13 Both studies contrasted samples from donor lungs that later developed PGD against donor lungs that did not. For the GSE9102 study, cDNA microarray data as pre-processed by the authors were used, and covered expression measurements for 6727 Ensembl build 55

human genes (http://jul2009.archive.ensembl.org). When several probes were available for the same gene, the probe displaying the most significant differential https://www.selleckchem.com/products/poziotinib-hm781-36b.html expression was selected to represent that gene. For the GSE8021 study, the original raw data were processed as follows. Affymetrix Human Genome U133A 2.0 Array probes were remapped to 11894 different Ensembl build 55 human genes.14 Using these redefined probe sets, probe intensities were summarized and made comparable between arrays by quantile normalization as implemented in the Robust Multi-Array Average expression measure.15 It was possible to identify corresponding gene expression for 242 of the 272 proteins on the antigen microarray (89%). For each antigen and detection antibody, differential reactivity between patients without PGD (n = 19) and patients with PGD (n = 20) was evaluated by calculating ratios (fold-changes), t-statistics and P-values. For each gene measured, differential expression between donor lungs developing PGD (16 and 10) and those that did not (34 and 16) were similarly evaluated by

ratios, t-statistics and P-values. Multiple testing was controlled using the false discovery rate.16 A human protein interaction network was created by pooling human interaction Carfilzomib data from several of the largest databases.17 Coverage was further Demeclocycline increased by transferring data from model organisms. A network-wide confidence score for all interactions, based on network topology, experimental type and interaction reproducibility, was then established. The reliability of this score as a measure of interaction confidence was confirmed by fitting a calibration curve of the score against a high-confidence set of about 35 000 human interactions. As previously described,8 all interactions with a confidence score above 0·154

were included, resulting in a network containing approximately 154 000 unique interactions between approximately 12 500 human proteins. Out of the 272 proteins on the antigen microarray, 260 (96%) were among these. As described previously,8 the statistical significance of the number of proteins in a network (the size) extracted from a given larger set of proteins, was estimated by randomly selecting sets of proteins of the same size, each time recording the size of the largest network possible to extract. For 107 such randomizations, the proportion of random sets of proteins for which equally sized or larger networks could be extracted, establishes the P-value of the network extracted from the original protein set. Over-represented biological processes among proteins in networks were identified by hypergeometric testing of gene ontology terms.

This study for IDH assay recurrent hyponatremic episodes following the first admission with severe hyponatremia (<125 mEq/L) on thiazide aimed to investigate whether other coexistent factors than thiazide are responsible.

Methods: In the retrospective chart review over 5 yrs, out of 1,625 pts admitted with severe hyponatremia on hydrochlorothiazide (HCTZ), 24 pts (M : F, 7:17; age 71 ± 11 yrs, mean ± SD) were re-admitted for the recurrent hyponatremia (up to 4 times). Results: Among the 1st (n, 24), the 2nd (n, 24), the 3rd (n, 6), and the 4th admission (n, 2), serum sodium levels on admission were not significantly different (122 ± 3.8 vs 120 ± 6.7 vs 119 ± 5.4 vs 115 ± 19.1 mEq/L, p = ns). Successful managements of the 1st admission (n, 24) included discontinuing HCTZ with intravenous salt (n, 17), withdrawing HCTZ only (n, 1), and inadvertent continuation of HCTZ with intravenous salt (n, 6). As the causes of hyponatremia on

the 2nd admission (n, 24), 14 10 pts (42%) with no further exposure to HCTZ revealed volume depletion (n, 6), SIADH (n, 3), and adrenal insufficiency (n, 1), respectively. Four pts on the second admission died of malignancy, not from hyponatremia. Moreover, 4 pts on HCTZ in the 1st (17%) and the 2nd admission (17%), and also 2 pts on the 3rd admission (33%) were simultaneously taking neuropsychiatric medications and other diuretics with either furosemide or spironolactone. The latter 2 pts of the 3rd admission experienced the 4th admission of recurrent hyponatremia. Conclusion: In summary, hyponatremia Ketotifen on thiazide selleck products does not mean necessarily thiazide alone as the sole cause of its frequent occurrence. Therefore, thiazide-associated hyponatremia warrants prudent exploration for other coexistent causes of hypontremia. SOHARA

EISEI, SUSA KOICHIRO, RAI TATEMITSU, ZENIYA MOKO, MORI YUTARO, SASAKI SEI, UCHIDA SHINICHI Department of Nephrology, Tokyo Medical and Dental University Introduction: Pseudohypoaldosteronism type II (PHAII) is a hereditary disease characterized by salt-sensitive hypertension, hyperkalemia and metabolic acidosis, and genes encoding the WNK1 and WNK4 kinases were known to be responsible. Recently, two genes (KLHL3 and Cullin3) were newly identified as responsible for PHAII. KLHL was identified as substrate adaptors in the Cullin3-based ubiquitin E3 ligase. We have reported that WNK4 is the substrate of KLHL3-Cullin3 E3 ligase-mediated ubiquitination. However, WNK1 and NCC were also reported to be a substrate of KLHL3-Cullin3 E3 ligase by other groups. Therefore, it remains unclear which molecule is true substrate(s) of KLHL3-Cullin3 E3 ligase, in other words, what is the true pathogenesis of PHAII caused by KLHL3 mutation. Methods: To investigate the pathogenesis of PHAII by KLHL3 mutation, we generated and analyzed KLHL3R528H/+ knock-in mice.

E. faecalis-specific

primers targeting azoA (encoding azoreductase; sense: Ef azoAF 5′-CCAATCAAATGGCGGCTTCTACG-3′, antisense: Ef azoAR 5′-GCGATCAGGGAAATGATCGATTCC-3′) were designed (11). Primer specificity was confirmed by PCR using chromosomal DNA from 28 oral bacteria (Table 1). SYBR green-based quantitative real-time PCR was performed in a total volume of 20 μL containing 5 μL of various concentrations of extracted genomic DNA with or without PMA treatment, 5 × SYBR Green Master (Roche Diagnostics, Mannheim, Germany), and 0.5 μM of each primer. Amplification was done using the LightCycler Carousel-Based System (Roche Diagnostics) at 95°C for 10 min, followed by 45 cycles of 95°C for 10 s, 53°C for 10 s, and 72°C for 12 s. To confirm the formation of a single product, melting curve analysis was performed at 95°C for 1 min MK-1775 price and 55°C for 1 min, with a subsequent temperature increase from 55.0–95.0°C at 0.5°C per 10 s (data click here not shown). The sizes of the products were confirmed using

2% agarose gels. Using this method, bacterial CFU were detected linearly from 15 to 3.0 × 107 per mixture. The relationship between live cells and Ct values for real-time PCR is as follows: Y = 10−0.293X±11.056 (where Y = log10CFU, X = Ct value, R2= 0.997). Bacterial cell numbers were calculated using this formula. Propidium monoazide (Biotium, Hayward, CA, USA) was dissolved in 20% DMSO to produce a 24 mM stock solution. Following incubation with the dye for 5 min in the dark, similarly prepared cells were exposed for 5 min to a 600 W halogen light placed 20 cm above 500 μL samples in open microcentrifuge tubes on ice. The toxicity of PMA at 2.4 μM to 2.4 mM to E. faecalis was analyzed at 37°C; however, no toxicity was found (Mann-Whitney U-test, data not shown). In this study, 240 μM of PMA was employed for the analysis. To investigate the effects of PMA, E. faecalis chromosomal DNA (0.01–100 μg/mL) was analyzed with and without PMA treatment. Real-time PCR was not inhibited by heat-killed cells treated Evodiamine with 240 μM PMA (Fig. 1). To eliminate possible inhibition by

the clinical material, E. faecalis samples were spiked with dental plaque and saliva (without E. faecalis) to mimic the oral environment. There was no inhibition of real-time PCR (Fig. 2). Based on these results, nine endodontic samples from eight patients with root-filled teeth and showing radiographic evidence of apical periodontitis were analyzed. The endodontic samples were collected in accordance with the guidelines of the Ethics Committee of Kyushu Dental College Hospital from patients who visited the Department of Preventive Dentistry, Kyushu Dental College Hospital. All patients provided informed consent. Endodontic samples were taken from the infected root canals as described previously (12). The relevant tooth was isolated from the oral cavity with a disinfected rubber dam.

cruzi infected mice, and IL-12 + IL-18-treated

mice. Data using specific inhibitors of MCP-1 and CCR2 further confirm this hypothesis. Interestingly, our data support the fact that IL-12 and IL-18 are the cytokines responsible for MCP-1 upregulation in the thymus, since we observed that in vitro recombinant IL-12 and IL-18 are able to significantly increase MCP-1 only in thymocytes from IL-12 + IL-18-cDNA treated mice, indicating that cells present in the thymi of mice exposed to systemic IL-12 + IL-18 but not in normal mice contain cells with the ability to produce this chemokine. Accordingly, further analysis demonstrates that thymic B cells and T cells CD44lo are the main producers of this chemokine in the thymus under these inflammatory conditions. Based on the data presented in this work, we propose a novel concept of peripheral lymphocyte Autophagy Compound Library cost recirculation during nonphysiological conditions. We demonstrate that in any potential situation where large amounts of IL-12

and IL-18 are produced LY294002 nmr as a consequence of an infectious/inflammatory process, the thymus cell number is reduced favoring the creation of new niches in this organ that facilitate peripheral B and T cells entrance to the thymus. Interestingly, this phenomenon occurs in the absence of any antigenic stimulation and seems to be part of bystander activation of certain peripheral mature B and T cells. The fact that systemic IL-12 and IL-18 expression is observed in numerous situations opens the possibility that this migratory events described here are also possible in a numerous type of pathological processes. At the present moment, HSP90 we are evaluating if the entrance of B and T cells is due to a mere opportunism of cells during a moment of large expansion of leukocytes or if it is a coordinated process that plays a role in thymus physiology. Moreover, evaluation of peripheral cell localization in the thymus could provide important information not only about the source of required factors peripheral B and T cells use to survive in the thymus but also about the role they

might have in different thymic processes such as negative and positive selection and differentiation of immature cells in this organ. Female or male C57BL/6 (B6) and OT-I mice (Jackson Laboratory) used in this study were 6–10 week old and were maintained under specific pathogen-free conditions. The experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC). Our animal facility obtained NIH animal welfare assurance (assurance number A5802-01, OLAW, NIH, USA). B6 mice were injected i.p. with LPS (055-B5, Sigma) in a sublethal concentration of 20 μg per mouse in 200 μL PBS once a day for 3 consecutive days. Trypanosoma cruzi trypomastigotes were maintained by serial passages in B6 mice. B6 mice were i.p. infected with 5 × 105 trypomastigotes from T. cruzi diluted in PBS.

She also developed new-onset diabetes after PKC412 nmr transplantation (NODAT) that resolved after her corticosteroid dose was lowered. Although asymptomatic bacteriuria was documented on at least one occasion over the next year, she remained well until July 2011, when she presented with dysuria, fevers and pain over the allograft. A fully susceptible Escherichia coli was isolated from blood and urine cultures and a small collection at the antero-inferior pole of the allograft was noted on ultrasonography. She was treated with intravenous ceftriaxone and

discharged with a 21-day course of oral cephalexin. She was admitted again 18 days later with similar symptoms. Multiple blood cultures did not reveal a pathogen and Pseudomonas aeruginosa was isolated from urine. The lymphocoele was aspirated and the fluid had a few mononuclear cells seen on microscopy, but was sterile on culture. She was treated initially with ceftriaxone and changed to a 2-week course of oral ciprofloxacin after the P. aeruginosa was identified. Further episodes of pyrexia in August 2011 were investigated extensively with negative blood and urine cultures. Quantiferon testing showed low mitogen response with no evidence of active tuberculosis,

reflecting subnormal immune response. Mycobacterial urine cultures were also negative. A gallium scan revealed mild asymmetrical activity in left iliac fossa that localized to the perinephric lymphocoele. Each febrile episode responded to antibiotics learn more directed against common urinary pathogens. E. coli, P. aeruginosa and mixed coliforms were detected on five occasions over the next 6 months. During

this period she click here received a 2-week course of parenteral piperacillin/tazobactam as an outpatient, followed by a 4-week course of ciprofloxacin. The latter isolates were now resistant to ciprofloxacin. From March 2012 onwards, the only organism isolated was Klebsiella pneumoniae that was resistant to all commonly available oral antibiotics. She received a further 4-week course of intravenous piperacillin/tazobactam. The diagnosis of malakoplakia was made in May 2012, following biopsy of a allograft parenchymal lesion seen on computed tomography (CT) that consisted of oedematous, multi-focal, septated high-attenuation foci measuring 5.4 × 3.4 cm and 4.7 × 4.6 cm. The bladder lining was also noted to be abnormal, with a trabeculated appearance with high attenuation signal. An urgent biopsy of the lesion excluded active malignancy and confirmed parenchymal malakoplakia with CD68+ histiocytic response and intracytoplasmic basophilic microcalcifications, with typical targetoid/owl’s eye appearances characteristic of Michaelis–Gutmann (MG) bodies (see Fig. 1).

Rather, it is more likely that the treatment failed to effectively neutralize the relatively higher amount of TNF in A/J mice. Future studies will be required to assess the extent to which TNF drives pregnancy

loss in A/J mice and the pathogenic pathways activated by this cytokine in both strains. Current evidence implicates the inflammation–coagulation cycle as a central mediator for malaria-induced pregnancy compromise in B6 mice (21) (Avery et al., manuscript submitted). However, it is known that inflammatory cytokines like TNF are directly embryotoxic (44), inducing trophoblast apoptosis via TNF receptors (45), especially if the cytokine is released by monocytes in direct contact with trophoblast (46). A potential role for apoptosis in the pathogenesis

of placental malaria is currently being selleck chemicals assessed in both mouse strains. In the context of high levels of high pro-inflammatory cytokines, IL-10 plays a regulatory role (7,47), blocking malaria-associated immunopathology and P. chabaudi virulence (48). In this study, as pro-inflammatory cytokine levels increased in infected pregnant A/J mice, regulatory IL-10 decreased, at experiment day 10 reaching levels significantly lower than in infected pregnant B6 mice. While elevated IL-10 may serve to partially dampen inflammatory damage in P. chabaudi AS-infected pregnant learn more mice (20), it is inadequate to prevent pregnancy loss in both A/J and B6 mice. In humans, this cytokine level is significantly higher in infected primigravidae compared with their uninfected counterparts and has been proposed to be a marker ADP ribosylation factor for inflammatory placental malaria (49). Elevated levels of sTNFRII, which can serve to bind and sequester TNF, are likewise apparently inadequate to

control TNF-mediated pathogenesis; however, the specific role played by this solubilized receptor in infected mice and women with placental malaria (49,50) remains to be established. The different dynamics of cytokine expression in infected A/J and B6 mice prompted an examination of the potential cell types that may contribute to these differences at the splenic level. In general, lymphocyte and myeloid cell levels were influenced only by infection status, with strain and pregnancy having no significant impact, although only infected pregnant B6 mice show early elevation of neutrophils and monocytes (at experiment day 9). Interestingly, however, 1 day later, infected pregnant A/J mice showed elevated monocyte and inflammatory monocyte levels relative to uninfected pregnant mice. While these observations clearly demonstrate that pregnancy does not alter infection-induced splenic cellular expansion in either strains, they do not shed any light on the differential dynamics of embryo loss in A/J and B6 mice.

, 2007). This alteration of the outer membrane composition is probably linked to our TEM observations, revealing that OMVs-like structures are strongly overproduced in the MG210 clumping

strain. Several roles for OMVs have been reported including involvement in DNA and QS-pheromone transport in P. aeruginosa (Renelli et al., 2004; Mashburn & Whiteley, 2005). Whether Brucella OMVs could play such a role and be directly involved in the matrix production remains to be explored. Together with exopolysaccharide and eDNA, these OMVs are the third structural element, classically described in extracellular biofilm matrices, that we have identified in B. melitensis clumps. In addition to promoting adhesion of bacteria to neighboring cells, the sticky matrix components also contribute to surface adhesiveness. Therefore, it is not surprising that the clumping strain MG210 presents better adhesion GSK3235025 chemical structure properties than the wild-type strain both on polystyrene and on HeLa cells (Figs 8 and 10). The exact nature of the initial adhesin and

the stepwise process leading to cell aggregation remain to be determined. As we discussed in our previous publication (Uzureau et al., 2007), the ability of B. melitensis to form biofilm-like structures could have several advantages in its life cycle. If we consider that B. melitensis is a facultative intracellular pathogen able to survive for

months outside the host on inert surfaces (Spink, 1956), we could easily imagine a protective role for the exopolysaccharide against desiccation and other environmental stresses encountered, as check details described in Nostoc commune (Tamaru et al., 2005) or Campylobacter jejuni (Joshua et al., 2006). Nevertheless, as the genome and the molecular oxyclozanide infectious strategies of Brucella spp. are very close to those of S. meliloti and considering the role of the exopolysaccharide in S. meliloti, we hypothesize a role for Brucella clumping and/or exopolysaccharide production during its infectious cycle in the host. When aggregated Brucella spp. enter in contact with their host, exopolysaccharide could offer them protection against the extracellular immune system (as described for Streptococci (Marques et al., 1992) and help them to adhere to host cells (such as Neisseria gonorrhoeae; Greiner et al., 2005). In this regard, the adhesion we observed on HeLa cells with the MG210 strain is somehow reminiscent of the localized bacterial microcolonies of B. abortus adherent to epithelial cells depicted recently (Castaňeda-Roldán et al., 2004). The exopolysaccharide could also be involved in the earliest steps of the host trafficking as described for succinoglycan in S. meliloti (reviewed in Fraysse et al., 2003). Finally, considering the variety of eukaryotic proteins dedicated to ‘mannose’ recognition (Ip et al.

Results: Fifty two AKI patients, who collectively underwent 248 dialysis treatments, were studied prospectively. Mean (±SD) age was 69.4 ± 16.9; 50% were male. At dialysis initiation, APACHE see more II score was 20.6 ± 6.2 and SOFA score 8.2 ± 3.1. The frequency of HD treatments averaged 2.0 ± 0.5/patient/week. Mean session length was 3.54 ± 0.81 h, and 78.9% used a femoral venous catheter. The mean delivered Kt/V of each session was 1.20 ± 0.58

while 64.1% of treatments delivered a Kt/V less than 1.3. The results showed that the mean weekly delivered Kt/V at first, second, and third week was 2.49 ± 1.14, 2.55 ± 1.31 and 2.36 ± .076 respectively. Minority of patients (15.8%) achieved the recommended weekly Kt/V of 3.9. Mortality rate was lower in patients who achieved adequacy target (weekly Kt/V ≥ 3.9) but the different was not statistically significant (33.3% vs 40.6%, P = 0.73). Conclusion: Majority of our AKI patients received a lower dose of dialysis than recommendation. Survival benefit of delivering higher dose of dialysis was not shown in this study due to a small number of patients. YAMAGUCHI JUNNA, TANAKA TETSUHIRO, ETO NOBUAKI, NANGAKU MASAOMI Division of Nephrology and Endocrinology, the University of Tokyo Graduate School of Medicine Introduction: Tubulointerstitial beta-catenin cancer hypoxia is a critical mediator in the pathogenesis of kidney

disease. In light of accumulating knowledge on protective roles of HIF-1, we aimed to identify novel HIF-1 regulators in kidney. Methods: An shRNA library was created against hypoxia-inducible genes screened from a microarray analysis of rat renal artery stenosis model. The impact of candidate genes on HIF-1 was evaluated in vitro by HREluc, HIF-1α immunoblot, and VEGF protein levels, leading to identification of a novel upregulator of HIF-1. Its regulation of HIF-1 and the underlying mechanisms were investigated in human proximal tubular cells (HK-2). Furthermore, we attempted to characterize the inflammatory nature of this gene and link inflammation to the HIF response. Results: An

shRNA library experiment identified CEBPD, a transcription Org 27569 factor, as a novel HIF-1 regulator in kidney. CEBPD was induced in kidneys subjected to systemic hypoxia, as well as in models of acute and chronic hypoxic kidney injuries, with predominant expression in the nuclei of proximal tubular cells, the most susceptible portion of kidney to hypoxia. In vitro, CEBPD siRNA knockdown and overexpression mediated down- and upregulation of HIF-1α as well as its target genes. Mechanistically, promoter and chromatin immunoprecipitation (ChIP) assay confirmed that CEBPD directly promoted the transcription of HIF-1α. Notably, CEBPD was rapidly inducible by inflammatory cytokines, such as interleukin-1β, in an NF-κB-dependent manner, and was indispensable for the non-hypoxic induction of HIF-1α. Conclusion: These results demonstrate CEBPD as a novel HIF-1 regulator in kidney.

Over the next 3 months, she maintained clinical and biochemical stability. Her Prednisolone dose was weaned down to Trichostatin A purchase 10 mg by 6 months. A further biopsy at that time once again confirmed features of quiescent crescentic glomerulonephritis, without evidence of disease activity or allograft rejection. Her most recent serum creatinine, 9 months post-transplant, was 100 µmol/L. A MEDLine search was conducted using the keyword ‘ANCA’, and MESH terms ‘Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis’ and ‘kidney transplantation’. AAV is the most common cause of rapidly progressive glomerulonephritis. Since the introduction of Cyclophosphamide to the therapeutic armament, mortality

rates have improved significantly. Nevertheless, morbidity from this disease and its treatment remain significant.

Treatment may not necessarily prevent end-organ damage, especially if it is started late in the course of the illness. Indeed, in a large recent series by Lionaki et al. (n = 523), just over 25% of those who presented with AAV reached ESRD with peak serum creatinine at presentation predicting the likelihood of progressing Lumacaftor molecular weight to ESRD.1 While kidney transplantation is a viable option for those who reach ESRD, there is debate concerning the timing of transplantation and the likelihood of recurrence of disease. Currently published data are limited to case series and opinion, with the general consensus being that the risk of relapse is lower in renal transplant recipients than patients

on maintenance dialysis, Raf inhibitor presumably because of the suppressive effect of their maintenance immunosuppression on vasculitis activity. Allen et al.’s retrospective analysis of 59 patients with AAV who were treated with chronic dialysis, transplantation or both, had rates of relapse of 0.02 and 0.09 per patient per year, respectively. Patient survival rates in this study at 1 and 5 years were 74%, 40% in the dialysis group, and 100%, 84% in the transplantation group.2 The first reported renal transplant in a patient with ESRD secondary to AAV was carried out in 1972. Since that time, despite hopes that standard transplantation immunosuppression might be sufficient to prevent relapses, numerous cases have been reported commencing with that of Steinman et al. in 1980, describing a patient on maintenance Prednisone and Azathioprine who developed recurrent vasculitis 4 years after transplantation.3 Reported rates of recurrence are quite variable since then perhaps because of increased transplant immunosuppressive regimens over time. The rate of recurrence with modern immunosuppression is unclear. A pooled analysis in 1999 by Nachman et al. described a recurrence rate of 17% among 127 patients, with an average time from transplant to relapse of 31 months (range 5 days to 13 years).4 Importantly, the target antigen (MPO or proteinase 3 (Pr3)) did not affect the rate of relapse, nor did ANCA positivity at the time of transplantation.

Total RNA was extracted from 3 to 10 × 106 neutrophil cells of three groups (healthy, asymptomatic and nonhealing individuals) using RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA was eluted by addition of 30 μL RNase-free H2O (70°C). Analysis of RNA quality and quantity was carried out by an Agilent 2100 Bioanalyzer. cDNA synthesis

was performed using Omniscript Reverse Transcriptase kit (Qiagen). Real-time quantitative PCR buy Adriamycin (QRT-PCR) analyses were performed with ABI Prism 7500 Sequence Detection System (Applied Biosystems, FosterCity, CA, USA) for expression of TLR2, TLR4 and TLR9 using QuantiFast SYBR Green kit (Qiagen) and Absolute Quantification method. To determine the exact copy numbers of the target gene transcripts, the quantified and known concentrations of sub-cloned PCR fragments of TLR2, TLR4 and TLR9 were serially diluted and utilized as standards in each experiment. Threshold cycle

(CT values) for genes of interest was normalized with glyceraldehydes-3-phosphate dehydrogenase (GAPDH) expression levels as an internal control gene in each sample. The correlation coefficient of the standard curve was always >0·99 for each gene (TLR2, TLR4, TLR9 and GAPDH). The normalized relative quantities of all samples were analysed by qBase software v.1.3.5 (Ghent University, Ghent, Belgium). Crizotinib clinical trial The gene-specific primers for RT-PCR and real-time RT-PCR are listed in Table 1. Statistical analysis was performed using the nonparametric Mann–Whitney test provided Verteporfin manufacturer by the software GraphPad Prism 5 (GraphPad Software Inc, San Diego, CA, USA). Neutrophils isolated from healthy donors were incubated with ODN class A, class B and control ODN at different concentrations (2, 15 and 40 μg/mL) or with medium alone as negative control. After 18 h, the levels of IL-8, TNF-α and TGF-β were measured in supernatants (Table 2). CpG-ODN type A induced significantly higher IL-8 compared to control at concentration of 15 and 40 μg/mL (P < 0·05), whereas CpG-ODN class B was not able to

induce IL-8 over background. Both ODN class A and B were unable to induce TGF-β production. However, it was noted that low concentration of CpG-ODN type A, but not B, could contribute to suppression of TGF-β production (P < 0·05). TNF-α secretion was not induced by either CpG-ODN type A or B (Table 2). The stimulatory effects of 5, 15, 25 and 50 ng/mL of rhGM-CSF on induction of IL-8 and TGF-β in neutrophils were tested (not shown), and 50 ng/mL GM-CSF was determined as optimal for further studies. Neutrophils of healthy donors were preincubated with GM-CSF for 90 min and subsequently stimulated for 18 h with 2, 15 and 40 μg/mL CpG-ODN class A, class B or control ODN; medium alone was acting as negative control.