As Fig. 3C demonstrates the increase in IFN-γ production associated with LLT1 activation becomes significant after 6 h and remains significant through 18 h post-stimulation. The same NK92 (rested overnight without IL-2):K562-CD161 IFN-γ production assay detailed selleck products earlier was now repeated in the presence of various

pharmacological inhibitors specific for various signalling mechanisms. As expected, inhibition of all cellular transcription using actinomycin D completely abrogated detectable production from our system (Fig. 4). This may be because of the inhibition of transcription of IFN-γ, or of various other gene products required for IFN-γ secretion or of both. Inhibition of Src-PTK with PP2 also abrogated IFN-γ production (Fig. 4). This was expected as Src-PTK acts to phosphorylate ITAMs on the accessory proteins associated with NK activating receptors, one of which LLT1 is likely to associate with [17]. Inhibition of the PKC pathway NVP-BEZ235 using bisindoylmaleimide I failed to significantly reduce IFN-γ production compared to the same reaction incubated with DMSO alone (Fig. 4). Additionally, inhibition of calcineurin using ascomycin and PI3K using LY294002 also failed to reduce IFN-γ production. When we inhibited the p38 MAPK pathway using SB203580, IFN-γ production was significantly reduced but not eliminated. This was also observed

when the MEK/ERK pathway was inhibited using PD98059 (Fig. 4). These results suggested that both the p38 and MEK/ERK pathways may be associated with LLT1-induced IFN-γ production. Use of pharmacological inhibitors on IFN-γ production suggested

that the p38 and MEK/ERK signalling pathways are associated with CD161 ligation of LLT1. Therefore, we hypothesized pheromone that upon binding NK92 with CD161 expressing target cells, we would observe increased phosphorylation of both p38 and ERK proteins compared to NK92 incubated with CD161 lacking target cells (Fig. 5A). Western blots were analysed by densitometry to confirm this increase in phospho-ERK associated with K562-CD161 and the results clearly demonstrate the increase in P-ERK over time associated with LLT1 ligation. (Fig. 5B). However, our western blot analysis was only capable of detecting an increase in phospho-ERK associated with K562-CD161 target cells. Phospho-p38 was detected in both NK92:K562-CD161 and NK92:K562-pCI-neo reactions (Fig. 5A). This does not entirely rule out the possibility that p38 is specifically associated with LLT1 downstream signalling. Our current LLT1 ligation system requires CD161 expressed on the surface of K562 to activate LLT1. As phospho-p38 is detectable in NK92 incubated with K562 targets lacking CD161, it is possible that any p38 phosphorylation associated with LLT1 ligation by CD161 is masked by p38 phosphorylation associated with the engagement of K562 by NK92. Note that because of paraformaldehyde fixing of K562-CD161/-pCI-neo, proteins detected via western blot are only from NK92.

CFDA and propidium iodide fluorescence were detected by flow cytometry. Proliferation was calculated by relating the mean fluorescence intensity to cells grown in IL-2 alone (100 % proliferation) and cells cultured in the presence of the mitosis inhibitor Colcemide (50 ng/mL, Biochrom) (0 % proliferation). This study was supported by grant HA 4318/37hyphen;3

from Deutsche Forschungsgemeinschaft (DFG). 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 Cabozantinib cost available as submitted by the authors. “
“The prevalence of OXA-type carbapenemase genes, ISAba1 insertion sequence, carbapenem resistance, biofilm forming ability and genetic heterogeneity in clinical isolates of Acinetobacter spp. from hospitals in Mangalore, South India was studied. Based on the presence of the blaOXA-51-like gene, the 62 isolates of Acinetobacter spp. were identified as 48 A. baumannii and 14 other Acinetobacter spp. The prevalence of blaOXA-23-like, blaOXA-24-like and blaOXA-58-like

genes in A. baumannii was 47.9%, 22.9% and 4.2%, while in other Acinetobacter spp. it was 28.5%, 64.3% and 35.7% respectively. Several A. baumannii isolates (16/48) harbored the insertion sequence ISAba1 in the upstream region of the blaOXA-23-like Sirolimus mw gene. Resistance to meropenem was seen in 39.6% and 14.2% of A. baumannii and other Acinetobacter spp. isolates, respectively. The ability to form biofilm was observed to be higher among A. baumannii in comparison to other Acinetobacter spp. The present study shows that blaOXA-23-like

DOK2 genes are more common in A. baumannii,whereas blaOXA-24-like genes are common to other Acinetobacter spp. The study revealed genetic heterogeneity among the isolates, indicating multiple sources in the hospitals. Acinetobacter spp., particularly, Acinetobacter baumannii have emerged as an important nosocomial pathogen worldwide (1), multiple drug resistance posing a serious problem in the clinical management of infections caused by them. Surveys by the British Society for Antimicrobial Chemotherapy in 2005 showed that >30% of bacteremia isolates were resistant to gentamicin and other drugs, resistance to imipenem being low (2). Carbapenem resistance mechanisms are mediated by beta-lactamases which are classified by two general schemes: the Ambler molecular scheme and the Bush-Jacoby-Medieros scheme (3, 4). In the Ambler molecular scheme, beta-lactamases are classified into four major types, A – D, based on protein homology. This excludes the phenotypic basis that is commonly used in any laboratory. Classes A, C and D are serine-beta-lactamases while class B includes metallo-beta-lactamases.

Methods:  Lipopolysaccharide (LPS)-treated mice were used as an animal model of albuminuria. We evaluated the effect of HGF on slit proteins using immunohistochemistry, western blotting and real-time polymerase chain reaction. Results: 

Albuminuria occurred 36 h after LPS treatment in mice. This albuminuria did not involve podocyte loss, but was associated with a decrease in nephrin and its key anchor, synaptopodin. In these processes, c-Met tyrosine phosphorylation, which represented HGF signal activation, occurred in glomerular cells including podocytes. When recombinant HGF was administrated to nephritic mice, c-Met tyrosine phosphorylation became Fludarabine datasheet evident in podocytes. The enhancement of the HGF-c-Met signal was associated with increases in nephrin and synaptopodin. An electron microscopic examination revealed that LPS induced the foot process effacement of podocytes, while HGF injections suppressed the foot process injury. Overall, albuminuria was attenuated in the LPS-treated mice after HGF administration. Conclusion:  HGF protects podocytes from a loss of nephrin, at least in part, through maintaining synaptopodin. As a result, HGF was shown to sustain foot process structure, and albuminuria was attenuated under inflammation. “
“Kidney disease develops to renal failure over a period of days, months or years, hence, clinical markers that indicate

the real-time renal pathophysiological conditions is important. Liver type fatty acid binding protein (L-FABP) is see more a 14 kDa molecule predominantly expressed buy KPT-330 in human proximal tubules. Clinical studies demonstrate that urinary excretion of L-FABP derived from the proximal tubules is an excellent biomarker for predicting and monitoring deterioration of renal function or for early detection of kidney

disease. However, in order to clarify the pathophysiological roles or dynamics of renal L-FABP in diseased settings, in vivo experimental studies of kidney diseases are indispensable. Since L-FABP is not endogenously expressed in murine kidneys, a transgenic (Tg) mouse model with expression of the human L-FABP gene was established. This review article summarizes the findings on the pathophysiological roles and dynamics of renal human L-FABP in the recent experimental studies performed using this Tg mouse model. The progression of kidney disease leads to renal failure, which requires renal replacement therapy with poor outcomes and at a high cost. Moreover, kidney disease is associated with the development and progression of cardiovascular1 or cerebrovascular disease.2 Therefore, clinical markers that accurately reflect the pathophysiological conditions of kidney disease are important in order to administer appropriate treatments and suppress the progression of kidney disease. Renal tubulointerstitial injury has been noted to have an important impact on the progression of kidney disease.

88 Chemotaxis and chemorepulsion Tanespimycin research buy in the context of T-cell trafficking have been studied in the process of thymic emigration. Egress of mature thymocytes from the medulla to the periphery has been shown to be orchestrated by chemoattraction exerted by S1P and a simultaneous fugetactic function of CXCL12, which induces cells to leave the thymus.81,89 A bimodal effect of chemokines on memory T-cell trafficking has also been demonstrated in cancer. Certain growing tumours initially generate the chemokine CXCL12 at a level that induces T-cell chemoattraction, but ultimately

establish an immune-privileged site through the chemorepellent effect of high levels of CXCL12 on tumour-specific T cells. In this setting, T-cell chemorepulsion impairs cytotoxic T lymphocyte-mediated lysis of tumour cells, which requires that the effector makes direct contact

with the target cell.90 Fugetaxis and chemorepulsion may coexist in situations where the concentration of the chemokine drives cells from chemotaxis to fugetaxis, but dual receptor engagement may take place. In fact, it has been shown that the chemokine CXCL12 mediates a concentration-dependent chemorepulsive effect on diabetogenic T cells by altering firm adhesion. As this effect is G-protein-coupled receptor dependent but is only partially reversed by CXCR4 blockade, it has been suggested that alternative downstream CXCL12 signalling pathways mediated by protein coupled receptor 1 (RDC1)/CXCR7 learn more may trigger chemorepulsion.91 Memory plasma cells reside on CXCL12-expressing stromal cells of bone marrow and rest there for a long periods.92–94 Until recently, evidence demonstrating the existence of survival niches for memory CD4 T cells has been elusive.95,96 In immune reactions characterized by long-term antigen persistence (virus or adjuvants), memory-phenotype

CD4 T cells are found in the spleen and lymph nodes for long periods.97,98 In contrast, following immunization in the presence of soluble adjuvants (lipopolysaccharide GPX6 or monophosphoryl lipid A), memory CD4 T cells in the spleen or lymph nodes substantially decrease in number 1 week after immunization.99,100 These T cells have been shown to locate to the bone marrow and rest on IL-7-expressing stromal cells of the bone marrow.99 The relocation of antigen-experienced CD4 T cells to the bone marrow is dependent on integrin α2β1, a collagen receptor. Inhibition of integrin α2β1 on primed CD4 T cells results in defective relocation of antigen-specific CD4 T cells to the bone marrow and reduced B-cell help (e.g. reduced affinity maturation). It is still unknown how the memory T cells migrate to their survival niches in the bone marrow, although they express CCR2 and CXCR6.99 The bone marrow is presumably the most best tissue for long-term localization of CD4 T cells primed by blood-borne antigen.

Furthermore, when injected intravenously into immunoglobulin-free mice, they deposited in the glomeruli, accompanied by murine complement C3. The kidneys showed mesangial proliferation and matrix expansion, thus reproducing pathologic changes characteristic of the human disease. These results support the multi-hit hypothesis wherein Gd-IgA1, the key autoantigen in IgA nephropathy, is produced as a result of dysregulation of multiple enzymes in IgA1-producing cells and

forms nephritogenic immune complexes Nivolumab with anti-glycan autoantibodies. These findings provide insight into the mechanisms of disease in IgA nephropathy and offer clues for future development of disease-specific therapy and biomarkers. SUZUKI YUSUKE, SUZUKI HITOSHI, MUTO MASAHIRO, OKAZAKI KEIKO, NAKATA JUNICHIRO, TOMINO YASUHIKO Juntendo University Faculty of Medicine, Japan Impaired immune regulation along the “mucosa-bone marrow axis” has been postulated

to play an important role in the pathogenesis of IgA nephropathy (IgAN) (1). Accumulating evidence from experimental approaches with animal models suggests that there is dysregulation of innate immunity in IgAN resulting in changes in the mucosal immune system (2, 3). Our recent experimental studies with IgAN prone mice revealed that mucosal activation of Toll like receptors (TLR) in B cells and dendritic cells are involved in the production of nephritogenic IgA and IgA immune complex (IC) (4–6). On the other hand, the nephritogenic roles of galactose-deficient Erlotinib ic50 IgA1 (Gd-IgA1) and Gd-IgA1 bound with anti-glycan IgG in IC (IgA/IgG-IC) have been L-gulonolactone oxidase discussed in human IgAN (7). Although many clinical studies indeed show serum elevation of GdIgA1 and IgA/IgG IC in IgAN patients and association between these serum levels and the disease activity (8), their production sites and relevant

cell types remain unclear. Our recent clinical studies indicate that the tonsils may be one of major sites for the production of GdIgA1 and tonsillar TLR9 activation may contribute to the extent of glomerular injury via the GdIgA1 production (5, 9). Moreover, we also recently found that aberrant overexpression of B cell related cytokines such as a proliferation-inducing ligand (APRIL) and its receptors are involved in the IgA/IgG IC formation. In this symposium, we would like to discuss the pathological roles of palatine tonsils and underlying molecular mechanisms in IgAN. 1. Suzuki Y, Tomino Y. The mucosa-bone-marrow axis in IgA nephropathy. Contrib Nephrol. 2007;157:70–79. 2. Suzuki Y, Tomino Y. Potential immunopathogenic role of the mucosa-bone marrow axis in IgA nephropathy: insights from animal models. Semin Nephrol. 2008;28:66–77. 3. Suzuki Y, Suzuki H, Sato D et al. Reevaluation of the mucosa-bone marrow axis in IgA nephropathy with animal models. Adv Otorhinolaryngol. 2011; 72:64–67. 4. Suzuki H, Suzuki Y, Narita I, et al.

These data show

that like other lymphocyte populations, including NK cells, iNKT cells are sensitive to the immunosuppressive effects of adenosine. Adenosine-related compounds cause the simultaneous engagement of Gs- and Gi-coupled adenosine receptors. We therefore asked whether ligation of the predominant high-affinity A2aR during TCR-mediated stimulation would modulate activation or effector functions, i.e. BMN 673 molecular weight cytokine production, of iNKT cells. To exclude a lack of costimulatory molecules accounting for a lack of IFN-γ secretion, we next used BMDC at day of culture, typically consisting of both immature and mature cells. To exclude responses of the BMDC to A2aR modulation, cells were fixed upon α-galactosylceramide (α-GalCer)-loading. Enriched iNKT cell preparations were thus stimulated in the presence of a specific A2aR agonist or antagonist. Comparable to the effects of the stable adenosine analogue, the exposure to A2aR agonist CGS21680 during the stimulation period inhibited the production of IFN-γ by iNKT cells. In striking contrast, CGS21680 led to a significant increase in IL-4 production. Conversely, the A2aR antagonist ZM241485 inhibited the iNKT cell-mediated MG-132 in vitro secretion of IL-4 and concomitantly increased the production of IFN-γ (Fig. 2B), markedly skewing the Th1/Th2 ratio of cytokines produced by iNKT cells toward IFN-γ. These data were corroborated

by a similar analysis of a human iNKT cell line (Fig. 2C). The requirement of A2aR signaling for IL-4 production clearly is in opposition to the effects of A2aR ligation on conventional T cells, which are inhibited non-selectively 24. These data also provide an explanation for the phenomenon Beldi et al. recently described 17, in which iNKT cells lacking the ecto-enzyme CD39, and hence unable to generate adenosine, were not able to produce IL-4 upon CD1d-mediated activation. To determine

the physiological in vivo significance of these findings, we asked whether iNKT cells in mice lacking the predominant A2aR would be functionally altered. We injected A2aR KO mice or WT mice with α-GalCer and tested the cytokine production 90 min and 5 h later, reflecting the time of appearance science in serum. The production of IL-4 and IL-10 upon α-GalCer administration can be observed early after activation, whereas IFN-γ secretion by iNKT cells requires IL-12 produced by DC upon maturation and hence are detectable later after injection. We detected increases in all four tested cytokines (IL-4, IL-10, TGF-β and IFN-γ) in the serum of α-GalCer-injected WT mice compared to un-injected mice (data not shown). Comparable with the in vitro results, iNKT cells in the absence of A2aR produced significantly lower levels of IL-4 upon α-GalCer injection (Fig. 3A). The expression of another Th2 cytokine IL-10 was also markedly decreased in the A2aR KO mice. In marked contrast, but also comparable with the in vitro results, IFN-γ was increased in the A2aR-deficient mice.

Luteolysis,[38] removal of the ovaries,[39] or administration of antiprogestational agents[40] leads to uterine activation with increased effective signaling through oxytocin or other

receptors and parturition. The difference in serum levels before parturition in mice and rats is said to make these animal a poor model for progesterone regulation in humans. However, further understanding of local progesterone 5-Fluoracil purchase metabolism and responsiveness is likely to reveal mechanisms that are to some extent important in humans and may be a natural stand in for women who do not respond to exogenous progesterone in the prevention of preterm birth. Rats also express an inhibitory receptor that increases in expression before parturition.[41] In guinea pigs, in which early pregnancy can be disrupted by antiprogestins,[42] maternal serum levels, similar to what is seen in humans, rise from the time of conception

to a peak in early gestation followed by a transient decrease in late gestation and increasing levels from that point beyond the time of parturition.[25] Rabbits and sheep in contrast have very low levels of progesterone in the serum as compared to humans, and in these animals, pregnancy brings a slight increase in serum progesterone and a rapid fall before parturition.[25] Another www.selleckchem.com/products/abt-199.html important endocrine system related to pregnancy is the hypothalamic pituitary adrenal axis,[43] both of the mother and the fetus. Activation of the HPA axis by stress or other factors initiates a cascade involving release of corticotropin-releasing hormone (CRH) from the hypothalamus, secretion of corticotropin (ACTH) from the anterior pituitary, and action of ACTH on the adrenal to release cortisol and other glucocorticoids which can then exert feedback suppression on their release. This system not only interacts with the immune system,

but is also thought to be part of the mechanism underlying poor pregnancy outcomes related to emotional or physiologic stress.[44, 45] CRH, a principle mediator of the HPA axis, is produced by the placenta and fetal membranes[45] and may be a mediator of local estrogen production. In pregnant women, the possibility for multiple sources of increased systemic Leukotriene-A4 hydrolase CRH presents an ongoing challenge in understanding the interaction between maternal stress, fetal stress, and normal HPA development in the generation of parturition or preterm birth.[46] Animal models are likely critical in the examination of this issue in that they can be used to isolate and understand the potential importance of maternal versus fetal HPA and other factors[47, 48] in this process. In related non-human primates, the placenta also expresses CRH, and development of the fetal adrenal and activation of the fetal HPA axis generate important support signals for normal labor.

1e,h, Table 1). Collagen deposition is observed in the airways of patients with asthma, therefore, experiments aimed at quantifying collagen deposition within the murine airway wall were performed. The areas of peribronchial trichrome staining were significantly greater in the OVA group than in the Control group (21·66 ± 3·34 versus 4·03 ± 0·73 μm2/μm,

Fig. 1i,j, Table 2, P < 0·01). Administration of triptolide significantly reduced the areas of peribronchial trichrome Selleck PI3K Inhibitor Library compared with the OVA group (13·61 ± 1·16 versus 21·66 ± 3·34 μm2/μm, Fig. 1j–k, Table 2, P < 0·01). Dexamethasone also decreased the areas of peribronchial trichrome staining compared with the OVA-sensitized/challenged animals (13·08 ± 0·68 versus 21·66 ± 3·34 μm2/μm, Fig. 1j,l, Table 2, P < 0·01). There was no significant difference in subepithelial fibrosis between the TRP group and DXM group (13·61 ± 1·16 versus 13·08 ± 0·68 μm2/μm, Fig. 1k–l, Table 2, P > 0·05). see more In view of recent studies showing that triptolide inhibits activation-induced cytokine gene transcription,24 RT-PCR was used to quantify levels of the mRNAs for constituent chains of TGF-β1 in the lungs of mice exposed for 8 weeks to OVA aerosol. Data were normalized to the levels of β-actin mRNA, a prototypical ‘housekeeping gene’, in the same isolated airway preparations.

We observed that, after an 8-week OVA-challenge, TGF-β1 mRNA expression in the OVA group was significantly increased RG7420 clinical trial compared with the Control group, whereas TGF-β1 mRNA expression in the TRP and DEX groups was significantly decreased compared with that in the OVA group (0·42 ± 0·04 and 0·44 ± 0·04 versus 0·54 ± 0·05, Fig. 2, Table 2, both P < 0·05). There was no significant difference in TGF-β1 mRNA expressions among mice treated with triptolide and dexamethasone (0·42 ± 0·04 versus 0·44 ± 0·04, Fig. 2, Table 2, P > 0·05). The immunostaining area of peribronchial TGF-β1 was quantified by image analysis and expressed as corrected average optical density. Positive staining showed TGF-β1 expression in the epithelium, macrophage leucocyte and smooth muscle. The immunostaining areas

of peribronchial TGF-β1 in the OVA group was significantly greater than those in the Control group (0·324 ± 0·00795 versus 0·0839 ± 0·00743, Fig. 3a,b, Table 2, P < 0·05). Administration of triptolide and dexamethasone in repetitively OVA-challenged mice both significantly reduced the immunostaining area of TGF-β1 compared with that in the OVA group (0·1152 ± 0·00740 and 0·1141 ± 0·00959 versus 0·324 ± 0·00795, Fig. 3b–d, Table 2, P < 0·05). There was no significant difference of TGF-β1 expression in mice treated with triptolide and dexamethasone. As TGF-β1 is able to induce epithelial hyperplasia, we measured levels of these cytokines in the BALF. Levels of TGF-β1 were significantly increased in the OVA group compared with those in the Control group (734 ± 56 versus 248 ± 53 pg/ml, Fig. 4, P < 0·05).

Amino acid sequence identity between these tropomyosins ranges from 73% to 74%, and some regions predicted as IgE-binding epitopes in shrimp tropomyosin Selleckchem Smoothened Agonist were found to be identical in these molecules. We also found that IgE antibodies to rAsc l 3 represent a high proportion (∼50%) of the total IgE response to an unfractionated parasite extract, and there was allergenic equivalence between rAsc l 3 and the native counterpart in the A. lumbricoides extract. Moreover, the anti-tropomyosin

IgE antibodies of sensitized subjects reacted against A. lumbricoides tropomyosin and induced mediator release in effector cells, both in vivo and in vitro. The clinical impact of these findings relies on the particular environmental conditions of the tropics (especially urbanized areas of low income), BGB324 concentration where perennial exposure to high concentrations of mite allergens and intermittent infections with A. lumbricoides are common. In this setting, allergenic stimulation by cross-reacting tropomyosins may provide signals for sustaining IgE synthesis and perpetuate the allergic inflammation.

Supporting this hypothesis, our mite-allergic patients with asthma are more frequently and more strongly sensitized to rAsc l 3 than controls, both groups being sensitized to the Ascaris extract (Figure 3). Because the main risk factor for asthma in the tropics is specific IgE to mites, it is possible that this pattern of reactivity is attributable to the exposure to cross-reactive tropomyosins (144). This mechanism may also explain, at a population level, why in tropical

environments from Africa and South America, tropomyosins from mite and other invertebrates (e.g. cockroaches) constitute very important allergens, with sensitization frequencies above 50% (145,146), while PI-1840 in developed regions among mite-sensitized patients, tropomyosin is a minor allergen (5–16%) (130,147,148), probably because of the low concentrations of this allergen in the mite body. These findings suggest that Asc l 3 influences the patterns of IgE responses to mite tropomyosins and may not be restricted to this allergen because Ascaris extract has at least 7 IgE cross-reactive components (200, 116, 77, 58, 40, 33 and 23 kDa) that may exert similar enhancer effects (24). Conventionally, the diagnosis of Ascaris infection is achieved by the identification of parasite eggs in stool samples. However, the evaluation of A. suum infection in pigs shows that egg counts in faeces greatly underestimate the proportion of exposed individuals compared with anti-Ascaris IgG titration by ELISA (149). Similar findings were obtained in humans, where serodiagnosis of ascariasis, as detected by Ascaris-positive IgE, is three times the positive egg prevalence (150,151).

In our experiment, Ag85A (5 μg/ml) and ConA (10 μg/ml) were used as a specific stimulator MK-1775 in vitro and a polyclonal stimulator of T cells, respectively. As shown in Fig. 3, a low background level of T cell proliferation was observed in vector control group and pcDNA3-ub group. A significant increase in T cells proliferation (P < 0.01) was observed in pcDNA3-Ag85A group compared with vector group or pcDNA3-ub group. The ubiquitinated Ag85A DNA vaccine significantly enhanced Th cell proliferation responses compared with non-ubiquitinated Ag85A DNA vaccine (P < 0.05). As a specific indicator of CD4+ T cell activation, the cytokines were also detected. Th1 cytokines (IL-2,

IFN-γ) and Th2 cytokines (IL-4, IL-5 and IL-10) are major parameters in our understanding of the polarization of immune responses. Th1 immune responses this website are thought to drive induction of cellular immunity, whereas Th2 immune responses preferentially drive humoral immunity. In this study, the level of IFN-γ and IL-4 was examined. As demonstrated in Fig. 4, the level of IFN-γ was significantly higher in Ag85A DNA vaccine group than that in pcDNA3 group or in pcDNA3-ub group. The secretion of IFN-γ significantly increased in UbGR-Ag85A fusion DNA vaccine group (P < 0.01) compared with Ag85A DNA vaccine group. However, the level of IL-4 was lower in fusion DNA vaccine group than that in non-fusion

vaccine group (P < 0.01). In Ag85A DNA vaccine group, the level of IFN-γ was higher than that of IL-4, which indicated the Ag85A DNA vaccine elicited a Th1-profile immune response. The ub fusion DNA vaccine increased the secretion of IFN-γ and decreased the level of IL-4, which demonstrated that the ub fusion enhanced the Th1-type immune response. As IFN-γ is clearly a key molecule in the anti-tuberculosis protective response, the role of CD4+ and CD8+ T cell for secreting IFN-γ was investigated by intracellular staining. As shown in Fig. 5, the frequency of IFN-γ+ CD4 T cells and IFN-γ+ CD8 T cells was higher in Ag85A DNA vaccine group than those in pcDNA3 vector group or in pcDNA3-ub group. The frequency of IFN-γ+ CD8

T cells was much higher in the spleen of the UbGR-Ag85A fusion DNA vaccine group than that in Ag85A DOK2 DNA vaccine group (P < 0.01). Although to a lesser extent, the frequency of IFN-γ+ CD4 T cells was also higher in the UbGR-Ag85A fusion DNA vaccine group, compared with the Ag85A DNA vaccine group (P < 0.05). Overall, UbGR-Ag85A fusion DNA vaccine induced more antigen-specific CD8+ T cells than CD4+ T cells. These results indicated that UbGR-Ag85A fusion DNA vaccine activated CD4+ and CD8+ T cells, particularly CD8+ T cells. Cytotoxic T cell responses were determined with a LDH release assay, after in vitro restimulation, against the target cell line P815-Ag85A, which stably expressed the Ag85A protein. P815 cell was used as a negative control. As shown in Fig.