1 mM nonessential amino acids, and 1 mM sodium pyruvate (Life Technologies, Grand Island, NY)]. All cultures were maintained at 37 °C in a humidity-controlled incubator with 5% CO2 and were grown to confluence over 5–6 days before addition of pathogenic bacteria C. rodentium. The cells were washed and placed in antibiotic-free medium for 1 h. Confluent stock monolayers were subcultured by trypsinization. In this study, we utilized mouse intestinal epithelial cell line CMT93 to better elucidate cell

signaling responses to enteric pathogens in vitro. Nine experiments were conducted independently with similar results. To determine the time-dependent intracellular changes of NF-κB and Smad 7 in response to pathogen exposure, CMT93 cells were exposed with Cr (2.5 × 107 CFU per well) for 1 h https://www.selleckchem.com/products/dinaciclib-sch727965.html in Obeticholic Acid solubility dmso antibiotic-free DMEM at 37 °C. Subsequently, the media and cell lysates were collected at 0, 15, 30, 60, 90, and 120 min and 14 and 24 h postpathogen exposure. Cells were washed and lysed [(1% Triton-X-100 supplemented with 0.1 μM phenylmethylsulphonyl fluoride, 0.1 μM sodium orthovanadate, and Halt protease inhibitor (10 μL mL−1,

Pierce cat# 78410, Thermo Scientific, Rockford, IL)]. The lysates were kept at −80 °C for future Western blot analysis. The culture supernatants were stored at −20 °C for future measurement of TNF-α cytokine production. Total RNA was isolated from frozen colonic tissue (distal part of the colon) and treated CMT93 cells using TRIzol (Invitrogen Life Technologies, Carlsbad, CA) following the manufacturer’s protocol. First-strand cDNA was synthesized using 2 μg of extracted total RNA (Ready-to-Go kit; Amersham Pharmacia Biotech, Piscataway, NJ). IL-10 and TGF-β colonic expression was determined by real-time PCR using QuantiTect SYBR green real-time PCR kit (Qiagen, Valencia, CA) on the Opticon II DNA thermocylcer (MJ Research, Waltham, MA). A PCR master mix was prepared according to the manufacturer’s Methane monooxygenase protocol with a reaction volume of 50 μL, using the following real-time cycler conditions: 95 °C for 15 min, 94 °C

for 15 s, 55 °C for 30 s, 72 °C for 30 s for 38 cycles. GAPDH was used as internal controls. LightCycler relative quantification software was used to normalize data to the same GAPDH mRNA level. Samples were run in duplicate. Mouse IL-10 and TGF-β commercially available PCR primers were purchased from Biosource International, Inc. (Camarillo, CA) for detection, while GAPDH commercially available upstream and downstream PCR primers were utilized for detection (R&D Systems, Minneapolis, MN). Mouse colonic tissue and treated CMT93 cells were homogenized with lysis buffer prepared as previously mentioned. The suspensions were centrifuged at 4 °C, and the supernatant was collected, and protein content was determined using DC protein assay (Bio-Rad Laboratories, Hercules, CA).

CD8 DCs are considered the classic cross-presenting DC and, for a long time, have been assumed to be the only mouse DC population with the ability to cross-present cell-associated antigens to CD8+ T cells. CD8 DCs display more efficient phagocytic uptake of dead cells and loading of antigenic

peptides into MHC class I than many other DC populations. In addition, CD8 DCs are able to produce high levels of bioactive IL-12p70 that helps in their induction of Th1/Tc1 responses. Acalabrutinib solubility dmso However, their capacity to present antigens in MHC class II to CD4+ T cells under conditions of limiting antigen is relatively poor (reviewed in [52]). Our studies show that FLT3L treatment greatly expanded the recently described mcDC population, that potently primes both CD4+ and CD8+ T cell to cell-associated antigens [12,23]. Importantly, T cells primed to cell-associated antigens by mcDC displayed greater primary expansion and development into memory cells than those primed by other DC populations.

The superior T cell priming capacity of mcDC can be contributed to several mechanisms. mcDC store phagoytosed materials in non-acid organelles and use this as an antigen depot which allows for prolonged antigen presentation [24]. Increasing the length of antigenic stimulation has been shown to positively affect T cell expansion, acquisition of effector functions and memory development [53–56]. Secondly, the type I IFN production by mcDC upon Selleckchem BMN 673 uptake of apoptotic material is likely to provide an adjuvant effect in both an autocrine and paracrine Selleckchem Fludarabine fashion (manuscript in preparation). Moreover, our previous observations indicated that mice deficient in type I IFN sensing failed to induce protective CD8+ T cell responses when treated with autologous tumour vaccines [12,23]. Besides the production of type I IFN, the mcDCs capacity to prime strong CD4+ T cell responses to cell-associated antigens

is also instrumental in the induction of anti-tumour CD8+ T cell responses. We and others have shown that CD4+ T cell help during priming of CD8+ T cells is required for optimal CD8+ T cell activation, primary expansion, acquisition of effector function and the development of memory [42,57,58]. Supportively, increasing CD4+ T cell help through transfer of (transgenic) CD4+ T cells or preimmunization of mice enhances the induction of CD8+ T cell responses [59,60]. In addition, ample studies indicate that CD4+ T cell help plays a supporting role in the maintenance, reactivation and expansion of existing memory cells [61–63]. FLT3L was shown recently to increase a DC population that had the ability to cross-present cell-associated antigens to CD8+ T cells without the need to express CD8α[64].

Tissue suspected of being infected with Mucorales should be minced into small pieces with a scalpel or single edge razor blade before inoculation onto media; grinding or homogenisation of tissue specimens may destroy the delicate hyphae rendering cultures negative. Colonies of Mucorales usually appear within 24–48 h unless residual antifungal agents, which can suppress growth. Most species demonstrate a greyish white, aerial mycelium with a wooly texture and fill a culture dish within 3–5 days. This study will therefore utilise morphological, physiological

and molecular methods for identification of organisms in culture and, where feasible, in paraffin-embedded tissue. Development of an archive of organisms recovered from patients with documented mucormycosis LY2109761 manufacturer is essential Protein Tyrosine Kinase inhibitor to achieving objective III. There are now several molecular and antigenic assays that detect the presence of Mucorales in laboratory animal models of mucormycosis.[14, 15] Other systems have not been studied in animal model systems but also exhibit analytical sensitivity and specificity

for the Mucorales.[16-19] Although one report describes the analytical performance of a three quantitative polymerase chain reaction assays using hydro-lysis learn more probes in 10 patients, the small number of cases and complexity of the molecular diagnostic platform limit regulatory review or extrapolation to other laboratories.[20] To enable candidate assays to become widely available for early diagnosis of mucormycosis and to improve patient outcome, an archive of specimens for

mucormycosis is critically required. As these assays must be validated in human specimens of mucormycosis for scientific, clinical and regulatory acceptance, the development of this archive (IMAS) is critical. This specimen archive will consist of the clinical samples (Table 3), where feasible and applicable, from each patient enrolled into ZWG2. Each investigator will store the specimens at his or her centre. At a designated time, specimens will be divided in equal amounts by the investigator and shipped to two central facilities under the care of Dr. Olivier Lortholary at the ZWG Archive Center in Paris and Dr. Thomas Walsh at the ZWG Archive Center in New York City. Storage in two geographically distinct locations assures preservation of specimens in the event of natural or human-made disasters. Following review of candidate assays, specimens will then be shipped to investigators conducting laboratory diagnostic projects approved by the ZWG Steering Committee.

Several serological studies from Sweden

(Dahlquist et al., 1995a, b) and Finland (Hyöty et al., 1985; Elfving et al., 2008) support a relationship between maternal enterovirus infection during pregnancy and T1D in the offspring, established by the age of 10 years or even later. However, enterovirus infections in the 1st trimester were not a risk factor (Viskari et al., 2002), which is not in accordance with the outcome of our experimental study. The present study shows for the first time that infection of outbred mice by oral route during gestation Selleckchem CH5424802 results in enhanced pathology upon postnatal challenge of the offspring with the homologous virus strain. The pathology was mainly confined to the pancreas and resulted in hyperglycemia. This observation provides a new model for study of the still enigmatic cause of T1D. The funding Midostaurin is provided by the Norwegian financial support mechanism, Mechanism EEA, and Slovak Government – Project SK0082 and received by S.B. The authors declare no conflict of interest. We thank Bill Coleman, Texas, USA, for proof reading and suggestions. Permission for the animal work was

obtained from the Ethics Committee of the Slovak Health University and the State Veterinary and Food Control Authority of the Slovak Republic. “
“Retinoic acid (RA), which is the biologically active form of vitamin A, acts through the nuclear hormone receptor RAR (RA receptor) to induce either gene activation or repression. RA production and its effects have been linked to macrophages,

dendritic cells, T and B cells, and iNKT cells in the immune system and play pro- as well as anti-inflammatory roles depending on the cell type and the immune context. In this issue of the European Journal of Immunology, Lee et al. [Eur. J. Immunol. much 2012. 42: 1685–1694] show that RA ameliorates Con A-induced murine hepatitis by selectively downmodulating IFN-γ and IL-4 production in disease-causing NKT cells in the liver. Remarkably, this effect is restricted to this liver disease model and does not apply to αGalCer-induced murine liver injury, which is driven by other cytokines. The study identifies retinoid signaling as an important endogenous mechanism controlling immune reactions and also as a potential pharmacological target for treatment of hepatic liver injury. Furthermore, the study by Lee et al. provides additional support for the concept of metabolic regulation of immune function. Presently there is an increased understanding and appreciation of the role that metabolic and lipid signaling plays in immune regulatory processes in multiple cell types (reviewed in [1]). For example, the orange pigment of carrots, beta-carotene, contributes to vitamin A levels in the body.

Ab stimulations were performed via crosslinking of the stimulating Abs (CD3 [0.5 μg/mL OKT3], CD28 [5 μg/mL CD28.2] or CD2 [3PTH9, 10 μg/mL] with 7.25 μg/mL goat anti-mouse Ab at 4°C). For the stimulation, T cells were set at a cell density of 4×106/mL. To analyze immune synapses, untransformed human T cells were purified from peripheral blood and incubated with SEB-loaded APCs (Raji B-cells; 5 μg/mL SEB) essentially as described previously 5, 8, 16. Briefly, T cells and APCs that were loaded with or without 5 μg/mL SEB were mixed at a ratio of 1:2 and centrifuged Microbiology inhibitor at 200×g for 3 min and suspended in 400 μL medium. After an incubation at 37°C for 45 min, the cells were fixed by adding 1.5 mL 1.5% PFA. The cells were

washed (PBS, 0.5% BSA) and stained for surface molecules with anti CD3-PeTxR and CD18 coupled to FITC (or PE if EGFP-expressing cells were used). Thereafter, cells were washed (PBS, 0.5% BSA, 0.07%NaN3) and permeabilized (PBS, 0.5% BSA, 0.07% NaN3, 0.05% Saponin) and stained for F-actin (Phalloidin-AF647) and nuclei (Hoechst33342). After extensive washing, the cells were suspended in 60 μL PBS for the ImageStream analysis. For MIFC analysis, cells were acquired using an ImageStream™ analyzer (IS100)

and selleck INSPIRE software (Amnis, Seattle, WA, USA). The ImageStream combines flow cytometry and microscopy using a 40× objective (0.75 NA). To analyze receptor accumulation in the T-cell/APC interface, MIFC was used as described recently 5. Briefly, cells were stained as described above and then acquired using an ImageStream™ analyzer (IS100) and INSPIRE software (Amnis). The cell classifier was adjusted in a way that APC singlets were not acquired. Image data were analyzed in a batch operation using IDEAS 3.0 software (Amnis). Fluorescence intensities were quantified in spatially defined regions of interest (masks) that specified the T cell or the T-cell/APC interface. Thus, a valley mask that was created between the Hoechst33342 stain of the T cells and the APCs

was defined as an intercell region. This valley mask was combined with a CD3-dependent T-cell mask resulting in the immune synapse mask. Thereafter, protein accumulation was calculated as the ratio between the pixel intensity of the respective protein in the immune synapse mask and the intensity selleck chemical of the same protein in the T-cell mask. If the ratio is bigger than 1, the respective protein is enriched in the immune synapse. We set a ratio threshold for protein enrichment at 1.2, to assure a significant degree of enrichment of the proteins in the immune synapse. To quantify the F-actin content in T cells, the phalloidin staining (MPI) within the T-cell mask was assessed 5. For lysate preparation, PB T cells were washed with phosphate-buffered saline (PBS) and lysed on ice for 30 min using TKM lysis buffer (50 mM Tris-HCl, pH 7.5, 1%NP40, 25 mM KCl, 5 mM MgCl2, 1 mM NaVO4, 5 mM NaF, 20 μg/mL Leupeptin/Aprotinin each).

The authors have no conflicts of interest. “
“Arachidonate 5-lipoxygenase-activating protein (ALOX5AP) plays a role in the 5-lipoxygenase (LO) pathway, which includes the LTC4, LTD4, LTE4 and LTB4. These leukotrienes are known causative factors of asthma, allergy, atopy and cardiovascular diseases. ALOX5AP lacks enzyme activity and acts by helping 5-LO function. In this study, healthy and general subjects who live in rural and urban areas of Korea were tested for the association of ALOX5AP polymorphisms with lung function. Lung function was also estimated by calculating the predicted values

for forced expiratory volume in one second (FEV1_%PRED) and the proportion of the forced vital capacity exhaled in the first second (FEV1/FVC_PRED). The linear regression was adjusted for residence area, gender, age, height and smoking status. The analysis revealed associations between FEV1 and the single-nucleotide polymorphism Saracatinib in vivo (SNP) rs9506352 and the haplotype TCAC (permuted P-value < 0.05). The linkage disequilibrium block that included the significant SNPs overlapped with SNPs that were revealed previously to associate with myocardial infarction and asthma and to affect lung function. This study is the first to demonstrate the association between lung function and ALOX5AP polymorphisms in a healthy and general population. Lung function is assessed for

two selleckchem purposes; epidemiologic and clinical evaluation of respiratory health. Two spirometric measures, namely forced expiratory volume in one second (FEV1) and the proportion of the forced vital capacity (FVC) exhaled in the first second (FEV1/FVC), are used as indices of the degree of airflow obstruction [1]. A reduced FEV1/FVC means the presence of airflow obstruction that correlates with the diagnosis of asthma; moreover, FEV1 serves as an objective index of asthma severity

[2]. In addition, FEV1 and FEV1/FVC are important factors for the diagnosis of chronic obstructive pulmonary disease (COPD) and decision of the disease severity [3]. Lung function is affected by environmental and genetic factors. Ethnic differences in lung function also were demonstrated in the Asia–Pacific region [4]. In particular, A previous study has examined the association between single-nucleotide polymorphisms (SNPs) in an asthma-susceptibility gene called disintegrin and metallo1protease 33 (ADAM33) and lung function in a general population. The results showed that ADAM33 SNPs are associated with lung function decline and can serve as risk factors for COPD [5]. Besides, polymorphisms in NFE2L2, KEAP1 and TIMP1 gene were associated with FEV1 in a general population of Vlagtwedde-Vlaardingen cohort [6, 7]. Obeidat et al. [8] found that association between FEV1 and rs3748312 in SERPINA1 gene on ever-smokers, although it was not passed a defined significance threshold.

To further investigate the immunomodulatory potential of DX5+CD4+ T cells, we now examined their effects on DC maturation and their ability to instruct DCs to modulate the outcome of T-cell responses. To this end, we first

incubated DX5+CD4+ T cells, which were isolated from mice that have received three injections with immature DCs [18, 19, 21, 22] with fresh bone marrow-derived DCs from naïve animals. Interestingly, we observed that DCs matured with LPS for 2 days in the presence of DX5+CD4+ T cells produced significantly less IL-12 p40 as compared to DCs cultured in the absence of these T cells. In contrast, DCs cultured in the presence of DX5−CD4+ T cells maintained their IL-12 production (Fig. 1A). These data indicate that DX5+CD4+ T cells can modulate the activation of DCs by inhibiting their IL-12 production. To assess whether cell–cell contact or a soluble factor is responsible for the Osimertinib mw suppression of IL-12 production, we next collected supernatant of either DX5+CD4+ or DX5−CD4+ T cells stimulated with anti-CD3 and anti-CD28 for 3 days. Addition of this supernatant to fresh DCs cultures

revealed that DX5+CD4+ T-cell supernatant, but not supernatant from DX5−CD4+ T cells, reduced the production of IL-12 Midostaurin price by DCs (Fig. 1B). Together, these data indicate that a soluble factor derived from DX5+CD4+ T cells can functionally modulate DCs by inhibiting IL-12 production. To explore the possibility that DX5+CD4+ T cells also modulate the cell-surface expression of molecules involved in T-cell activation, we next analyzed the expression of various surface molecules (PDL-1, PDL-2, CD80, CD86, CD40, and MHC class II) on DCs after culture with the supernatant of DX5+CD4+ T cells. The data show that

the supernatant of DX5+CD4+ T cells cultures is able to enhance the expression levels of the inhibitory molecules PDL-1 and PDL-2 on the surface of DCs. Likewise, the expression of CD80, CD86, CD40, and MHC class II was also increased after incubation of DCs with DX5+CD4+ supernatant (Fig. 2 and Supporting Information Resveratrol Fig. 2). These effects were not observed when DCs were cultured with DX5−CD4+ supernatant or were left in medium alone. These data show that phenotypic changes of DCs installed by CD4+DX5+ T cells are caused by (a) soluble factor(s) secreted by DX5+CD4+ T cells. Together, these data demonstrate the ability of DX5+CD4+ T cells to modulate the expression of cell surface molecules on DCs and cytokine production by DCs that are involved in setting the outcome of T-cell responses. We next wished to identify the soluble factor responsible for the suppression of IL-12 production. To this end, we used the results of the analysis of cytokine production of DX5+CD4+ T cells as published recently [19, 21]. Of the cytokines produced by DX5+CD4+ T cells, especially IL-4 and IL-10 [19, 21] (Supporting Information Fig.

In conclusion this case highlights the relevance, in selected cases, of sural nerve biopsy to orient the genetic/molecular tests, while in vitro analyses may strengthen the pathogenic role of novel mutations. “
“The characterization of molecular responses following cerebral ischemia-induced changes in animal models capable of undergoing real-time analysis is an important goal for stroke research. DMXAA in vitro In this study, we use transgenic mice to examine the activation of two different promoters in a firefly luciferase reporter mouse analyzable through a non-invasive bioluminescent imaging

system. In the first model, we examine the middle cerebral artery occlusion (MCAO)-induced activation of Smad-binding elements (SBE), a downstream target of Smad 1/2/3 transcription factors, in which SBEs regulate the expression of the fluc reporter. We observed that MCAO induces a bilateral activation (i.e., both ipsilateral and contralateral brain hemispheres) of the SBE-luc reporter with a peak at 24 h. In the second model, we examined MCAO-induced activation of the osmolarity-sensitive promoter nuclear factor of activated T-cell 5 (NFAT5) and identified a peak reporter expression 72 h post-MCAO in the ipsilateral GDC-0068 in vitro but not contralateral hemisphere. In each of these models, the assessment of

post-MCAO fluc-expression provided both a quantitative measure (i.e., radiance in photons/sec/cm2/steradian) as well as qualitative localization this website of the molecular

response following focal ischemic injury. “
“Extrapleural solitary fibrous tumors are uncommon mesenchymal neoplasms frequently observed in middle-aged adults and are classified, according to the WHO classification of soft tissue tumors, as part of the hemangiopericytoma tumor group. However, these two entities remain separated in the WHO classification of tumors of the central nervous system. In fact, meningeal solitary fibrous tumors are believed to be benign lesion and only in a minority of cases local relapses have been described, although detailed survival clinical studies on solitary fibrous tumors of meninges are rare. In contrast to hemangiopericytoma, which frequently shows distant extracranial metastases, such an event is exceptional in patients with meningeal solitary fibrous tumors and has been clinically reported in a handful of cases only and their histopathological features have not been investigated in detail. In this report, we describe the detailed clinico-pathological features of a meningeal solitary fibrous tumor presenting during a 17-year follow-up period, multiple intra-, extracranial relapses and lung metastases. “
“Thanatophoric dysplasia is a lethal form of chondrodysplastic dwarfism in which the cerebral cortex displays a unique and complex malformation.

Such a correlation would be consistent with the hypothesis that the natural autoantibody repertoire reflects the immunogenic body state – the immunological find more homunculus.11 We took an integrative, systems-level analysis approach by evaluating 39 patients, for whom autoantibody profiles were already available, for PGD based on chest radiographs and oxygenation data. We found that 19 patients had no indication of PGD whereas 20 patients manifested PGD grade 1 or higher. We paired the autoantibody

profiles with gene expression profiles from two recent studies comparing donor lungs that developed PGD with those that did not. We report that PGD can be differentiated by a profile of differentially reactive autoantibodies, most of which are connected in a protein–protein interaction network involved in proliferative processes such as regulation of development and cell communication. Furthermore, for the implicated proteins, we observed significant positive correlation between differential IgM reactivity and differential gene expression levels in the presence Temozolomide supplier or absence of PGD (increased expression associated with increased reactivity and vice versa). Patients attending scheduled visits during a half-year period in the out-patient clinic

at the Danish National Lung Transplant Programme were included in the study. The transplant programme has been described in detail previously.8,12 For 39 patients, PGD could be evaluated retrospectively from chest radiographs and oxygenation data pertaining to the first 72 post-operative hours. Table 1 presents clinical characteristics for this patient cohort. An additional

nine patients for whom reactivity data were also available, but whose original chest radiographs had been discarded, were set aside for validation. In this validation cohort, the presence or absence of PGD was mafosfamide ascertained from patient journals (which included day-to-day observations from chest radiographs describing the presence or absence of pulmonary oedema and infiltrates during the first 72 hr as well as documentation for treatment with nasal oxygen when this had been used). Reactivity data for IgG and IgM antibody binding in sera from these patients were retrieved from http://www.nanotech.dtu.dk/Research/Theory/SSS/Research/LungTransplant.aspx. Antigen microarray preparation, incubation of serum and fluorescent anti-IgG and anti-IgM antibodies, laser scanning and data pre-processing have been described previously.8 Briefly, 504 antigens were judged positive for IgG antibody binding (signal-to-noise ratio > 2 in at least four patients) and 610 antigens were judged positive for IgM antibody binding (473 antigens overlapping). These antigens cover 272 recombinant proteins and synthetic peptides from the sequences of key proteins. The log2-transformed, median centred, measured intensity of an antigen is denoted the reactivity of the antigen.

Two patients with Mod-PTB had unilateral pleural disease, while one had lymph node involvement. Overall, 32 of 36 patients with PTB were AFB smear/culture and/or radiology positive. Four patients were diagnosed based on radiology, clinical diagnosis and response BTK inhibitor chemical structure to treatment. Tuberculous lymphadenitis (LNTB) was diagnosed by histopathological

staining of fine needle aspirate or excision biopsy with AFB smear and culture. Pleural TB was diagnosed on the basis of pleural fluid biochemical findings, AFB culture, histopathological findings on pleural biopsy, supportive radiological evidence on X-rays and/or contrast-enhanced CT scan and response to antituberculous treatment. Diagnosis of meningeal TB was based on CSF biochemical findings, supported by AFB culture and findings on contrast-enhanced CT and/or MRI [25]. Patients with L-ETB comprised those with LNTB (n = 19) and unilateral pleural disease (n = 12). PI3K inhibitor Patients with D-ETB comprised those with tuberculous meningitis (n = 1), bilateral pleurisy (n = 1),

abdominal (n = 2), spinal (n = 4) and miliary TB (n = 2). Bacille Calmette-Guerin (BCG)-vaccinated asymptomatic healthy volunteers who were staff of AKU were used as endemic controls (ECs). Tuberculin skin testing (TST) was assessed by administering five tuberculin units on the volar surface of the right arm subcutaneously and read by a Erastin cost single reader at 48 h. An induration of ≥10 mm was used as a cut-off for positive responses (TST+), which are considered to be indicative latent infection. TST+ (n = 21) and TST− (n = 21) ECs were included in the study. Reagents. Mycobacterium tuberculosis H37Rv whole cell sonicate (MTBs) and recombinant antigens ESAT6 and CFP10 were provided through the NIH Tuberculosis vaccine testing and reagent material contract (NO1-A1-40091) awarded to Colorado State University, USA. Whole blood assay.  Heparinized venous blood was diluted 1 : 10 in RPMI-1640 medium each and set up in 96-well tissue

culture plates as per protocol [26]. Cells were stimulated with MTBs (10 μg/ml) and ESAT-6 and CFP10 (5 μg/ml each) and cultured for up to 5 days. Supernatants were collected for cytokine measurements at 2 and 5 days post-stimulation, spun to collect cellular debris and stored at −70 °C until tested. ELISA for IFNγ, CXCL9, CXCL10, CCL2 and IL10.  IFNγ standards and monoclonal antibody pairs for capture and detection were obtained from Pharmingen, San Diego, CA, USA. Reagents for CCL2, CXCL9 and CXCL10 were obtained from R&D Systems, Minneapolis, MN, USA. All measurements were carried out according to the manufacturer’s recommendations and as described previously [16]. The limit of detection for each read-out was following: IFNγ, 15.6 pg/ml; IL10, 7.8 pg/ml; CXCL9, 69 pg/ml; CXCL10, 23 pg/ml and CCL2, 23 pg/ml, respectively. Statistical analysis.