While the innate signals triggered by TLR4 engagement are well studied, the contribution selleck kinase inhibitor of SE remains unclear. To better understand the effect of SE on the adjuvant properties of GLA-SE, we compared the innate and adaptive immune responses elicited by immunization with different formulations: GLA without oil, SE alone or the combination, GLA-SE, in mice. Within the innate response to adjuvants, only GLA-SE displayed features of inflammasome activation, evidenced by early IL-18 secretion and IFN-γ production in memory CD8+ T cells and neutrophils. Such early IFN-γ production was ablated in caspase-1/11−/− mice

and in IL-18R1−/− mice. Furthermore, caspase-1/11 and IL-18 were also required for full Th1 CD4+ T-cell induction via GLA-SE. Thus, we demonstrate that IL-18 and caspase-1/11 are components of the response to immunization with the TLR4 agonist/squalene oil-in-water based adjuvant, GLA-SE, providing implications for other adjuvants that combine oils with TLR agonists. “
“Activation of naïve T cells requires costimulation via TCR/CD3 plus accessory receptors, which enables the dynamic rearrangement Erlotinib of the actin cytoskeleton and immune synapse maturation. Signaling events induced

following costimulation may thus be valuable targets for therapeutic immunosuppression. Phosphorylation of the actin-bundling protein L-plastin represents such a costimulatory signal in primary human T cells. Phosphorylated L-plastin Abiraterone in vitro has a higher affinity toward F-actin. However, the importance of the L-plastin phosphorylation for actin cytoskeleton regulation upon antigen recognition remained unclear. Here, we demonstrate that phosphorylation of L-plastin is important for immune synapse maturation. Thus, expression of nonphosphorylatable L-plastin in untransformed human peripheral blood T cells leads to reduced accumulation of LFA-1 in the immune synapse

and to a diminished F-actin increase upon T-cell activation. Interestingly, L-plastin phosphorylation is inhibited by the glucocorticoid dexamethasone. In line with this finding, dexamethasone treatment leads to a reduced F-actin content in stimulated T cells and prevents maturation of the immune synapse. This inhibitory effect of dexamethasone could be reverted by expression of a phospho-mimicking L-plastin mutant. In conclusion, our data introduce costimulation-induced L-plastin phosphorylation as an important event for immune synapse formation and its inhibition by dexamethasone as a novel mode of function of this immunosuppressive glucocorticoid. Activation and inactivation of antigen-specific T cells is the basis of a functional adaptive immune system. To become fully activated, primary T cells need at least one secondary stimulus besides the antigen-specific TCR/CD3 signal; a process called costimulation.

Treatment of N9 cells with increasing concentrations of LPS (0·1, 0·5 and 1 μg/ml) showed a significant dose-dependent induction of miR-155 expression, which reached a 25-fold increase in miR-155 levels for the highest LPS concentration tested (Fig. 1a). A similar result was obtained in primary microglia cultures, where it was possible to observe a 12-fold or 21-fold increase in the expression of miR-155 following incubation

with 0·1 or 1 μg/ml LPS, respectively (Fig. 1b). To establish a time–course for this event, changes in miR-155 levels were monitored by qRT-PCR at different time-points (30 min, 1, 2, 4, 18 and 24 hr), following stimulation of N9 cells with Fulvestrant solubility dmso the lowest concentration of LPS (0·1 μg/ml). The levels of miR-155 remained constant until 4 hr after the beginning of the stimulus, when a significant increase was observed with respect to control levels (Fig. 1c). Levels of miR-155 continued to increase, reaching a maximum at 18 hr, but showed a tendency to decrease after

an incubation period of 24 hr. To confirm the results obtained by qRT-PCR, in situ hybridization studies were performed in primary microglia cultures exposed to 0·1 or 1 μg/ml LPS, using an LNA BEZ235 price probe specific for the mature form of miR-155 (Fig. 2). The miR-155 labelling was significantly more intense in the cytoplasm of microglia cells incubated with LPS than in control cells. Since the probe only recognizes the Anidulafungin (LY303366) mature form of this miRNA, these results further validate the qRT-PCR data presented in Fig. 1(b) and confirm that, under inflammatory conditions, miR-155 expression increases not only in N9 microglia cells but also in microglia primary cells. Primary microglia cells are not easily obtained with high yield, are extremely difficult to transfect and are easily activated by cell culture procedures, also, the responses of N9 cells and primary microglia cultures to LPS treatment are similar, so the subsequent

studies were performed in N9 cells. This cell line, which comprises immortalized mouse-derived microglia cells, has been described as mimicking the behaviour of primary microglia regarding TLR expression, cytokine release and NO production, and has been employed in several studies as an in vitro model to study microglia activation.24–26 The miRNAs exert their regulatory effects mainly at the post-transcriptional level, by targeting complementary or partly complementary mRNAs and inducing mRNA cleavage or translation repression. To identify potential targets of miR-155 that might be relevant in the microglia immune response, we screened the mouse and human miR-155 sequences using the miRBase and PicTar miRNA target identification programmes.

IFNγ responses regulate CXCL10, which directs migration and stimulation of activated T cells by binding to the CXCR3 receptor [38]. CXCL10 has been proposed a marker of TB infection in children where specific immunity to M. tuberculosis assessed by CD4 T cell responses would be unreliable [12, 38, 39]. Here, Ponatinib we

show for the first time that CXCL10 levels can differentiate severity in TB. The lowered CXCL10 levels observed in patients with far advanced PTB may be attributed to decreased IFNγ levels and may result in limited recruitment of leucocytes, adversely affecting granuloma formation in advanced disease TB [12]. We observed that patients with localized extrapulmonary TB had higher MTBs-induced IFNγ levels in lymph node as compared with pleural disease. While both lymphadenitis and pleurisy are forms of localized TB, the cellular composition at these sites is different and may influence the cytokine/chemokine levels. It is reported

that the pleural involvement with pulmonary disease results in an increase in the systemic levels of cytokines as compared with those who have pulmonary disease only [40, 41]. In M. tuberculosis infection of the pleura, T cells are localized in the pleural fluid and it was observed that IFNγ and chemokines are increased in the fluid [42]. In the lymph node, M. tuberculosis can be restricted in localized granulomas by appropriate T cell-driven chemokine responses. Thus, site-specific LDE225 in vivo differences in IFNγ secretion at lymph node and pleural site probably reflect the efficacy of T cell recruitment and activation responses. This increased antigen-induced IFNγ observed in whole blood cell responses of patients with lymph node TB support the hypothesis of a higher IFNγ/IL10 ratio in less-severe forms of TB [27]. We found that MTBs-stimulated CCL2 levels were raised in pulmonary as compared with extrapulmonary TB. This is in agreement with studies in which increased CCL2 Exoribonuclease was observed in PTB as compared with ETB in response to BCG stimulation [26]. However, we found that MTBs-induced CCL2 levels were reduced in

patients with ETB as compared with ECs. Previously, it has been shown that BCG and M. tuberculosis stimulation of PBMCs results in increased CCL2 secretion in patients with TB[17]. This may indicate a differential response related to differences between live Mycobacterium–stimulated response and those to whole sonicate antigen and that live M. tuberculosis and BCG may be more potent activators of CCL2 than the sonicate used in this study. We observed that MTBs-induced IL10 levels were greater in pulmonary as compared with extrapulmonary TB and were also higher in patients with localized as compared with disseminated ETB. IL10 is an immunosuppressive cytokine shown to be increased in TB [21]. Infections such as those caused by M.

Consistent with previous studies in other cell types, Fig. 4A demonstrates that both Syk kinase and PI3K are required for phagocytosis in RBL cells [1,

2]. Our data also indicate that FcγRIIA mediated serotonin secretion is dependent on PI3K for full activity. We observed that see more inhibition of PI3K by the inhibitor wortmannin, at the low concentrations that are sufficient to abolish phagocytosis, also reduces FcγRIIA-mediated serotonin secretion by nearly 50% (Fig. 4B). Inhibition of Syk with the Syk-selective inhibitor piceatannol did not significantly inhibit serotonin release by cells expressing WT FcγRIIA, despite the fact that the concentration used (25 μg/ml) had been previously shown to reduce other Syk functions in RBL cells, including serotonin secretion mediated by other Fc receptor isotypes [21, 24]. These data indicate that signaling for FcγRIIA-mediated serotonin release can bypass Syk kinase in RBL cells. In B cells, Syk kinase acts proximal to PI3K [25]. Likewise, in neutrophils stimulated through their IgG receptors, piceatannol treatment blocked the activation of PI3K, indicating that Syk acts proximal to PI3K [25, 26]. Our observation that FcγRIIA-mediated serotonin

release is sensitive to PI3K inhibition but independent of Syk thus appears at odds with a current concept that Syk kinase, recruited early to the phosphorylated ITAM, must serve as an adapter to recruit PI3K for FcγR signaling. Rather, our data suggest that stimulation of FcγRIIA Idasanutlin in vitro may directly engage PI3K and that this event is sufficient to initiate serotonin release. This sequence of events is consistent with other studies that indicate that PI3K can specifically bind to a phosphorylated ITAM without prior involvement of Syk kinase [27]. On the the basis of their discovery that PI3K can bind the phosphorylated ITAM independently of Syk, Cooney et al. proposed a model for phagocytic signaling whereby

Syk and PI3K function in parallel [27]. It is highly possible that their discovery of PI3K’s direct recruitment to the phosphorylated ITAM of FcγRIIA has significant implications for secretion signaling. Our current studies likewise suggest a direct signaling role for PI3K. Since pharmacologic blockade of Syk does not reduce secretion, signaling via Syk appears less involved. While we cannot yet definitively state that there is a direct interaction between FcγRIIA and PI3K, our experiments clearly demonstrate that FcγRIIA-mediated signaling for secretion utilizes ITAM tyrosines and downstream signaling agents different from those required for phagocytosis [3, 27]. These observations are consistent with evidence that the ITAM requirements for FcγRIIA triggered phagocytosis and endocytosis are very different. Specifically, mutations of ITAM tyrosines that completely block phagocytosis do not effect endocytosis.

1 and 2). This relative stability

of the CD277 surface expression prompted us to further investigate the potential action of the CD277 engagement in immune cells. The role of CD277 engagement was investigated on TCR-induced cytokine production. Purified CD4+ T cells from healthy donors were cultured during 24–72 h with CD3+CD28 mAbs or CD3+CD277 mAbs or CD3 mAb+IgG1 (control condition). After 24 h of culture, IL-2 and IFN-γ production by CD4+ T cells were measured by ELISA. As expected, these two cytokines were secreted in large amounts after CD3+CD28 stimulation by comparison with the control condition (Fig. 1A: IL-2, 120 pg/mL, p=0.0079; Fig. 1B: IFN-γ, 7000 pg/mL, p=0.0317). Although the IL-2 levels produced by the CD3+CD277 co-activated CD4+ T cells were lower than the IL-2 levels obtained with CD3+CD28 Epigenetics inhibitor co-stimulation, the quantity of IL-2 induced by CD3+CD277

co-activation was significantly higher than that induced with the IgG1 control (Fig. 1A: IL-2, 40 pg/mL, p=0.0159). Moreover, click here IFN-γ secretion was strongly enhanced by CD3+CD277 co-activation (Fig. 1B: IFN-γ, 9000 pg/mL, p=0.0159) compared with the control situation, and, surprisingly, the production was even greater than that obtained after CD3+CD28 co-activation. A similar effect was obtained regarding the expression profile of the activation marker CD25 under CD3+CD277 co-stimulation (Fig. 1C). Altogether, these results suggest that the CD277 molecule acts as a T-cell co-stimulatory molecule for cytokine

production. To investigate whether similar co-stimulatory effects are obtained in NK cells, CD107 expression under P815-redirected cytotoxicity (Fig. 1D) and IFN-γ assays (Fig. 1E) were performed. The NK cells are stimulated via two different activation receptors, CD16 or NKp46, using specific mAbs, in the presence of isotypic control, CD277 mAb, anti-DNAM (positive control for a co-stimulation of the activation receptors) or anti-NKG2A (positive control for a co-inhibition of the activation receptors). The CD277 triggering tuclazepam alone did not induce any effect on NK cell stimulation. Moreover, in contrast to DNAM (co-stimulation) or NKG2A (co-inhibition), CD277 engagement fails to modulate CD16- or NKp46-induced NK cell activation, both for degranulation as evaluated by CD107a/b staining and IFN-γ secretion. These results show that CD277 is not involved in the regulation of NK cell activation, contrary to that which was observed with T cells. The BTN3/CD277-mediated positive signals shown in T-cell cytokine production (Fig. 1A and B) are not in accordance with previous work in which another CD277 mAb clone has been used 13. To further test the robustness of our results, we investigated the capacity of CD277 triggering to regulate TCR-induced early T-cell events such as signaling pathways.

D.). 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. “
“The signal transducer and activator of transcription 3 (STAT3) transcription factor pathway plays an important role in many biological phenomena. STAT3 transcription is triggered by cytokine-associated signals. Here, we use isolated human B cells to analyse the role of STAT3 in interleukin (IL)-10 induced terminal B cell differentiation and in immunoglobulin (Ig)A production

selleck chemicals as a characteristic readout of IL-10 signalling. We identified optimal conditions for inducing in-vitro IgA production by purified blood naive B cells using IL-10 and soluble CD40L. We show that soluble CD40L consistently induces the phosphorylation of nuclear factor (NF)-κB p65 but not of STAT3, while IL-10 induces the phosphorylation of STAT3 but not of NF-κB p65. Interestingly, while soluble CD40L and IL-10 were synergistic in driving the terminal maturation of B cells into IgA-producing plasma cells, they did not Epacadostat in vitro co-operate earlier in the pathway with regard to the transcription factors NF-κB p65 or STAT3. Blocking either NF-κB p65 or STAT3 profoundly altered the production of IgA and mRNA for activation-induced cytidine deaminase (AID), an enzyme strictly

necessary for Ig heavy chain recombination. Finally, C-X-C chemokine receptor type 7 (CXCR-7) the STAT3 pathway was directly activated by IL-10, while IL-6, the main cytokine otherwise known for activating the STAT3 pathway, did not appear to be involved in IL-10-induced-STAT3 activation. Our results suggest that STAT3 and NF-κB pathways co-operate in IgA production, with soluble CD40L rapidly activating the NF-κB pathway, probably rendering STAT3 probably more reactive to IL-10 signalling. This novel role for STAT3 in B cell development reveals a potential therapeutic or vaccine target for eliciting IgA humoral responses at mucosal interfaces. Naive mature B cells express both immunoglobulins (Ig) M and D. Antigen and T cell-dependent or -independent activation induces class switch recombination (CSR) of differentiated B cell genes, a molecular mechanism involving Ig heavy chain (CH) gene rearrangements. After such activation, B cells produce IgG, IgA or IgE antibodies [1]. Whatever the mechanism, antibody production involves activation-induced cytidine deaminase (AID), an enzyme strictly necessary for Ig heavy chain recombination [2]. IgA constitutes the most abundant antibody class in the gut, where it contributes to immune protection against certain pathogens. Within the gut, low- and high-affinity IgA is produced in the lamina propria (LP) and Peyer’s patches, respectively [3].

This observation underlines the need to be cautious to extrapolate in vitro studies with Tregs to in vivo situations. Besides the issue of level of FOXP3 expression, duration of expression may be an important facet determining the function of

induced Tregs. The reduced effectiveness of the induced FOXP3 T cells may be time-dependent as earlier in vitro studies report that continuous levels of FOXP3 are required to convert naive T cells into Tregs with full effectiveness 6. In this setting of systemic inflammation, 24 h seems to be too short to procure the full molecular and transcriptional changes necessary for suppression. On the other hand, it does seem to be sufficient to inhibit the cell from dividing after TCR stimulation in vitro. Accordingly, FOXP3 may act as an intrinsic regulator during inflammation, preventing collateral damage by temporarily silencing activated T DNA Methyltransferas inhibitor cells. In conclusion, during systemic inflammation due to cardiac surgery in children, FOXP3+ T cells lose suppressive capacity. While these cells are anergic to TCR stimulation, the transiently increased expressed FOXP3 is not capable of taking on a suppressive function. Furthermore, the inflammatory milieu in which Tregs exert their action after cardiac surgery inhibits

their suppressive activity. This study illustrates the functionality of FOXP3+ T cells in a human model of inflammation and underlines the requirement of more human in vivo systems to understand the properties and potential of induced FOXP3+ Tregs in human disease. Children admitted to our hospital 5-Fluoracil datasheet for surgical repair of either a VSD or an ASD were enrolled in this study. Patients were excluded from the study if at the time of admission they had received steroids within 2 wk before surgery, had signs of infection or had a documented immunodeficiency. Informed consent was obtained from the parents of children Thiamet G participating in the study. The medical ethics committee approved this study (METC 03/049-K, UMC Utrecht, The Netherlands). General anesthesia was always implemented using a standard technique

involving high-dose sufentanil, midazolam, pancuronium, dopamine and milrinone. All patients were given a single dose of dexamethason (1 mg/kg) after induction of anesthesia. Non-pulsatile CPB was used, the standard pump flow rate was 2.8 L/m2/min. Combined alpha and pH stat management of acid–base status was used during CPB. The cardioplegia procedure was standardized using St. Thomas’ solution. After weaning from CPB, all patients remained intubated and ventilated and were admitted to the pediatric intensive care for further management. All patients were treated by the same surgical team. Blood samples were obtained from a central venous catheter at the following time points: immediately after insertion during anesthetic induction (T1), at the end of the CPB (T2) and at 4 h (T3), 24 h (T4) and 48 h after surgery (T5).

e. able to induce full T-cell differentiation 27, 38, 39. BALB/c ByJ and OT-I TCR-transgenic (Charles Rivers), C57BL/6J (Janvier), and ubiquitin–GFP-expressing mice 23 (Jackson) were housed and bred CH5424802 supplier in our SPF animal facility. Unless otherwise specified in the legend of the figures, wt C57BL/6 mice were used in the experiments. This study was carried out in strict accordance with the recommendations in the Guide

for the Care and Use of Laboratory Animals of the Commitee of Animal Care and Use of the Regional Cote d’Azur. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Institut de Pharmacologie Moléculaire et Cellulaire (Permit Number: B-06-152-5, delivered by the Veterinary Services of the Alpes-Maritimes Prefecture) and by the animal use committees at the Albert Einstein

College of Medicine. All efforts were made to minimize suffering and provide humane treatment to the animals included in the study. We used the L. monocytogenes 10403s background strain in all experiments, either wt or deleted in the secA2 gene, expressing or not GFP 16. Wt Lm-OVA was a kind gift from Hao Shen (University of Pennsylvania, PA, USA). For infections, Lm were grown to log phase (OD600∼0.05–0.15) in broth heart infusion (BHI) medium (Sigma-Aldrich), diluted in PBS and injected in the lateral tail vein. For Lm titers, organs were Vadimezan solubility dmso dissociated on metal screens (water 0.1% Triton X-100), and serial dilutions plated onto broth heart infusion plates. Spleens were digested 20 min at 37°C in HBSS (Invitrogen) containing 4000 U/mL collagenase I (Invitrogen) and 0.1 mg/mL

DNase I (Roche). Red blood cells were lysed for 5 min in 170 mM NH4Cl, 17 M Tris-HCl and pH 7.4. All fluorochrome-labeled mAbs are listed in the Supporting Information Table S1. PE-conjugated LLO91-99/H2-Kd Urease tetramers were obtained from the NIH tetramer core facility. Splenocytes were stained with the specified antibodies in PBS containing 0.5% BSA (FACS buffer). For surface staining, cells were incubated for 20 min on ice. For intracellular staining, splenocytes were incubated for 4 h at 37°C, 5% CO2 in RPMI1640 (Invitrogen) 5% FBS, 2 μg/mL Golgi Plug (BD) with or without 100 nM LLO91–99 peptide (Mimotopes), fixed in 1% paraformaldehyde/FACS buffer 10 min, incubated 20 min in 1× Perm/Wash (BD). Cells were analyzed on a FACSCalibur cytofluorometer (BD). When indicated, cells were sorted on a FACSVantage SE cell sorter (BD). Organs were homogenized in PBS containing a complete protease inhibitor cocktail (Roche), centrifuged 10 min 12 000×g. The supernatants were incubated with the BD Cytometric Bead Assay Flex Sets and analyzed using a FACS Array (BD).

S1). In our next experiments, we used live FITC-conjugated S. aureus (strain SH1000) to investigate

the effect of PAR2-cAP alone or together with IFN-γ on the phagocytic activity of human monocytes and neutrophils APO866 against viable bacteria. We found that PAR2-cAP (1 × 10−4 m) or IFN-γ (100 ng/ml) alone enhanced phagocytic activity (Fig. 1a–d; a,b for neutrophils and c,d for monocytes). Although IFN-γ already appeared to stimulate phagocytic activity of monocytes at a concentration of 10 ng/ml, these effects were not statistically significant (Fig. 1c,d). Interferon-γ at a higher concentration (100 ng/ml) also enhanced phagocytic activity of human monocytes and neutrophils. The effects of IFN-γ at a concentration of 100 ng/ml reached statistical significance MK0683 (Fig. 1a–d). Stimulation with IFN-γ increased the number of FITC-positive human monocytes (49 ± 13% of change compared with untreated cells) and FITC-positive human neutrophils (41 ± 7% of change compared with untreated cells). The MFI also increased in IFN-γ-treated human monocytes (increased by 53 ± 14%) and neutrophils (increased by 80 ± 18%) compared with untreated controls. PAR2-cAP led to an increase in the amount of FITC-positive monocytes (increased by 35 ± 7%) and FITC-positive

neutrophils (increased by 24 ± 4%) compared with untreated samples. The MFI also increased in monocytes treated with PAR2-cAP (increased by 38 ± 8%) and in neutrophils (increased by 38 ± 4%) compared with untreated control samples.

The combined action of PAR2-cAP and IFN-γ using the same concentrations did not enhance the phagocytic activity of neutrophils or monocytes beyond that triggered by either agonist acting alone (Fig. 1a–d). Interferon-γ is a well-known endogenous modulator of phagocytic bacteria killing and secretory activity of human neutrophils and human monocytes.25,26 As an exogenous activator, LPS also affects phagocytic activity of both cell types. We wondered whether PAR2-cAP stimulation might interfere with LPS-modulated phagocytic activity of human neutrophils and monocytes. However, PAR2-cAP stimulation of human neutrophils as well MycoClean Mycoplasma Removal Kit as monocytes did not enhance the LPS-induced phagocytic activity against S. aureus (see supplementary material, Fig. S2). Hence, despite the fact that PAR2-cAP alone up-regulates the phagocytic activity of human neutrophils and monocytes against S. aureus, this agonist failed to enhance IFN-γ-induced and LPS-induced phagocytic activity. We next investigated whether treatment of isolated human neutrophils with PAR2-cAP alone or in combination with IFN-γ affects the bactericidal activity of these phagocytes. In accordance with biosafety limitations, we used live E. coli bacteria in our experiments to estimate neutrophil killing activity.

30 To determine the CD74/MIF downstream signalling cascade in B cells from SLE-afflicted mice, we tested the production of the anti-apoptotic molecules https://www.selleckchem.com/products/jq1.html Bcl-2 and Bcl-xL, and of the pro-apoptotic molecule Caspase-8 and assessed the effect of treatment with hCDR1 on their expression. Figure 4(a)

presents the mean levels of the anti-apoptotic Bcl-2 and Bcl-xL gene expression relative to the expression in the vehicle-treated group. As shown, the expression of Bcl-2 and Bcl-xL was significantly reduced in B cells of hCDR1-treated mice, compared with B cells of vehicle-treated (P = 0·03 and P = 0·01) or control peptide-treated (P = 0·0001 and P = 0·05) mice, respectively. The down-regulating effect of hCDR1 on the latter genes was also confirmed at the protein level by Western blot analysis. Figure 4(b)

shows that the expression of the pro-apoptotic Caspase-8 was significantly up-regulated in B cells of hCDR1-treated mice (P = 0·001 and 0·02, respectively), compared with B cells of vehicle-treated or control-peptide-treated mice. The up-regulating effect of hCDR1 on Caspase-8 was also confirmed at the protein level using Western blot analysis. Hence, hCDR1 down-regulates the anti-apoptotic molecules Bcl-2 and Bcl-xL, which were elevated, and up-regulates the pro-apoptotic molecule Caspase-8, GDC-0068 purchase which was diminished, in B cells of SLE-afflicted mice. We further investigated the association Phosphatidylethanolamine N-methyltransferase between the expression levels of the anti-apoptotic and pro-apoptotic molecules and the rates of apoptosis in B cells from the experimental mice. Figure 5(a) shows the B220+ cells

that were stained for Annexin-V out of the propidium iodide-negative cells. An increase in the percentage of Annexin-V-positive cells was found in B cells of hCDR1-treated mice compared with the vehicle-treated and control-peptide-treated mice. To directly demonstrate whether the up-regulation of B-cell apoptosis by hCDR1 was mediated through the down-regulation of MIF (Fig. 2), we incubated spleen cells isolated from vehicle-treated or hCDR1-treated mice in the presence or absence of MIF and analysed them by Annexin-V staining. It can be seen that the addition of MIF to the vehicle-treated B cells induced almost no change in the low levels of apoptosis, as determined by the Annexin-V staining. The figure shows that the number of Annexin-V-positive cells was increased in the hCDR1-treated cells but the addition of MIF to the latter cells resulted in a significant reduction of B-cell apoptosis. Hence, MIF, CD74 and CD44 regulate B-cell survival in SLE-afflicted mice and following hCDR1 treatment the expression of these molecules and their downstream cascade are diminished. Kidney and central nervous system (CNS) involvement are common in SLE. We demonstrated previously that treatment with hCDR1 ameliorated kidney damage,4,6,7,31–33 CNS pathology and cognitive behaviour5 in the SLE-afflicted mice.