The macrophages appear large, polygonal with foamy eosinophillic cytoplasm PS-341 nmr – the so-called von Hansemann cell. Attempts to correct the abnormal ratio of cGMP to cyclic adenosine monophosphate (cAMP) with the cholinergic agonist bethanechol chloride and ascorbic acid have had mixed results. Due to the protean nature of presentation and histopathological findings, it is likely the disease is under-recognized. A positive result from renal biopsy may yield the correct diagnosis in only 30% of cases. The disorder
most commonly associates with recurrent E. coli infection (80% of cases), with the exception of those cases related to human immunodeficiency virus (HIV), wherein infection with Rhodococcus equi is the rule. In some cases, inciting organisms have been cultured from biopsy tissue, just as we were able to demonstrate K. pneumoniae in the bladder biopsies in our patient, despite sterile urine. This suggests that the local environment may be permissive for bacterial survival and provide a viable reservoir for the ongoing aberrant inflammatory process. Malakoplakia can present with SCH772984 infection at multiple sites but expresses particular affinity for the genitourinary tract, especially
in females, with 58% cases involving this organ system. The kidney is the predominant site of involvement in 15% of cases, but has only been reported in renal allografts on fewer than 20 occasions. In the kidney, the enlarging parenchymal nodules can sometimes be mistaken for malignancy, with the diagnosis only made following transplant nephrectomy. The gastrointestinal tract is the second most common site with a spectrum of presentations possible, from an incidental 3-oxoacyl-(acyl-carrier-protein) reductase finding to haemorrhage or obstruction. Historically, malakoplakia was associated with poor outcomes, with a 6-month mortality rate above 50%. The development of quinolone antibiotics in the 1990s, agents with high bioavailability within macrophages, has improved the outlook. Sulphonamides are similarly active against malakoplakia. However, despite the success of these agents, malakoplakia has resulted in permanent
loss of renal function through graft failure, transplant nephrectomy and salt losing nephropathy over time.[2, 5] Patients with bilateral disease tend to fare especially poorly. These cases pose a difficult question as to whether treating nephrologists should pursue repeat transplantation, given the risk of recurrence on long-term immunosuppression is unknown. However, successful outcomes with preserved renal function have been documented. In our case, and in a few recent case reports, a strategy of minimization of immunosuppressive medications and prolonged antibiotic therapy has resulted in patient and allograft survival. In particular, the use of purine synthesis inhibitors such as azathioprine or mycophenolate mofetil might relate to poor outcomes through suppression of monocyte function.
The innate immune response is critical in shaping the subsequent acquired immune response. As individuals living in endemic areas are liable to be exposed to infectious cercariae on multiple occasions during domestic, recreational, or occupational water contacts, it has been suggested that repeated exposure to E/S antigens Epigenetic Reader Domain inhibitor released by invading cercariae may modulate the host’s immune response . Indeed, in an experimental murine model, multiple infection
with S. mansoni cercariae down-modulated CD4+ T-cell responses in the skin-draining lymph nodes . Multiple infection also down-regulated the development of egg-specific responses in distant lymphoid tissues and modulated the size buy GDC-0068 of egg-induced granulomas in the liver . Therefore, human immune responsiveness to larval E/S material warrants investigation. Unfortunately, human immune responses to cercarial antigens have been infrequently investigated and have been restricted to preparations comprising the soluble fraction of whole cercariae (termed CAP or SCAP) [11-15]. This preparation is dominated by cytosolic components
recovered from the disrupted cercarial bodies and is therefore not reflective of larval E/S material. Analysis of human immune responses specifically to cercarial E/S material is unprecedented. The study presented here undertook to make an initial analysis of innate/early immune responsiveness to cercarial E/S (i.e. 0–3 h RP) in a cohort of patients from an area endemic
for schistosomiasis in northern Senegal. Specifically, the early cytokine response at 24 h of whole-blood (WB) cultures stimulated with 0–3 h RP was examined. The cytokines studied (i.e. IL-8, TNFα and IL-10) were chosen as ones typically released by innate immune cells such as macrophages and monocytes upon activation. Cytokine responses were compared Phosphatidylethanolamine N-methyltransferase between individuals who did not harbour patent schistosome infection, those infected with S. mansoni alone, and those co-infected with S. mansoni and S. haematobium to investigate whether responsiveness to larval E/S products is influenced by current infection status. We report that cercarial E/S antigens stimulated the release of greater quantities of regulatory IL-10, but not pro-inflammatory TNFα or IL-8, in participants infected with schistosomes compared with uninfected controls. This study was conducted in 2009 as part of a larger investigation (SCHISTOINIR) examining immune responses in three endemic countries , for which approval was obtained by the review board of the Institute of Tropical Medicine in Antwerp, the ethical committee of the Antwerp University Hospital and ‘Le Comité National d’Ethique de la Recherche en Santé’ in Dakar, Senegal. Informed and written consent were obtained from all participants; for children, informed consent was obtained from their parents or legal guardant.
The difference was considered statistically significant when P ≤ 0.05. Leica Microscopy system was used to take the picture, and magnification used was 40 with numerical aperture of the objectives, at temperature room. The slides were mounted using Vectashield mounting medium (Vector laboratories), and Alexa 488 fluorochrome was used to detect the positive signal (Invitrogen). As a first step, we designed recombinant adenovirus vectors containing ESAT-6 with and without calreticulin to determine whether calreticulin increased the immune response to the antigen. AdESAT-6 and AdCRT–ESAT-6 were created as described in the Materials and methods. Expression of ESAT-6 in both constructs was under the control of a cytomegalovirus promoter (Fig. 1A–C). The capacity of these constructs to express ESAT-6 was first verified by immunoblot https://www.selleckchem.com/products/gsk1120212-jtp-74057.html analyses of HEK293 cells transfected with one of the recombinant vectors (data not shown). ESAT-6 protein expression was also demonstrated by immunofluorescence analysis of HEK293 cells transfected with AdESAT-6, AdCRT-ESAT-6 or AdLacZ (Fig. 1D). As shown in Fig. 1D, only cells transfected with
AdESAT-6 and AdCRT-ESAT-6 express ESAT-6. Therefore, our recombinant adenovirus constructs were proven to be capable of producing ESAT-6. To test the ability of AdCRT–ESAT-6 or AdESAT-6 to generate ESAT-6-specific cellular immune responses in vivo, mice Selinexor molecular weight were immunized by the intranasal route with the adenovirus constructs.
At 4 weeks post-vaccination, splenocyte cultures were prepared Protein kinase N1 and restimulated with ESAT-6, and the resultant cytokine responses were analysed. It was found that while splenocytes from mice immunized with the antigen alone (AdESAT-6) showed no differences in cytokine production compared to splenocytes from LacZ-immunized mice (controls), there were significant inductions of IFN-γ and TNF-α (measured by ELISPOT and ELISA, respectively) in splenocytes from mice immunized with the antigen ESAT-6 fused to calreticulin (AdCRT–ESAT-6) (Fig. 2A,B). Taken together, these data demonstrate that immunization with ESAT-6 linked to calreticulin is an effective approach to generate potent immune responses. It has been previously shown that fusion of ESTA-6 with CFP-10 enhances the immune response. Hence, using the same strategy, we expressed a calreticulin–ESAT-6–CFP10 fusion protein (AdCRT–ESAT-6–CFP10) and compared its ability to induce a cytokine response against AdCRT–ESAT-6. The expression of the fusion protein was demonstrated by immunoblot analysis of lysates of cells transfected with the fusion vector using an anti-CFP10 polyclonal mouse antibody (Fig. 3A). While no reaction was observed in the uninfected HEK293 cell lysates, a single antibody-reactive band of approximately 90 kDa was detected in the AdCRT–ESAT-6–CFP10 cell lysates. The size of the reactive band correlated with the predicted size of the CRT-ESAT-6–CFP10 fusion protein.
The broth was incubated at 37 °C until the culture equalled 0.5 McFarland standard. A McFarland 0.5 turbidity standard corresponded to an inoculum of 1 × 108 CFU mL−1 (Acar & Goldstein, 1991). Usually, 2–8 h were required to reach this standard. A sterile cotton swab was dipped in the inoculum and the excess was removed by rotating
the swab several times against the inside wall of the tube above the level of the fluid. Mueller–Hinton agar (MHA) medium supplemented with 2% NaCl was used for this study. The surface of this plate was inoculated by streaking the swab cancer metabolism inhibitor over the surface. Streaking was repeated three times and each time the plate was rotated 60°. The antibiotic disks of methicillin
(10 μg), penicillin (10 U) and vancomycin (30 μg) were applied with forceps. To ensure complete contact of the disks with the agar surface, the disks were pressed down with a slight pressure. Inoculated plates were inverted and incubated at 37 °C for 18 h. After the incubation period, the diameter of zone of inhibition was measured and results were interpreted according to the standards of Clinical and Laboratory Standards Institute (2005). For the preliminary screening, the paper disk diffusion method was used to determine the antimicrobial activity of endophytic fungal extract Saracatinib molecular weight (Acar & Goldstein, 1996). Sterile disks (6 mm) were impregnated with 10 μL of extract at a concentration of 1 mg mL−1. For bacteria, the microorganisms were swabbed on the surface of MHA; for fungi, PDA was used. Tetracycline 10 μg per disk for Gram-positive bacteria, chloramphenicol 10 μg per disk for Gram-negative bacteria and nystatin 10 U per disk for fungi were used as a positive controls. Paper disks treated with 10% Dipeptidyl peptidase DMSO were used as negative controls. The plates were incubated at 37 °C for 18 and 48 h
for bacteria and fungi, respectively. The diameter of the inhibition zone around each disk was measured at the end of the incubation time. Experiments were performed in triplicate and the antimicrobial activity was expressed as the average of inhibition zone diameters (in mm) produced by the endophytic fungal extract. MIC of methanol extract was determined based on a broth microdilution method in a 96-well microplate (Al-Bayati, 2008). Briefly, S. aureus strains (1–10) were cultured overnight at 37 °C on Mueller–Hinton (MH) broth and adjusted to a final density of 108 CFU mL−1 by 0.5 McFarland standards. The methanol extract (1 mg mL−1) was dissolved in DMSO, and twofold serial dilutions were made in the concentration range from 7.8 to 1000 μg mL−1. In the 96-well plate, each well had 90 μL of MH broth supplemented with 2% NaCl, 10 μL of bacterial inoculum and 10 μL of different concentrations of fungal extract. The plate was incubated at 37 °C for 18 h.
HESNs were defined collectively as individuals lacking anti-HIV-1 IgG seropositivity
or evidence of infection despite frequent exposure to HIV-1 and/or repeated high-risk behaviour in areas with high HIV-1 prevalence. The seronegative description addresses the possibility that some HESN subjects may have mucosal immunoglobulin (Ig)A responses to HIV-1, but by definition all HESN subjects must be anti-HIV-1 IgG seronegative and are often also tested for the presence of HIV-1 by ultra-sensitive polymerase chain reaction (PCR). In terms of documenting exposure to HIV-1, studies of HIV-1 discordant couples and haemophiliacs have had the advantage of known exposures to quantifiable amounts of HIV-1 . Nevertheless, studies of commercial sex workers SAHA HDAC clinical trial and i.v. drug users have inferred exposure to HIV-1 based upon mathematical models of the frequency of high-risk activity and the prevalence of HIV-1 in the community being studied [1,18,22]. Throughout this review, we will compare and contrast the evidence for adaptive and innate responses as correlates of resistance in high-risk HESN subjects. We will also explore how mechanism(s) of innate resistance to HIV-1 in HESN subjects intersect or differ with mechanisms
of control over HIV-1 https://www.selleckchem.com/products/nu7441.html replication during chronic infection. Since the first identification of HIV-specific T cell responses in HESN subjects , HIV-specific T cell responses have been identified in a number of high-risk uninfected individuals from multiple cohorts [3–5,14,24]. Subsequent reports confirmed the presence of antigen-specific T cell responses to HIV-1 in HESN subjects while characterizing the functional and proliferative capacity of HIV-specific T cells in these subjects [7,25–27]. Genetically, both major histocompatibility complex (MHC) class I  and human leucocyte
antigen (HLA) class II  alleles have been associated with a reduced risk of infection with HIV-1. In terms of protection, the anti-viral mechanisms utilized by T cells against HIV-1 may come in the form of direct lysis of virally C59 infected cells or through the secretion of anti-viral factors such as chemokines/cytokines or other CD8 non-cytolytic anti-viral factors (CNAR) . Together with the description of anti-HIV-specific responses in HIV-infected long-term non-progressor subjects controlling viral replication [31,32], these findings raised hope that the generation of antigen-specific T cell immune responses to HIV-1 following high-risk contact could result in protection from HIV-1 in subsequent exposures.
Recently, several reports have suggested that the amount of mitochondria in mature cells
may be, in part, controlled by autophagy, a process usually inhibited by mTOR activity 23–25. Because of the altered mTOR activity in TSC1KO T cells, we sought to determine whether TSC1-deficiency in T cells might deregulate the normal induction of autophagy. Using the colocalization of LC3 molecules within a cell GSK-3 inhibition as a readout of the induction of autophagy 26, we observed a slight increase in autophagy in TSC1KO T cells in a nutrient-sufficient environment compared with WT T cells. When starved, autophagy in both WT and TSC1KO T cells was increased. However, there was no obvious difference between these two types of cells (Fig. 4C and D). Thus, in the TSC1 deficiency setting,
increased mTORC1 activity does not inhibit autophagy. Further studies are needed to understand mechanisms that may counter-balance with mTORC1 signaling to regulate autophagy in TSC1KO T cells. ROS is a byproduct of mitochondrial energy production and is toxic to T cells in excess amounts 27. Although mitochondrial content is reduced in TSC1KO T cells, they produced elevated amounts of ROS, ABT 199 which correlated to their positive staining for dead cells (Fig. 4E). The fluorescent dye DiOC6 has been utilized to measure mitochondrial potential. Its dilution is indicative of loss of mitochondrial membrane potential, a precursor to membrane permeabilization 28. Both CD4+ and CD8+ TSC1KO T cells displayed diluted DiOC6 staining indicating decreased mitochondrial membrane potential
and increased mitochondrial membrane permeabilization in these cells (Fig. 4F). An increase in mitochondrial membrane permeability can result in the release of cytochrome C Oxymatrine to the cytosol to trigger the activation of the intrinsic cell death pathway 22. Increased cleaved caspase-9 (initiator caspase) and caspase-3 (effector caspase) were detected in TSC1KO T cells before and after anti-CD3 stimulation as compared with WT T cells, demonstrating activation of the intrinsic cell death pathway in TSC1KO T cells (Fig. 4G). Thus, TSC1 has a pro-survival function in T cells by maintaining mitochondrial membrane integrity and preventing the activation of the intrinsic death pathway. To investigate the mechanisms that promote death in TSC1KO T cells, we measured expression of several key pro-apoptotic and pro-survival proteins. No obvious decreases in pro-survival molecules, Bcl-2, Bcl-XL, Mcl-1, or increases in pro-apoptotic proteins, Bim, Puma, Bid, or Bax were observed in TSC1KO T cells (Fig. 5A). Noxa, another pro-apoptotic molecule was actually decreased in TSC1KO T cells. Whether the decreased Noxa expression contributes to TSC1KO T-cell death remains to be investigated. Akt is downstream of both PI3K and mTORC2, and plays critical roles for cell survival. mTORC2 phosphorylates Akt at serine 473 (S473) to promote Akt activation 29.
The IgG is
then released into the fetal circulation.4 The FcRn is also expressed on the intestinal epithelium and mediates the transepithelial transfer of the IgG1 present in the maternal milk to the circulation of the progeny.5 Transplacentally acquired maternal IgG is important for protection of infants in the early KPT-330 price months of life from bacterial or viral infections. The transfer of maternal antigen-specific IgG has also been shown to influence antigen-specific immune responses later in the life of the progeny. Hence, the transfer of maternal IgG bearing a κ light chain to κ-light-chain-deficient fetuses has been shown to alter in an antigen-dependent manner the repertoires of T lymphocytes.6 Further, the transfer of maternal anti-idiotypic IgG directed against anti-phosphorylcholine (PC) antibodies has been shown to skew the repertoires of PC-specific B lymphocytes after immunization of the offspring with PC later in life.7 In addition, the passive transfer
of maternal IgG during pregnancy has been occasionally shown to impair vaccination in early infancy, probably as the result of the neutralization of the immunogen by the transferred IgG.8 Here, using a mouse model of haemophilia Selleckchem IWR 1 A, we investigated whether maternal anti-FVIII IgG transferred during the ontogeny of the immune system of the progeny may modulate the capacity to develop an anti-FVIII immune response later in adulthood. Mice were 7- to 10-week-old inbred 129 × C57BL/6 (H-2Db background) exon 16 FVIII-deficient males and females (a gift from Prof. Kazazian, University of Pennsylvania School of Medicine, Philadelphia). Animals were handled in agreement with local ethical authorities (Comité regional d’éthique p3/2008/024). Mice were administered human recombinant FVIII (1 IU; Helixate®, CSL-Behring, Marburg, Germany) diluted in phosphate-buffered saline (PBS) or PBS only by retro-orbital intravenous injection once a week for up to 6 weeks. Alternatively, mice were immunized by a subcutaneous injection of ovalbumin
(OVA, 50 μg, grade VII; Sigma, St Louis, MO) in complete Freund’s adjuvant Sirolimus ic50 followed by two injections of OVA (50 μg) in incomplete Freund’s adjuvant with a weekly interval. Blood was collected by retro-orbital puncture 5 days after each administration of FVIII or the last immunization with OVA. Serum was kept at − 20° until use. Groups of five to seven mice were used in each set of experiments. Plates for enzyme-linked immunosorbent assay (ELISA; Nunc, Roskilde, Denmark) were coated with rFVIII (2 μg/ml; Recombinate®, Baxter, Maurepas, France) or with OVA (2 μg/ml, grade V; Sigma) overnight at 4°, and blocked with PBS, 1% bovine serum albumin or with PBS, 1% milk, respectively. Serum dilutions were then incubated for 1 hr at 37°. Bound IgG was revealed using a horseradish peroxidase-coupled monoclonal anti-mouse IgG (Southern Biotech, Anaheim, CA, USA) and substrate.
Amplicons were detected by electrophoresis (Bio-Rad) on a 2% agarose gel (NuSieve, Rockland, ME). Four sets of 24 species-specific primers were designed based on the rRNA gene ITS region of P. marneffeiSUMS0152 (AB353913) (Liu et al., 2007; Xi et al., 2007) using primerexplorer v4 software (http://primerexplorer.jp). A set of six species-specific LAMP primers was selected as follows: forward outer primer (F3): CCG AGC GTC ATT TCT GCC, reverse outer (B3): AGT TCA GCG GGT AAC TCC T, forward inner primer (FIP): TCG AGG ACC AGA CGG ACG TCT TTT TCA AGC ACG GCT TGT GTG, reverse inner (BIP): TAT GGG GCT CTG TCA CTC
GCT CTT TTA CCT GAT CCG AGG TCA selleck screening library ACC, loop forward (LF): GTT GGT CAC CAC CAT ATT TAC CA and loop reverse (LB): TGC CTT TCG GGC AGG TC. LAMP was performed in 25-μL reaction volumes containing 0.25 μM of F3 and B3 each, 1.0 μM of FIP and BIP each, 0.5 μM of LF and LB each, 1.0 mM dNTPs, 1 M betaine (Sigma), 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 4 mM MgSO4, 0.1% Triton X-100 and 8 U of Bst DNA large
fragment polymerase (New England Biolabs), with 2 μL of crude DNA extract as the template. The reaction mixture, except Bst DNA polymerase, was denatured at 95 °C for 5 min and cooled on ice, followed by the addition of 1 μL Bst polymerase and incubation at 65 °C in KU-57788 supplier a water bath for 60 min and final heating at 85 °C for 2 min to terminate the reaction. DNAs of 40 P. marneffei and 46 reference strains were used as templates to evaluate the specificity of the LAMP assay. DNA of strain SUMS0152 was used as a positive control; reaction mixtures without P. marneffei DNA, i.e. healthy human skin DNA, healthy bamboo rat DNA and DNAs from Penicillium purpurogenum, Penicillium funiculosum and other biverticillate penicillia taxonomically close to P. marneffei were used as negative controls. A recombinant plasmid (pT-IT12) was constructed as a template for establishing the detection limit of the LAMP assay. The ITS region of P. marneffei (603 bp) was amplified from SUMS0152 HAS1 genomic DNA using primers ITS4 and ITS5 and subcloned into the
pGEM-T Easy vector (Promega) according to the manufacturer’s instructions. Detection limits were evaluated using 10-fold serial dilutions of plasmid pT-IT12. The plasmid DNA (0.32 μg μL−1, equivalent to 8.067 × 1010 copies μL−1) was 10-fold serially diluted and 2 μL of each dilution was used as a template for the LAMP reaction. DNA of P. marneffeiSUMS0152 was used as a positive control; the reaction mixture without DNA was used as a negative control. To evaluate the inhibition of nontarget DNA in the LAMP assay, 2 μL crude DNA extract each of P. marneffei was added to the LAMP-negative samples, and then tested by LAMP again. Amplified products were analyzed by electrophoresis on 1% agarose gels, stained with ethidium bromide and photographed. A 100-bp DNA ladder was used as the molecular weight standard. LAMP reaction products were made visible by the addition of 2.
In addition to that, a stretch of sequence upstream of the primate CLEC9A coding region shows high homology to CLEC-2. Therefore, we hypothesize that this inversion took place after a partial duplication check details of the gene encoding CLEC-2 in the genome of a common primate ancestor. The additional genes CLEC9A and CLEC12B show
all typical characteristics of C-type lectin-like genes as far as amino acid sequences, exon–intron structure and corresponding protein domains are concerned. CLEC9A is unusual as far as it contains three non-coding upstream exons, probably originating from duplication of part of the CLEC-2 gene. CLEC12B has been reported recently to function as inhibitory receptor in macrophages by recruiting the phosphatases SHP-1 and SHP-2 through its immunoreceptor tyrosine-based inhibition motif (ITIM) . Our analysis found CLEC12B to be differentially spliced. In addition to mRNA coding for a regular lectin-like protein, three additional splice variants were identified resulting from two independent alternative splicing events. All these differential splicing CX 5461 events lead to truncations and probably non-functional proteins. Alternatively spliced isoforms have been described for other receptors of this complex. In particular, mature mRNA
of DECTIN-1 and CD94 have been demonstrated to be generated by multiple splicing events leading to various isoforms, some of which code for truncated and potentially non-functional proteins [43–45]. Moreover, functional isoforms lacking the stalk exon of NKG2A, known as NKG2B, DECTIN-1 and CD94 have been shown to be expressed [43, 45, 46]. Curiously, in the case of CLEC12B these truncated mRNA that probably encode non-functional proteins constitute the majority of transcripts in most cell types why tested. It is however possible that mRNA coding for full-length CLEC12B are transcribed only in certain cell types or upon certain kinds of stimulation not tested in this study. Because both CLEC12B and CLEC9A share all major characteristics with
the other lectin-like receptors encoded by genes of the myeloid cluster, it is possible that these proteins fulfill similar functions. However, the pattern of expression of these two genes shows some differences when compared to the other members of the myeloid subfamily. CLEC9A expression was recently described to be present on BDCA3+ DC and on a small subset of CD14+ CD16− monocytes . Although in our hands CLEC12B and CLEC9A are expressed in cells of the myeloid lineage similar to CLEC-1, CLEC-2 and DECTIN-1, highest expression was detected in the T-cell line CCRF-CEM. Moreover, neither CLEC12B nor CLEC9A expression is significantly downregulated upon stimulation of DC using different stimuli, a feature common to other C-type lectin-like receptors of the myeloid subfamily.
analysis was conducted using ImageJ software (NIH). Student’s unpaired t-test was used to measure statistical significance between two groups and one-way ANOVA with Dunnet’s multiple comparison test was used to determine statistical significance between multiple groups against WT control. All statistical analyses were performed by Prism 5 (Graphpad Software). We thank Dr. Clifford Lowell for providing Itgb2−/− mice and Dr. Hua Gu and Dr. Phil Greenberg for providing Cblb−/− mice. We would also like to acknowledge Dr. Amy Weinmann for advice on chromatin immunoprecipitation MK-8669 solubility dmso and thank members of our laboratory for helpful discussions and review of the manuscript. This work was supported by NIH grants R01AI073441 and R01AI081948, an Investigator Award from the Cancer Research Institute, a pilot award from the Alliance for Lupus Research and DOD grant W81XWH-10-1-0149 (to J.A.H). N.Y. was supported in part by NIH training grant 5T32CA09537. The authors declare no financial or commercial conflict of interest. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.
Figure S1. Phenotypic characterization of Itgb2-/- macrophages. (A) The expression of integrin alpha subunits, CD11a, CD11b, CD11c and F4/80 was determined on bone marrow-derived macrophages by flow cytometry. Selleck AZD9291 (B) Macrophages were stimulated with the indicated concentrations of IFNγ for 48 hours and MHC II expression was assessed by flow cytometry. (C) Macrophage surface expression of TLR4, TLR2 and Dectin-1 was determined by flow cytometry. (D) TLR9 mRNA expression was determined
by qPCR, with levels normalized to GAPDH. The data are shown as mean +/- SD of triplicate wells and representative of 3 experiments.! Figure S2. Itgb2-/- macrophages are GNA12 hypersensitive to TLR stimulation. (A) Representative data of the results shown in Fig. 1A. Macrophages were stimulated with the indicated TLR agonists and supernatant cytokine concentrations were determined by ELISA 24 hours later. Results are displayed as mean +/- SD of independently stimulated wells from one experiment. (B) Expression of IL-23 p19 and IL-12 p35 was determined by qPCR, with values normalized to GAPDH. Results are representative of 2 experiments and shown as mean +/- SD of triplicate wells. (C) Representative data of the results shown in Fig. 1B. Kinetics of cytokine secretion as assessed by ELISA. Results are shown as mean +/- SD independently stimulated triplicate wells from one experiment. * p < 0.05, ** p < 0.01, *** p < 0.001! Figure S3. Isolation of thioglycollate-elicited macrophages (A) Mice were injected i.p.