘SFG’, and a final diagnosis of R.

We previously demonstrated that IC negatively regulates TLR4-triggered inflammatory

response in macrophages through FcγRIIb 27. We here demonstrate that in the presence of IC, FcγRIIb overexpression promotes resistance of immature DCs to TLR-triggered maturation induction and also increases DC tolerogenecity. Accordingly, IC-stimulated, FcγRIIb-overexpressing DCs (DC-FcγRIIb) can downregulate immune response more significantly both in vitro and in vivo, thus attenuating the progression of disease in lupus-prone mice. To investigate whether IC/Ig could inhibit TLR-induced maturation of DCs via FcγRIIb, Dinaciclib mw immature DCs derived from WT or FcγRIIb−/− mice were incubated with IC (OVA plus anti-OVA)/Ig (anti-OVA) for 24 h before these DCs were stimulated with LPS or CpG ODN for another 24 h. As for WT DCs, IC alone (whereas not Ig) slightly upregulated the expression of I-Ab, CD40, CD80

and CD86. Interestingly, IC pretreatment significantly inhibited the LPS or CpG ODN-induced upregulation Ferroptosis inhibitor of I-Ab, CD40, CD80 and CD86 expression, and Ig pretreatment significantly inhibited the LPS-induced upregulation of the four molecules and the CpG ODN-induced upregulation of CD80 and CD86 expressions (Fig. 1A). In contrast, neither IC nor Ig pretreatment had significantly inhibitory effect on the LPS or CpG ODN-induced upregulation of I-Ab, CD40, CD80 and CD86 expression on FcγRIIb−/− DCs (Fig. 1A). These data indicate that FcγRIIb mediates the inhibitory effect of IC/Ig above. IC alone could stimulate WT DCs as well as FcγRIIb−/− DCs to secrete TNF-α and IL-1β to some degree; however, IC pretreatment obviously suppressed LPS or CpG ODN-induced TNF-α and IL-1β secretion from WT DCs (Fig. 1B). In contrast, IC pretreatment could not suppress TNF-α and IL-1β secretion by LPS-stimulated FcγRIIb−/− DCs, and even promoted TNF-α and IL-1β secretion by CpG ODN-stimulated

FcγRIIb−/− DCs (Fig. 1B). OVA alone had no effect on the phenotype and cytokine Endonuclease secretion of WT DCs and FcγRIIb−/− DCs with or without stimulation with LPS, CpG ODN (data not shown). Considering that the Ig (anti-OVA mAb) we used, also exhibited inhibitory effect on LPS-induced DC maturation (in a similar manner to IC), we speculated that anti-OVA mAb might have some aggregated Ig, because aggregated Ig has a higher binding affinity to FcγRIIb than monomeric Ig. Therefore, we compared the differences between monomeric and aggregated Ig. As expected, the aggregated Ig significantly inhibited WT DCs to express I-Ab and CD40 and to secrete TNF-α in response to LPS stimulation, whereas monomeric Ig did not. However, neither aggregated Ig nor monomeric Ig had such inhibitory effect on LPS-induced FcγRIIb−/− DC maturation (Supporting Information Fig.

7 years follow up, was examined. CKD was measured by using estimated glomerular filtration rate or dipstick proteinuria (1+). The association between MetS or combination patterns of MetS abnormalities and CKD was evaluated using Cox models with adjustment for confounders. Results:  The incidence of CKD was 288/10 000 person-years (95% confidence interval (CI), 283–293). The findings showed that central obesity (OB), high blood pressure (BP) and high triglyceride were considered

to be the major metabolic events in the study cohort. Incidences and hazard ratios (HR) on CKD had evidently increasing trends with the number of MetS components. The multivariable-adjusted HR for CKD associated with ATP-III-MetS was 1.30 Daporinad concentration (95% CI, 1.24–1.36). Equivalent HR for IDF-MetS were 1.37 (95% CI, 1.30–1.44). The associations were still observed when analyzing by stratifying incident diabetes and adjusting hypertension status. Conclusion:  MetS induces KU-60019 mouse an increased risk for CKD independent of baseline confounding factors and subsequent incident diabetes modified the associations lightly. The mechanism through which MetS may cause CKD in this population likely is the development

of multiple metabolic pathogenic processes together. “
“Immunoglobulin (Ig)A nephropathy is one of the major causes of chronic kidney disease (CKD) in Japan. Despite statutory urinalysis of industrial workers and school children, Japan unfortunately still ranks among the countries with the highest CKD-5D prevalence in the world. Topics of this review are as follow: (i) early diagnosis and treatment; (ii) influence of the period from onset to medical

intervention on renal prognosis; and (iii) epidemiology of IgA nephropathy patients in Japan. Some investigators have discussed the possibility of predicting the diagnosis and prognosis of this disease. We indicated that the frequency of various casts in urinary sediments and total numbers of each type of urinary cast should provide highly convincing data for prediction of the prognosis in IgA nephropathy Atezolizumab patients prior to renal biopsy. Furthermore, early medical intervention (anti-platelet agents, anticoagulants, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, corticosteroids and/or tonsillectomy) may lead to better renal prognosis in patients with IgA nephropathy. In a nationwide survey on IgA nephropathy in Japan, predictive factors after 10 years were as follows: (i) male sex; (ii) under 30 years old; (iii) diastolic hypertension; (iv) heavy proteinuria; (v) mild haematuria; (vi) low serum albumin; and (vii) elevated serum creatinine and impaired renal pathology.

Differences of PBDC and tissue-infiltrated DC counts by duration time of the clinical

course in patients with Sicca syndrome were calculated Selleck Kinase Inhibitor Library by Pearson’s correlation coefficient. These tests were used for statistical analysis using a Statview statistical program (Abacus Concepts, Berkeley, CA, USA). Differences were considered significant when P-values were less than 0·05. The clinical characteristics of the patients are shown in Tables 1 and 2. All but seven patients with secondary SS (three overlapping with SLE and four overlapping with RA) and none of the normal volunteers received medication of corticosteroids and immunosuppressants during the study (Table 2). The clinical characteristics of secondary SS and primary SS patients are shown in Table 1. Five of the 24 secondary SS patients had an overlapping

SLE. The SLE disease activity index (SLEDAI) [19] in these patients was 6, 12, 13, 22 and 26, respectively, at the time of the examination. In two patients, the symptoms of SLE and those of SS developed almost simultaneously. In the remaining three patients, SLE symptoms preceded those of SS. These three patients were receiving 5 mg/day Atezolizumab of prednisolone at the time of the examination. Barnett classification is an evaluation system for the severity of SSc determined by the extent of skin sclerotization caused by this disease. When skin sclerotization is localized only at the fingers and hands, the case is classified as class I (B-I). Conversely, when skin sclerotization is extended to the face or further to the trunk the classification of B-II or B-III is made, respectively. According to the Barnett classification, the eight secondary SS patients

who had an overlapping SSc were classified into four B-I, three B-II and one B-III. The onset profile of the symptoms was variable among patients with SSc-merged secondary SS. The symptoms of SSc and those of SS almost appeared simultaneously in three patients. In two patients the symptoms of SSc preceded those of SS, while in the remaining three patients Sicca syndrome appeared first and skin sclerotization developed several years later. Eleven 3-mercaptopyruvate sulfurtransferase secondary SS patients had an overlapping RA. Two of the 11 patients were diagnosed as having RA-merged secondary SS at the initial presentation. On the other hand, five of the 11 patients were diagnosed originally as primary SS and subsequently as RA-merged secondary SS when the RA symptoms developed later. By contrast, in the remaining four patients, Sicca syndrome appeared after the diagnosis of RA was established. Disease modified anti-rheumatic drugs (methotrexate 6 mg/week, 8 mg/week, bucillamine 50 mg/day and salazosulphapyridine 1000 mg/day, respectively) had been administered to four patients whose RA preceded SS. SLE patients showed low white blood cell (WBC) numbers (normal control: mean 4822/µl, range 3800–10 200; SLE: mean 3864, range 1900–8400) (Table 1).

Thus, we aimed to more closely replicate the in vivo situation of antigen presentation during allergic lung hypersensitivity. The purified lung DC obtained from B6 mice were given serum containing either anti-OVA IgG (obtained from OVA+Alum sensitized mice) or anti-BSA IgG (obtained from BSA+Alum sensitized mice) together with increasing OVA concentrations. The resulting antigen-specific T-cell stimulation was determined using CFSE-labeled OT-II cells after 60 h of culture. As depicted in Fig. 5C, serum of OVA+Alum

sensitized mice yielded a significant three- to fourfold increased antigen-specific T-cell proliferation induced by lung DC, as compared to serum of BSA- or non-sensitized mice. To further prove selleck chemical the specificity of this observation, lung DC from FcγR-deficient mice were used as a control, revealing no increase in T-cell GSI-IX in vitro proliferation even at the highest OVA concentration tested and exposure to serum of OVA+Alum sensitized mice (Fig. 5D). These data strongly suggest that anti-OVA IgG-IC formation through increased DC-mediated antigen-specific T-cell proliferation is able to contribute to allergic airway hyperresponsiveness. Our study provides experimental evidence that allergen-specific IgG, generated during sensitization, can lead to IC formation

upon antigen challenge and result in enhanced FcγR-mediated antigen presentation. This augmented antigen presentation and Th2 T-cell proliferation, possibly in concert with enhanced DC activation 17, 18, promotes the manifestation of pulmonary allergic hypersensitivity reaction during the effector phase. These findings expand significantly upon previous reports on the role of FcγR and allergen-specific IgG in allergic Interleukin-3 receptor asthma 13, 14 in that we now show a novel mechanism and impact of FcγR during the airway challenge phase. Previous reports suggested a specific role for FcγRIII signaling in the regulation of optimal Th2 cell differentiation in allergy during

sensitization, regulated by IL-10 production from the DC. Moreover, Kitamura et al. 13 demonstrated that expression of FcγR, most likely FcγRI, on DC is important during the sensitization phase for the development of allergic airway inflammation. Other studies indirectly suggested that activating FcγR could contribute to inflammation through the activation of Syk, a downstream kinase by which FcγR are known to augment antigen presentation 17, 19, 20. The reduced eosinophilia in FcR γ-chain deficient mice, which do not express FcγRI, FcγRIII, FcγRIV and FcεRI, corroborates a previous report 13 and could be a result of effects other than antigen presentation. Signaling via FcγRIII on mast cells has been demonstrated to induce the release of soluble mediators that have a role in the regulation of Th2 differentiation.

Activation of Tregs during infection with PyL requires TLR9 signaling in DCs 10. It is quite possible that Tregs are not activated in IDA mice due to insufficient TLR9 signaling because IDA erythrocytes contain much less hemoglobin/heme (data not shown), the source of a known malaria-derived TLR9 ligand, hemozoin 11. Thus, we analyzed the immune responses in IDA mice. First, we assessed the number of cells

PI3K inhibitor involved in protection against malaria in the spleen 6 days after infection with PyL (Fig. 3A). Infection with PyL clearly increased the population of spleen cells. Unexpectedly, the number of whole splenocytes and splenic CD4+CD25– T cells in IDA mice was less than that in control mice. There was no increase in the number of macrophages. IFN-γ production by whole spleen cells in response to ConA

was evaluated using ELISA. Infection of control mice with PyL markedly reduced the production of IFN-γ; however, infection selleck chemical of IDA mice reduced it to an even greater degree (Fig. 3B). The production of IgG antibodies specific for the malaria parasite was also assessed. Humoral immunity to the malaria parasite was induced after infection with PyL in iron-sufficient mice. However, IDA mice had much lower total IgG levels (Fig. 3C). Thus, neither humoral nor cellular responses were enhanced in IDA mice. We further evaluated the functional properties of splenic Tregs by investigating the suppression of TCR-driven T-cell proliferation. CD4+CD25+ T cells isolated from IDA mice were Progesterone cultured with CD4+CD25− T cells from uninfected mice in the presence of ConA. Tregs from uninfected mice suppressed proliferation in a dose-dependent manner. Infection of iron-sufficient mice with PyL markedly enhanced the suppressive function of Tregs, reflecting Treg activation

(Fig. 3D). Tregs in IDA mice had much stronger suppressive abilities (Fig. 3D), presumably resulting in reduced immune responses in these mice. Again, we saw no evidence for the enhancement of acquired immunity in IDA mice. Finally, to analyze whether acquired immunity is involved in the resistance of IDA mice to malaria, we infected T-cell and iron-deficient athymic nude mice with PyL. As shown previously, IDA euthymic mice showed lower levels of parasitemia and prolonged survival compared with euthymic mice fed with an iron-sufficient diet (Fig. 3E). IDA athymic mice clearly showed lower levels of parasitemia than mice fed with an iron-sufficient diet although they still succumbed to infection with PyL. These results suggest that acquired immunity, in which T cells play a central role, is required to survive infection by PyL, but it is not involved in IDA-associated resistance to malaria during the early phase of infection.

2b). This indicates

that the weak cytotoxic activities of these mutants are due to attenuation of the affinity of mutant alpha-toxin to the GPI-anchored protein. Because WDW_W is the most important sequence in the tryptophan-rich region, hydrophobicity and electrical charge in the side chain of these four amino acids would affect cytotoxic activity. Researchers have shown that the cytotoxic mechanisms and primary structure of C. septicum alpha-toxin are similar to those of Aeromonas hydrophila aerolysin [6, 8]. Although the receptor of aerolysin on cell membranes is also a GPI-anchored protein [24], N-glycan on GPI-anchored proteins is required for efficient binding of aerolysin; however, binding of alpha-toxin is independent of N-glycan [27]. Aerolysin has a tryptophan-rich region (GEVKWWDWNWT) JAK inhibitor that is similar to that of alpha-toxin near the C-terminus, and possesses the same sequence in this

region, WDW_W, which should be an important sequence for binding of alpha-toxin to cell receptors. With the exception of WDW_W, the amino acid sequence in Inhibitor Library ic50 the tryptophan-rich region of alpha-toxin does not exhibit identity with that of aerolysin. Therefore, this difference may determine whether N-glycan is indispensable for binding of alpha-toxin and aerolysin to GPI-anchored proteins. This work was supported in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology in Japan. The authors have no conflicts of interest associated with this study. “
“B-cell-activating Alanine-glyoxylate transaminase factor (BAFF) influences peripheral B-cell survival, maturation and immunoglobulin class-switch recombination and has a range of potential clinical implications. Biological functions of BAFF and its relevance in various clinical disorders including currently investigated BAFF-targeting therapies are reviewed and discussed based on PubMed search of relevant articles. Serum levels of BAFF are increased in autoimmune diseases including autoimmune hepatitis and primary biliary cirrhosis where BAFF concentrations are

related to titres of autoantibodies and disease progression. Increased BAFF levels are found in synovial, bronchoalveolar and gut lavage fluids, suggesting local class switching and immunoglobulin production. Clinical relevance and diagnostic potential of BAFF are also noted in patients with allergic diseases, malignancies and infections including hepatitis C virus. BAFF antagonists are promising new therapeutic agents, currently being tried in B-cell-related autoimmune diseases. Serum level of BAFF may indicate disease mechanisms and the degree of activity. Determination of BAFF in different body compartments like synovium, airways and gut may also have clinical implications. Results of ongoing clinical trials with BAFF antagonists are eagerly awaited. B-cell lymphocytes play a major role in the humoral immune response.

3 ± 7.9 v.s. 43.9 ± 9.5/ × 40 field; Nestin(+) area: 13.8 ± 7.1 v.s. 20.0 ± 7.7 × 103 pixel/ × 40 field, p < 0.01). In vitro, CS-KS significantly suppressed cisplatin-induced cell apoptosis in both adult kidney cells (NRK-52E cells) and KS cells. Moreover, CS-KS treatment for NRK-52E cells significantly increased Histone H3(+) cells and Nestin(+) cells. These results suggested that CS-KS could promote not only cell proliferation but also dedifferentiation toward immature lineage. Conclusion: Adult kidney stem/progenitor cells make important roles such as renal stem/progenitor cells' direct differentiation into mature renal composed cells, secreting protective

factors and the effect toward dedifferentiation from renal composed mature cells to immature cells. YOKOTE SHINYA1,2,3, YAMANAKA SHUICHIRO1,2,3, KATSUOKA YUICHI2,3, H 89 molecular weight IZUHARA LUNA2,3, OGURA MAKOTO1, YOKOO TAKASHI1,2 1Division of Nephrology and Hypertension, Department of Internal Medicine, Jikei University School of Medicine; 2Project Laboratory for Kidney Regeneration, Jikei University School of Medicine; 3Division of Regenerative Medicine, Jikei University

School of Medicine Introduction: Previous studies have investigated using mesenchymal stem cells (MSCs) to treat damaged kidneys. However, the effect of adipose-derived MSCs (ADMSCs) on vascular calcification in adenine-induced AZD2014 concentration kidney disease is still poorly understood. In the present study, we explored the potential of ADMSCs as an alternative source for the treatment of kidney disease and vascular calcification. Methods: Chronic renal failure was induced in 12-week-old male Sprague-Dawley rats (n = 16) by feeding a diet containing 0.75% adenine for 4 weeks. Time course changes in serum inorganic phosphorus (iP), calcium, and creatinine were measured. Rats were randomized into two groups: control (phosphate buffered saline, n = 9); and ADMSC (intravenous transplantation of 5 × 105 autologous

ADMSCs on Days 0, 7, 14, CYTH4 21 and 28 following adenine-feeding, n = 7). At the end of the study, vascular calcification was evaluated by Von Kossa-stained sections and calcium and P content in the aorta. Results: Histopathology of the kidney (hematoxylin & eosin) showed a greater dilation of tubular lumens in the control group than the ADMSC group. Creatinine clearance rate in the ADMSC group is higher than the control group (P < 0.05). ADMSC transplantation significantly reduced serum iP compared with control (P < 0.05). Calcium and P content of the aorta in the ADMSC group was lower than the control group (P < 0.05). Von Kossa staining of the thoracic aorta media also revealed that ADMSC transplantation suppressed vascular calcification compared with the control group.

Each 20 µl PCR reaction contained 2 µl of DNA and 18 µl of mastermix containing FastStart DNA Master SybrGreen I, 4 mM MgCl2 (both from Roche), 0·5 µM of each primer and sterile dH2O. The PCR was performed as follows: one cycle of denaturation at 95°C for 10 min, 45 cycles of amplification consisting of denaturation at 95°C for 10 s, primer annealing at 72–62°C for 5 s (0·5°C drop in Selumetinib price each cycle for 20 cycles) and extension at 72°C for 6 s, followed by melting at 95°C for 0 s, 62°C for 10 s and 95° for 0 s (0·1°C/s temperature increase) and ending by cooling at 40°C for 30 s. Frozen mucosal tissue, preincubated

in RNAlater, as well as frozen thymic tissue obtained from human infants undergoing cardiac surgery, was homogenized with a mortar and pestle in liquid nitrogen and then added to RLT buffer (RNeasy mini kit, Qiagen). Frozen cells, preincubated in RNAlater, were placed in RLT buffer directly and both tissues and cells were homogenized by passing the lysate through a blunt 20-gauge needle. RNA was purified by the RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. The RNA concentrations in all samples were determined by ultraviolet spectrophotometry at 260 and 280 nm and the purity and

integrity of extracted RNA was confirmed by electrophoresis in a 1% agarose gel. Fifty ng/µl RNA was reverse-transcribed to cDNA using QuantiTect reverse transcription kit (Qiagen) according to the manufacturer’s instructions Alpelisib ic50 and then stored at −20°C. The amount of RAG1 and pre-TCR-α mRNA relative to the amount of the reference gene CD3γ mRNA was determined by real-time PCR (LightCycler480; Roche Diagnostics GmbH), using specific primers and human universal probes as specified below for detection of specific products. The PCR primers were purchased from Invitrogen, Paisley, UK. The sequences were as follows: RAG1 forward: 5′-ATT GCA GAC ATC TCA ACA CTT TG-3′ and reverse: 5′-GAA AGA GGC TGC CAT GCT-3′; pre-TCR-α forward: 5′-TCC TGC CTC CTT CCG AGT-3′

and reverse: 5′-CCA GAG AAG GAA AGG GTG TG-3′; CD3γ forward: 5′-TTG GGG TCT ACT TCA TTG CTG-3′ and reverse: 5′-AAC AGA GTC Cediranib (AZD2171) TGC TTG TCT GAA GC-3′. These primers generated specific products of 74, 111 and 70 bp, respectively. Each 15 µl PCR reaction contained 80 ng of cDNA in a volume of 5 µl, 5 µl LightCycler480 Probes Master and 0·2 µl human universal probe number; 27 (RAG1-primer), 2 (pre-TCR-α-primer) or 58 (CD3γ-primer), 0·2 µl (20 µM) of each primer and 4·4 µl RNase free dH20. The PCR was performed as follows: denaturation at 95°C for 10 min, 45 cycles of amplification at 95°C for 10 s, annealing at 60°C for 30 s and extension at 72°C for 1 s, before the samples were cooled at 40°C for 30 s.

epidermidis stain harboring PQG56 (spx antisense knock-down plasmid) is increased substantially, in accordance with the phenotype in the homologous spx mutant strain of S. aureus (Pamp et al., 2006). This observation further supports that spx is an important regulator mediating the biofilm formation of S. epidermidis. Biofilm formation by S. epidermidis

is generally considered as a two-step process, including primary attachment and biofilm accumulation. To investigate ACP-196 research buy which step is affected by Spx, we first compared the attachment ability of the Spx-overexpressing strain (harboring pQG55) and the vector control strain (harboring pQG53). In primary attachment assays, the Spx-overexpressing strain showed decreased attachment ability (about 34-fold) to polystyrene compared with the WT strain, whereas the strains carrying either pQG53 or pQG54 showed no difference in primary attachment (Fig. 3a and b). To investigate whether the transcription of atlE was affected INCB018424 chemical structure by Spx, quantitative RT-PCR was performed. The result indicates that the transcriptional level of atlE in the Spx-overexpressing strain carrying pQG55 shows no difference compared with the other three strains (Fig. 3c). This indicates that Spx does not affect the attachment ability by regulating

atlE. We then compared the primary attachment on 96-well polyethylene plates between WT and ica-negative strains isolated from our previous work (Li et al., 2005), and no significant difference was found (data not shown). PIA is a key factor in the biofilm accumulation of S. epidermidis (Rupp et al., 1999). To investigate whether the production of PIA was affected by Spx, immuno-dot blot Dehydratase assays were performed. The Spx-overexpressing strain was found to produce significantly less PIA compared with the vector control strain (Fig. 4a). The transcription of the icaADBC operon and its repressor icaR among different strains was further examined by quantitative RT-PCR. Decreased

icaADBC, but comparable icaR transcriptional levels were found in the Spx-overexpressing strain compared with the vector control strain (Fig. 4b and c). This result indicates that Spx affects PIA production by regulating the transcription of icaADBC in an icaR-independent manner. In B. subtilis and S. aureus, Spx plays an important role in the oxidative-stress adaptation. The B. subtilis and S. aureus spx mutant strains were hypersensitive to diamide, a thiol-specific oxidant (Nakano et al., 2003a; Pamp et al., 2006). To study whether the overexpression of Spx affects S. epidermidis in the adaptation to diamide, the diamide sensitivity of the Spx-overexpressing strain (harboring pQG55) and the control strain (harboring pQG53) was compared using disk diffusion tests.