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

mice Data

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

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

and IL-18 are produced Selleckchem LY2835219 as a consequence of an infectious/inflammatory process, the thymus cell number is reduced favoring the creation of new niches in this organ that facilitate peripheral B and T cells entrance to the thymus. Interestingly, this phenomenon occurs in the absence of any antigenic stimulation and seems to be part of bystander activation of certain peripheral mature B and T cells. The fact that systemic IL-12 and IL-18 expression is observed in numerous situations opens the possibility that this migratory events described here are also possible in a numerous type of pathological processes. At the present moment, very we are evaluating if the entrance of B and T cells is due to a mere opportunism of cells during a moment of large expansion of leukocytes or if it is a coordinated process that plays a role in thymus physiology. Moreover, evaluation of peripheral cell localization in the thymus could provide important information not only about the source of required factors peripheral B and T cells use to survive in the thymus but also about the role they

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

“Cranial fasciitis is a rare lesion of young children char

“Cranial fasciitis is a rare lesion of young children characterized by proliferation of fibroblastic spindle cells. Most are scalp masses and are only rarely intracranial, where an association with radiation therapy is exceptional. We report a 32-month-old toddler

with a facial rhabdomyosarcoma, diagnosed at 3 months of age, and treated with surgery, chemotherapy and brachytherapy. Brain MRI at 28 months revealed a large, left parasagittal, dural-based, T2 hyperintense and T1 hypointense enhancing mass with superior sagittal sinus compression and bony hyperostosis. The mass was completely resected during an open craniotomy. Histologically, the lesion was comprised of loosely and haphazardly arranged bland spindle cells embedded in a myxoid background. Thick hyalinized collagen bundles were especially prominent. The spindle cells reacted for vimentin but not SMA, Sorafenib myogenin, MyoD1 or EMA. A diagnosis of cranial fasciitis was rendered. The role of radiation therapy in the pathogenesis of intracranial cranial fasciitis is discussed. “
“JC virus (JCV) granular neuronopathy remains an under-appreciated

phenomenon whereby JCV inhabits neurons in the granular layer of the cerebellum causing neuronal loss, gliosis and a clinical cerebellar syndrome. The following ITF2357 chemical structure case describes a man with sarcoidosis and idiopathic leukopenia who developed a clinical cerebellar syndrome due to JCV granular neuronopathy, followed by neurological decline due to rhombencephalic progressive multifocal leukoencephalopathy. This case reminds us of the ability of JCV to produce dual neuropathology which includes JCV granular neuronopathy, and the pathogenesis and clinical implications for this phenomenon are discussed. “
“An unusual case of intraparenchymal

myofibromatosis of the brain occurring in a 29-year-old woman is described. Preoperative CT and MRI examinations revealed two well-circumscribed nodular masses localized in the wall of the left lateral ventricle and right temporal lobe, respectively. Both masses were completely resected, and the patient remains disease-free 2 years post-surgery. Histopathologically, the lesions were characterized by stratification. From outer Cyclic nucleotide phosphodiesterase to inner, there was a reactive glial component, lamellated well-differentiated muscle-like cells, densely compact collagen fibers and cellular tumor with nodular and hemangiopericytoma-like patterns, respectively. The myofibroblastic nature of this tumor was verified by immunohistochemical staining and ultrastructural analysis. Intraparenchymal myofibromatosis may be confused with, and should be distinguished from, meningioma, myopericytoma, solitary fibrous tumor, leiomyoma and inflammatory myofibroblastic tumor for accurate diagnosis and optimal treatment. “
“A 68-year-old Japanese man gradually showed abnormal behavior and gait disturbance with bradykinesia.

In vitro studies demonstrate WKN4 mutations leading to decreased

In vitro studies demonstrate WKN4 mutations leading to decreased expression of ROMK, and lead to increase chloride permeability. Treatment with hydrochlorothiazide not only improves biochemical parameters, it has also reportedly improved growth & pubertal development, highlighting the need for early diagnosis. This case highlights the challenge of patients who pose a diagnostic dilemma, and the need for overall review of a patient, especially when

individual specialists are treating individual symptoms. 284 IS RENAL BIOPSY NECESSARY IN HIGH RISK TGF-beta inhibitor LUPUS PATIENTS? A CASE REPORT P SANGHI, B HIREMAGALUR, J KURTKOTI Gold Coast University Hospital, Australia Introduction: Early renal biopsy in Lupus nephritis (LN) not only

helps in diagnosis but guides management & prognosis too. However bleeding remains foremost concern following the procedure in these patients. Hence biopsy should be deferred if the management is not going to be altered. Case: A 23 year old with known class IV/V LN being treated with cyclosporine & prednisone along with warfarin for positive lupus anticoagulant state, presented with 3 day history of pleuritic chest pain, vomiting, & abdominal distension. She was heamodynamically stable with ascites on clinical examination. Her investigations showed anemia, elevated INR, low compliments, elevated double stranded DNA & acute CP-868596 molecular weight renal failure along with haemoproteinuria. A diagnosis

of flare of lupus was made & her immunosuppression was increased. Follow up: She was commenced on daily plasma exchange (PE) with albumin & fresh frozen plasma. She underwent a renal biopsy & was discharged after 2 weeks of completing PE. She was readmitted again Pomalidomide research buy with 3 day history of severe abdominal pain and hypotension. Initial CT angiogram revealed large left sided retroperitoneal haematoma requiring urgent coiling & embolization. She was discharged home after 3 weeks stay in hospital with regular renal follow up. Conclusions: Although relapse after therapy would prompt a repeat biopsy, in patients with known class III/IV even in a flare state repeating biopsy may not be required. Our patient already had 3 renal biopsies in the past with evidence of global sclerosis. This case highlights the bleeding complications involved with biopsy in high risk lupus patients which can add to their morbidity. Hence we recommend that repeat renal biopsy is unnecessary & should be better avoided in high risk lupus patients.

However, identification of the

JAK responsible for the th

However, identification of the

JAK responsible for the therapeutic effectiveness of JAK inhibitors against rheumatoid synovitis remains a key question. CP-690,550 and INCB028050 both blocked OSM-induced JAK-1/-2/-3 phosphorylation, as well as STAT-3 activation and subsequent acute-phase SAA mRNA expression. In contrast, the JAK-3-selective inhibitor, PF-956980, failed to inhibit OSM-induced STAT-3 activation and acute-phase SAA mRNA expression. In addition to STAT-3, STAT-1 and STAT-5 have also been shown to exert potent immune-activation actions and to contribute to rheumatoid synovitis [29]. In agreement with previous reports, this study showed that JAK-3 plays an important role in downstream PD-332991 STAT-1/-5 activation and subsequent MCP-I mRNA expression [20]. However, JAK-3 inhibition alone was insufficient to control STAT-3-mediated proinflammatory cascades. JAKs are fundamental components of diverse signalling

pathways, ALK inhibitor drugs including immune cells [30]. It appears likely that this new class of immunomodulatory drug will have an impact on the treatment of immune-mediated diseases. In relation to JAK-specific inhibition, CP-690,550 was reported recently to have modest selectivity against JAK-1/-2 in addition to JAK-3 [16], while the JAK-1- and JAK-2-selective inhibitor INCB028050 has also demonstrated efficacy in an RA mouse model mice, as well as in the treatment of RA [17]. These findings suggest that JAK-1/-2 signalling may also contribute to the rheumatoid proinflammatory process, and that pan-JAK inhibitors also effectively suppress STAT-3-mediated rheumatoid inflammation. Our results revealed that selective inhibition of JAK-3 alone resulted Amrubicin in abortive STAT-1/-5 activation in rheumatoid synoviocytes, but did not affect OSM-induced STAT-3

activation. Additionally, JAK-3-selective inhibition did not down-regulate OSM-induced acute-phase SAA mRNA expression, in which STAT-3 activation plays a critical role [22]. Research into JAK inhibitors is at an interesting phase, with several selective and non-selective inhibitors in various stages of clinical trials [31]. It seems logical to target a single JAK, if possible, in order to minimize the adverse effects [32]. However, non-selective JAK inhibitors may have advantages against multi-factorial disorders with proinflammatory characteristics. In conclusion, the results of this study indicate that JAK inhibition can affect multiple steps of cytokine-induced proinflammatory pathways by targeting downstream STATs in rheumatoid synovial fibroblasts. However, suppression of JAK-3 alone did not affect STAT-3 activation or STAT-3-dependent proinflammatory gene expression. These results suggest that the proinflammatory responses induced by IL-6-type cytokines may be blocked by non-selective JAK inhibitors such as CP-690,550 and INCB028050.

In three groups a nerve defect of 20 mm was bridged with a vein g

In three groups a nerve defect of 20 mm was bridged with a vein graft. Our first experimental group comprized

an empty venous graft, in group II the venous nerve graft was filled with saline where as in group III the venous nerve graft was filled with BMSC. The animals were tested for functional recovery up to 3 months post repair. Our results show that the BMSC filled venous graft resulted in significantly better regeneration of the nerve defect compared to controls, as confirmed by the functional recovery measured by somatosensory evoked potentials, toe spread, pin prick, and gastrocnemius muscle index. Conclusively, the results confirm that the vein graft supported with BMSC is associated with better functional selleck screening library nerve regeneration. © 2010 Wiley-Liss, Inc. Microsurgery, 2010. “
“The aim of this study was to

evaluate the effect of Platelet Rich Plasma (PRP) and Platelet ICG-001 Rich Fibrin (PRF) on peripheral nerve repair. Thirty-two Wistar rats were randomly divided into four equal treatments groups: autologous nerve grafts (ANG), silicon tube plus saline solution (SS), silicon tube plus PRP, and silicon tube plus PRF. In ANG group, 10 mm segment from sciatic nerve was excised and reimplanted between the nerve stumps. In the SS, PRP, and PRF groups, 5 mm segment from sciatic nerve was excised and bridged with a 12 mm silicone conduit to create a 10 mm nerve gap. The conduit was filled in accordance with the different treatments. Walking track analysis was performed periodically and on the 90th post-operative day histomorphometric analysis was performed. The ANG, PRF, and PRP groups presented a significant functional improvement

in relation to the SS group (P = 0.001) on 90 days after surgery. Histomorphometric analysis demonstrated that the Casein kinase 1 ANG group achieved a larger nerve fiber diameter in proximal stump while comparing with the SS group (P =0.037) and showed larger fiber diameter in median stump in comparison to the PRP group (P = 0.002) and PRF group (P = 0.001). Axonal diameter and myelin sheath thickness showed no statistical significant difference between the groups in the three stumps (P ≥ 0.05). This study suggests that PRP and PRF have positive effects on the functional nerve recovery; however, these groups don’t achieve a significant improvement on the histomorphometric analysis. © 2013 Wiley Periodicals, Inc. Microsurgery 33:383–390, 2013. “
“Among possible causes of a condition of immunodeficiency, we have to consider the presence of a serious chylous dysplasia, due to the great loss of proteins through the intestinal lumen. A 20-year-old male, suffered from diarrhoea (2–4 times a day), weight loss (8 kilos in 5 years), and malnutrition (hypogammaglobulinemia, hypoalbuminemia, leukocytopenia with lymphocytopenia).

In situ, IL-33 was highly expressed in the inner nuclear cells of

In situ, IL-33 was highly expressed in the inner nuclear cells of the retina of naïve mice, and its expression was elevated in EAU mice. ST2-deficient mice developed exacerbated EAU compared with WT mice, and administration of IL-33 to WT

mice significantly reduced EAU severity. The attenuated EAU in IL-33-treated mice was accompanied by decreased frequency of IFN-γ+ and IL-17+ CD4+ T cells and reduced IFN-γ and IL-17 production but with increased frequency of IL-5+ and IL-4+ CD4 T cells and IL-5 production in the draining lymph node and spleen. Macrophages from the IL-33-treated mice show a significantly higher polarization toward an alternatively activated macrophage phenotype. Our results therefore demonstrate that the endogenous IL-33/ST2 pathway plays an important role in EAU, and suggest that IL-33 represents a potential option for treatment of uveitis. “
“Interleukin-7 (IL-7) is essential for T cell development in the thymus and maintenance of peripheral T cells. The α-chain of the IL-7R is polymorphic with the existence of SNPs that give rise to non-synonymous amino acid substitutions. We previously found an association between donor genotypes and increased treatment-related mortality (TRM) (rs1494555G) and acute graft versus host disease (aGvHD) (rs1494555G and rs1494558T) after hematopoietic cell transplantation

(HCT). Some studies have confirmed an association between MK-2206 price rs6897932C and multiple sclerosis. In this study, we evaluated the prognostic significance of IL-7Rα SNP genotypes in 590-recipient/donor pairs that received HLA-matched unrelated donor HCT for haematological malignancies. Consistent with the primary studies, the rs1494555GG and rs1494558TT genotypes of the donor were associated with aGvHD and chronic GvHD in the univariate analysis. The Tallele of rs6897932 was suggestive of an association with increased frequency

of relapse by univariate analysis (P = 0.017) and multivariate analysis (P = 0.015). In conclusion, this study provides further evidence of a role of the IL-7 pathway and IL-7Rα SNPs in HCT. Interleukin-7 ID-8 (IL-7) is essential for T cell development in the thymus [1] and maintenance of peripheral T cells [2]. IL-7 receptor (IL-7R) consists of the common gamma-chain (CD132) as well as an α-chain (CD127). The α-chain of the IL-7R is polymorphic with the existence of four non-synonymous single nucleotide polymorphisms (SNPs) in the exons; rs1494558 (+510C/T in exon 2), rs1494555 (+1237A/G in exon 4), rs6897932 (+2087T/C in exon 6) and rs3194051 (+3101A/G in exon 8) that all give rise to amino acid substitutions [3, 4]. The α-chain is also used by the receptor of thymic stromal lymphopoietin (TSLP), a cytokine with complex effects on cytokine profiles, including stimulation of TNF production by dendritic cells (DC) and the induction of Th2 cytokines [3, 5, 6].

S3) This suggests that modulation of DCs by B10 cells observed i

S3). This suggests that modulation of DCs by B10 cells observed in other tissue compartments [17] does not occur in the liver. Having demonstrated that hepatic B cells comprise fewer regulatory subsets than splenic B cells, a question not addressed in this study is why Bregs appear not to contribute to the overall tolerogenic liver environment. One possibility may be to prevent overinhibition of immune responses in the liver. As shown in this report and by others [15-17], the TLR-4 ligand LPS, a normal constituent of portal venous blood, is a potent stimulator of B10 cells. The absence of B10 cells and the presence of B cells with proinflammatory potential in an overall tolerogenic liver environment

could help to balance the hepatic capacities of immune tolerance and immune stimulation. Our data presented here show that the absence of hepatic B cells compromises further the capacity of mDCs to respond to LPS (Fig. 3). To obtain sufficient numbers of liver Fulvestrant purchase mDCs for

analysis, Flt3L-treated mice were used in Fig. 3 and Supplementary Figs S2 and S3. We are aware of the caveat that Flt3L might modify the composition of mDC subsets as well as other cells. Extended experiments using animal models are BEZ235 clinical trial needed to confirm the positive regulation of liver mDCs and liver immune responses by hepatic B cells. Future research to understand more clearly the mechanisms underlying hepatic B cell activation and function is merited, and may lead to improved understanding and therapy of different liver-related pathological conditions. The authors thank Dr David Rothstein for the gift of IL-10 reporter mice and Thomson laboratory members for helpful discussion. The work was supported by NIH grant P01AI81678 (A.W.T.), Anidulafungin (LY303366) grant (874279717) from the Roche Organ Transplantation Research Foundation (A.W.T.) and by an American Society of Transplantation Basic Science Fellowship awarded to Hong Zhang. Hong Zhang did most of the experiments and wrote the manuscript, Donna

Beer Stoltz performed immunofluorescence, Geetha Chalasani provided direction for B cell subset analysis and Angus W. Thomson provided intellectual input and guided the preparation of the manuscript. The authors declare no financial or commercial conflicts of interest. Fig. S1. Expression of cell surface activation markers on murine B cells following in-vivo poly I:C administration. C57BL/6 (B6) mice were injected intraperitoneally (i.p.) with lipopolysaccharide (LPS). On days 0 and 1 post-injection, the mice were examined for the expression of the indicated surface molecules on spleen versus hepatic B cells; n = 4 mice per group. On day 1, both liver and splenic B cells up-regulated expression of CD39, CD40, CD80 and CD86; *P < 0·05. No significant difference was observed between the liver and spleen. Data are representative of two independent experiments. Fig. S2. Close proximity of B cells (CD19+) and dendritic cells (DCs) (CD11c+) in liver parenchyma.

G Dranoff, Dana-Farber Cancer Institute, Boston, MA, USA), repla

G. Dranoff, Dana-Farber Cancer Institute, Boston, MA, USA), replaced every other day. On day 6, BMDC were detached with enzyme-free digestion buffer (Sigma-Aldrich, St. Louis, MO, USA). BMDC pulsed with α-GalCer (200 ng/mL, Kirin) or vehicle (Tween-20) in medium for 3 h at 37°C. BMDC were subsequently washed with PBS and

fixed with 0.02% glutaraldehyde (Sigma-Aldrich) for 1 min click here before being used in experiments. Single cell suspensions from spleens were prepared by standard techniques. Liver MNC were isolated as previously described 17 without prior Collagenase digestion. Briefly, livers were perfused with PBS, minced and iNKT cells were enriched by centrifugation in a two-step Percoll gradient. Enriched populations typically contained 20–30% iNKT cells. Human iNKT cell lines were

established by sorting PBMC with iNKT-mAb 6B11 and expanding with mitogen as described 26. Lines were maintained by periodic re-stimulations and purity checked with Vα24 mAb 26. iNKT cells from livers were stimulated in the presence of either plate-bound PBS57-loaded CD1d monomers or α-GalCer-pulsed and Glutaraldehyde-fixed BMDC. PBS57-loaded CD1d monomers were plate-bound overnight in PBS at 4°C, blocked and washed with complete culture medium before cells were added. Cytokine-specific ELISA assays (eBioscience, San Diego, CA, USA) were performed following the manufacturers instructions. Sera were diluted 1:10 in PBS/1% BSA. RNA isolations using TRIzol (Invitrogen, Carlsbad, CA, USA) and RT reactions were performed as described 27. Real-time

NADPH-cytochrome-c2 reductase PCR using 1/20 volume of reverse BIBW2992 price transcription reactions and primers specific for adenosine receptors A1R (F, 5′-CATTGGGCCACAGACCTACT-3′, R, 5′- CAAGGGAGAGAATCCAGCAG-3′), A2aR (F, 5′- CACGCAGAGTTCCATCTTCA-3′, R, 5′-ATGGGTACCACGTCCTCAAA-3′), A2b (F, 5′- TGCTCACACAGAGCTCCATC-3′ R, 5′- AGTCAATCCAATGCCAAAGG-3′), A3R (F 5′-GCTGATCTTCACCCATGCTT-3′, R, 5′- ATCCAAACTGACCACGGAAC-3′), and GAPDH (F, 5′-aactttggcattgt-3′, 5′-acacatttgggggta-3′) were performed using Quantitect SYBR Green in a Corbett (Qiagen, Valencia, CA, USA). Target gene expression was normalized against levels of GAPDH and normalized against standards with known copy numbers (102–105/reaction) of adenosine receptors. Subsequent to blocking with anti-CD16/32 mAb cells were stained with CD3-FITC, NK1.1-PE and CD1d tetramer-APC. NKT cells were gated as CD3+NK1.1+CD1d-tetramer+ and sorted to purities >95% using a FACSAria (all BD Biosciences, San Jose, CA, USA). Intracellular stainings for IL-4 and IFN-γ were performed using Cytofix/cytoperm (BD Biosciences) according to manufacturer’s instructions. Results are expressed mean±SD. For statistical analyses, the one-way-ANOVA with Newman-Keuls post-test was used. Values of p<0.05 were considered as significant.

After centrifugation at 5000 g 10 min, supernatants were frozen a

After centrifugation at 5000 g 10 min, supernatants were frozen at −80°C until used. Extracts (50 µg protein/lane) subjected to 10% SDS-PAGE were immunoblotted with antibodies to total Bad, phosphorylated Bad (Santa Cruz Biotechnology) and revealed by enhanced chemiluminescence (ECL) detection system (Pierce). Densitometric analysis of protein levels was performed with ImageQuant software. The frequency of

apoptotic acini cells was also assessed by flow cytometry analysis with Annexin V/IP double staining following the manufacturer’s recommendations (BD). Flow cytometry data were acquired in a FACSAria cytometer® and results analysed using WinMDI software®. For bax expression assays, acinar cells were homogenized either freshly or after induction with TNF-α and RT–PCR experiments were carried out as indicated FG-4592 manufacturer above and previously [16]. Statistical significance of differences was determined by the two-tailed t-test check details for independent populations. When multiple comparisons were necessary, the Student–Newman–Keuls test was used after analysis of variance. Differences between groups were considered significant at P < 0·05. Figure 1a shows the expression kinetics of VIP and their receptors in submandibular

glands isolated from NOD mice of different ages from postnatal day 2 to 20 weeks of age. Compared to normal mice, NOD mice showed the highest level of VIP expression at 4 weeks of age and decreased thereafter. The progressive decrease in VIP expression from the fourth week takes place with no changes in VPAC1 and VPAC2 receptors. A clear reduction

of VIP levels was evident in NOD submandibular glands at 16 weeks ever of age (Fig. 1a), which was confirmed by qRT–PCR (Fig. 1b). The decline in VIP/VPAC1 relative expression over time is similar to the kinetics of neural nitric oxide synthase (nNOS) activity and salivary secretion loss shown previously [12]. NF-κB appears as an intracellular pivotal determining factor that conditions the apoptotic or survival fate of TNF-α-stimulated cells [28]. Thus, we analysed NF-κB activation and apoptosis in response to TNF-α in NOD acinar cells. As shown in Fig. 2a, acinar cells from NOD glands present a basal translocation of p65 of NF-κB to the nucleus (merge image with PI-stained nuclei) that is not seen in normal BALB/c mice. Consistent with this, WB analysis of I-κB in the cytosolic fraction or p65 in the nuclear fraction revealed that p65 appeared located to the nucleus, while I-κB expression was increased in cytosol of acini in basal conditions (Fig. 2b). Moreover, when treated in vitro with TNF-α, NOD mice acinar cells showed an abnormal NF-κB activation kinetics compared with BALB/c acinar cells (Fig. 2a,b).

e able to induce full T-cell differentiation 27, 38, 39 BALB/c

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 BIBW2992 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 Ensartinib mw 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 Fludarabine 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).