pallidum. The response of NK cells producing IFN-γ against microbial stimulation is highly significantly associated with KIR genotype [32]. Artavanis-Tsakonas et al. [23] reported that there was a significant association between KIR genotype and NK cell response to Plasmodium falciparum-infected erythrocytes and found that induction of IFN-γ synthesis https://www.selleckchem.com/products/bay-57-1293.html was dependent on the direct contact between NK cells and the infected erythrocytes. Therefore, we speculate that IFN-γ productions

in response to syphilis maybe have association with KIR genotype, but this needs to be confirmed further. Here, we put forward two hypotheses by which KIR genotype might affect NK cell producing IFN-γ in responses to syphilis. First, signals produced from the interaction of KIRs with their ligands during T. pallidum infection might modulate the degree of activation of NK cells. Alternatively, binding of KIR by relevant HLA ligands during NK cell ontogeny might lead to greater or lesser education of NK cells. Additional studies examining KIR–HLA class I interactions in patients and

controls may be fruitful. “
“Activation of NK cells is a hallmark of infections with intracellular pathogens. We previously showed that the BMS-777607 protozoan parasite Leishmania infantum triggered a rapid NK-cell response in mice that required TLR9-positive myeloid DC and IL-12, but no IFN-α/β. Here, we investigated whether IL-15 or IL-18 mediate the activity of IL-12 or function as independent activators of NK cells. In contrast to earlier studies that described IL-15 as crucial for NK-cell priming in response to TLR ligands, the expression of IFN-γ, FasL, perforin and granzyme B by NK cells in L. infantum-infected mice was completely preserved in the absence of IL-15, whereas the proliferative capacity of NK cells was lower than in WT mice. IFN-γ secretion, cytotoxicity ZD1839 research buy and FasL expression of NK cells from infected IL-18−/− mice were significantly reduced compared with controls, but, unlike IL-12, IL-18 was not essential for NK-cell effector functions.

Part of the NK-cell-stimulatory effect of IL-12 was dependent on IL-18. We conclude that IL-15 is not functioning as a universal NK-cell priming signal and that IL-18 contributes to the NK-cell response in visceral leishmaniasis. The cytokine requirements for NK-cell activation appear to differ contingent upon the infectious pathogen. “
“Tumour-loaded dendritic cells (DCs) from patients with chronic lymphocytic leukaemia (CLL) matured using an α-type 1-polarized DC cocktail (IL-1β/TNF-α/IFN-α/IFN-γ/poly-I:C;αDC1) were recently shown to induce more functional CD8+ T cells against autologous tumour cells in vitro than DCs matured with the ‘standard’ cocktail (IL-1β/TNF-α/IL-6/PGE2;PGE2DCs).

Indeed, when purified ASC−/− CD4+ and Selleck LEE011 CD8+ T cells were stimulated for 2 days with anti-CD3/CD28 in a co-culture assay, T-cell proliferation was inhibited compared with similarly activated ASC+/+ CD4+ and CD8+ T-cell co-cultures (Fig. 2a). Working on the hypothesis that in the co-culture set-up one ASC−/− T-cell subset is able to suppress the proliferation of the other when activated, we next attempted to identify this suppressive ASC−/− T-cell subset. ASC+/+ and ASC−/− CD4+ and CD8+ T cells were purified and co-cultured with different purified T-cell fractions under activation conditions (anti-CD3/CD28 stimulation) (Fig. 2b). In this set up, significant

inhibition of proliferation was observed in co-cultures that included Selleckchem PFT�� ASC−/− CD4+ T cells. A slight, but significant reduction was also noted in some co-cultures that included ASC−/− CD8+ T cells. When the expression of CD25 (Fig. 2c), CD44 and CD62L (data not shown) were assessed in co-cultures where T-cell proliferation was impaired, no activation-induced differences were observed. Collectively, these results suggest that activated ASC−/− CD4+ T cells are able to suppress activation-induced proliferation of other neighbouring activated T cells. Furthermore, as no changes in cell surface

expression of T-cell activation markers were noted following anti-CD3/CD28 stimulation we speculate that T-cell activation in the presence ASC−/− CD4+ T cells occurs normally and that inhibition of proliferative responses occurs at the phase of T-cell clonal expansion. One possible mechanism for the

observed suppression of T-cell proliferation after CD3/CD28 stimulation in the presence of activated ASC−/− CD4+ T cells could be the secretion of suppressive soluble factor(s). To test this hypothesis we used WT CD4+ (Fig. 3a) and CD8+ T cells (Fig. 3b) as effector T cells. These cells were then activated (anti-CD3/CD28 stimulation) in the presence of supernatant derived from activated WT or ASC−/− CD4+ T cells. T cells stimulated in the presence of activated ASC−/− CD4+ T-cell-derived supernatant proliferated significantly less than those stimulated in the presence of supernatants derived from ASC+/+ CD4+ Masitinib (AB1010) T cells. These results suggest that ASC−/− CD4+ T cells once activated secrete soluble factor(s) that have suppressive potential. To characterize the suppressive factor(s) involved in ASC−/− CD4+ T-cell mediated suppression, we compared the cytokine secretion profile of activated ASC+/+ and ASC−/− CD4+ T cells. Interestingly, we found that anti-CD3/CD28-activated ASC−/− CD4+ T cells produced significantly less interferon-γ over a 4-day time–course experiment when compared with their ASC+/+ counterparts (Fig. 3c). Interleukin-2 concentrations were also decreased in activated ASC−/− CD4+ T-cell cultures at day 2, which represented peak secretion of IL-2 for WT controls.

Unveiling novel mechanisms will undoubtedly provide new insights into T-cell-mediated diseases. This work was supported by NHMRC Project Grant awarded to S.R. and also by a UC PDF Fellowship awarded to P.S.L. in S.R.’s laboratory. This work was supported by the UTas Rising Star award to A.F.H. The authors declare no conflict of interest. selleck chemicals “
“Tregs are crucial in controlling inflammation. Although the transcription factor FOXP3 is the most

applicable phenotype marker of Tregs, it does not indisputably characterize suppressive function during T-cell activation in vitro. A question that remains is: what is the functionality of FOXP3+ T cells during inflammation in vivo? We studied FOXP3+ T cells in a human model of acute inflammation due to cardiac surgery. Twenty-five children who underwent cardiac surgery for correction of a septum defect were included. Following surgery, we observed a transient systemic EPZ-6438 chemical structure inflammatory response accompanied by an increased proportion of CD25bright T cells with sustained Treg phenotype. During this transient

immune activation, both the percentage of CD4+FOXP3+ cells and the level of expression of FOXP3 in the CD4+CD25brightCD127low population increased. While Tregs remained present during systemic inflammation and continued to be anergic, the capacity to suppress effector T cells was reduced. The reduced suppressive state of Tregs could be induced in vitro by plasma obtained during the peak of inflammation after surgery. These data show that inflammation inhibits Treg function through soluble factors present in plasma. These results underscore the functional role

of FOXP3+ Tregs during inflammation in vivo. Tregs have an important role in the maintenance of immune tolerance in both mice and humans. Besides a central role in autoimmunity and transplantation medicine, these cells have left their mark as regulators of inflammation such as in tumor immunology, allergy and infectious diseases. While the functionality Y-27632 2HCl of Tregs is indisputable in animal models, defining their in vivo role in humans is problematic. For example, most markers associated with Tregs have been shown to be upregulated after in vitro T-cell activation without necessarily qualifying the cells as suppressive Tregs. Therefore, measurement of Tregs in human disease is generally biased when conducted during inflammation. In the following study, we describe the functionality of CD4+CD25+FOXP3+ T cells during the systemic inflammatory response in children undergoing cardiac surgery. Cardiac surgery with the use of cardiopulmonary bypass (CPB) induces a systemic inflammatory response 1–4. Factors involved in triggering an immune response include anesthesia, surgical trauma and contact of immune competent cells with surface of extra-corporeal circuit. In uncomplicated cases, this is a temporary event.

Databases searched: MeSH terms and text words for type 1 and type 2 diabetes mellitus were combined with MeSH terms and text words for renal replacement therapy and dialysis. The search was carried out in Medline (1950–March, Week 3, 2008). The Cochrane Renal Group Trials Register was also searched for trials

not indexed in Medline. Date of search/es: 2 April 2008. A prospective study was conducted by Villaret al.5 in order to examine the epidemiology and long-term survival of patients with incident end-stage kidney disease (ESKD) by diabetes status in Australia and New Zealand. The ANZDATA Registry was used to identify patients ≥16 years of age who began dialysis from 1 April 1991 to 31 December 2005. Data collection consisted of information on patient demographics, comorbidites and multiple other parameters FK506 cell line (Table 1). This study included 1284 patients with type 1 diabetes (4.5%), 8560 patients with type 2 diabetes (30.0%) and 18 704 non-diabetic patients (65.5%). Rates of coronary artery, peripheral vascular and cerebrovascular disease were higher in diabetic than in non-diabetic patients (Table 1) (P < 0.0001). Multivariate survival analysis showed the risk for death BYL719 after the first dialysis treatment was 64.0% (HR 1.64 (1.47–1.84)

greater in type 1 diabetic (P < 0.0001) and 13.0% (HR 1.13 (1.06–1.20) higher in type 2 diabetic (P < 0.0001) patients versus non-diabetic patients. Sex was not associated with survival in type 1 diabetics or PDK4 in non-diabetics; however, older (≥60 years) type 2 diabetic women

had a worse outcome than older type 2 diabetic men, and this difference did not appear to be explained by different comorbid conditions. In type 1 diabetic patients, survival did not alter over time (adjusted HR 0.94 (0.83–1.07) per 5-year period, P = 0.36 but it improved significantly by 9.0% per 5-year period in type 2 diabetics (0.91 (0.87–0.95), P < 0.0001) and by 5% in non-diabetic patients (0.95 (0.92–0.98), P = 0.001). In the DOPPS, a prospective observational study of haemodialysis practices and clinical outcomes among patients treated at randomly selected dialysis facilities in France, Germany, Italy, Japan, Spain, UK and the USA (2004), diabetes was associated with a significantly higher relative risk of mortality (RR = 1.55, P < 0.001).6 Similarly, from the USRDS database, the 5-year survival in diabetic haemodialysis patients is 20% compared with 50% in non-diabetic patients.7 The percentage of all deaths attributed to cardiovascular disease (CVD) in diabetic haemodialysis patients varies from 23% to 54%.

4 This review was not limited to people with type 2 diabetes. Based on review of clinical trials and estimates of the performance characteristics of tests for proteinuria, it was estimated that screening of 20 000 Australians (>50 years) would lead to subsequent treatment of 100 prescribed with ACEi and prevention of 1.3 cases of ESKD over 2–3 years. A cost benefit evaluation indicated a net cost saving for the health care system assuming a one-off dipstick screening program in men and women over 55 based on assumed prevention of 205 cases of ESKD, 100% compliance with screening and best estimates of unit costs for screening and treatment. However,

the cost-effectiveness was quite sensitive to screening

costs with a reversal point noted occurring at $2 per person compared with a base assumption of $0.50. selleck compound Overall savings on the base assumptions were estimated at $A70 000 (2–3 years treatment costs for ESKD). Given the sensitivity of the estimates to key areas of uncertainty with respect to ESKD risk factors in the general population including, performance of screening tests and the benefits of ACEi treatment in screen-detected low risk-subjects, it remains unclear whether population wide screening for kidney disease would do ‘more harm than good’. Presumably these uncertainties would be lower in the selleck compound library higher risk type 2 diabetes sub group favouring adoption of screening and treatment in this setting. Cass et al.,3 Craig et al.4 and Palmer et al.1 determined, that given microalbuminuria does not directly cause morbidity or mortality, the effectiveness of treating microalbuminuria can be assessed by comparing the cost of treatment to the savings resulting from the presumed

prevention of ESKD. However, it should be emphasized that no study has followed the effects of ACEi or other intervention in normotensive, microalbuminuric people with type 2 diabetes until the development of ESKD. Nevertheless, such analysis can aid in determining which of several approaches provides the most cost-effective treatment of microalbuminuria. It should be noted that treatment of microalbuminuria is only one of several prophylactic Edoxaban programs that may benefit people with diabetes, and cost-benefit analysis provides a useful tool in the efficient allocation of limited health resources. The alternatives to screening for and treating diabetic microalbuminuria with ACEi or ARBs are to wait until elevated BP (BP > 130/85) or gross proteinuria develops before instigating therapy, or to treat all people with type 2 diabetes with ACEi or ARBs regardless of their urinary protein excretion. Palmer et al. considered the costs and benefits for screening for albuminuria and subsequent treatment with an ARB and discussed above.1 Golan et al.

Peritoneal macrophages of Roxadustat concentration caspase-1

knockout mice were stimulated for 24 h with either B. afzelii or B. burgdorferi. Both strains were able to induce IL-1β and IL-6 in peritoneal macrophages of WT mice. Macrophages from caspase-1-deficient mice showed significantly decreased levels of IL-1β, while the production of IL-6 by Borrelia was not affected in caspase-1-deficient cells. Although a slight increase in IL-6 in caspase-1 mice was found, this difference was not statistically significant (Fig. 1C). Borrelia is able to elicit IL-β and IL-6 production, cytokines that are often associated with inflammatory processes. In addition, production of IL-17 and IFN-γ by Th17 and Th1 subsets, respectively, has been suggested to play a role in the immune response against Borrelia 9, 22. To investigate whether spleen cells of naïve mice are able to produce IL-17 and IFN-γ after Borrelia exposure, spleen cells of WT mice were stimulated for 5 days with 1×106/mL spirochetes. A significant amount of IL-17 production after Borrelia stimulation

could be detected (Fig. 2A). In addition, IFN-γ production was also potently induced after exposure to Borrelia (Fig. 2A). Since it was shown that Borrelia activates caspase-1, the contribution of caspase-1 in the induction of IFN-γ and IL-17 was investigated. A significant decrease in both IL-17 and IFN-γ production Hydroxychloroquine order was detected in spleen cells of caspase-1 gene-deficient mice stimulated with Borrelia spp. (Fig. 2B). Since we know that caspase-1 plays an important role in the induction of cytokines, we examined the role of caspase-1 in vivo.

Borrelia spirochetes were injected directly into knee joints of naïve (WT) and caspase-1 knockout mice. After 4 h, patellae were collected and Immune system cytokine levels were measured in patella washouts. Highly significant differences in IL-1β, IL-6 and KC production could be detected when WT patellae were compared with caspase-1 gene-deficient patellae (Fig. 3A). In addition, the influx of inflammatory cells into the joint cavity of caspase-1 KO mice were decreased as compared to WT mice. Lower amounts of PMN could be seen in caspase-1−/− mice as well as less thickening of the synovial lining (Fig. 3B). When we counted the cell influx, we were able to see approximately 30% reduction in cell influx in all examined joints (n=10) of the caspase-1-deficient animals in comparison to the WT animals (n=10), which was found to be significant (Fig. 3C). We explored whether IL-1β might play a role in the induction of IL-17 during Borrelia host defense. Peritoneal macrophages and spleen cells of IL-1β gene-deficient mice were stimulated with 1×106/mL B. afzelii and B. burgdorferi for 24 h or 5 days, respectively. No differences in IL-6 production could be observed between the WT and IL-1β-deficient cells (Fig. 4A).

DDX3 as well as IPS-1 were expressed even without any stimulation (Fig. 2C and 4A and B) and bound each other in the cytoplasm (Fig. 2C). Hence, DDX3 is a cytoplasmic molecule that can detect viral RNA produced in infected cells. Knockdown studies suggested that polyI:C-mediated IFN promoter activation was abrogated in DDX3-deficient cells even in the presence of overexpressed RIG-I or MDA5 (Fig. 5). DDX3 silencing happened with two different siRNA. Thus, DDX3

may enable RIG-I and IPS-1 to confer activation of the cytoplasmic RNA-sensing pathway on virus-infected cells. The IFN-β-inducing pathway involves IRF-3 kinases TBK1 and IKKε, which may be targets of DDX3 15, 16. By in vitro reporter analysis, increasing amounts of DDX3 barely

affected IFN-β promoter Z-VAD-FMK price activation by TBK1 and IKKε (Fig. 6A and B). Slight TBK1-enhancing activity could manage to be detected with DDX3 when decreasing amounts of TBK1 was used in the assay (Fig. 6C and D). HeLa cells induced the mRNA of RIG-I and IFN-β in response to polyI:C Ixazomib order stimulation within 1 h (Fig. 4A). More exactly, IFN-β induction was ∼30 min faster than RIG-I induction in response to polyI:C. IFN-β mRNA induction was peaked around 3 h post stimulation, while RIG-I induction continued to increase>3 h (Fig. 4A). When HEK293 cells were infected with vesicular stomatitis virus (VSV) (a RIG-I-stimulating virus), the IFN-β mRNA was induced from 6 h, and by that time no RIG-I

message was generated (Fig. 4B–D). The RIG-I message began to appear>8 h and was markedly increased (Fig. 4B and D). In either case, no up-regulation was observed with DDX3 but sufficiently present in the cytoplasm (Fig. 4C). PLEK2 Furthermore, overexpression of DDX3 in HeLa cells resulted in potential prevention of VSV propagation (Fig. 7). However, the distribution profiles of DDX3 and IPS-1 were barely altered in response to polyI:C stimulation (Fig. 2C). The results allow us to interpret that when viral RNA enter the cytoplasm of infected cells, the RNA first induce a small amount of IFN-β in conjunction with the complex containing trace RIG-I and then the induced IFN-β fosters intensive RIG-I/MDA5 induction. The complex is reconstituted together with upcoming RIG-I/MDA5 to amplify the cytoplasmic IFN-inducing pathway. Although the molecular reconstitution was not visible with overexpressed proteins by confocal analysis, DDX3 may act as an enhancing factor for initial RNA-sensing by the IPS-1 complex and conducts the rapid response to viral RNA to facilitate the IPS-1 signaling. We identified DDX3 as a protein that bound to the IPS-1 CARD region, duplexed RNA and RLR. Although the DDX3 helicase domain is a DEAD box type similar to those of RIG-I and MDA5, DDX3 does not have a signaling domain corresponding to the CARD domain.

The azoles interact with other medicines primarily by inhibiting biotransformation or by affecting drug distribution and elimination. The echinocandins have the lowest propensity to interact with other medicines. The clinical relevance of antifungal–drug interactions

varies substantially. While certain interactions are benign and result in little or no untoward clinical outcomes, others can produce significant toxicity or compromise efficacy if not properly managed through monitoring and dosage adjustment. However, certain interactions produce significant toxicity or compromise efficacy to ABT-888 datasheet such an extent that they cannot be managed and the particular combination of antifungal and interacting medicine should be avoided. With the continued expansion of the antifungal drug class, clinicians have a much wider variety of choices in the prevention or management of systemic fungal infections. This expansion has allowed clinicians to more clearly distinguish the advantages and disadvantages of using a particular agent in a given case. For example, existing polyenes (the amphotericin B formulations) are active against a broad spectrum of fungal pathogens, but their toxicity Peptide 17 mouse may limit their use in certain patients. Moreover, existing polyenes are only available intravenously (i.v.), which often precludes their use in the primary care setting. Although the echinocandins

are generally devoid of significant drug interactions or toxicity, they are active against only Candida and Aspergillus species, which are significant opportunistic pathogens, but they are devoid of activity against other important but less common opportunistic pathogens (i.e. pathogens of Zygomycetes, Cryptococcus, etc.) and the primary pathogens associated with endemic mycoses (Histoplasma, Blastomycetes, etc.). In addition to this comparatively

very narrow spectrum of activity, like the polyene agents, they are only available as i.v. products. As a class, the systemically acting azoles are safe, have a broad spectrum of activity and can be administered i.v. or orally. However, most agents have variable and unpredictable pharmacokinetics, undergo significant metabolism and therefore may interact with many medicines. When considering antifungal Fossariinae therapy, clinicians often either possess susceptibility data or are well versed in the spectrum of activity of a specific antifungal agent. Similarly, they often are well aware of the potential toxicities of antifungal agents. However, the potential for antifungal agents to interact with other medications is vast and may be difficult for clinicians to recognise it consistently. Failure to recognise a drug–drug interaction involving an antifungal agent may produce deleterious consequences to the patient, including enhanced toxicity of the concomitant medications or ineffective treatment of the invasive fungal infection.

Counts of eosinophils and globule leucocytes were not normally distributed, were transformed as ln(count + 1), and were analysed using the general linear models procedure of SAS. The model included fixed effects of breed, group (infection status by day of sacrifice, with two infected and three control groups) and breed by group interaction. Results are presented as back-transformed means and SE. Serum

immunoglobulin concentrations were analysed within infection status using the model used for the repeat-measures analysis of variance of FEC and PCV. Bortezomib supplier Lymph node IgE concentrations at sacrifice were analysed using the model applied to the abomasal cell counts. Simple correlations (r) were calculated between measurements taken in infected animals at sacrifice at 3 and 27 days p.i. (i.e. in the presence of larvae and adult worms respectively). Reported correlation coefficients differed from zero (P < 0·05) unless stated otherwise. No parasite eggs were seen in the

faeces of control animals throughout the study, but all experimentally infected lambs had measurable FEC by 16 days p.i. (Figure 2). The mean FEC of wool sheep was similar to that of hair sheep on day 16, but was 2·8-fold higher at day 21 (3647 ± 770 vs. 1280 ± 867 respectively), and 2·5-fold higher at day 27 (3136 ± 1599 selleck screening library vs. 1267 ± 837) than that of wool sheep (P = 0·12 when mean FEC were averaged across days 21 and 27). Abomasa of control sheep were free of adult H. contortus, whereas worms were present in all challenged sheep. On day 27 p.i., the mean number of adult H. contortus in infected hair sheep (2491 ± 753) was lower (P = 0·07) than

that in wool sheep (4535 ± 690). Lower worm counts were correlated with higher PCV (r = −0·53, P = 0·08) and lower FEC (r = 0·71, P = 0·01). The average PCV of control hair (36·3 ± 0·7) and wool (35·5 ± 0·5) sheep were similar and did not differ between days. However, infection was associated with lower PCV in both breeds at days 16 and 21, followed by an increase in PCV in both breeds at day 27 (Figure 2). In infected animals, PCV were Dolichyl-phosphate-mannose-protein mannosyltransferase higher in hair compared with wool sheep; this difference approached significance (P < 0·10) at day 21 p.i. The day of peak FEC corresponded to the time of lowest PCV and FEC and PCV were negatively correlated (r = −0·78, P = 0·07). Breed differences in abomasal lymph node weight were not observed in control animals, but lymph nodes from infected hair sheep were heavier than those of infected wool sheep (P = 0·04, Table 1). Lymph nodes of infected animals of both breeds were likewise heavier (P < 0·001) than those of corresponding control animals. Lymph node weights at sacrifice were favourably associated with PCV on days 0 (r = 0·58), 16 (r = 0·61) and 21 p.i. (r = 0·56).

Strain oxyR::CAT/oxyR−/rpoS− was produced by conjugation between strains oxyR::CAT/oxyR− (9) and rpoS− (7) with selection by chloramphenicol and tetracycline.

Strain oxyR::CAT/rpoS− was produced by conjugation Selleck beta-catenin inhibitor between strains rpoS− (7) and oxyR::CAT (9) and selection on tetracycline, chloramphenicol and trimethoprim. Strain oxyR::CAT/rpoS−/RpoS was produced by conjugation between strains rpoS− with a strain carrying the complement rpoS gene, represented as RpoS (7) and oxyR::CAT (9) and selection on tetracycline, chloramphenicol, trimethoprim, and spectinomycin. Strains katG::CAT/oxyR−, katG::CAT/rpoS− and katG::oxyR−/rpoS− were produced by conjugation between strain katG::CAT (10) and strains oxyR− (9), rpoS− (7) and oxyR−/rpoS− (above) respectively,

with selection on trimethoprim and tetracycline (katG::CAT/oxyR− and katG::CAT/rpoS) or trimethoprim, chloramphenicol and tetracycline (katG::CAT/oxyR−/rpoS−). Strains dpsA::lacZ/oxyR−, dspA::lacZ/rpoS− and dpsA::lacZ/oxyR−/rpoS− were produced by conjugation between strain dpsA::lacZ (10) and strains oxyR− (9), rpoS− (7) and oxyR−/rpoS− (above) respectively, with selection on trimethoprim and tetracycline (dpsA::lacZ/oxyR−, dpsA::lacZ−/rpoS−) or trimethoprim, chloramphenicol and tetracycline (dpsA::lacZ/oxyR−/rpoS−). Strain rpoS::lacZ/oxyR− was produced by conjugation between strain oxyR− (9) and rpoS:: lacZ (7) and selection on tetracycline and trimethoprim. After antibiotics selection, the genotypes

of all constructed mutants were confirmed by the PCR method using specific primers as previously described (7, 9). Overnight selleck screening library cultures of B. pseudomallei were subcultured (OD600∼0.1) and grown in LB at 37°C. During the mid-exponential phase cells were treated with 0.5 mM H2O2 every 10 min for 1 hr Dolutegravir clinical trial or 0.5 mM menadione for 1 hr before harvesting during the log phase (4 hr), early stationary phase (12 hr), or late stationary phase (24, 48 and 72 hr). Cell lysates were prepared and assayed for CAT activity using acetyl-CoA and 5, 5′-dithio-bis (2-nitro-benzoic acid), or for β-galactosidase activity using O-nitrophenyl-β-D-galactoside as the substrate as previously described (11, 12). Protein concentrations were determined by the Bradford Assay (13). All cultures were assayed in triplicate, and reported values are averages from at least three independent experiments. Total RNA was extracted using the modified hot acid phenol method as described elsewhere (14). For RT-PCR experiments DNA contamination was removed by incubation with 1 U DNase I per μg RNA for 30 min at 37˚C. RT-PCR was undertaken using the Qiagen OneStep RT-PCR kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s recommendations. The semi-quantitative RT-PCR reaction was performed in a final volume of 50 μl containing 200 ng of B. pseudomallei total RNA, 0.