abortus rough strain RB51 and smooth strain 2308 to stimulate mur

abortus rough strain RB51 and smooth strain 2308 to stimulate murine bone marrow-derived DC (BMDC) activation and function based on the cell surface expression of costimulatory molecule and cytokine production. This study assessed simultaneously, for the first time, the differential ability of live, HK and IR rough and smooth strains of B. abortus KU-57788 chemical structure at the same doses to stimulate DC activation and function. Female 6–8-week-old BALB/c mice were obtained from Charles River Laboratories Inc. (Wilmington, MA). Mice were used under animal care protocols approved by Institutional Animal Care and Use Committee at Virginia Tech. BMDCs were generated,

as described previously (Inaba et al., 1992). Briefly, tibias and fibulas of 7–8-week-old BALB/c mice were incised and bone marrow (BM) cells were removed. Following red blood cell lysis and filtration, the cells were resuspended and plated in RPMI 1640 complete media with 10% non-heat-inactivated fetal bovine serum and 20 ng mL−1 rGM-CSF (recombinant Granulocyte colony stimulating factor; Invitrogen, Carlsbad, CA). The cells were incubated at 37 °C in 5% CO2. Fresh media containing rGM-CSF was added at days 2, 4 and 5 and harvested on day 6. The cells harvested on day 6 were typically 70% CD11c+ and displayed low levels of major

histocompatibility complex (MHC) class II, CD40 and CD86 expression, consistent with immature DCs. Flow cytometry was performed to confirm DC activation status (Inaba et al., 1992). Stock cultures of live-attenuated rough B. abortus vaccine strain RB51 and virulent smooth selleck chemical strain 2308 from our culture collection (Schurig et al., 1991; Vemulapalli et al., 2000) were stored at −80 °C. An aliquot each of strain RB51 and strain

2308 were subjected to γ-irradiation using a 60Co source irradiator with a radiation output of 2200 rads min−1 (Model 109-68R by J.L. Shepherd and Associates, San Fernando, CA) for 3 h (396 krads SDHB of γ-radiation). Aliquots of strain RB51 and strain 2308 were subjected to heat killing by incubating in an 80 °C water bath for 60 min. IR and HK bacterial preparations were confirmed to be nonviable by plating aliquots on TSA plates and confirming lack of growth following 4 days of incubation. All experiments with Brucella were performed in our CDC-approved (C2003 1120-0016) Biosafety Level-3 facility. On day 6, DCs were harvested and plated at 5 × 105 cells per well in 24-well plates and stimulated with live, IR or HK strain RB51 or strain 2308 at 1 : 10 (DC : Brucella) or 1 : 100 CFUs per well (i.e. 5 × 106 or 5 × 107 CFU equivalents per well of IR or HK B. abortus). Stimulation was enhanced by a short spin at 400 g for 5 min at room temperature. The stimulated cells were incubated for 4 h at 37 °C in 5% CO2. Then cells were washed with media containing gentamicin (Sigma, St. Louis, MO) 30 μg mL−1. The stimulated cells were incubated for an additional 20 h in complete media with 10 ng mL−1 rGM-CSF and 30 μg mL−1 gentamicin.

We found no clear difference between the efficiencies of propagat

We found no clear difference between the efficiencies of propagation of each strain in NA cells (Fig. 5a). In addition, the growth curves of the RC-HL and R(G 242/255/268) strains in other neural cell lines, such as human neuroblastoma SYM-I and SK-N-SH cells, were almost identical (data not shown). These results indicate that the propagation efficiency of the RC-HL strain in vitro is almost identical to that of the R(G 242/255/268) strain. On the other hand, inconsistent with these Y-27632 results, it was found that the RC-HL strain grew less efficiently in the mouse brain than did the R(G 242/255/268) strain (18),

suggesting that another factor is involved in their different efficiencies in in vivo propagation. Interestingly, we found that infection with the RC-HL strain induces inflammation in the

infected mouse brain more strongly than does infection with the Nishigahara strain (unpublished data). Therefore, it is possible that infection with the R(G 242/255/268) strain induces host immune responses less efficiently than does infection with the RC-HL strain, resulting in more restricted propagation of the RC-HL strain in the mouse brain. We conclude that amino acid substitutions at 242, 255 and 268 in rabies virus G protein affect the efficiencies of cell-to-cell spread, resulting in different distributions of RC-HL and R(G 242/255/268) strain-infected cells in the mouse brain and, consequently, distinct pathogenicities. Although the molecular mechanisms Raf inhibitor Acyl CoA dehydrogenase remain to be elucidated, we clarified here important biological characteristics related to the different pathogenicities of the Nishigahara and RC-HL strains. We believe that this study provides basic information for understanding the pathogenicity of rabies virus, and also for establishing

an antiviral therapy for rabies. This study was partially supported by a grant (Project Code No., I-AD14-2009-11-01) from the National Veterinary Research & Quarantine Service, Ministry for Food, Agriculture, Forestry and Fisheries, Korea in 2008 to M.S. “
“To express the 56-kDa protein of O. tsutsugamushi strain Karp, this protein gene was cloned into pET30a(+) before transforming into host bacteria, E. coli Rossetta. Specificity of the recombinant protein was assessed by ELISA using rabbit sera against common members of the order Rickettsiae and 10 other pathogenic bacteria. After IPTG induction, SDS-PAGE analysis of isolated protein demonstrated a band at approximately 46-kDa. Western blot and mass spectrometry analysis proved that the recombinant protein was expressed successfully. Specificity analysis demonstrated that all sera were negative, except sera against O. tsutsugamushi strains TA763, TH1817 and Kato, B. quintana, A. phagocytophilum, E. chaffeensis and B. bacilliformis.

Indirect allorecognition (i e involving recipient APCs) and dire

Indirect allorecognition (i.e. involving recipient APCs) and direct allorecognition (i.e. involving donor APCs) occur in chronic and acute rejection, respectively 15. Thus, to analyze allograft-derived donor APCs in acute rejection process, we transplanted WT and CalpTG skin allografts onto BALB/C mice and examined the skin allograft survival. The survival of the C57BL/6 skin allograft was not affected by the presence of the transgene under these conditions (10 d for allografts derived from both WT and CalpTG donors;

n=5 and 6, respectively). To further assess whether the defective recruitment of T cells in CalpTG recipients was explained by a direct effect of calpastatin transgene in T cells, we transplanted BALB/C skin allografts onto recipient mice lacking T cells (RAG-1−/− mice) and reconstituted

with either WT or CalpTG spleen lymphocytes. At Ulixertinib day 8, allograft infiltration by CD3+ cells was significantly reduced after adoptive transfer of lymphocytes from CalpTG as compared with WT mice (59.6±15.0 versus 508.8±102.6 cells/high power field (HPF); n=4; p<0.004). Thus, calpastatin transgene expression in lymphocytes is sufficient to limit markedly Adriamycin research buy skin allograft infiltration by these cells. Prior to gain insight onto how calpastatin transgene might affect T-cell recruitment, we verified the ability of calpastatin transgene to limit calpain activity in T cells. As assessed by measuring the calpain-specific cleavage of fluorescent 7-Amino-4-methylcoumarin (AMC) (Fig. 3A) and by measuring the 145/150-kDa spectrin BDP expression by Western Inositol monophosphatase 1 blotting (Fig. 3B), calpastatin excess had no effect on calpain activity in resting T cells, but limited TCR-dependent calpain activation in

T cells exposed to αCD3 mAb. These data are consistent with a model in which calpains and calpastatin are not co-localized within the cell at rest. Calpastatin diffusion after calcium-related cell stimulation allows calpastatin to interact with calpains, thereby modulating its activity 13. Given that the calpain activity is involved in the activation of NF-κB and NFATc1 6, 9, two pathways leading to the generation of effector T cells 16, the nuclear expression of these transcription factors was also determined in T cells from WT and CalpTG. As shown in Fig. 3C and D, αCD3 mAb-induced nuclear translocation of NF-κB and NFATc1 was not modified by calpastatin transgene expression. These data suggest that the activation of NF-κB and NFATc1 is not essential for the control of T-cell recruitment by calpastatin transgene. Failure of T-cell recruitment into skin allograft is potentially explained by sequestration of circulating T cells into secondary lymphoid tissues and/or impairment in T-cell adhesion, migration and proliferation. We first determined by flow cytometry the number of CD3+ cells in spleen and graft-draining lymph nodes, 8 days after skin transplantation.

[1, 2] Sodium chloride concentration in tubular

[1, 2] Sodium chloride concentration in tubular selleck compound library fluid mostly depends on the volume and speed of the glomerular filtrate through a tubular system. When this concentration decreases (usually the result of decreased glomerular filtration rate), signals from MDC lead to the increased renin release from juxtaglomerular cells (located in arteriole walls), as well as vasodilatation of afferent arterioles that supplies the glomerulus with blood. Both these events result in increased glomerular hydrostatic pressure, which returns glomerular filtration rate to normal levels. Macula densa cells are also important contributors to the activity of renin-angiotensin-aldosterone system (RAAS), mainly

through their regulation of renin release in juxtaglomerular cells.[1-3] In postnatal development, it is known that kidney as an organ undergoes various changes in its structural organization and function. These changes reflect on glomerular filtration rate, Proteases inhibitor renal blood flow, glomerular basement membrane (GBM) permeability and overall ability of kidney to concentrate urine.[4, 5] These changes are primarily the consequence of age-related processes occurring in renal cortex. Neonatal kidney has certain unique characteristics that distinguish it from adult organ.[6, 7] Mechanisms and/or the rate of ion transport

in tubular system, such as sodium/hydrogen exchange and sodium/phosphate transport significantly change in postnatal development.[5, 8] Developmental changes occur both in transcellular and paracellular ion transport.[9, 10] Also,

reactivity of neonatal tubular system to certain hormones, such as vasopressin is smaller when compared with adult kidney.[5, 11] This significantly decreases the urine concentration capability of neonatal kidney. Unlike other parts of the nephron, in macula densa cells, it is unclear what kind of structural and functional changes occur during postnatal development either in humans or in experimental animal models. In recent years, there have been many research efforts to apply various imaging methods in kidney research. Fractal analysis (FA) is today one of the modern imaging techniques that are commonly used to detect structural and ultrastructural changes in cell and tissues.[12] In nephrology, so far, it has been successfully applied Interleukin-2 receptor in complexity quantification of kidney microvascular morphology, by determining two major FA parameters: fractal dimension and lacunarity. Microvascular morphology was evaluated on digital tissue images after conversion to binary format, using modern image analysis software, such as ImageJ and MATLAB.[13] A similar approach has been used to analyze vascular networks in renal carcinomas.[14, 15] In this article, we present evidence that the complexity of chromatin structure of macula densa cells decreases during postnatal development in mice.

Furthermore, the doxycycline-induced proliferative expansion of p

Furthermore, the doxycycline-induced proliferative expansion of pre-B- and immature B-cell pools is reversible upon termination of Pim1/Myc overexpression. To test whether Pim1/Myc overexpression could also induce propagation of mature B cells

after in vivo maturation, 5×106 Pim1/Myc double-transgenic pre-BI cells were transplanted into sublethally irradiated Rag1−/− mice. After 2–4 weeks, expression of Pim1 and Myc was switched on in these matured cells by feeding doxycycline. Two and four weeks after the start of doxycycline PD0325901 manufacturer treatment, mice were sacrificed and B-lineage cells of the BM (1 femur, 1 tibia), spleen and peritoneal cavity were analyzed by FACS (see Fig. 4A for an outline of the experiment). As shown in Fig. 4B, total B-cell numbers did not increase in BM, spleen or peritoneum when overexpression of Pim1 and Myc in B cells was induced 2 weeks after transplantation. Furthermore, there was no change in the percentage of immature, CD93+ cells versus mature, CD93−IgM+ cells in the spleen (Fig. 4C) and peritoneum. Transplantation of Pim1/Myc transgenic pre-B cells into sublethally irradiated Rag1−/− mice in the absence of doxycycline allows the development of CD93+IgM+ immature and of CD93−IgM+ mature B cells in the spleen of the transplanted mice. Thus, four weeks after maturation of these

transplanted cells, IgM+ cells were prepared from the spleens of these mice by magnetic MACS beads Navitoclax in vitro (see Fig. 5A for an outline of the experiment). The cells, which had a purity of ∼97%, were induced for 18 h with doxycycline or left in medium alone. Then, RNA was prepared, transcribed into cDNA, and cDNA levels of Pim1, Myc and Gapdh were analyzed by semiquantitative PCR (Fig. 5B). The results of the RT-PCR analyses show that doxycycline successfully induced the overexpression of transgenic Pim1 and Myc in IgM+ sorted splenic B cells ex vivo. However, in spite of the induced overexpression of both oncogenes, there were no significant changes in the survival and proliferation of IgM+ or CD19+ splenic B cells (Fig. 5C). CFSE labeling of splenic CD19+ B

cells cultured for 4 days FAD in the absence of mitogens revealed that overexpression of Pim1 and Myc did not induce cell cycle entry in these cells (Fig. 5D). Next, we tested these resting, mature B cells for their capacities to proliferate in response to polyclonal B-cell activators. Hence, the sorted IgM+ splenic B cells were cultured either in the absence or in the presence of polyclonal B-cell activators such as CD40-specific mAbs, IL-4, IL-5, IgM-specific mAbs, LPS, or combinations thereof. To rule out the possibility that the purification step with anti-IgM antibodies had an anergizing or otherwise detrimental effect on the sorted B cells, the experiment was repeated with CD19+ MACS-sorted B cells and erythrocyte-depleted total splenic cells. Data in Fig.

It will also explore the role of pre-existing renal disease in ca

It will also explore the role of pre-existing renal disease in causing preeclampsia and the potential for new biomarkers, both serum and urinary, to inform clinical practice with regard to differentiating preeclampsia from pre-existing renal disease. Recommendations about the future of women who have had preeclampsia

are unclear but the general consensus is that there are future cardiovascular risks, and to a lesser extent, future renal risks in these women. Regular review of proteinuria and glomerular filtration rate as well as overall cardiovascular risk status seems a logical step. Hypertension is the commonest medical complication in pregnancy and falls into four categories; gestational hypertension, preeclampsia, chronic hypertension (including www.selleckchem.com/products/BIBW2992.html essential and secondary hypertension) and preeclampsia superimposed on chronic hypertension. Hypertension in pregnancy is defined as a blood pressure elevation greater than 140 mmHg systolic or

90 mmHg diastolic, which is confirmed with repeated measures over several hours. The hypertension of preeclampsia (de novo or superimposed) and gestational hypertension occurs after 20 weeks of gestation and resolves typically by 3 months post-partum.1 Chronic hypertension occurs when the blood pressure is elevated outside of these time constraints. Preeclampsia and superimposed preeclampsia, however, Selleckchem Palbociclib are multisystem disorders, and as 4-Aminobutyrate aminotransferase such are characterized by hypertension and evidence of involvement by one or more other organs.2 Other organ involvement commonly, but not always, involves the kidneys

and presents as proteinuria (>300 mg/24 h or spot urinary protein: creatinine ratio of ≥30 mg/mmol), elevated plasma creatinine >90 µmol/L or oliguria. Other organ involvement includes haematological changes (thrombocytopaenia, haemolysis, disseminated intravascular coagulation), liver disease (elevated serum transaminases, severe epigastric or right upper quadrant pain), neurological effects (convulsions, hyperreflexia, visual disturbances, stroke or headache), pulmonary oedema, foetal growth restriction or placental abruption. Maternal renal adaptation is characterized by an increase in glomerular filtration rate (GFR) to about 50% above pre-pregnancy states.3,4 An increase in renal plasma flow as well as an increase in the fractional excretion of urate is due to a decrease in renovascular resistance.5 The fractional excretion of sodium declines in pregnancy resulting in a net increase in total body water and sodium. These changes are initiated very early in pregnancy (prior to the first missed period) and are fully established by the end of the first trimester.3 They are maintained until the last 6 weeks prior to delivery when a reduction to pre-pregnancy creatinine clearance has been shown.

Furthermore, mechanistic studies have revealed that virally encod

Furthermore, mechanistic studies have revealed that virally encoded suppressors can act at different steps in the silencing pathway, including Dicer-2 processing and Ago2 slicing [4],

suggesting that indeed, the entire pathway is required for defense. In contrast to RNA viruses, very little is known about the interactions of DNA viruses with the antiviral RNA-silencing machinery, particularly in arthropods. If these selleck inhibitor viruses were restricted by the RNAi machinery, the DNA genome could not be targeted directly; rather, RNA transcripts from the viral genome would form structures with double-stranded character that would be recognized and processed by Dicer-2 (Fig. 1A). In Drosophila, a recent study by Bronkhorst et al. [15] found that overlapping bidirectional transcription of the dsDNA virus invertebrate iridescent virus 6 (IIV-6) likely leads to the formation of dsRNA in trans, which

is processed by Dicer-2 into small RNAs. Conversely, small RNAs produced in wild-caught mosquitoes infected with a ssDNA densovirus, which has no overlapping convergent transcripts, map predominantly to the viral RNA transcripts, suggesting that local interactions within a single-stranded RNA strand form dsRNA in cis that are targeted by antiviral RNAi [16]. Peptide 17 However, the mechanism by which the insect RNAi pathway restricts infection of DNA viruses remains poorly understood, and is an important subject of future study. Shrimp are arthropods of agricultural and ecological importance, and white spot syndrome virus (WSSV) is a highly pathogenic dsDNA virus that impacts aquaculture and is thought to have caused over $15 billion in losses [17]. It has been demonstrated that sequence-specific long dsRNAs could confer antiviral immunity against WSSV, as well as against the shrimp RNA virus Taura syndrome virus [18]. Moreover, injection of a synthetic siRNA against WSSV VP28, a viral envelope protein, conferred sequence-specific antiviral resistance [19]. Therefore, both long dsRNAs and synthetic siRNAs induce sequence-specific antiviral immunity in shrimp. Whether the shrimp RNAi pathway

naturally targets RNA or DNA viral pathogens remained unclear. However, in this issue of the European Journal of Immunology, Huang and Zhang examine whether the RNAi pathway directs an antiviral immune response against the dsDNA virus WSSV in shrimp [20]. Since a synthetic siRNA designed to target VP28 (vp28-siRNA) Fossariinae is capable of controlling infection, Huang and Zhang first asked whether vp28-siRNA is produced naturally during infection of the shrimp Marsupenaeus japonicus with WSSV. Indeed, vp28-siRNA can be detected by northern blotting and small RNA sequencing of infected tissues. Expression of vp28-siRNA in various shrimp tissues is dependent upon WSSV infection, as the siRNA cannot be detected in tissues where WSSV does not replicate to detectable levels. Thus, vp28-siRNA is a virus-derived small RNA that is generated from WSSV transcripts during infection.

Of note, these occurrences are likely polygenic, pertaining to ge

Of note, these occurrences are likely polygenic, pertaining to genes such as the genes of human leucocyte antigen (HLA), KIR and class cytokine receptor [12]. NK cells can play a crucial role in the innate response to infection by lysis of infected cells and by secretion of proinflammatory cytokines (such as IFN-γ) that

promote phagocytic clearance of microbes [13]. NK cell activity is regulated by an extensive repertoire of regulatory receptors. The most polymorphic receptors belong to the KIR family [14]. A number of studies implicated KIR diversity in susceptibility selleck screening library to both infectious and non-infectious diseases [14, 15]. KIR genes provide activating or inhibitory MG-132 purchase signals to regulate the activation of NK cells and T cells and play an important role in anti-micro-organism immunity [15]. The combination of maternal and paternal haplotypes with distinct gene content produces diversity among individuals in their KIR gene content profile (KIR genotype), which may influence the individuals’ immunity and susceptibility or resistance to diseases. Interestingly, several clinical studies have shown associations between diseases and KIR genotypes. For example, individuals with KIR genotype A/A were reported to relatively protect against chronic inflammatory diseases [16,

17], and individuals with genotype A/B were significantly more likely to remain seronegative than those with genotype A/A among long-term HIV-exposed subjects [18]. However, the role of overall KIR genotype in patients with syphilis remains unclear up to now. The objective of this study was to examine whether the KIR genotypes and haplotypes influence susceptibility or resistance to syphilis. Therefore, we analysed KIR genes in a Chinese Han population of 190 patients with syphilis and 192 healthy controls by means of polymerase chain reaction with sequence-specific

primers (PCR–SSP). Patients and controls.  One hundred and ninety unrelated patients with syphilis, who were diagnosed at Jinan Hospital of Dermatosis, were enrolled as the case group. The diagnosis of syphilis was based on the criteria for syphilis of the Health Ministry of the People’s Republic of China (WS 273-2007). The toluidine red unheated serum test Nintedanib (BIBF 1120) (TRUST) and the T. pallidum particle agglutination assay test (TPPA) were performed for all patients. Both TRUST and TPPA were positive for the patients, and the TRUST titre ranged from 1:4 to 1:128. Of these patients, 108 were men and 82 were women, and their ages ranged from 19 to 55 years, with an average age of 34 years. Meanwhile, 192 healthy subjects were from Chinese marrow donors consisted of 159 men and 33 women, and their ages ranged from 18 to 44 years, with an average age of 28 years. Both TRUST and TPPA were negative for all controls.

This crude material was dialyzed in distilled water The water so

This crude material was dialyzed in distilled water. The water solution was then lyophilized to obtain CMWS. Polysaccharides were completely hydrolyzed in 2.0 M CF3CO2H (115°C, 1.5 hr). The sugars were converted to alditol acetates by reduction followed by treatment with acetic anhydride in an equal volume of pyridine (100°C, 1 hr), and then analyzed by GLC using a GC-2014AF instrument (Shimadzu, Kyoto, Japan) equipped with a flame ionization detector and a 30 m × 0.25 mm (0.25 mM) DB-225 capillary column (J&W Scientific, Folsom, CA,

USA). The total carbohydrate concentration was determined by the phenol-sulfuric acid method using a mixture of d-mannose and d-glucose as selleck a standard. Total protein was determined by using the BCA Protein Assay Regent kit (Pierce Biotechnology, Rockford, IL, USA), with BSA as a standard. Endotoxin content was determined by the Toxicolor LS-50M Set (Seikagaku Biobusiness, Tokyo, Japan). We used the DBA/2 mouse strain in this experiment because this strain shows the most serious coronary arteritis after treatment with the CAWS that is secreted into the culture supernatant by Candida albicans (11). In week1, CMWS (4 mg/mouse) was administered intraperitoneally for 5 consecutive days to each mouse. The hearts of the animals were fixed with 10% neutral formalin and embedded in paraffin blocks. Tissue sections were stained with HE. Preparation of paraffin blocks and HE staining was done by Japan SLC. The incidence

and severity of rapid Selleck PD98059 Lck anaphylactoid shock was assessed within 1 hr of i.v. injection (0.1 mL/10 g body weight) of CMWS (8 mg/kg) into ICR mice. The subsequent mortality (in the first hour after injection) was recorded. The reactivity

of cell wall extracts to serum factors from Candida Check, which consists of rabbit polyclonal antibodies against Candida cell wall mannan (23–25), was assessed by ELISA. A solution of cell wall extracts in 50 mM carbonate buffer (pH 9.6) was coated onto Nunc immunoplates (Roskilde, Denmark), which were then incubated at 4°C overnight. The plates were washed extensively with PBST; unbound sites were blocked by the addition of BPBST to wells for 40 min at 37°C; and then the wells were washed six times with PBST. Candida serum factors serially diluted with BPBST were added and incubated for 60 min at 37°C. After six washes with PBST, the wells were treated with peroxidase-conjugated goat anti-rabbit IgG and the TMB microwell peroxidase substrate system (KPL, Gaithersburg, MD, USA). After 45 min, the reaction was stopped with 1 N H3PO4. The optical density of each well was then read at 450 nm on an automatic microplate reader. The reactions were evaluated as positive when the maximum optical density was over 1.0 at an 80-fold dilution ratio of Candida serum factor because Candida serum factors are polyclonal antibodies. Exchangeable protons were removed by dissolving cell wall extracts in D2O, and samples were then lyophilized. This exchange process was repeated three times.

4b, upper panel) By

4b, upper panel). By VX-770 molecular weight contrast, Ku70 staining was faint and nuclear staining was nearly undetectable in CD40L/IL-4-stimulated B cells (Fig. 4b, lower panel), a finding that coincided with the absence of proliferation

(Fig. 1b) and B-cell blast formation under these stimulatory conditions.[17] Full-blown proliferative responses as observed with CpG ODN stimulation might, therefore favour nuclear translocation of Ku70/80, but do not seem to be a prerequisite for RAG re-expression, because RAG-1 was detectable in CD40L/IL-4-stimulated B cells, whereas BCR stimulation failed to trigger RAG-1 expression (Fig. 2d). Having confirmed these molecular prerequisites for receptor revision we sought functional evidence for RAG activity. We postulated that re-expression of RAG in peripheral B cells enables Igκ/Igλ rearrangement in response to TLR9 ligation. To prove this hypothesis we purified Igκ+ B cells, and compared Igκ/Igλ expression in B cells stimulated with CpGPTO or CD40L/rhIL-4,

two stimuli that result in comparable cellular survival and autocrine Ceritinib IL-6 but that differ in the extent of proliferation. Despite the absence of Igλ+ cells in sorted Igκ+ B cells (Fig. 5a), unstimulated and CD40L/rhIL-4-stimulated B cells, a small population of Igκ-negative Igλ+ B cells became detectable after TLR9 stimulation for 4–6 days (Fig. 5b). Moreover, co-expression of Igκ and Igλ on a subset of B cells (Fig. 5b) was interpreted as indicative for ongoing Igκ/Igλ rearrangement. Staining with the isotype control proved the specificity of the anti-Igλ staining (Fig. 5c). Importantly, the low frequency of the evolving Igλ+ population (Fig. 5b), e.g. for CpGPTO: 0·4 ± 0·2% (n = 6) and for CD40L/IL4: 0·03 ± 0·04% (n = 4) makes Igκ/Igλ rearrangement a rare event, a finding that is compatible with the overall low expression of TLR9-induced RAG-1 and selective accumulation of RAG-1 and Ku70 in a small B-cell subfraction. Taken together, these results provided the notion this website that stimulation with TLR9-active ODN triggers RAG re-expression and consecutively catalyses LC rearrangements in a subfraction of B cells, so proving functional

integrity of TLR9-induced RAG proteins in these cells. The current understanding of receptor editing and revision implies that these processes must be initiated by binding of an autoantigen to the BCR. Of note, earlier reports described binding of CpGPTO to the BCR,[22] which raised the notion that CpGPTO could act as unselective BCR stimuli or might even mimic autoantigens. In a previous report we further demonstrated that stimulation of TLR9 with PTO-modified ODN selects IgM+ B cells for proliferation and differentiation.[17] As depicted in Fig. 6(a), CpGPTO-induced B-cell blasts originate from IgM+ CD27+ B cells because blast formation in response to CpGPTO is restricted to CD27+ and IgM+ B-cell fractions and is absent in CD27− and IgM− (class switched) B-cell fractions.