1 This encapsulated yeast is also able to persist in healthy host

1 This encapsulated yeast is also able to persist in healthy hosts, thus causing dormant infections that may later be reactivated under an immunosuppressive disease.2 Cryptococcal infections in rats have been shown to have similarities with human cryptococcosis,

revealing a strong granulomatous response and a low susceptibility to disseminated infections.3 T-cell-mediated immunity is a critical component of protective immunity against infection with C. neoformans. Both CD4+ and CD8+ T cells are required for effective immune pulmonary clearance and prevention of extrapulmonary dissemination.4 The cells recruited during the inflammatory response include neutrophils, eosinophils, Ixazomib nmr monocyte/macrophages (Mφ), dendritic cells and lymphocytes [CD4+ T cells, CD8+ T cells, B cells

and natural killer (NK) cells]. Of these cells, activated Mφ, neutrophils and lymphocytes are all capable of in vitro killing or growth inhibition of C. neoformans.5 Related to this, previous studies in our laboratory have shown that Mφ from infected rats appear to be able to kill C. neoformans, principally by generating nitric oxide (NO).6 Moreover, the Obeticholic Acid order NADPH oxidase system was also found to be very important in the mechanism of C. neoformans killing by rat peritoneal cells, with the superoxide anion, hydrogen peroxide (H2O2) Lepirudin and the hydroxyl radical being involved in this process.7 Eosinophils,

in contrast, are implicated as effector cells in helminthic infections, releasing their many cytoplasmic granules, containing toxic molecules, in response to antigenic stimuli.8 Moreover, they notably contribute to allergic inflammation at airway mucosal sites.9 Recent studies have also demonstrated that eosinophils are able to function as antigen-presenting cells (APCs). The eosinophils express major histocompatibility complex (MHC) class I and class II, and the costimulatory molecules CD28, CD40, CD80 and CD86, suggesting that these cells can directly communicate with T cells to regulate immune responses. In addition, eosinophils also secrete a range of cytokines that are not only proinflammatory, but also function as growth factors, stimulants and chemoattractants [e.g. interleukin (IL)-2, IL-4, IL-5, IL-10, IL-12, IL-16, interferon-γ (IFN-γ) and regulated on activation, normal, T-cell expressed, and secreted (RANTES)] for T cells.10 In this sense, eosinophils were demonstrated to present antigens to primed T cells, thus increasing T-helper 2 (Th2) cytokine production.10–14 Furthermore, antigen-loaded eosinophils migrate into local lymph nodes and localize in the T-cell-rich paracortical zones, where they stimulate the expansion of CD4+ T cells.

Neonates have limited exposure to antigens in utero, thus leaving

Neonates have limited exposure to antigens in utero, thus leaving them with the immaturity in adaptive immunity during infancy [4, 13]. Mounting evidence has shown several deficiencies of adaptive immunity in neonates and infants for both cell- and antibody-mediated responses. For example, in addition to low numbers of effector-memory Selleck LY2157299 T cells (CD45RA−CD45RO+) and memory-effector B cells (CD27+), large numbers of both recent thymic emigrants of T cells recently produced by the thymus and transitional B cells produced recently

in the BM are present in the circulation during infancy [4, 16, 17]. These recent thymic emigrants exhibit a defect in their acquisition of the Th1 function, whereas transitional B cells are less functionally effective than mature naive B cells. Thus, the predominance of both transitional T cells and B cells may contribute to the vulnerability of neonates and infants to infection with intracellular pathogens. Furthermore, the adaptive immune system of neonates and infants is also characterized with defective NK cell activities, slow development of the CD4+ T-cell response, delayed, shortened, and reduced antibody responses, less efficient in the production of Th1-polarizing cytokines including type I IFN (or bias

to the Th2-type response), and decreased MHC class II expression on APCs [13, 15, 18-20]. Due to the immature state of the adaptive immune system, neonates and infants are thought to rely more heavily on their innate immunity against microbial infection [4, 21]. Furthermore, a recent study revealed that survival from LY2606368 nmr polymicrobial sepsis Chlormezanone in murine neonates was neither dependent on an intact adaptive immune system nor affected by the T-cell–directed adaptive immune modulation [22], which highlights the increased importance of the innate immune response during microbial sepsis in neonates

and infants. Both macrophages and PMNs, the professional phagocytes, are highly specialized innate effector cells and have evolved for the killing of microbial pathogens. The innate immunity-mediated antimicrobial response to bacterial infection is initiated by the receptor-associated recognition of invading pathogens, and subsequently, these invaded pathogens are engulfed by the professional phagocytes including macrophages and PMNs via phagocytic receptors and killed within the phagocyte through a process of phagosome/lysosome fusion, which is essential for host innate immunity to limit microbial infection [23-25]. However, the innate immunity-mediated antimicrobial response during microbial sepsis remains poorly defined in neonates and infants. Here, we show that infant PMNs, characterized with reduced expression of the chemokine receptor CXCR2, exhibit diminished in vitro chemotaxis and in vivo recruitment, whereas infant macrophages display impaired phagosome maturation and reduced killing of the ingested bacteria.

The most robust

human immune model is generated by implan

The most robust

human immune model is generated by implantation of human fetal thymic and liver tissues in irradiated recipients followed by intravenous injection of autologous fetal liver haematopoietic stem cells [often referred to as the BLT (bone marrow, liver, thymus) model]. To evaluate the non-obese diabetic (NOD)-scid IL2rγnull (NSG)–BLT model, we have assessed various engraftment parameters and how these parameters influence the longevity of NSG–BLT mice. We observed that irradiation and subrenal capsule implantation of thymus/liver fragments was optimal for generating human immune systems. However, after 4 months, a high number of NSG–BLT mice develop a fatal graft-versus-host disease (GVHD)-like syndrome, which correlates with the activation of human T cells and increased levels of human immunoglobulin (Ig). Onset of GVHD was not delayed in NSG mice lacking murine major histocompatibility RXDX-106 complex (MHC) classes I or II and was not associated with a loss of human regulatory T cells or absence of intrathymic cells of mouse origin (mouse CD45+). Our findings demonstrate that NSG–BLT mice develop robust human immune systems, but that the experimental window for these mice may be limited by the development of GVHD-like

pathological changes. Immunodeficient mice engrafted with human immune systems represent a promising alternative for the in-vivo study of human immune systems without Epothilone B (EPO906, Patupilone) placing patients at risk [1-4]. These ‘humanized’ mice are created by the engraftment of immunodeficient mice with mature human immune cell populations, human selleck chemicals haematopoietic stem cells (HSC) or human fetal tissues [5-7]. Early humanized models using immunodeficient mice bearing the Prkdcscid (scid) recombination activating gene

1 (Rag1null) or 2 (Rag2null) mutations were limited by low levels of systemic engraftment of human immune cells, variability in the overall levels of human cell survival and limited functionality of the human immune system [8]. The limitations of these initial immunodeficient mouse models were largely overcome by the introduction of targeted mutations in the interleukin (IL)-2 receptor common gamma chain (IL2rg) gene [8]. The IL-2rγ-chain is required for high-affinity ligand binding and signalling through multiple cytokine receptors, including those for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 [9]. Immunodeficient mice bearing a targeted mutation within the IL2rg gene support higher levels of human haematolymphoid engraftment than all previous immunodeficient stocks and permit the engraftment of functional human immune systems [10-19]. Although a number of engraftment strategies are currently being used to produce humanized mice [8], the implantation of human fetal thymic and liver tissues accompanied by intravenous (i.v.

However, a direct immunostimulatory effect of anthelmintic treatm

However, a direct immunostimulatory effect of anthelmintic treatment cannot be excluded (53) and may be stronger in hair lambs. “
“Urinary catheters are standard medical devices utilized in both hospital and nursing home settings, but are associated with a high frequency of catheter-associated GSK2126458 mouse urinary tract infections (CAUTI).

In particular, biofilm formation on the catheter surface by uropathogens such as Klebsiella pneumoniae causes severe problems. Here we demonstrate that type 1 and type 3 fimbriae expressed by K. pneumoniae enhance biofilm formation on urinary catheters in a catheterized bladder model that mirrors the physico-chemical conditions present in catheterized patients. Furthermore, we show that both fimbrial types are able to functionally compensate for each other during biofilm formation on urinary catheters. In situ monitoring of fimbrial expression revealed that neither of the two fimbrial types is expressed when cells are grown planktonically. Interestingly, during biofilm formation on catheters, both fimbrial types are expressed, suggesting that they are both important in promoting

biofilm formation on catheters. Additionally, transformed into and expressed by a nonfimbriated Escherichia coli strain, both fimbrial types significantly increased biofilm formation on catheters compared with the wild-type E. coli strain. The widespread stiripentol occurrence of the two fimbrial

Selleckchem GSK1120212 types in different species of pathogenic bacteria stresses the need for further assessment of their role during urinary tract infections. “
“The extravasation of CD4+ effector/memory T cells (TEM) across the blood-brain barrier (BBB) is a crucial step in the pathogenesis of experimental autoimmune encephalomyelitis (EAE)or multiple sclerosis (MS).Endothelial ICAM-1 and ICAM-2 are essential for CD4+ TEM cells crawlingon the BBBprior todiapedesis. Here, weinvestigated the influence of cell surface levels of endothelial ICAM-1 in determining the cellular route of CD4+ TEM-cell diapedesis across cytokine treatedprimary mouse BBB endothelial cells under physiological flow. Inflammatory conditions inducing high levels of endothelial ICAM-1 promoted rapid initiation of transcellulardiapedesis of CD4+ T cells across the BBB, while intermediate levels of endothelial ICAM-1 favored paracellular CD4+T-celldiapedesis.Importantly, the route of T-celldiapedesis across the BBB was independent of loss of BBB barrier properties. Unexpectedly, a low number of CD4+ TEM cells was found to cross the inflamed BBB in the absence of endothelial ICAM-1 and ICAM-2 via an obviously alternatively regulated transcellular pathway.In vivo, this translated tothe development of ameliorated EAE in ICAM-1null//ICAM-2−/−C57BL/6J mice.

Louis, MO) diluted in dimethylsulphoxide plus

saline was

Louis, MO) diluted in dimethylsulphoxide plus

saline was injected intravenously into mice 6 hr before splenocyte harvest, and subjected to cell surface and intracellular cytokine staining as described.33,34 The CD8+ T-cell response to OVA257–264 was examined with H-2Kb dimer X (BD Biosciences, San Jose, CA) loaded with OVA257–264 peptide.30 Antibodies for cell surface and reagents for intracellular cytokine staining were purchased from BD Biosciences. For quantifying cytokine production by L. monocytogenes-specific T cells, splenocytes XL765 were plated into 96-well round bottom plates (5 × 106 cells/ml), and stimulated with the H-2Kb major histocompatibility complex (MHC) class I OVA257–264 or I-Ab MHC class II listeriolysin O (LLO)189–201 peptides (1 μm) in media supplemented with brefeldin https://www.selleckchem.com/ATM.html A (Golgi-plug reagent).30,31 The concentration of IFN-γ

in serum was quantified by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN). The differences in geometric mean CFUs, number and percentage of T cells between groups of mice were evaluated using the Student’s t-test with P < 0·05 taken as statistically significant (GraphPad Prism software, La Jolla, CA). Based on the potency whereby IL-21 controls the activation and differentiation of NK and T cells,1 and the protective roles for each of these cell types in innate L. monocytogenes host defence, the impact conferred by IL-21 deficiency on early susceptibility to L. monocytogenes infection was enumerated. After infection with 1 50% lethal dose (LD50; 105 CFUs in control B6 mice), both IL-21-deficient and control B6 mice each contained similar numbers

of recoverable L. monocytogenes CFUs within the first 72 hr after infection (Fig. 1a). Moreover by 72 hr post-infection, the remaining mice in each group uniformly became moribund. Therefore, no apparent defects in innate susceptibility based on the degree of bacterial proliferation and time to death were found for IL-21-deficient compared with control mice after high-dose L. monocytogenes infection. Erythromycin In similar experiments, the susceptibility of IL-21-deficient mice was also enumerated after infection with reduced L. monocytogenes inocula (103 CFUs) to more precisely characterize the potential requirement for IL-21 in innate host defence. With this reduced L. monocytogenes inocula, IL-21-deficient and control mice both appeared healthy and did not become moribund. Furthermore, no significant differences in L. monocytogenes bacterial burden were identified for IL-21-deficient mice compared with control mice at each time-point within the first 7 days post-infection even with this reduced L. monocytogenes dose (Fig. 1b). In both groups of mice, the bacterial burden was sustained over the first 72 hr after infection, and then declined to levels that approached the limits of detection by day 5 post-infection.

The stained cells were washed with saponin buffer twice, suspende

The stained cells were washed with saponin buffer twice, suspended in isoflow, and analysed by flow cytometry. Production of LTB4 was analysed in the supernatants of CD11c+ cells purified from the lungs (1·5 × 105 cells/200 μl cultured for 18 hr) by enzyme-linked immunosorbent assay (ELISA) (IBL Internat.; IBL-America Minniapolis, MN). Differences between means

were analysed using Student’s t-test, and values of P < 0·05 were considered to indicate statistical significance. All calculations were performed with GraphPad Prism 4 for Windows Roscovitine (GraphPad Software; La Jolla, CA). Airway inflammation was induced in BALB/c mice by i.p. administration of OVA followed by challenge with aerosolized OVA, as described in the Materials and Methods.

Control mice were challenged with saline instead of OVA. Five days after the challenge with aerosolized OVA, we collected the BAL to confirm the development of the allergic process. This was confirmed by the high number of eosinophils found in the BAL of allergic mice (4·6 ± 2·3 × 105 cells/ml; eosinophil percentage 47 ± 9%) but not in control mice (2·8 ± 1·2 × 104 cells/ml; eosinophil percentage 2·3 ± 1·9%) [mean ± standard error of the mean (SEM), n = 6, P < 0·001, for allergic versus control mice]. Also revealing the development of the allergic status, we found high levels of serum IgE antibodies directed to OVA (Fig. 1a). DCs were differentiated from bone marrow precursors, as described in the Materials and Methods. Figure 1(b) shows the phenotype of these DCs, while Fig. 1c LEE011 cell line shows that i.t. inoculated DCs effectively arrived to lung tissues 6 hr after inoculation. We then investigated whether i.t. inoculation of histamine-treated DCs pulsed with OVA was able to modulate lung infiltration by T cells in allergic mice. Airway inflammation was induced in BALB/c mice as described in the Materials and Methods. Histamine-treated DCs (DCHISs) were prepared by incubating DCs and histamine (1 μm) for 30 min at 37°. Then, either control DCs (DCs) or

DCHISs were pulsed with OVA (100 μg/ml) for 3 hr at 37° and, after washing, they were injected i.t. into BALB/c mice 3 days after OVA challenge. Control mice were inoculated i.t. with PBS instead of DCs. Lung tissues were collected in all cases 2 weeks later. Cell suspensions were obtained from the lungs after Ponatinib supplier collagenase treatment, and T cells were purified by magnetic isolation, using a monoclonal antibody directed to CD3 coupled to magnetic beads (> 80% purity). The total number of T cells purified from the lungs was similar for mice inoculated with PBS, DCs or DCHISs (not shown). Interestingly, a significant increase in the percentage of CD8+ T cells was observed in T cells purified from the lungs of DCHIS-treated mice (Fig. 2a,b) compared with T cells from mice treated with either PBS or control DCs. No changes in the percentage of CD4+ T cells were detected (Fig. 2c,d). We then analysed the pattern of cytokine production by lung CD8+ T cells.

Transfer of 7 × 107 donor B6 splenocytes, depleted of CD25+ cells

Transfer of 7 × 107 donor B6 splenocytes, depleted of CD25+ cells to eliminate endogenous Treg-cell activity, into CB6F1 recipients resulted in lethal aGVHD

in approximately 50% of mice within 25 ± 10 days (Fig. 1A). Acute disease was due to the high precursor F1 reactive cytotoxic lymphocyte frequency within donor inoculums, and also due to removal of Treg-cell activity [30, 31]. Therefore to develop a cGVHD model, B6 splenocytes were also depleted of CD8+ T cells, which resulted in no weight loss or lethality over the experimental duration (Fig. 1A), and animals surviving for greater than 15 weeks. In addition to hair loss (data BMS-354825 clinical trial not shown), analysis of peripheral blood and splenocytes showed consistent and long-term donor cell engraftment over 7 INK 128 order weeks following GVHD induction (Fig. 1B). Detected splenomegaly in cGVHD animals (Fig. 1C) was a consequence of both donor cell engraftment (Fig. 1D) and hyperproliferation of recipient lymphocyte compartments (Fig. 1E). Donor cells composed on average 7.0% (range 0.72–17.8%) of total splenocytes, and consisted predominantly of donor CD4+ T lymphocytes (3.4 ± 1.2%) with lower levels of B220+ B cells (0.63 ±

0.59%) (Fig. 1D). Of particular relevance to this disease model, donor cell transfer also resulted in an increase in the proportion of recipient splenic CD4+ T cells (cGVHD versus PBS, p = 0.004) and B cells (cGVHD versus PBS p = 0.02) (Fig. 1E). This was due to expansion of recipient

lymphocytes as evidenced by a mean 3.2- ± 1.1-fold increase in absolute numbers of recipient cells isolated from cGVHD spleens compared with those in sham-treated mice (Table 1), and lymphocyte hyperactivity as detected upon ex vivo re-stimulation (Fig. 1F). No differences in splenic composition of recipient CD3+CD4− T cells were detected (not shown). Donor engraftment and Rebamipide recipient hyperproliferation correlated with elevated serum IgG1 and IgG2a anti-single-stranded DNA autoantibodies and IgG immune complex deposition within kidney glomeruli (Fig. 1G and H). In concordance with previous reports [13], donor-derived B cells were not the main drivers of glomerulonephropathy as evidenced by maintenance of elevated serum autoantibody levels when using donor inoculates pre-depleted of B cells for cGVHD induction (Fig. 1G). Thus transfer of naïve B6 donor T cells induced an alloreactive response against recipient H-2d alloantigens presented via the direct and indirect pathways of alloantigen presentation, both of which are constitutively active within this model (Fig. 1I), resulting in autoimmune cGVHD pathology. Detection of IgG class switched antibodies indicated a T-cell dependent mechanism of B-cell activation was predominant.


(2010) check details demonstrating a significant reduction in intestinal pro-inflammatory TNF-α expression in synbiotic-treated patients. Moreover, the results from this investigation provide evidence to suggest that early treatment with synbiotic combination of probiotic La and prebiotic inulin can effectively prevent pathogen-induced intestinal inflammation

by affecting NF-κB and Smad 7 signaling within the intestinal epithelium. Prebiotics are known to help colonization of beneficial probiotics. While early administration of a synbiotic combination of probiotic La and prebiotic inulin attenuated the secretion and expression of pro-inflammatory cytokines and inflammation, supporting a potential indirect role of prebiotic inulin in regulating mucosal immune response

by modulating the colonic microbial communities. Our results are supported by previous observations showing that a diet supplemented with Fructooligosaccharides (FOS) and inulin can trigger and stimulate the gut mucosal immune system (Benyacoub et al., 2008). Our observations also are in line with the results of randomized controlled trials, which provide evidence to click here suggest that synbiotic therapy can be more effective in the treatment IBD than therapies limited to probiotics or prebiotics (Fujimori et al., 2009; Macfarlane et al., 2009; Steed et al., 2010). In the current study, we found that prebiotic (inulin) treatment of young mice resulted in a reduction in fecal C. rodentium output after the bacterial infection (Fig. 2b and c). It was reported previously that feeding rats with an inulin-oligofructose diet resulted in reduced numbers of Salmonella Typhimurium in the content of ileum and cecum (Kleessen & Blaut, 2005). However, contradicting results have also been reported. Petersen et al. (2009) reported that BALB/c mice fed diets containing prebiotics (FOS or xylo-oligosaccharide) had significantly higher

numbers of S. Typhimurium, translocated into liver, spleen, and MLN compared with mice fed with control diet. In contrast, no increased translocation of S. Typhimurium was found in mice fed inulin (Petersen next et al., 2009), in that same study. Nevertheless, most prebiotics and/or probiotics have not been shown to cause illness, but additional research is needed to determine the safety of prebiotics and probiotics in young children or people whose immune system is compromised. The observations showing an enhanced colonic TGF-β and IL-10 responses in mice with early synbiotic or probiotic treatments provided evidence to support the idea that these treatments may modulate gut mucosal inflammatory responses by promoting immunological regulatory mechanisms, which parallel results by Roller et al.

05 were considered as significant (*) This work was supported by

05 were considered as significant (*). This work was supported by grants from the Chilean government FONDECYT 1070954 (R.Q.) and Scholarship for Postgraduate Studies 21050679 (F.M.) and by grants of the Deutsche Forschungsgemeinschaft DFG-PR 727/3-1 (I.P.) and SFB621-A14 (I.P.). The authors thank Andreas Krueger and Nadja Bakočević for critically reading the manuscript and Mathias Herberg for animal care. 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. “

human T helper 17 cells Sotrastaurin concentration in health and disease. Clin Exp Immunol 2009; doi:10.1111/j.1365-2249.2009.04037.x selleck inhibitor Induction of interleukin-17 production by regulatory T cells. Clin Exp Immunol 2009; doi:10.1111/j.1365-2249.2009.04038.x Are T helper 17 cells really pathogenic in autoimmunity? Clin Exp Immunol 2009; doi:10.1111/j.1365-2249.2009.04039.x Development of mouse and human T helper 17 cells. Clin Exp Immunol 2009; doi:10.1111/j.1365-2249.2009.04041.x CD4+ T cells display considerable flexibility in their effector functions, allowing them to tackle most effectively the range of pathogenic infections with which we are challenged. The classical T helper (Th) 1 and Th2 subsets have been joined recently by the Th17 lineage. If not controlled, the potent effector functions (chiefly cytokine production) of which these different cells are capable can lead to (sometimes fatal) autoimmune and allergic inflammation. The primary cell population tasked with providing this control appears to be CD4+ regulatory T (Treg) cells expressing the forkhead box P3 (FoxP3) transcription factor. Here we consider the comparative capacity of FoxP3+ Tregs to influence the polarization, expansion and effector function of Th1, Th2 and Th17 cells in vitro and in vivo as well as in relation to human disease. This remains a particularly challenging series

of interactions to understand, especially given our evolving understanding of Treg and T effector interrelationships, as well as recent insights into functional plasticity that cast doubt upon the wisdom of a strict categorization of T effector cells based Immune system on cytokine production. The study of CD+ T cells has been greatly facilitated by their division into functional subsets. The basis for this division was the identification of distinct cytokine production profiles among T cell clones, giving rise to T helper (Th) 1 and Th2 subsets [1]. The developmental and functional relationship between these prototypic Th subsets was subject to intense study and provided the framework for classifying T cell responses for almost two decades. These ‘classical’ subsets exemplify the characteristics required to claim subset status.

gingivalis was inserted into the p-MAL plasmid pMD157, followed b

gingivalis was inserted into the p-MAL plasmid pMD157, followed by transfection to E. coli and incubation. After 1 or 2 days of incubation, the E. coli suspension was centrifuged and the pellet was homogenized. The homogenized suspension

was subjected to the dialysis treatment, gel-filtration chromatography, and ion-exchange chromatography. Finally, isolation buy GSK126 of the antigen was performed using amylose resin column affinity chromatography, and 25k-hagA was obtained via cleavage treatment of 25k-hagA-MBP using Factor Xa (New England BioLabs, Ipswich, MA). For sublingual immunization on days 0, 7, and 14, mice were anesthetized with pentobarbital, and 30 μL of phosphate-buffered saline (PBS) containing 50 μg of 25k-hagA-MBP was delivered with a micropipette applied against the ventral side of the tongue while directed toward the floor of the mouth. Mice were immunized with 7.5 μL of antigen four times (total volume = 30 μL). Ten minutes of interval were set between each administrations. A nonimmunized

group was PBS treated. Animals were maintained with their heads placed in ante flexion for 30 s during each delivery. Serum and saliva were collected from each group to examine the 25k-hagA-MBP-specific Ab responses. Ab titers were detected using an enzyme-linked immunosorbent assay (ELISA) as described previously (Maeba et al., 2005). Briefly, plates were coated with 25k-hagA-MBP (5 μg mL−1). After APO866 in vivo washing with PBS containing 0.05% Tween 20, plates were blocked with PBS containing 1% bovine serum albumin. Next, serial dilutions of serum or saliva samples were added in duplicate. The starting dilution of the serum was 1 : 26, while that of the saliva was 1 : 22. The plates were incubated for 5 h at room temperature, washed, and then incubated with horseradish peroxidase-labeled goat anti-mouse heavy chain γ, γ1, γ2a, γ2b, γ3, or α-specific antibodies (Southern Biotechnology Associates, Birmingham,

AL) at 4 °C for 20 h. Finally, 2,2′-azino-bis (3-ethylbenz-thiazoline-6-sulfonic http://www.selleck.co.jp/products/BIBF1120.html acid) (ABTS) with H2O2 (Moss, Inc., Pasadena, MD) was added for color development. Endpoint titers were expressed as the reciprocal log2 of the last dilution, which gave an optical density at 415 nm of 0.1 greater than that of nonimmunized control samples after 15 min of incubation. Single-cell suspensions were obtained from the salivary gland 7 days after the last immunization. Briefly, salivary glands were carefully extracted, teased apart, and dissociated using 0.3 mg mL−1 collagenase (Nitta Gelatin Co. Ltd, Osaka, Japan) in RPMI-1640 (Wako Pure Chemical Industries Ltd, Osaka Japan). Mononuclear cells were obtained at the interface of the 50% and 75% layers of a discontinuous Percoll gradient (GE Healthcare UK, Ltd, Little Chalfont, Buckinghamshire, UK) (Maeba et al., 2005). To assess the numbers of antigen-specific AFCs, an enzyme-linked immunospot (ELISPOT) assay was performed as described previously (Yamamoto et al., 1997).