Thereafter, genomic sequencing of the non-O1, non-O139 V. cholerae strain AM-19226 revealed that V. cholerae carry T3SS genes related to V. parahaemolyticus T3SS2 in VPI-2 [8]. Additionally, in the infant mouse model T3SS in V. cholerae is needed for efficient intestinal colonization; the effector proteins have

already been characterized [9-11]. Therefore, in addition to CT, T3SS in V. cholerae is another possible virulence determinant. The T3SS gene cluster is distributed among various non-O1, non-O139 strains [8, 12] and a phylogenetic analysis of T3SS-related genes implied horizontal gene transfer of a T3SS gene cluster among Vibrio species [13, 14]. Up to now, however, there has been no experimental evidence of horizontal transfer of the T3SS-related genes. We herein examined the distribution of T3SS-related genes among various serogroups of V. cholerae isolates and found that the cassette selleck chemical of T3SS-related genes was transferrable among V. cholerae isolates by transformation, and that these subsequently integrated into a VPI-2. V. cholerae strains used in this study were isolated from natural surface water (environmental; 110 isolates) and diarrhea patients (clinical;

14 isolates) in Bangladesh. These V. cholerae isolates were obtained from the culture collection GDC-941 of the International Center for Diarrhoeal Disease Research, Bangladesh. All 124 isolates, which were primarily confirmed as cholera toxin gene (ctxAB) negative V. cholerae serogroups non-O1/non-O139, were screened by PCR assays with three sets of primer pairs (T3SS-1, T3SS-2 and T3SS-3; Table 1) to detect T3SS-related genes. The primer pair of T3SS-1 amplified a target gene of A33_1670, which encodes structural protein. The primer pairs of T3SS-2 and T3SS-3 targeted genes for translocated effector proteins of A33_1684 and A33_1697, respectively. All primers were designed in the conserved sequence of each gene. The PCR conditions were as follows: after initial denaturation at 95°C for 2 mins,

25 cycles of denaturation at 95°C for 10 s, annealing at 55°C for 20 s and extension at 72°C for 1 mins; and final extension at 72°C Edoxaban for 3 mins with TaKaRa Ex Taq (Takara Bio, Shiga, Japan). The amplified fragments were detected by agarose gel electrophoresis after staining with ethidium bromide. Strains producing the three amplicons from the three primer pairs were defined as positive for T3SS-related genes. Subsequently, strains positive for T3SS-related genes were serogrouped by slide agglutination using a panel of specific antisera for each serogroup of V. cholerae. To evaluate the genetic similarity between T3SS-related gene regions, a PCR-RFLP analysis was performed with the positive strains identified as described above. Because the long length of the whole locus precluded its amplification with one primer pair, it was divided it into seven regions (ca. 5–10 kb) to ensure successful amplification with seven sets of primer pairs (RFLP-1 to RFLP-7; Table 1).

Background: MK is a novel cytokine, which is pathologically

implicated in a number of inflammatory disease processes including kidney disease. It has potential as both a biomarker and a biological therapeutic target in acute and CKD. To date there is little data on MK levels in humans with CKD. Method: This is a prospective, observational study. Plasma, serum and urine samples were simultaneously obtained from CKD outpatients and healthy 3Methyladenine volunteers (HV), stored at −70°C, and assayed for MK levels using a commercially available MK-ELISA kit (Cellmid Ltd, Sydney, Australia). MK levels were compared between 2 severity groups, divided as HV and stage 1–2, compared with a second group of stage 3–5. Result: Samples were obtained from 20 HV and 126 CKD patients. Serum MK levels were significantly higher in the CKD stage 3–5 group than the HV or CKD 1–2 group (3009 (SD = 1942) vs 870 pg/mL (SD = 384) P < 0.001). Urine MK levels were significantly higher in the CKD stage 3–5 group than the HV or CKD 1–2 group (6008 (SD = 13462) vs 654 pg/mL (SD = 1517) P ≤ 0.001). Conclusion: Serum and urine Midkine levels are elevated in stage 3–5 CKD patients compared to non-CKD or lesser stages 1–2. Whether this is association, or reflecting

part of the pathological process Talazoparib ic50 requires further exploration. 161 MIDKINE LEVELS CAN BE MEASURED IN EITHER PLASMA OR SERUM V CAMPBELL1,2,3, NA GRAY1,3, C ANSTEY2,3, R GATELY1, C CLARK1,2, E NOBLE1, K MAHADEVAN1,2, PR HOLLETT1,2, A POLLOCK1, D JONES4, S HALL5 1Renal Unit, Nambour General Hospital, Nambour, Queensland; 2Sunshine Coast Clinical School, The University of Queensland, Nambour, Queensland; 3Intensive Care Unit, Nambour General hospital, Nambour, Queensland, Australia; 4Cellmid Ltd; 5Pathology North – Hunter New England Aim: To compare Midkine Phosphoprotein phosphatase (MK) levels when measured in plasma and serum. Background: Midkine is a novel cytokine, which is pathologically implicated in a number of inflammatory and malignant disease processes. Levels have usually been measured in serum, however protein assays can be performed on either plasma or serum. Because of the increasing number of both

serum and plasma banks being stored as part of large clinical trials, validating the assay in both sample types would allow further investigation of this cytokine. Methods: Plasma and serum samples were simultaneously obtained from chronic kidney disease (CKD) outpatients and healthy volunteers (HV), stored at −70°C, and assayed for MK levels using a commercially available MK-ELISA kit (Cellmid Ltd, Sydney, Australia). Data were analysed using multivariate linear regression. Results: Samples were obtained from 20 HV and 126 CKD patients. The causes of CKD included 26% diabetes, 37% hypertension/vascular, 9% glomerulonephritis, 5% polycystic disease, and 24% other. The CKD stages ranged from 1–5, with the majority being stage 3–4.

After initial T-cell–DC contacts, T cells migrate again and sample several other DCs. However, T-cell migration is diminished appreciably in the presence of an antigen with high affinity for a given TCR that elicits a relatively strong Ca2+ signal in T cells. The continued use of intracellular dyes that change their fluorescence properties upon binding to Ca2+ will advance our investigation of this crucial role of Ca2+ signalling in T-cell migration and antigen recognition. Hence, 2P microscopy coupled with the quantification of intracellular Ca2+ signalling by T cells activated by different antigens in vivo can be informative GSI-IX mouse about the relative strength of T-cell–DC interactions

and the immune responses that follow under conditions of health and disease. The relative strength of TCR signalling in vivo can also be measured

by following the shedding of CD62L from the surface of T cells.[93] A few minutes after TCR activation in a T cell, the CD62L extracellular domain is cleaved by the protease ADAM17 (a disintegrin and metalloproteinase domain-containing protein 17). The extent of CD62L shedding reflects TCR signal strength, i.e. a strong TCR signal elicits increased shedding of CD62L. Hence, T-cell dynamics in vivo may be tracked together with TCR signals by measuring the disappearance of CD62L after in vivo staining with fluorescent anti-CD62 antibody Fab fragments. The functional role of NKT cells has been analysed in mice selleck using CD1d−/− (lack both type I and type II NKT cells) and Jα18−/− (lack only type I NKT cells) mice as well as using blocking or depleting antibodies reactive to CD1d and the semi-invariant TCR. The combined use of both of these mouse strains and antibodies has allowed us to ascribe the outcome of specific immune responses to the effect of either type I NKT cells or type old II NKT cells. However, various compensating

mechanisms, such as an altered conventional TCR repertoire, may control NKT cell function in such knockout mouse environments. Our understanding of the roles of NKT cells in the induction and/or protection from autoimmune disease has taken advantage of analyses of NKT cells in such diseases that either arise spontaneously or are antigen-induced (Table 4). It is important to note while αGalCer has been informative about type I NKT cell activation and function, it has not revealed a comprehensive understanding of the physiological role of type I NKT cells. A role for type I NKT cells in the regulation of autoimmune disease was provided by observations that fewer type I NKT cells are found in both spontaneous autoimmune disease models, type 1 diabetes in NOD mice and systemic lupus erythematosus in MRL/lpr mice.[94, 95] However, CD1d deficiency did not result in potentiation of disease, as expected in all models.

MHC class II molecules are functionally dedicated to the presentation of exogenous antigens internalized by DC receptors and processed into endosomal/lysosomal compartments

(46). This function requires the integrity of a class Erlotinib ic50 II molecule biosynthesis process and the formation of MHC class II (I-a)–peptide complexes. These molecular events occurred following a cascade of reactions involving (CIITA, li, H-2Ma and Cat-S) molecules acting at different compartment (organelles) of DCs (14,47). We observed that a down-regulation of the relative mRNA levels of molecules (CIITA, li, H-2Ma and Cat-S) implicated in the pathway used by MHC class II (I-a) molecules, corroborated with the reduced expression level of (I-a)-β on pe-DCs from AE-infected mice. The down-regulation of CIITA, the key molecule that initiate (I-a) gene expression, might be attributed to the high level of TGF-β expressed either by AE-pe-DCs or by CD4+ pe-T Venetoclax supplier cells. Others have found that TGF-β attenuates CIITA gene expression and consequently inhibits HLA-DRA expression (48). The invariant chain that binds to newly synthesized MHC class II α/β heterodimers in the endoplasmic reticulum prevented their premature association

with endogenous polypeptides, assisted in their folding and intracellular moving to endosomal/lysosomal compartments (49). In our study, the relative level of li expression was found to be significantly decreased, which may have as consequence a reduction in the amount of MHC class II (I-a)–li complexes within endosomal/lysosomal compartments. It had been demonstrated that the invariant chain might be degraded by noncysteine proteases and cysteine for proteases including Cat-S that has a critical role in the late stage of li degradation, leading to the formation of MHC class II–CLIP complex in B cells, DCs and to a lesser degree in macrophages (50).

Thereafter, CLIP is dislodged, leading to the loading of the antigenic peptides and the formation of MHC class II (I-a)–peptide complexes. However, Cat-S alone can also degrade full-length li in vitro (51). In our work, the relative Cat-S expression level in AE-pe-DCs was significantly down-regulated. In vivo Cat-S proteolytic effects take place in endosomal/lysosomal compartments, rich in antigenic peptides and H-2 m molecules (52). The class II-like molecule, H-2M, which uniquely resides in endosomal/lysosomal compartments, was shown to catalyse the exchange of antigenic peptides following the high dissociation rate of CLIP (53). It acts also as chaperon preventing isolated empty class II dimers from unfolding or aggregation at low pH (54). We showed that the relative H-2M expression level was decreased in pe-DCs of AE-infected mice in comparison with naive pe-DCs. The consequence of H-2M deficiency includes a profound defect in the presentation of exogenous antigens (55).

, manuscript selleck chemical in preparation). We and Berlier et al.72 have demonstrated that SP also induces the expression of CCL20, a key chemotactic factor involved in recruitment and maturation of Langerhans cells and dendritic cells, which, together with intraepithelial T lymphocytes, are considered to be the first target cells for HIV genital mucosal infection.73–75 A common gene overexpressed in pathological conditions involving mucosal inflammation is cyclooxygenase (COX)-2. Semen exposure leads to overexpression of COX-2

in pig and mare endometrium.76,77 COX-2 catalyzes the rate-limiting step in the synthesis of prostaglandins from arachidonic acid.78 Prostaglandins are considered to be important biological modulators of inflammation. They attract immune cells to the area of inflammation. They also act in an autocrine/paracrine manner to elevate COX-2 expression.79,80 Seminal plasma contains 1000-fold higher concentration of prostaglandins, mainly PGE2, compared to normal endometrium.81 Seminal plasma PGE2 has been reported to induce

COX-2 in immortalized human endocervical cells.82 This induction is because learn more of the intracellular activation of cAMP pathway via PGE2 receptor subtypes, EP2 and EP4. Our laboratory has demonstrated that SP also induces COX-2 in human vaginal cells (Joseph et al., manuscript in preparation). Furthermore, it potentiates COX-2 induction by microbial products such as bacterial lipopeptides (Fig. 1). This enhanced expression of COX-2 could be one of the main causes of inflammation associated with STIs and CV infections. In addition, SP has been shown

to activate multiple signal transduction pathways, which are involved in inflammatory responses. In cervical cells, SP induces the phosphorylation of extracellular signal-regulated kinase (ERK1/2) via EP4 receptor.83 In endometrial cells, SP induces the phosphorylation of c-Src, ERK, and activation of cAMP pathway via EP2 receptor.84 SP has also been shown to activate NF-kB signaling pathway in vaginal cells. This pathway is considered central to inflammation and is involved in the control of numerous proinflammatory genes including COX-2 and multiple chemokines and Tolmetin cytokines. NF-kB activation has also been linked to the enhancement of HIV replication.11 The role of semen in HIV-1 transmission is defined by a complex array of factors and processes involved in semen, virus, and female genital tract interactions. Semen carries CF and CA virus and is believed to be the main vector for HIV-1 in male-to-female sexual transmission. Seminal viral load varies with multiple factors such as stage of infection and disease in the male, presence of reproductive tract inflammation, and whether or not the man is on antiretroviral therapy. However, semen is more than a carrier for HIV.

Twenty-four patients were enrolled. Following a 4-week run in period, patients were randomized

into two groups. They were assigned to receive dialysis using either the second generation high-flux dialyzer or to continue on low-flux dialyzers for 12 week period. Data on serum phosphorus, calcium, haemoglobin and albumin were collected at baseline and after 12 weeks. The statistical analysis was www.selleckchem.com/JAK.html done on the normally distributed data by SPSS version 17 using the t test for equality of means. Results: At 12 weeks, there was no significant difference in serum phosphate reduction between high flux and low flux dialyzers (P = 0.88). The mean serum phosphate in the high flux- was 7.05 ± 1.59 g/dl at baseline and 5.73 ± 1.20 g/dl RAD001 manufacturer at study termination. While in the low-flux dialysis group it was 7.14 ± 1.15 g/dl at baseline and 5.70 ± 1.05 g/dl at the end of study. The same held true with haemoglobin (P = 0.47) and albumin (P = 0.39). Conclusion: The second generation high flux dialyzers did not reveal an increased phosphate clearance as compared to low flux dialyzers in the short term in this study. CHOI SU JIN, KIM YOUNG SOO, YOON SUN AE, KIM YOUNG OK Uijeongbu St. Mary’s Hospital

Introduction: Vascular calcification, which is independent risk factor of cardiovascular mortality, and anemia are very common in hemodialysis (HD) patients. Some uremic milieu such as inflammation, oxidative stress, and mineral bone disturbance may contribute to these conditions. Non-specific serine/threonine protein kinase The aim of this study was to evaluate the relationship between arterial micro-calcification (AMC)

and ESA hypo-responsiveness in hemodialysis (HD) patients. Methods: Eighty-four patients received with ESAs for anemia without iron deficiency were evaluated. We assessed ESA hypo-responsiveness of patients using ESA hypo-responsiveness index (EHRI), defined as the weekly ESA dose per kilogram of body weight divided by the hemoglobin level. The AMC was diagnosed by pathologic examination of arterial specimen by von Kossa stain, which was acquired during the vascular access surgery. Results: AMC was detected in 35 (41.7%) patients. There were no significant differences between patients with and without AMC with respect to clinical characteristics except for age and the presence of diabetes, including sex, body mass index, HD duration, and medications with phosphate binder and vitamin D. Among the 35 patients with AMC, 28 (80.0%) patients had diabetes compared with 16 (32.7%) of 49 patients without AMC (p = 0.001). The following laboratory values did not differ between two groups: hemoglobin, iron, ferritin, transferrin saturation, C-reactive protein, triglyceride, alkaline phosphatase, and calcium. The serum levels of albumin and total cholesterol were higher in patients without AMC than in patients with AMC (p = 0.048 and 0.014).

Thus, these studies suggest that the overall

B cell compartment and its functions are suppressed www.selleckchem.com/products/Tipifarnib(R115777).html partially during normal human pregnancy. The full biological significance of such suppression is unclear, but is believed to enable immune tolerance. Aberrant B cell numbers and functions are associated with obstetric complications [42-59]. Earlier studies have shown that complicated pregnancies exhibit an abnormal increase in the frequencies or absolute numbers of circulating maternal B cells (Table 1). For instance, CD5+ B cell counts are significantly higher in patients with anti-phospholipid syndrome (APS) and recurrent spontaneous abortion (RSA) groups than in healthy controls [43, 45-50]. This B cell subset is also increased in placental tissues of RSA patients [50]. The absolute number and percentages of CD19+ B cells are also increased in pregnancy complications Cabozantinib manufacturer [43, 51-59], and a higher number of CD19+IgD+ B cell numbers are observed in APS mothers with associated risks of thrombotic events [42]. Increases in B cell activation markers and functions have also been reported in pre-eclampsia, intrauterine growth

restriction (IUGR) and pregnancy-induced hypertension (PIH) cases in human studies [52, 58, 60, 61]. Collectively, these studies present the evidence of an association between human pregnancy complications and an abnormal increase in B cell-activated functions and/or numbers. It is not exactly clear what causes these anomalies in the B cell compartment of adverse pregnancies, and whether they simply represent an exacerbation of the pre-existing autoimmune conditions of the mother

that is triggered by the physiological state of pregnancy. Under normal conditions, B lymphopoiesis is suppressed and autoreactive B cells are deleted during pregnancy to maintain maternal–fetal immune tolerance [25-27]. However, these normal regulatory mechanisms are impaired in autoimmunity leading to the expansion of autoreactive B cell subsets and deleterious autoantibody production. This notion is supported strongly by observations of an abnormally increased number of CD19+CD5+, mature CD19+CD27+ and CD19+IgD+ B cells in a number of obstetric conditions (Table 1). Indeed, Olopatadine these B cell subsets are well-known producers of autoantibodies such as rheumatoid factors, anti-thyroid, anti-ssDNA, anti-histone and anti-phospholipid autoantibodies [14, 43, 48, 62-65]. In particular, the autoantibody-producing CD19+CD5+ B cell populations, which possibly include both human B1-like or activated B2 cells, are often expanded in autoimmune conditions such as APS, systemic lupus erythematosus (SLE) and primary Sjögren’s syndrome [43, 65, 66], which are often exacerbated by pregnancy and linked strongly to risks of obstetric complications [9, 10]. Thus, the strong link between CD19+CD5+ B cells and autoimmunity make them a prime candidate for further investigation in pregnancy conditions.

While these data suggest a potential utility of testing for the HPV DNA and antibody status before vaccinating older women who have already initiated sexual contacts [61],

current guidelines do not recommend screening with HPV testing because very few women have Romidepsin cell line been exposed to all types in the vaccine, and protection against other vaccine types is not affected by the presence of infection with one vaccine type. Moreover, there is no evidence of clinical utility for HPV genotyping at young ages (<25 years), as nearly all HPV infections will clear spontaneously and unnecessary HPV testing could generate over-diagnosis and treatment [62,63]. Immunization of males.  Immunization of boys with VLPs elicits a serum immune response similar to that in girls. Because genital HPV infection is sexually transmitted, immunization of men may help to prevent infection of women. Modelling studies on herd immunity, i.e. indirect protection of those who remain susceptible, owing to a reduced prevalence of infections in the risk group for disease, have been published this website [64–66]. The utility of immunization of males depends upon the assumed population coverage of vaccination, with successively smaller additional benefits seen in scenarios with high population coverage [67]. Modelling of programmes with high population coverage (90%) have found that addition of male vaccination gives a more rapid infection control

and have suggested that both sex vaccination programmes may be required to achieve an ultimate eradication of the infection [60]. Vaccination programme strategies as a randomized health-care policy.  Design of HPV vaccination programmes has been based upon estimations of the impact of HPV vaccination on the burden of cervical cancer incidence and mortality using mathematical modelling of projected effects from the observed surrogate endpoint effects [59,67,68]. Whereas

clinical end-points are essential for estimates of effects on health economy, the control of HPV infections is a more immediately relevant ifenprodil end-point in models that compare different programme designs [60]. For programme design issues that are ambiguous, notably which age groups should be targeted and whether vaccination of males is required, randomization of vaccination programmes is an interesting option. That the incidence of cervical and other HPV-associated cancers does eventually decrease in vaccinated populations should then be verified by monitoring HPV incidences in sexually active youth groups and incidences of HPV-associated diseases by registry-based follow-up [69–72]. HPV types.  Antibody responses elicited by VLP immunization are, in general, specific for the individual HPV type. However, lower titre cross-reactivity is noted for closely related HPV types [31,33,45,52] as well as partial protection against disease end-points associated with these non-vaccine types [35,73].

Interestingly, we also noted TGF-β secretion, which was lost in A2aR KO mice, suggesting that TGF-β may be produced by iNKT cells and enhanced through adenosine stimulating A2aR. However, TGF-β production has not been described in iNKT cells and could have been indirectly from other cells. We therefore activated sorted iNKT cells with

plate-bound CD1d molecules and assessed their TGF-β production. As Fig. 3B shows, iNKT cells directly produced TGF-β in the active form in response to CD1d-mediated activation. To further confirm selleckchem that the cytokines observed in sera were from NKT cells, we injected WT and A2aR KO mice with α-GalCer and tested NKT and NK cells for their intracellular cytokine content. NKT cells from A2aR KO mice produced significantly more IFN-γ compared to stimulated WT counterparts. Additionally, NK cells known to be transactivated by NKT cells produced significantly more IFN-γ in the absence of an A2aR (Fig. 3C, bottom), however, no IL-4 could be detected in these cells (data not shown). Supporting the serum data

(Fig. 3A), we observed a clear trend to a lower IL-4 production in A2aR−/− NKT cells, although not reaching statistical significance (n=3). Collectively, our data suggest that the secretion of type-2 cytokines IL-4, IL-10 and Cilomilast cost TGF-β by iNKT cells requires signaling through the A2aR since blocking or genetic ablation of this receptor efficiently abrogates Buspirone HCl their secretion. In contrast, ligation of the same receptor abrogates the production of IFN-γ. Pharmacological ligation of the high-affinity A2aR might reflect the situation in vivo with low

adenosine concentrations skewing the cytokine production of iNKT cells toward a Th2-type phenotype. Increased levels of adenosine, such as found in tumors might then additionally ligate the low-affinity A2bR and thus inhibit the activation of iNKT cells, comparable to other cell types. Conceivably, the manipulation of the A2aR on iNKT cells might control their activation and support host defense and immunotherapeutic approaches in both malignancy and tolerance. C57BL/6J were purchased from Jackson Laboratories (Bar Harbor, MA, USA). Mice deficient the A2aR were previously described and backcrossed to C57BL/6 background 8. Mice were housed under specific pathogen-free conditions. Animal experiments were performed in accordance to protocols approved by Institutional Animal Care and Use Committee. Six- to eight-week-old C57BL/6J mice were used for experiments. PBS57-loaded or empty CD1d monomers and tetramers were provided by the NIH tetramer facility (Emory Vaccine Center, Atlanta, GA, USA). CADO, CGS21680, and ZM241485 were purchased from Tocris (Ellisville, MO, USA). Cells were cultured in RPMI-1640 supplemented with penicillin, streptomycin (Mediatech, Manassas, VA, USA) and 5% FBS (Hyclone, Logan, UT, USA). DC were generated from mouse BM in the presence of GM-CSF as described in 25 with modifications.

As predicted from the previous studies with non-Tg

B cells 19, R2+AM14 B cells displayed an attenuated response to GAMIG when compared with R2− AM14 B cells although they responded comparably to increasing concentrations of F(ab′)2 fragments of GAMIG (Fig. 1). Expression of FcγRIIB did not affect the responses to standard TLR ligands; R2+ and R2− AM14 and non-transgenic B cells responded comparably to ligands known to engage both the cell surface (LPS) and the endosomal (CpG 1826 and R848) TLR (Fig. 1 and results not shown). Although selleck chemical FcγRIIB−/− mice on the C57Bl/6-deficient background can develop spontaneous autoimmune disease 3, all the mice used for these studies were between 6- to 8-wk of age and these data demonstrate that they maintained normal responses

to BCR, TLR9 and TLR7 engagement. AM14 B cells express a receptor specificity commonly produced by spontaneously activated autoreactive B cells 20 that reacts weakly with IgG2a 21. Briefly, Everolimus solubility dmso 20.8.3 BCR Tg B cells express a higher affinity receptor for IgG2a, initially elicited by an allotype-disparate immunization 22. In contrast to 20.8.3 B cells, AM14 B cells do not proliferate when stimulated with IC consisting of IgG2a bound to proteins 11. Protein IC do, however, induce upregulation of activation markers in AM14 B cells 23, although this signal is insufficient to stimulate cell cycle entry, possibly due to engagement of the inhibitory FcγRIIB. To determine whether the loss FcγRIIB would enable AM14 B cells to proliferate in response to protein IC, R2+ and R2− AM14 B cells were stimulated with IC consisting of biotinylated-BSA bound by the IgG2a anti-biotin mAb 1D4. Even in the absence of the inhibitory receptor, AM14 B cells failed to proliferate in response to these protein IC. By Reverse transcriptase comparison, 1D4/Bio-BSA IC, but not 1D4 or Bio-BSA alone, did induce 20.8.3 B-cell proliferation (Fig. 2 and data not shown). These results demonstrate that the inability of AM14 B cells to proliferate in response to protein IC is not simply due to engagement of FcγRIIB. The chromatin-reactive mAb PL2-3 binds

uncharacterized DNAse-sensitive components of cell debris and strongly activates AM14 B cells through a mechanism dependent on both the BCR and the TLR9. To evaluate the role of FcγRIIB in the regulation of AM14 B-cell responses to these chromatin IC, R2+ and R2−, AM14 B cells were stimulated with increasing concentrations of PL2-3. However, in multiple experiments, we found that the dose–response curves for these two populations were essentially identical (Fig. 2A). These results were similar to those obtained previously with the PL2-3-activated 20.8.3 cells and appeared to further support the notion that FcγRIIB did not regulate optimal responses emanating from an endosomal TLR when ligated in conjunction with BCR engagement.