, Montgomery, TX, USA) for 30 min on ice and finally washed with 1% BSA–PBS. Multi-colour flow cytometry was performed on a fluorescence activated cell sorter (FACS)Canto,
interfaced PF-562271 price to a FacsDiva software (BD Biosciences, San Jose, CA, USA) and analysed through Flow-Jo software version 8·8·3 (Three Star Inc., Ashland, OR, USA). The binding of the antibody to the cells incubated with the different plasma samples was measured and the percentage of binding-inhibition calculated according to the background staining (cells incubated without plasma). A cartoon showing the principles of the assay is presented in Fig. 1. Purified PBMCs were thawed and stained with the following conjugated monoclonal antibodies: CD19-Alexa 488, interleukin (IL)-21R-phycoerythrin (PE), CD27-peridinin chlorophyll-cyanin 5·5 (PerCP-Cy5·5), selleck chemical CD21-allophycocyanin (APC), IgD-H7 (all from BD Biosciences) and the CD10-PE-Cy7 (Biolegend, San Diego, CA, USA). The frequencies of MA (defined as CD10–CD21–) and DN (defined as CD27–IgD–) B cell subpopulations were calculated from total CD19+ B cells. Multi-colour flow cytometry was performed on a FACSCanto,
interfaced to a FacsDiva software (BD Biosciences) and analysed through Flow-Jo software version 8·8·3 (Three Star Inc.). Plasma IL-21 titres were measured using the human IL-21 platinum ELISA kit (eBioscience, San Diego, CA, USA), following the manufacturer’s instructions. The Mann–Whitney U-test and Spearman’s correlation were used for all analyses. A P-value <0·05 was considered statistically significant. GraphPad Prism software for Windows was used to perform the analyses. The ALA titres before and after flu vaccination were quantitated as described in the Materials and methods and in Fig. 1. Before vaccination, significantly lower ALA titres were found in the
HIV group compared to KT and HC Acesulfame Potassium (P < 0·0001) (Fig. 2a), while no significant difference was found between the KT and the HC groups (P > 0·05) (Fig. 2a). Interestingly, after vaccination individuals in both the HIV and KT groups increased ALA titres substantially compared to HC (P = 0·0001 and P = 0·0002, respectively) (Fig. 2b). Between HIV and KT, the biggest increase was recorded in the HIV group (P = 0·0008) (Fig. 2c). HC increased ALA titres only slightly compared to HIV and KT (P = 0·0001 and P = 0·0003, respectively (Fig. 2c). Fifteen per cent of the HIV-1-infected individuals (10 of 65) were having a viraemic blip at the time of vaccination (Table 1). However, this did not relate to any of the parameters analysed as confirmed by Spearman’s correlation (P > 0·05). Moreover, the CD4+ T cell counts were similar in the viramic and aviraemic patients (P > 0·05).
After Fer-1 transplantation the quantity of TFH-cells was the highest in patients with pre-existent DSA. In kidney biopsies taken during rejection, TFH-cells co-localized with B cells and immunoglobulins in follicular-like structures. Our data on TFH-cells in kidney-transplantation demonstrate that TFH-cells may mediate humoral alloreactivity, which is also seen in the immunosuppressed milieu. “
“Compared with HLA-DR molecules, the specificities of HLA-DP and HLA-DQ molecules have only been studied to a limited extent. The description of the binding motifs has been mostly anecdotal and does not provide a quantitative
measure of the importance of each position in the binding core and the relative weight of different amino acids at a given position. The recent publication of larger data sets of peptide-binding to
DP and DQ molecules opens the possibility of using data-driven bioinformatics methods to accurately define the binding motifs of these molecules. Using the Selleckchem Idasanutlin neural network-based method NNAlign, we characterized the binding specificities of five HLA-DP and six HLA-DQ among the most frequent in the human population. The identified binding motifs showed an overall concurrence with earlier studies but revealed subtle differences. The DP molecules revealed a large overlap in the pattern of amino acid preferences at core positions, with conserved hydrophobic/aromatic anchors at P1 and P6, and an additional hydrophobic anchor at P9 in some variants. These results confirm the existence of a previously hypothesized supertype encompassing the most common DP alleles. Conversely, the binding motifs for DQ molecules appear more divergent, displaying unconventional anchor positions and in some cases rather unspecific amino acid preferences. The MHC performs an essential role in the cellular immune system, and regulates
immune responses through presentation of processed antigens to T lymphocytes. The MHC is also widely studied because of its association with many autoimmune and inflammatory diseases, including type I diabetes, rheumatoid arthritis, multiple sclerosis and Crohn’s disease, STK38 and certain MHC alleles have been linked to susceptibility to infectious diseases such as malaria and HIV (reviewed in ref. 1). Unlike MHC class I, which samples peptides from cytosolic proteins, MHC class II molecules present short peptide sequences derived from extracellular proteins. Human MHC class II molecules are heterodimers consisting of an α-chain and a β-chain encoded on chromosome 6 in one of three HLA loci: DR, DP and DQ. Compared with DR molecules, the specificities of DP and DQ molecules have only been studied to a limited extent, and their binding motifs are poorly characterized and understood.
PWM was used in this study as a positive control. The assay tubes were incubated for 48 h at 37°C. At 12-, 24- and 48-h time-points, 50 μl of the supernatant was transferred into Eppendorf tubes and frozen immediately at −80°C for future cytokine analyses. By rarefying these small supernatant volumes, significant dilution effects could be minimized. Frozen supernatants were measured in a blinded fashion after thawing. Concentrations of
the prototypic T helper type 1 (Th1) cytokines IL-2, IFN-γ and TNF-α were analysed by LuminexxMAP® technology (Bioplex®) with commercially available reagents from BioRad Laboratories Inc. (Hercules, CA, USA), according to the manufacturer’s guidelines. Data were analysed using Bioplex software; the sensitivity threshold was at 2 pg/ml for the analysed cytokines. Biotinylated antibodies Torin 1 research buy against CD3 (BioLegend Europe, Uithoorn, the Netherlands) were applied to lithium-heparinized
blood. After an incubation period of 10 min anti-biotin MACSiBeadTM particles (Miltenyi Biotec, Bergisch Gladbach, Germany) were added for 10 min. Mechanical cell separation took place in a cell separation magnet. Cell-depleted blood was transferred and added to the new cytokine release in-vitro test. Supernatant samples were taken after 24 and 48 h for further cytokine determination. To monitor and control the success of the T cell depletion, anti-CD3 fluorescein isothiocyanate (FITC)-marked antibodies were used subsequently to verify the T cell elimination by flow Z-VAD-FMK chemical structure cytometry. Immunostaining of cell surface antigens and intracellular
cytokines in T cells were performed according to the manufacturer’s guidelines. First, whole blood cultures with 1 ml total volume were treated for 6 h with 20 μl brefeldin A [1:10 Tyrosine-protein kinase BLK dilution, BD Cat. no. 347688; Becton Dickinson Immunocytometry Systems, Palo Alto, CA, USA]. One ml of 1:10-diluted fluorescence activated cell sorter (FACS) lysing solution (BD Cat. no. 349202) was added to 200 μl whole blood from in-vitro stimulation. After 10 min incubation, samples were centrifuged (500 g for 5 min) and the supernatant decanted; 500 μl ×1 FACS permeabilizing solution 2 (BD Cat. no. 340973) was added after ‘vortexing’ for 10 min incubation at room temperature. After washing with phosphate-buffered saline (PBS) containing 0·5% bovine serum albumin (BSA) and 0·1% NaN3 and 5 min centrifugation, 10 μl monoclonal antibodies were added and incubated for 30 min in the dark. Additional washing and resuspension of stained cells in PBS with 1% paraformaldehyde was performed. The following monoclonal antibodies (MAbs) directed against human leucocyte surface markers were used: FastImmune anti-interleukin (IL)-2/CD69/CD4/CD3 (BD Cat. no. 337188), CD4 peridinin chlorophyll (PerCP) (BD Cat. no. 345770) and CD3 allophycocyanin (APC) (BD Cat. no. 345767).
Survival data were analyzed using the log-rank test. All other data were analyzed using one-way or two-way ANOVA with the Bonferroni post-test. Statistical analyses were performed with GraphPad Prism version 5. p≤0.05 was considered significant. Venetoclax research buy We thank Leon Douglas for providing MHC I tetramers. This work was supported by grants from Cancer Research UK, Leukaemia and Lymphoma Research and the Association for International Cancer Research (to A. Al-S.). Conflict of interest: The authors declare no financial or commercial conflict of interest.
Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. “
“Suppressors of cytokine signalling (SOCS) proteins are induced in responses to many stimuli and by binding to cytokine receptors and associated janus kinase (JAK) proteins, directly regulate the activation of the signal transducers and activators of transcription (STATs). STAT proteins AUY-922 cell line regulate the expression of many genes required for the differentiation of various CD4+ T helper cell lineages, and there is now accumulating evidence that
SOCS also play essential roles in the regulation and maintenance of CD4+ T-cell polarization. As it is now clear that CD4+ T cells are more plastic than initially thought, it is of particular importance to understand the molecular mechanisms regulating CD4+ T-cell differentiation. Here we review the current understanding of how STATs and SOCS act in concert to influence the polarization of CD4+ T cells and highlight the relevance of this in disease.
After interaction with their cognate antigen, naive CD4+ T cells proliferate and, depending on the cytokine micro-environment, polarize towards different CD4+ lineages, which then shape the immune response. CD4+ lineages include T helper type 1 (Th1), which drives the immune response against intracellular pathogens, Th2, which promotes humoral responses, Th17 cells, which contribute to the elimination of extracellular pathogens, and Foxp3+ regulatory T (Treg) cells, which prevent the development of autoimmunity (Fig. 1a). The differentiation towards each lineage is associated with the Sucrase up-regulation of specific transcription factors that act as master regulators by controlling the expression of a panel of genes, conferring a specific phenotype1 (Fig. 1a). There is accumulating evidence that CD4+ T-cell lineages are not as stable as initially thought, but rather, in specific environments, secrete cytokines and co-express master regulators specific for other lineages.2 The factors that control CD4+ T-cell stability versus plasticity are currently poorly understood, but it is clear that signal transducer and activator of transcription (STAT) proteins play key roles in this process.
IL-5 and GM-CSF were determined in supernatants using specific ELISA
Kit assays (eBiosciences). The results are expressed as the mean±SD. Data were analyzed using Student’s t-test (Prism AZD6244 ic50 GraphPad Software, San Diego, CA, USA). This work was supported by grants from the Italian Ministry of Health, Associazione Italiana Ricerca sul Cancro, Ministero dell’Istruzione, Università e Ricerca (PRIN 2005), Fondazione Cariplo, Agenzia Spaziale Italiana (Progetto OSMA), LR.26 del Friuli Venezia Giulia. The authors thank Silvia Piconese and Mario Colombo (Istituto Tumori, Milan, Italy) for providing OX40-deficient Tregs. They are grateful to Francesco Vitrani for helpful suggestions. Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance LY2109761 cell line to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted
by the authors. “
“Interleukin-19 (IL-19) plays an important role in asthma by stimulating T helper type 2 (Th2) cytokine production. Interestingly, IL-4, a key Th2 cytokine, in turn up-regulates IL-19 expression in bronchial epithelial cells, so forming a positive feedback loop. In atopic dermatitis (AD), another Th2 disease closely related to asthma, IL-19 is up-regulated in the skin. We propose to use IL-4 transgenic (Tg) mice and human keratinocyte culture to delineate the molecular mechanisms involved in the up-regulation
of IL-19 in AD. IL-19 is similarly up-regulated in the skin of IL-4 Tg mice as in human AD. http://www.selleck.co.jp/products/Paclitaxel(Taxol).html Next we show that IL-4 up-regulates IL-19 expression in keratinocytes. Interestingly, the up-regulation was suppressed by a pan-Janus kinase (Jak) inhibitor, suggesting that the Jak–signal transducer and activator of transcription (Jak-STAT) pathway may be involved. Dominant negative studies further indicate that STAT6, but not other STATs, mediates the up-regulation. Serial 5′ deletion of the IL-19 promoter and mutagenesis studies demonstrate that IL-4 up-regulation of IL-19 in keratinocytes involves two imperfect STAT6 response elements. Finally, chromatin immunoprecipitation assay studies indicate that IL-4 increases the binding of STAT6 to its response elements in the IL-19 promoter. Taken together, we delineate the detailed molecular pathway for IL-4 up-regulation of IL-19 in keratinocytes, which may play an important role in AD pathogenesis. “
“The in vivo or in vitro formation of IgG4 hybrid molecules, wherein the immunoglobulins have exchanged half molecules, has previously been reported under experimental conditions. Here we estimate the incidence of polyclonal IgG4 hybrids in normal human serum and comment on the existence of IgG4 molecules with different immunoglobulin light chains.
1d) was significantly higher in NSG mice that were irradiated and implanted with fetal thymic and liver tissues. In the bone marrow (Fig. 1e), irradiated groups had higher percentages of human CD45+ cells compared to non-irradiated groups, although the difference in CD45 percentages for the non-irradiated recipients with or without thymic implants was not significant. CD34+/CD38–-positive human HSC (Fig. 1f, expressed as a percentage
of human CD45+ cells) were detectable in all groups of mice, with a slightly higher percentage in non-irradiated mice click here injected with HSC only. The increased percentage of CD34+ HSC in the bone marrow of non-irradiated mice injected with HSC only was attributed to the overall low levels of human CD45+ cells in the bone marrow. As described in Materials and methods, NSG recipient mice were injected with a range in number of CD34+ HSC (1 × 105–5 × 105), depending on cell recovery and number of mice implanted. www.selleckchem.com/products/PD-0332991.html To determine if this fivefold range influenced the levels of human cell engraftment, NSG mice
that were either non-irradiated or irradiated and then implanted with human fetal thymic and liver tissues and HSC were evaluated for human CD45+ chimerism in the peripheral blood at 12 weeks (Supporting information, Fig. S1). Surprisingly, there was no correlation between the number of HSC-injected and levels of CD45+ cells in the peripheral blood, suggesting that the inherent variability in human cell chimerism between individual donor tissues is not overcome by a fivefold increase in HSC
number for the BLT model. Together these results suggest that optimal human cell chimerism after implant of human HSC mice requires irradiation, but that a significant level of chimerism can be achieved by co-implantation of human thymic tissues in the absence of irradiation. In addition, we have compared the levels of human CD45+ cells at 12 weeks in the peripheral blood of female or male NSG mice that were irradiated and implanted with fetal thymus and liver tissues and L-NAME HCl HSC (standard BLT mice) from either male or female donors (Supporting information, Fig. S2). The data show that tissues from both male and female donors engraft NSG mice effectively. Moreover, for five of eight sets of tissues, female NSG recipients engrafted at slightly higher levels with human CD45+ cells compared to NSG male mice, as described previously for human umbilical cord blood-derived HSC . This preferential engraftment of female mice was evident for tissues from both female and male donors. The presence of human thymic tissue within the BLT model allows for high-level development of human T cells following injection of HSC [21, 59]. We next evaluated the importance of host mouse irradiation on T cell development in either NSG mice injected with human HSC only or in NSG mice implanted with human thymic and liver tissues and injected with autologous HSC.
AND TCR transgenic mice bear a Vα11Vβ3 TCR that recognizes pigeon cytochrome c peptide bound to MHC II H-2k and H-2b molecules 24. However, thymocytes that develop on the H-2k haplotype have small thymi with a reduction of DP thymocytes most likely due to selleck kinase inhibitor partial clonal
deletion and have therefore been utilized as a model of negative selection 29. We first compared the thymocyte profiles of WT and KSR1−/− AND mice on the positively selecting C57BL/6 background (H-2b) 24 (Fig. 4). There was a similar percentage and absolute number of DN, DP or SP thymocytes between WT and KSR1−/− mice. This was also true when we restricted our analysis to thymocytes expressing the transgenic AND receptor (TCR Vα11+) (Fig. 4). These data indicate that, similar to our results in HY TCR mice, KSR1 is dispensable for efficient positive selection of CD4+ AND T cells. To determine whether negative
selection is affected by the absence of KSR1 in the AND TCR mouse model, we analyzed the thymic selection of AND TCR transgenic thymocytes on the weakly negative-selecting H-2k haplotype 29, 30 (Fig. 5A and C). We observed similar percentages Selleckchem Fluorouracil and numbers of DN, DP and SP thymocytes between WT and KSR1−/− AND mice, indicating that negative selection in this model is unaffected by the loss of KSR1 (Fig. 5A and C). We also analyzed the selection of AND T cells in mice with the heterozygous H-2bxk haplotype, a background that should have a lower negative selection stimulus 29.
Again, the percentages and total numbers of the thymocyte populations were comparable between WT and KSR1−/−mice on this background (Fig. 5B). These data indicate that, unlike in HY male mice, negative selection in the AND TCR transgenic mouse model does Acesulfame Potassium not require KSR1-dependent ERK activation. Because we observed different results regarding negative selection in the absence of KSR1 in two different mouse models, we next analyzed negative selection of T cells in response to an endogenous superantigen. We used KSR1-deficient mice on the DBA1/LacJ background because they express the endogenous retroviral superantigen MMTV-7. MMTV-7 expression in WT mice results in deletion of T cells expressing the TCR Vβ-6, 7, 8.1 and 9 chains by negative selection 31. To determine if KSR1 is important for negative selection in this model, we compared the representation of these Vβ chains in splenocytes from WT or KSR1−/− on the DBA1/LacJ background (Fig. 6). These analyses showed that the representation of TCRVβ-6 and 7 in splenic T cells was not significantly different between WT and KSR1−/− mice. These data show that the negative selection mediated by endogenous superantigen on the DBA/LacJ background is not affected by the absence of KSR1. KSR1 is a scaffold that plays a role in facilitating ERK activation.
Our data further suggest that the production of ROS and NO is linked. Since transcriptional selleck chemicals regulation of iNOS is altered, this linkage is most likely at the level of the signaling pathways and ultimately NFκB associated. It remains unclear whether this effect is mediated by the ROS molecules themselves, the changes in vesicular pH, or another mechanism; however, our data are supported by findings in which the anti-inflammatory regulator Nrf2 was found to be defective
in CGD 42. This raises the possibility that increased iNOS transcription in CGD upon GlyAg stimulation could be a result of an inability to shut down the initial GlyAg-mediated TLR2-dependent signal 19 to activate iNOS synthesis in the first place. The difference between WT and CGD responses to an actual antigen like PSA from B. fragilis provides an ideal model system to explore the relationship between the control of ROS and NO production. Taken together, our findings suggested that NO in macrophages, but not neutrophils, is the primary mediator of hyperresponsiveness to GlyAg in CGD. Our adoptive transfer experimental ATM inhibitor data further suggest that the loss of ROS in the T-cell population, which has been linked to a switch between T effector and T regulatory cells 43, does not explain the enhanced GlyAg response. These interpretations were confirmed
in vivo using iNOS inhibition which completely prevented abscess formation in 6 of 14 animals while significantly reducing the abscess severity in the remaining mice. Since 1400W
did not appear to increase the risk of bacterial sepsis, this strategy may represent a new pathway of treatment for CGD patients, although far more stringent testing with more invasive organisms would be needed to confirm these initial findings. In contrast to the CGD T-cell studies in which non-specific anti-CD3/anti-CD28 stimulation of T cells was used 44, 45, our findings suggest a novel pathway responsible for CGD-associated recurring abscess formation that is centered upon professional APCs, increased GlyAg processing, Erythromycin and antigen-mediated T-cell activation. This pathway can be specifically targeted through inhibition of iNOS activity in vivo, resulting in attenuation of CGD-associated immune pathology arising from bacterial infection. This approach could significantly improve treatment outcomes for CGD patients through increasing antibiotic efficacy and reducing the need for surgical drainage of abscesses. WT (C57BL/6J, stock 000664) and X-linked gp91phox-deficient CGD (B6. 129S6-Cybbtm1Din/J, stock 002365) breeders were purchased from Jackson Labs and colonies were housed at CWRU Animal Resource Center. Experiments were performed in accordance with the guidelines of the National Institutes of Health (NIH) and protocols approved by the Institutional Animal Care and Use Committee. All experimental mice were at least 12 wk old.
Recently, a commercial complement kit containing standardized ELISA-based assays for the assessment of all three complement pathways in clinical laboratories has been released. In this kit the MBL LP pathway is measured in wells coated with mannan and the contribution from the CP is inhibited by the use of a blocking anti C1q antibody . Contribution from the AP is avoided by a minimal dilution of sera 1:101. These assays were validated in three different laboratories and they demonstrated high stability and reproducibility. However, one major concern BI 6727 cell line associated
with these assays is the interference of the AP when assessing the functional capacity of the LP. Using novel ELISA set-ups in the present study, the normal functional activity of the three complement activation pathways was determined using serum samples from 150 healthy Danish blood donors. The functional capacity of the CP, determined as deposition of C3 on immune complexes, showed a normal distribution with a mean activity of
101% (57·4–161·9%). The capacity for the AP was determined as the deposition of C3 on an LPS-coated surface and showed a range of to 54·8–129·2%, with a mean value of 91%. Because of AZD1152-HQPA molecular weight the normal distribution of the AP and the CP functional pathway activity, the lower cut-off value of normal activity was defined as the mean –1·96 × SD, resulting in a lower cut-off value of normal complement activity for the AP at 63·5% and 61% for CP. As expected, and in agreement with Garred et al.  and Seelen et al. , the complement activation capacity for the MBL pathway among healthy blood donors showed a large variation range with a bimodal distribution. This is due mainly to the variations in degrees of oligomerization of MBL as the concentration of functional MBL is the primary limiting factor for the LP activity. This was confirmed by a strong positive correlation between the MBL serum Calpain concentration
and the functional MBL pathway activity (r2 = 0·70, P < 0·0001). Given the relatively high frequency of individuals with MBL deficiency in the general population, it is important to define a normal MBL activity range. In attempt to define a pathway activity, it was decided to define the meaningful cut-off value for normal MBL cut-off activity level as the lowest activity level measured in an XA/O individual (selected from genotyping of individuals with MBL pathway activities between 0 and 43%). The highest MBL pathway activity level measured in a XA/O individual among the genotyped donors was 8% (Table 1), while all O/O individuals among the genotyped donors had no functional MBL pathway activity.
LAB have health-promoting effects, manifested through enhanced host immune responses due to increased production of NO and cytokines by macrophages (2). Thus, LAB are widely used as food supplements or therapeutic agents for several infectious diseases (3). Macrophages are phagocytes that reside within host tissues. These cells differentiate from monocytes and play an important role in host immune
responses (4). Various stimuli, including bacteria, LPS, lymphokines and interferons activate macrophages Galunisertib by (5). Activated macrophages regulate host immunity by secreting NO and inflammatory cytokines such as IL-1β and TNF-α (6, 7). NO, which is synthesized from L-arginine by the enzyme NO synthetases, is a short-lived mediator that either kills or inhibits the growth of bacteria and tumor cells (8, 9). IL-1β is a proinflammatory cytokine that induces a variety of cellular responses, including cell proliferation, differentiation, and apoptosis. It also triggers a cascade of immune responses by inducing expression/secretion Selleck Protease Inhibitor Library of other cytokines and chemokines (10, 11).
TNF-α has a broad spectrum of systemic and cellular activity and mediates resistance to infectious disease by suppressing intracellular pathogens and controlling inflammatory processes (12). Enterococcus faecium, Gram-positive cocci belonging to the genus Enterococcus, often occurs in pairs (diplococci) and are a commensal organism commonly found in the intestines. Enterococci are facultative anaerobic organisms, that is, they prefer to use oxygen but they can survive in the absence of oxygen when necessary (13). Administration of E. faecium enhances innate and acquired immune responses
in dogs and mice (14, 15). The immunomodulatory properties of LGG have been well-described. Early studies reported that LGG induces increased dendritic cell expression of IL-12, IL-17 and TNF-α (16) and that see more peripheral blood mononuclear cells or macrophages co-cultured with Mycobacterium tuberculosis antigen release NO and IFN-γ (17). The objective of the present study was to investigate the immunomodulatory properties of E. faecium strain JWS 833, and its possible use as a feed-additive. JWS 833 was killed by heating and its immunomodulatory properties regarding NO and cytokines production by C57BL/6 peritoneal macrophages examined in vitro. Furthermore, the protective effects of JWS 833 were examined in vivo using a murine model of L. monocytogenes. The effects in in vitro and in vivo were compared with those mediated by LGG (ATCC 53103). JWS 833 and LGG were each grown in MRS broth (BD, Sparks, MD, USA) at 37°C for 24 hrs and viable cells (cfu/mL) on the MRS agar plates counted (BD). The bacterial cells were collected by centrifugation at 14,300 g for 10 mins at 4°C and the culture supernatant discarded.