The CD25+ B-cell subset secrete higher levels of IL-6, IL-10 and

The CD25+ B-cell subset secrete higher levels of IL-6, IL-10 and INF-γ, are more efficient antigen-presenting cells, and a higher frequency of this subset also produced higher levels of immunoglobulins of IgA, IgG and IgM isotypes spontaneously compared with CD25− B cells. In addition, CD25+ B cells secrete higher levels of antigen-specific antibodies of especially IgM, but also IgG class following OVA immunization in vivo. They have the ability to migrate towards the CXCL13, and

a higher number of cells expressed selected homing receptors in the CD25+ B-cell population than CD25− B cells. We suggest that CD25 is a developmental marker of B cells, and the CD25+ B-cell population is functionally different from the CD25− population and might belong to the memory B-cell population. Knowledge PF-562271 solubility dmso about murine CD25+ B cells from

secondary lymphoid organs is scarce. It has been shown that B cells during their development in the bone marrow, at the pre-B-cell stage, express high levels of CD25 [8, 9]. The expression of CD25 is, however, down regulated, while the B cells mature and leave the bone marrow. Currently, CD25 together with CD69 is used as a marker for activated B cells in vitro, but there are to our knowledge no studies aiming to examine the functional properties of these cells in vivo. Although it is common knowledge that the major function find more of B cells is to produce antibodies, B cells also have the capacity to produce different spectrum of cytokines [14]. Harris et al. has shown that cytokine-producing B cells can be divided in to two effector subsets – Be1 (producing mainly IFN-γ, IL-12, LTα) and Be2 (producing IL-4, IL-6, IL-2). These cytokines click here have the ability to regulate the differentiation and expansion of naïve T cells in to the Th1 and Th2 subsets [15]. In addition, a third B-cell effector subset regulatory B cells (Breg) mainly produce IL-10 and has been shown to play a key role in controlling autoimmunity [16–19], allergy [20, 21] and chronic intestinal inflammation [22]. To reveal the cytokine production pattern, CD25+ B cells were stimulated

with the TLR2-, TLR4- and TLR9- agonists resulting in a high production of IL-6, IFN-γ, IL-10 and to some extend IL-4. Cytokines like IL-6 and IFN-γ may also function directly on B cells inducing differentiation of B cells into antibody producing cells [23–26], while the effects of IL-10 on murine B cells is still under discussion [27, 28]. No IL-2 could be detected and that may be a result of autocrine consumption, as CD25 expressing B cells express the high affinity IL-2 receptor and the CD25 negative B cells have the intermediate IL-2 receptor. We could detect a broad array cytokines produced by CD25+ B cells in response to different stimulatory agents. These findings suggest that the CD25+ subpopulation of B cells are an important source of cytokines and might have impact on the outcome of the immune response.


“Please cite this paper as: Pacella JJ, Kameneva MV, Brand


“Please cite this paper as: Pacella JJ, Kameneva MV, Brands J, Lipowsky HH, Vink H, Lavery LL, Villanueva

FS. Modulation of pre-capillary arteriolar pressure with drag-reducing polymers: a novel method for enhancing microvascular perfusion. Microcirculation 19: 580–585, 2012. Objective:  We have shown that drag-reducing polymers (DRP) enhance capillary perfusion during severe coronary stenosis and increase red blood cell velocity in capillaries, through uncertain mechanisms. We hypothesize that DRP decreases pressure loss from the aorta to Selleck Mitomycin C the arteriolar compartment. Methods:  Intravital microscopy of the rat cremaster muscle and measurement of pressure in arterioles (diameters 20–132 μm) was performed in 24 rats. DRP (polyethylene oxide, 1 ppm) was infused i.v. and measurements were made at baseline and 20 minutes after completion of DRP infusion. In a 10-rat subset, additional measurements were made three minutes after Selleckchem MG132 the start, and one to five and 10 minutes after completion of DRP. Results:  Twenty minutes after the completion of DRP, mean arteriolar pressure was 22% higher than baseline (from

42 ± 3 to 49 ± 3 mmHg, p < 0.005, n = 24). DRP decreased the pressure loss from the aorta to the arterioles by 24% (from 35 ± 6 to 27 ± 5 mmHg, p = 0.001, n = 10). In addition, there was a strong trend toward an increase in pressure at 10 minutes after the completion of DRP (n = 10). Conclusions:  Drag-reducing polymers diminish pressure loss between the aorta and the arterioles. This results in a higher pre-capillary pressure and probably explains the observed DRP enhancement in capillary perfusion. "
“Please cite this paper as: Sprague RS, Ellsworth ML. Erythrocyte-derived ATP and perfusion distribution: role of intracellular and intercellular communication. Microcirculation 19: 430–439, 2012.

In complex organisms, both intracellular and intercellular communication are critical for the appropriate regulation of the distribution of perfusion to assure optimal O2 delivery and organ function. The mobile erythrocyte is in a unique position in the circulation as it both senses and responds to a reduction in O2 tension in its environment. When erythrocytes enter a Nintedanib (BIBF 1120) region of the microcirculation in which O2 tension is reduced, they release both O2 and the vasodilator, ATP, via activation of a specific and dedicated signaling pathway that requires increases in cAMP, which are regulated by PDE3B. The ATP released initiates a conducted vasodilation that results in alterations in the distribution of perfusion to meet the tissue’s metabolic needs. This delivery mechanism is modulated by both positive and negative feedback regulators. Importantly, defects in low O2-induced ATP release from erythrocytes have been observed in several human disease states in which impaired vascular function is present.

The correlation analysis was performed with Pearson correlation a

The correlation analysis was performed with Pearson correlation after log transformation of the antibody results. All statistical analyses were performed with spss version 15.0 (IBM, Armonk, NY, USA). The characteristics of the patients are presented in Table 1. Their mean age (SD) was

64 years (10) ranging from 38 to 80; 96 were men and 45 women. To be able to investigate, if periodontal status or carriage of periodontal pathogens is associated with HSP60 antibody levels in the whole population, the putative effect of clarithromycin on the antibody levels was first examined. The median A. actinomycetemcomitans, P. gingivalis and HSP60 antibody levels at baseline and during the follow-up are presented in the whole population and buy Palbociclib separately for the placebo and clarithromycin groups in Table 2. All antibody levels remained remarkably stable during the follow-up and only minor changes were noticed. None of the antibody levels differed between the

placebo and the clarithromycin groups in the follow-up. CRP concentrations, however, decreased Kinase Inhibitor Library as expected (Table 2). Heat shock protein 60 IgA-class antibody levels had a moderate but significant positive correlation with A. actinomycetemcomitans and P. gingivalis IgA antibody levels at baseline with correlation coefficients of 0.237 and 0.295, respectively. HSP60 IgG-class antibody levels had a strong correlation with A. actinomycetemcomitans IgG antibody levels with a correlation coefficient of 0.489, but no statistically significant correlation with P. gingivalis IgG-class antibody levels (correlation coefficients 0.042). No significant correlations

were found between CRP and HSP60, A. actinomycetemcomitans or P. gingivalis antibody levels at baseline. The antibody levels to periodontal pathogens were divided into seronegative and seropositive results. The HSP60 IgG-class antibody levels were significantly higher in the IgA- or IgG-seropositive patients for A. actinomycetemcomitans compared to seronegative patients at baseline (Fig. 1). No such association was seen between HSP60 and Sodium butyrate P. gingivalis antibody levels. The results were similar throughout all time points. The HSP60 antibody levels did not differ between patients PCR-positive and -negative for salivary periodontal pathogens at baseline (Fig. 2). As expected, patients harbouring A. actinomycetemcomitans in their saliva had higher serum antibody levels against the pathogen than patients negative for it. Similar observations were performed for P. gingivalis positive and negative patients (Fig. 2). According to the panoramic tomograms taken at baseline, the patients were divided into edentulous (n = 34), non-periodontitis (n = 29) and periodontitis (n = 75) groups. The HSP60 IgA- or IgG-class antibody levels did not relate to the dental status (Table 3). As expected, the salivary occurrence of P.

Results from an animal model support that the higher levels of Ky

Results from an animal model support that the higher levels of Kyn in renal failure are attributed mainly to a combination of increased TDO activity and decreased kynureninase activity in the liver, and not to impaired renal excretion [16]. Conversely, the increased neopterin concentrations are attributed most probably to increased cellular immunity activation accompanying reduced renal function [18]. MLN8237 Overall, the examined lifestyle factors associated with inflammation [3, 22, 24, 25, 35] were weaker

determinants of circulating markers of cellular immune activation and kynurenines compared to the biological determinants. Despite the fact that obesity is related to increased IFN-γ activity [4], BMI was not associated with neopterin in this or in a previous study [19]. In contrast, some studies indicate a positive association of BMI with neopterin [12, 22, 23], and inconsistencies might relate partly to the different study designs; one of the studies included mainly overweight and obese participants [22], whereas another presented only crude associations [23]. In contrast to the null findings for neopterin, we observed that overweight and obesity were associated positively with KTR and all kynurenines, except

AA, which is in line with previous studies on KTR [20, 21]. Thus, it is possible that kynurenines are involved in obesity and/or obesity-related conditions. Interestingly, HAA and HK can induce the formation of free radicals selleck inhibitor [36] and thereby may mediate oxidative stress associated with obesity [37]. Furthermore, XA can react with insulin and therefore may lead potentially to insulin resistance [4], a condition related strongly to obesity [37]. Finally, we observed recently that KA is a strong predictor of pre-eclampsia in obese women [38]. It has been shown that physical activity has an anti-inflammatory effect [24] and is associated with a reduction in visceral fat mass. In

the present study, physical activity was not associated with neopterin, KTR or kynurenines, except for a weak inverse find more association between physical activity and KA. Previous studies on the short-term effect of intense exercise have reported an increase in both neopterin [39, 40] and Kyn [35]. Conceivably, short-term and habitual physical activity may have different effects on IFN-γ-mediated pathways, as demonstrated previously for several inflammatory markers [24]. In this community-based study we did not observe an association of current smoking with neopterin or KTR, as both Trp and Kyn were decreased slightly in moderate smokers and decreased further among heavy smokers; therefore, KTR was not changed in any of the groups. We also found a similar inverse association between smoking and all other kynurenines, except HK.

Previous immunization studies had shown that a particular idiotyp

Previous immunization studies had shown that a particular idiotype, C12, generates a large fraction of the virus-specific early response to influenza A/PR8 in BALB/c mice 24, 27 and an anti-C12 idiotype mAb had previously been generated 24. Infection of BALB/c mice with influenza A/PR8 showed that C12Id-expressing virus-specific serum Ab responses peaked rapidly, at around 2 wk after infection, consistent with the earlier immunization studies 24. The C12Id response peak preceded the overall virus-specific Ab response peak by roughly 2 wk (Fig. 1A). In contrast to

the C12Id-encoded responses induced by immunization, which rapidly disappeared 24, antiviral C12Id serum Ab were still measurable by day 60 following infection, albeit at levels reduced from their LY2606368 cost peak. The overall anti-viral serum Ab response reached plateau levels https://www.selleckchem.com/products/VX-765.html about one month after infection, after which time they were maintained over the lifetime of the mouse (Fig. 1A, right panel and data not shown). ELISPOT analysis on respiratory tract draining MedLN, spleen and lung identified the regional LN as the major site of early C12Id+ Ab production

(Fig. 1B). In contrast to the Ab responses in secondary lymphoid organs, the Ab secreting cells in the lung tissue indicated a steady accumulation. The rapid increase then decline of C12Id+ virus-specific serum Ab could not be explained by T cell-independent B-cell activation. T-cell-deficient nude mice showed greatly reduced antiviral C12Id+ serum Ab titers compared with WT BALB/c Urease mice (Fig. 1C). While the C12Id-encoded response was greatly diminished, however, it was still measurable and followed kinetics similar to responses

in WT mice. Together, the virus-specific C12Id+ responses showed response kinetics distinct from those of the overall infection-induced humoral responses (Fig. 1A). The magnitude of this C12Id response suggested that we could follow C12Id+ B cells elaborating this response as prototypic “early” responders in the context of non-genetically manipulated WT BALB/c mice. To study the characteristics of the rapidly differentiating C12Id+ B cells, we focused on regional LN, the site of strongest Ab production (Fig. 1B). C12Id-expressing B cells were easily identified in MedLN and peripheral LN (pooled inguinal and axillaries) of non-infected mice, where they represented a relatively high frequency of B cells (between 1 and 2% of B cells, Fig. 2A and data not shown). Their frequencies in MedLN of non-infected mice were not significantly different from those in peripheral LN. As MedLN are extremely small in non-infected mice, we therefore used the peripheral LN as control for all remaining studies. The relatively high frequency of C12Id+ B cells is consistent with previous findings of high titers non-HA-specific C12Id-encoded Ab in BALB/c mice prior to infection (24 and data not shown).

By now it is clear that Tregs consist of many different T-cell su

By now it is clear that Tregs consist of many different T-cell subsets that can be distinguished by their development: Naturally occurring CD4+CD25+ Foxp3+ Tregs arise during the normal process of maturation in the thymus, whereas inducible Tregs are generated in the periphery during immune responses 6, 22, 23, 29. The generation of Tregs by specific modes of stimulation, especially in a particular cytokine milieu, has been described by several groups 10, 30. For instance, Tr1 cells arise from naive precursors and can be differentiated both in vitro and in vivo by repeated TCR stimulation in the presence of IL-10 10, 30. Our data demonstrate

that DN T cells belong to the inducible Treg subset that Navitoclax cost exerts their suppressive activity exclusively after activation with APCs. Similar to our observation, Zhang et al. reported that murine DN T cells suppress transplant rejection only after previous in vivo activation induced by donor lymphocyte infusion 11, 13, 17, 19. Of note, once the regulatory function of DN T cells has been induced, they retain their suppressive activity Selleck BMN 673 upon repetitive stimulation.

Although human DN T cells are only present at low numbers 12, they can be induced to Tregs and expanded ex vivo for clinical application. The mechanisms by which Tregs mediate suppression are highly diverse: Several studies demonstrated that murine DN T cells acquire peptide-MHC-complexes from APCs and interact via transferred molecules with effector T cells 11, 31, 32. We have previously demonstrated that human DN T cells were also able to acquire MHC-complexes 12. By now it is clear that various cell populations can acquire membrane fragments from APCs, a process called trogocytosis. However, DN T-cell-mediated suppression was not affected when plate-bound anti-CD3 mAb or artificial APCs were used as stimulators. In addition, blocking trogocytosis via CMA was not able to abrogate the suppressive activity of human DN T cells (our unpublished data), indicating

that human DN T cells suppress responder T cells by other mechanisms. Several studies demonstrated that murine DN T cells mediate suppression by eliminating this website T cells through Fas/FasL interaction or via perforin/granzyme 11, 13, 15, 16, 19, 20. Previously, we have shown that human DN T cells induce apoptosis in highly activated CD8+ T-cell lines 12. However, in the prior study, we used DN T cells as suppressors that acquired peptide-MHC-complexes from APCs. Tsang et al. demonstrated that T cells can induce apoptosis in neighboring T cells following acquisition of MHC-complexes 33. Thus, induction of apoptosis might be a process that is not specific for the suppressive activity of human DN T cells.

The purity of the T cells analysed after labelling with CD3-PerCP

The purity of the T cells analysed after labelling with CD3-PerCP and CD45-FITC was > 97%. In selected experiments, the isolated SCH727965 T cells were labelled with CD45RA-FITC and CD45RO-APC to isolate naive and memory T cells by flow cytometric sorting. Monocytes were isolated using CD14 microbeads over an MS column. Purity was > 98%. For the generation of monocyte-derived DC (MoDC) monocytes (1 × 106/ml) were cultured in RPMI supplemented with 10% fetal calf serum (FCS), 50 ng/ml GM-CSF and 200 U/ml IL-4 for 7 days. On day 6, 500 ng/ml lipopolysaccharide (LPS) was added to stimulate MoDC maturation. Purified PDC (2 × 104/200 μl RPMI supplemented with 10% FCS) were stimulated in round-bottomed wells with 5 μg/ml CpG

A ODN2336 or 400 μM loxoribine in the absence or presence of 20 ng/ml rapamycin. In all conditions 10 U/ml IL-3 was added as a survival factor. Rapamycin, or dimethylsulphoxide (DMSO) vehicle in the case of non-stimulated cells, were added 1 h before addition of the stimuli. After 18 h supernatants were collected for quantification of cytokines, and the PDC immunophenotype was analysed. The following combinations of antibodies were used: CD80-FITC, anti-BDCA4-PE

and CD86-APC, anti-BDCA4-PE and CD40-APC, and anti-HLA-ABC-FITC, anti-BDCA4-PE and anti-HLA-DR-APC. Dead cells were excluded with 7-AAD. Cells were analysed on a Canto II flow cytometer using Diva version 6·0 software (Becton Dickinson) or a Calibur flow cytometer with CellQuest Pro version 5·2 software. Isotype-matched irrelevant mAb labelling was used to analyse expression Ku-0059436 molecular weight of these molecules appropriately. PDC were stimulated with CpG-A or loxoribine as described, and thereafter lysed in Laemmli buffer. The lysates were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted

on Immobilon-FL transfer membrane (Millipore, Billerica, MA, USA). The membranes were incubated with the appropriate antibodies, Vorinostat and for detection anti-rabbit or anti-mouse IRDye-conjugated secondary antibodies (Li-cor Biosciences, Lincoln, NE, USA) were used according to the manufacturer’s directions. The blots were scanned by Odyssey infrared imaging (LI-COR Biosciences). Results were visualized with Odyssey version 3·0 software. After stimulation of purified PDC (2 × 104/200 μl) for 18 h with CpG A ODN 2336 or loxoribine in the absence or presence of 20 ng/ml rapamycin, rapamycin was carefully washed away, and allogeneic CD3+ T cells were added (1 × 105/200 μl RPMI supplemented with 10% FCS). The cells were cultured at 37°C with 5% CO2. Proliferation was determined after 5 days of culture by measurement of incorporation of 0·5 μCi/well [3H]-thymidine (Radiochemical Centre, Amersham, Little Chalfont, UK) during the last 18 h of the culture. In all cultures, T cells stimulated with PHA (5 μg/ml) served as a positive control to assess their proliferative capacity.

Table S1 Results from multiple linear regression fitting age and

Table S1. Results from multiple linear regression fitting age and cytomegalovirus (CMV) status as co-variates. Table shows the unstandardized coefficient, significance and 95% confidence interval from the output of SPSS software for each CD45RA/CD27 subset. Unit of age is equal to 1 year. Table S2. Mean frequencies and the standard error of the mean of CD40 ligand (CD40L), interferon-γ (IFN-γ), interleukin-2 (IL-2) and tumour necrosis factor-α (TNF-α) in all possible combinations in each CD45RA/CD27 subset. “
“Hereditary angioedema (HAE) is a rare disease characterized by episodes of potentially

life-threatening angioedema. For affected children in the United Kingdom, there are relatively few data regarding disease prevalence, service organization and the humanistic burden of the disease. Romidepsin cell line To improve knowledge in these areas, we surveyed major providers of care for children with HAE. A questionnaire was sent to major paediatric centres to determine patient numbers, symptoms, diagnostic

difficulties, BTK inhibitor molecular weight management and available services. In addition, all patients at a single centre were given a questionnaire to determine the experiences of children and their families. Sixteen of 28 centres responded, caring for a total of 111 UK children. Seven children had experienced life-threatening crises. One-third of patients were on long-term prophylactic medication, including C1 inhibitor prophylaxis in four children. Eight centres reported patients who were initially misdiagnosed. Broad differences in management were noted, particularly regarding indications for long-term prophylaxis and treatment monitoring. We also noted substantial variation in the organization of services between centres, including the number of consultants contributing to patient care, ifenprodil the availability of specialist nurses, the availability of home therapy training and the provision of patient information. Ten of 12 patient/carer

questionnaires were returned, identifying three common themes: the need to access specialist knowledge, the importance of home therapy and concerns around the direct effect of angioedema on their life. To our knowledge, this study represents the first dedicated survey of paediatric HAE services in the United Kingdom and provides useful information to inform the optimization of services. “
“Galectin-3, an endogenous glycan-binding protein, plays essential roles during microbial infection by modulating innate and adaptive immunity. However, the role of galectin-3 within the CD4+CD25+Foxp3+ T regulatory (TREG) cell compartment has not yet been explored. Here, we found, in a model of Leishmania major infection, that galectin-3 deficiency increases the frequency of peripheral TREG cells both in draining lymph nodes (LNs) and sites of infection. These observations correlated with an increased severity of the disease, as shown by increased footpad swelling and parasite burden.

A careful preventive monitoring as well as an optimal blood press

A careful preventive monitoring as well as an optimal blood pressure control may reduce the risk of AD and improve the outcome of these patients. “
“Background:  Both the presence of peripheral arterial disease and chronic kidney disease has been reported to be independent

risk factors associating with poor prognosis. However, the impact of combination of peripheral arterial disease and chronic kidney disease remains unknown. Methods:  The long-term outcome in 715 consecutive patients who had undergone coronary angiogram for the evaluation of chest pain was analyzed. Patients on haemodialysis were excluded from this analysis. Cohort patients were divided into four groups according to the Selleck Panobinostat Ankle Brachial Index (ABI <0.9) and glomerular ICG-001 research buy filtration rate (GFR <60 mL/min per m2): group A (n= 498; ABI >0.9, GFR >60); B (n = 65, ABI <0.9, GFR >60); C (n = 99; ABI >0.9, GFR <60); and D (n = 53; ABI <0.9, GFR <60). The mean follow-up period was 620 ± 270 days and evaluated the major cardiac adverse events included survival, stroke, acute coronary syndrome and heart failure. Results:  The mean follow-up period was 620 ± 270 days. Total long-term event was present in 89 patients (groups A–D were 9.4%, 18.5%, 15.2% and 28.3%, respectively). Long-term event rate was 28.3% for patients with the presence of peripheral arterial disease and chronic kidney disease,

compared to 9.4% for those selleck without peripheral arterial disease and chronic kidney disease (P < 0.0001). Kaplan–Meier event-free survival curves also showed that the combination of peripheral arterial disease and chronic kidney disease predicted long-term event rate. Conclusion:  The combination of chronic kidney disease and ABI of less than 0.9 undergoing coronary angiogram is strongly associated with long-term event rate. "
“Sepsis has been shown to induce the expansion of CD4+CD25+ regulatory T cells (Tregs), and this paradoxical immune suppression has been suggested to

be closely associated with the development of sepsis-induced organ dysfunction. In the present study, we aimed to investigate the possible link between immune suppression and the development of septic acute kidney injury (AKI). We prospectively enrolled patients with a diagnosis of sepsis, with or without AKI and as well as patients with AKI but without sepsis. Serum and urine samples at the time of the diagnosis were collected to measure neutrophil gelatinase-associated lipocalin (NGAL), cytokines, and soluble CD25 (sCD25). Of the 82 patients enrolled, 44, 18, and 20 patients were classified into septic-AKI, sepsis-non AKI and non-septic AKI groups. There were no differences in the baseline characteristics in all three groups and the severity of infection in the two sepsis groups. Serum levels of interleukin (IL)-10 were significantly elevated in patients with septic-AKI compared to the other two groups.

5 mM MgCl2, DTT; pH8 7), 2 μl of Qiagen OneStep RT-PCR Enzyme Mix

5 mM MgCl2, DTT; pH8.7), 2 μl of Qiagen OneStep RT-PCR Enzyme Mix (Qiagen GmbH) and 10 U of RNase inhibitor. The thermal cycler program was carried out at 50°C for 30 min. A 15 min denaturation at 95°C was included prior to the initiation of PCR cycles for the Qiagen One-Step RT-PCR kit, since it contains a hot-start Taq polymerase. At the end of 27 cycles, the reaction-samples (5 μl) were analyzed on 1% agarose gels after amplification. For Northern analysis total RNA extracted from early stationary phase B. pseudomallei was separated by electrophoresis and transferred to solid matrix. Membrane was probed

with a 500-bp SphI-PstI fragment spanning dpsA labeling with α-32P-dCTP by a PCR labeling method and by X-ray exposure Selleck BMN-673 detection as previously described (14). To investigate whether expression of oxyR regulates rpoS, the B. pseudomallei strain rpoS::lacZ was conjugated with B. pseudomallei strain oxyR−. A mutant rpoS::lacZ, oxyR strain was then selected and designated rpoS::lacZ/oxyR−. The extent to which LacZ was expressed was investigated in the log, early Trametinib stationary

and late stationary growth phases in rpoS::lacZ and rpoS::lacZ/oxyR−. As can be seen in Figure 1a, both strains showed essentially the same response curve, although late stationary phase concentrations of lacZ were somewhat higher in the rpoS::lacZ/oxyR− strain. These results suggest that rpoS expression does not require oxyR. To determine the converse, whether rpoS regulates the expression of oxyR, the B. pseudomallei strain oxyR:: CAT, which contains a chromosomal oxyR::CAT transcriptional AMP deaminase fusion as well as an integrated mini-transposon containing oxyR (mtoxyR+) (9), was conjugated separately with B. pseudomallei strains rpoS− and the one which carries complement rpoS (rpoS−+ pBSS1[rpoS+]), represented as RpoS, resulting in production of strains oxyR::CAT/rpoS− (chromosomal oxyR::CAT/mtoxyR+/rpoS) and oxyR::CAT/rpoS−/RpoS

(chromosomal oxyR::CAT/mtoxyR+/rpoS, +pBSS1[rpoS+]), respectively. The extent of CAT expression was assessed during log phase growth (4 hr post subculture), and the early (12 hr) and late stationary phases (24, 48, 72 hr). As can be seen in Figure 1b, significant induction of CAT expression was observed during the log to early stationary phase of growth in both strains, oxyR::CAT and oxyR::CAT/rpoS−/RpoS, in both cases declining slowly during the late stationary phase. In contrast, no induction of CAT expression was observed in strain oxyR::CAT/rpoS− (which contains no RpoS), showing that RpoS is required for the induction of oxyR gene expression under normal growth conditions.