Expression Of CD86 Molecules Research Articles

Expression of CD1 molecules and colony-stimulating factor 1 receptor in indeterminate cell histiocytosis.

Indeterminate cell histiocytosis (ICH) and Langerhans cell histiocytosis (LCH) are rare histiocyte proliferating disorders with unknown etiologies. However, both tumor cells immunophenotypically share some features of Langerhans cells (LC), thereby expressing CD1a. Recent transcriptome analysis revealed that circulating CD1c+ myeloid dendritic cells are the potential precursor of LCH tumor cells. LC express CD1a as well as CD1c, but not CD1b. We discovered that both tumor cells express CD1c, but not CD1b, similar to LC. Moreover, like LC, both tumor cells express colony-stimulating factor 1 receptor (CSF-1R). Considering the crucial role of the interleukin (IL)-34/CSF-1R axis for the development and survival of LC, CSF-1R on both tumor cells might facilitate their survival and proliferation in situ. These data provide additional evidence to support the fact that ICH and LCH share immunophenotypical features with LC. In addition, we hypothesized that tumor cells in ICH and LCH survive and proliferate through IL-34-mediated CSF-1R signaling.

The antioxidant MnTBAP does not effectively downregulate CD4 expression in T cells in vivo

The antioxidant MnTBAP was previously shown to down-regulate the surface expression of CD4 molecule in T cells. This observation obviously holds great potential impact in a number of pathological human conditions, including autoimmunity. Three different single doses of MnTBAP reduced the frequency of CD4high cells. However, the median florescent intensity (MFI) was not different. Initiation of in vivo pharmacotherapy or vehicle control was performed inC57BL/6 mice that were actively immunized for experimental autoimmune encephalomyelitis (EAE). In contrast to published reports, the mean frequency of CD4high cells, and the median fluorescent intensity (MFI) of CD4 was similar in both treatment groups. 25-day survival following active immunization among the MnTBAP treated animals compared to vehicle controls was16.6 ± 6.9 days vs 23.6 ± 2.7 days; (P value <0.05). We conclude that MnTBAP (Sack and Herzog, 2009 (Sack and Herzog, 2009)) does not effectively downregulate CD4 expression in T cells in vivo, probably due to extensive mechanism that distinguishes it from an in vitro model (Harding, 1993 (Harding, 1993)) possesses toxic properties that may limit its clinic use in possible doses that could deliver the immunomodulation through down regulation of CD4 expression, and (Saizawa et al., 1987 (Saizawa et al., 1987)) has limited availability in specific tissues, including the CNS.

Identification and characterization of two CD4 alleles in Microminipigs.

BackgroundWe previously identified two phenotypes of CD4+ cells with and without reactions to anti-pig CD4 monoclonal antibodies by flow cytometry in a herd of Microminipigs. In this study, we analyzed the coding sequences of CD4 and certified the expression of CD4 molecules in order to identify the genetic sequence variants responsible for the positive and negative PBMCs reactivity to anti-pig CD4 monoclonal antibodies.ResultsWe identified two CD4 alleles, CD4.A and CD4.B, corresponding to antibody positive and negative, respectively, by nucleotide sequencing of PCR products using CD4 specific primer pairs. In comparison with the swine CD4 amino-acid sequence [GenBank: NP_001001908], CD4.A had seven amino-acid substitutions and CD4.B had 15 amino-acid substitutions. The amino-acid sequences within domain 1 of CD4.B were identical to the swine CD4.2 [GenBank: CAA46584] sequence that had been reported previously to be a modified CD4 molecule that had lost reactivity with an anti-pig CD4 antibody in NIH miniature pigs. Homozygous and heterozygous CD4.A and CD4.B alleles in the Microminipigs herd were characterised by using the RFLP technique with the restriction endonuclease, BseRI. The anti-pig CD4 antibody recognized pig PBMCs with CD4.AA and CD4.AB, but did not recognized those with CD4.BB. We transfected HeLa cells with the FLAG-tagged CD4.A or CD4.B vectors, and certified that transfected HeLa cells expressed FLAG in both vectors. The failure of cells to react with anti-CD4 antibodies in CD4.B pigs was associated to ten amino-acid substitutions in domain 1 and/or one amino-acid substitution in joining region 3 of CD4.B. We also found exon 8 was defective in some CD4.A and CD4.B resulting in the loss of the transmembrane domain, which implies that these CD4 proteins are secreted from helper T cells into the circulation.ConclusionsWe identified that amino-acids substitutions of domain 1 in CD4.B gave rise to the failure of some CD4 expressing cells to react with particular anti-pig CD4 monoclonal antibodies. In addition, we developed a PCR-RFLP method that enabled us to simply identify the CD4 sequence variant and the positive and negative PBMCs reactivity to our anti-pig CD4 monoclonal antibodies without the need to use flow cytometric analysis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-016-0856-8) contains supplementary material, which is available to authorized users.

Chlamydia muridarum respiratory tract infection induces the proliferation of γδT cells and the secretion of IFN-γ and IL-17

Objective To investigate the proliferation, activation and cytokine production of γδT cells during different periods of Chlamydia muridarum (Cm) respiratory tract infection. Methods C57BL/6 mice were inoculated intranasally with 3×103 inclusion-forming units(IFU)of Cm strains to induce the murine model of chlamydial pneumonitis. Mononuclear cells were isolated from lung tissue samples collected from mice at different time points after infection. Flow cytometry analysis was used to detect the percentages of CD3+ TCRγδ+ T cells in lung tissues and the expression of CD69 molecule. Intracellular cytokine staining was performed to analyze the secretion of IL-17 and IFN-γ by γδT cells. Results The mouse model of chlamydial pneumonitis was successfully established by intranasal inoculation of C57BL/6 mice with 3×103 IFU of Cm strains. Compared with the mice without Cm infection, the percentage and the absolute number of CD3+ TCRγδ+ T cells in mice lung tissues were significantly increased after Cm infection, the peak of which was reached on the 7th day, followed by a decline. The expression of CD69 molecule on γδT cells isolated from mice lung tissues reached to the highest level on the third day after Cm infection and declined slightly afterwards. Moreover, enhanced secretion of IL-17 and IFN-γ by γδT cells were observed in mice with Cm infection and the highest levels of the two cytokines were detected on the third day after infection. Most of the cytokines (IL-17 and IFN-γ) were secreted by γδT cells rather than by αβT cells in the early stage of Cm infection. However, the CD3+ CD4+ T cells were more capable of producing IFN-γ and IL-17 than γδT cells after three days of Cm infection. Conclusion Cm respiratory tract infection could induce the aggregation and activation of γδT cells at the infection site. The γδT cells were the predominant cells producing IL-17 and IFN-γ in the early stage of host anti-chlamydia response. Key words: Chlamydial pneumonitis; γδT cells; IFN-γ; IL-17

Analysis of human B cell response to recombinant Leishmania LPG3.

To evaluate the capability of recombinant Leishmania LPG3 and its fragments in the activation of B cells. In the present study, human B cells were purified from peripheral blood of 10 adult healthy subjects using magnetic-activated cell sorting technique. Subsequently, purified B cells were treated with recombinant LPG3, and its N-terminal and C-terminal fragments at different concentrations (2, 10 and 20μg/mL). B cell activation was assessed through expression of CD69 molecule by flow cytometry and secretion of IL-6, TNF-α and IL-10 cytokines via enzyme-linked immunosorbent assay following treatment with recombinant antigens. Our results showed that while the recombinant LPG-3 could significantly increase the production of IL-6 and TNF-α (P<0.05) in B cells, it had no effect on the secretion of IL-10 by B cells. Our study indicated that recombinant LPG-3 and especially its N-terminal fragment could stimulate B cell response as an important immune response component against leishmaniasis. Thus, it seems that it can be considered as an effective adjuvant in vaccine developments against leishmaniasis.

Three - (-) Catechin-O-Rhamnosides from the Eastern Nigeria Mistletoe with Potent Immunostimulatory and Antioxidant Activities

In an attempt to provide further convincing evidence for the variously reported immunomodulatory potentials of mistletoes, bioassay-guided fractionation of the eastern Nigeria mistletoe afforded three compounds: - (-) catechin- 7-O-rhamnoside (1), - (-) catechin-3-O- rhamnoside (2) and a 4′-methoxy-7-O-rhamnoside (3). Their effects on C57BL6 mice splenocytes proliferation and expression of CD69 molecule were determined using flow cytometry techniques and compared to Lipopolysaccharide (LPS; 10 μg/ml) and Concanavalin A (ConA; 2 μg/ml) as standards. The antioxidant study was by the DPPH model with ascorbic acid as standard. The compounds (1-3) at 100 μg/ml showed statistically significant (p<0.05) stimulatory activity with values of 91.49 ± 0.22%, 95.17 ± 0.01% and 94.23 ± 0.07% respectively compared to 2.65 ± 0.33% for the unstimulated control. However, the CD69 expression assay showed only moderate stimulation. Their measured antioxidant potentials (effective concentration; EC50) were high (≤ 55.42 ± 0.99 mg/ml) when compared to ascorbic acid (17.6 ± 1.78 mg/ml). Characterization of the compounds was achieved by chromatographic and spectroscopic methods.

CD1d and CD1c Expression in Human B Cells Is Regulated by Activation and Retinoic Acid Receptor Signaling

B cell activation and Ab production in response to protein Ags requires presentation of peptides for recruitment of T cell help. We and others have recently demonstrated that B cells can also acquire innate help by presenting lipid Ags via CD1d to NKT cells. Given the newfound contribution of NKT cells to humoral immunity, we sought to identify the pathways that regulate CD1 molecule expression in human B cells. We show that ex vivo, activated and memory B cells expressed lower levels of CD1d compared with resting, naive, and marginal zone-like B cells. In vitro, CD1d was downregulated by all forms of B cell activation, leaving a narrow temporal window in which B cells could activate NKT cells. CD1c expression and function also decreased following activation by CD40L alone, whereas activation via the BCR significantly upregulated CD1c, particularly on marginal zone-like B cells. We found that the CD40L-induced downregulation of CD1d and CD1c correlated with diminished expression of retinoic acid receptor α (RARα) response genes, an effect that was reversed by RARα agonists. However, BCR-induced upregulation of CD1c was independent of the RAR pathway. Our findings that both CD1d and CD1c are upregulated by RARα signaling in human B cells is distinct from effects reported in dendritic cells, in which CD1c is inversely downregulated. One functional consequence of CD1d upregulation by retinoic acid was NKT cell cytotoxicity toward B cells. These results are central to our understanding of how CD1-restricted T cells may control humoral immunity.

Functional modulation of the pathway between dendritic cells (DCs) and CD4+T cells by the neuropeptide: Methionine enkephalin (MENK)

MENK, the endogenous neuropeptide, is suggested to be involved in the regulatory loop between the immune and neuroendocrine systems, with modulation of various functions of cells related to both the innate and adaptive immune systems. Our present research findings show that MENK serves as an immune modulator to the pathway between DCs and CD4+T cells. We studied changes of DCs in key surface molecules, the activity of acid phosphatases (ACPs), the production of IL-12, and the effects on murine CD4+T cell expansion and their cytokine production by MENK alone, and in combination with interkeukin-2 (IL-2) or interferon-γ (IFN-γ). In fact, we found that MENK could markedly induce the maturation of DCs through the addition of surface molecules such as MHC class II, CD86, and CD40 on murine DCs, the production of IL-12, and the down-regulation of ACP inside DCs, (which occurs when phagocytosis of DCs is decreased, and antigen presentation increased with maturation). We also found that MENK alone or in combination with IL-2 or IFN-γ, could markedly up-regulate both CD4+T cell expansion and the CD4 molecule expression in vivo and in vitro and that MENK alone, or MENK + IL-2, could enhance the production of interferon-γ from CD4+T cells. Moreover, MENK alone, or MENK + IFN-γ, could enhance the production of IL-2 from CD4+T cells. It is therefore concluded that MENK can exert positive modulation to the pathway between dendtritic cells and CD4+T cells.

Role of extracellular calcium ions at early stages of human T-lymphocyte polyclonal activation in vitro

In this work we comparatively analyzed interleukin-2 (IL-2) and interferon gamma production (IFN-gamma) and also CD69 and CD25 expression by activated T-cells depending on extracellular calcium concentration ([Ca2+]e), which was varied with EGTA. The expression of CD69 molecules on the surface of T-cells depended only on the presence of phorbol myristate acetate, occurred at [Ca2+]e higher than 0.2 mM, and did not require the presence of ionomycin. The increase in [Ca2+]e by itself cannot induce expression of CD25 and CD69 molecules by activated cells. The values of [Ca2+]e, at which maximal fractions of CD3+CD69+(IL-2)+, CD3+CD69+(IFN-gamma)+, and CD3+CD25+ activated T-cells were reached, never coincided with mean values of [Ca2+]e for healthy donors and were different from each other. So, there is different [Ca2+]e dependence for initial stages of activated T-cells differentiation. The relation between T-cells activation parameters and their differentiation is discussed.

Splenic natural killer cell activity in two models of experimental neurodegenerative diseases

Natural killer (NK) cells are antitumour/anti-viral effectors and play important roles in shaping the immune system, but their role in neurodegenerative diseases is not clear. Here, we investigated the fate of these cells in two neurodegenerative diseases. In the first model, the activity of NK cells was examined in mice with experimental autoimmune encephalomyelitis (EAE) treated with glatiramer acetate (GA or Copaxone), a drug used to treat EAE in animals and multiple sclerosis in human. The second disease model is twitcher (Galc(twi)/Galc(twi)) mice, which represents an authentic model of human Krabbe's disease. Administration of GA ameliorated EAE in SJL mice corroborated with isolating NK cells that expressed higher killing than cells isolated from vehicle-dosed animals against immature or mature dendritic cells (DCs). However, this drug showed no effect on the numbers of NK cells or the expression of CD69 molecule. On the other hand, NK cells either disappeared from the spleens or were present in low numbers in the white pulp areas of Galc(twi)/Galc(twi) mice, which have increased D-galactosyl-beta1-1'-sphingosine (GalSph) levels. Analysis by confocal microscopy shows that NK cells found in the spleens of Galc(twi)/Galc(twi) mice were apoptotic. Incubating NK cells in vitro with GalSph induced the apoptosis in these cells, confirming the results of twitcher mice. Our results provide the first evidence showing that amelioration of EAE in mice is corroborated with NK cell lysis of antigen-presenting DCs, whereas NK cell distribution into the spleen is altered in a devastating lipid disorder corroborated with induction of their apoptosis.

Serum lipids regulate dendritic cell CD1 expression and function

Dendritic cells (DCs) are highly potent antigen-presenting cells (APCs) and play a vital role in stimulating naïve T cells. Treatment of human blood monocytes with the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 stimulates them to develop into immature dendritic cells (iDCs) in vitro. DCs generated by this pathway have a high capacity to prime and activate resting T cells and prominently express CD1 antigen-presenting molecules on the cell surface. The presence of human serum during the differentiation of iDCs from monocytes inhibits the expression of CD1a, CD1b and CD1c, but not CD1d. Correspondingly, T cells that are restricted by CD1c showed poor responses to DCs that were generated in the presence of human serum, while the responses of CD1d-restricted T cells were enhanced. We chemically fractionated human serum to isolate the bioactive factors that modulate surface expression of CD1 proteins during monocyte to DC differentiation. The human serum components that affected CD1 expression partitioned with polar organic soluble fractions. Lysophosphatidic acid and cardiolipin were identified as lipids present in normal human serum that potently modulate CD1 expression. Control of CD1 expression was mediated at the level of gene transcription and correlated with activation of the peroxisome proliferator-activated receptor (PPAR) nuclear hormone receptors. These findings indicate that the ability of human DCs to present lipid antigens to T cells through expression of CD1 molecules is sensitively regulated by lysophosphatidic acid and cardiolipin in serum, which are ligands that can activate PPAR transcription factors.

Soluble Jagged 1/Fc chimera protein induces the differentiation and maturation of bone marrow-derived dendritic cells

A soluble Jagged 1/Fc chimera protein (Jagged 1/Fc) was directly used to induce differentiation and maturation of bone marrow-derived dendritic cells (DCs) in mice in vitro. A model of inducing and amplifying DCs in vitro was established. The effect of Jagged 1/Fc on morphology of DCs induced by both rmGM-CSF and rmIL-4 was observed under a confocal microscope. A fluorescein-labeled monoclonal antibody staining combined with flow cytometry was applied to detect the effect of Jagged 1/Fc on the expression of CD11c, MHC-II, CD86, CD80 and CD40 molecules on the surface of DCs. The results showed that Jagged 1/Fc did not affect the morphological properties of DC differentiation induced by both rmGM-CSF and rmIL-4. But it could promote the differentiation and maturation of DCs induced by both. The effect of it was strikingly different in the expression profile of co-stimulating molecules and the morphologic properties of DCs from lipopolysaccharide (LPS). The levels of MHC-II and CD40 molecule expression on the surface of DCs stimulated by Jagged 1/Fc were significantly lower than those stimulated by LPS, and the level of CD80 expression on the surface of DCs induced by Jagged 1/Fc was near to that induced by LPS. Jagged 1/Fc had no influence on the expression of CD86 molecule on the surface of DCs. Jagged 1/Fc when used alone could not maintain the growth, differentiation and maturation of DCs. All the findings indicate that Jagged 1/Fc influences the differentiation and maturation of DCs, which is not markedly similar to LPS, providing important evidence for its development and application as a novel immunosuppressant.

Butyrate inhibits functional differentiation of human monocyte-derived dendritic cells

Dendritic cells (DCs) play a key role in immune function through antigen presentation by MHC and CD1, as well as cytokine production that shapes the immune response. Here we report that butyrate, a histone deacetylase inhibitor, inhibits the functional differentiation of human monocyte-derived DCs. Mature DCs were generated from monocytes in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), followed by 2 day LPS stimulation. Butyrate treatment throughout the culture period inhibited the expression of CD1 molecules, but not on CD83, CD86, and MHC molecules. The suppression was exerted at protein and mRNA levels. Butyrate-treated immature DCs also showed decreased expression of CD1 molecules. Moreover the butyrate-treated immature DCs showed lower production of IL-12 p40 and IL-6 in response to lipopolysaccharides and induced less Th1 cells in allogenic mixed lymphocyte reactions. Our results imply that histone acetylation is involved in regulating immune responses through regulating functional differentiation of DC. Thus HDAC may be one of the targets for controlling the immune response.

CD40/CD40L expression in leukocytes from chronic granulomatous disease patients

Chronic granulomatous disease (CGD) is an inherited disorder caused by defects in the NADPH oxidase complex, which generates superoxide, the precursor of hydrogen peroxide (H(2)O(2)) and other reactive oxygen derivatives with microbicidal activity. Because CGD patients are at risk of chronic inflammatory manifestations, including inflammatory bowel disease and autoimmune diseases, and it is not clear whether these pathologies are exclusively secondary to altered superoxide production, or whether distinct immunologic defects are involved, we explored cell proliferation, lymphocyte cell counts, immunoglobulin levels, presence of autoimmune antibodies and expression of costimulatory molecules in leukocytes from CGD patients. We found that CGD patients have a diminished phytohemagglutinin-induced proliferation of blood mononuclear cells. Following stimulation with PMA plus ionomycin, a reduced percentage of CD40L expression in T lymphocytes and a diminished expression of CD40 molecules in neutrophils were observed on leukocytes from these patients. Our results suggest an altered interplay between elements of innate and adaptive immunity in CGD patients, which may be reflected in an increased susceptibility to opportunistic infections.

Polymorphisms of CD1 genes in chronic dysimmune neuropathies

CD1 are MCH-like glycoproteins specialized in capturing and presenting glycolipid to T cells. Expression of CD1 molecules has been observed on endoneurial machrophages in patients with chronic inflammatory demyelinating polyneuropathy (CIDP) and vasculitis and polymorphisms of CID1A and CD1E genes have been associated with susceptibility to develop Guillain–Barré syndrome. In 46 patients with CIDP, in 13 patients with multifocal motor neuropathy and in 132 controls we genotyped exon 2 of CD1A and CD1E genes. We found no association between chronic dysimmune neuropathies, with or without anti-ganglioside antibodies, and polymorphisms of CD1A and CD1E genes.

Effects of Andrographitis Paniculata extracts on the expression of CD40 in endothelial cells

In order to investigate the expression of CD40 in endothelial cells (ECs) in a variety of injured conditions and the interventional role of Andrographitis Paniculata isolate (API(0134)), the thoracic aorta ECs of guinea pigs were cultured in vitro until the third passage, incubated in the presence of media containing xanthine oxidase (XO) and xanthine (Xan) which produced oxygen free radical (OFR group); oxidized-LDL (ox-LDL group); XO, Xan and API(0134) (OFR+API(0134) group); or ox-LDL and API(0134) (ox-LDL+API(0134) group). The expression of CD40 in ECs was detected by immunofluorescence assay and reverse transcription-PCR (RT-PCR). The results showed as compared with the control group, the expression of CD40 in ECs in OFR group and ox-LDL group was increased (P<0.01), but attenuated significantly in OFR+ API(0134) group and ox-LDL+API(0134) group (P<0.05). It was suggested that API(0134) could protect atherosclerosis by inhibiting the expression of CD40 molecule in injured ECs.

Cell wall-associated alpha-glucan is instrumental for Mycobacterium tuberculosis to block CD1 molecule expression and disable the function of dendritic cell derived from infected monocyte

We previously described an escape mechanism exploited by Mycobacterium tuberculosis (Mtb) to prevent the generation of fully competent dendritic cells (DC). We have now tested the effect of isolated mycobacterial components on human monocyte differentiation into DC and demonstrated that cell wall (CW)-associated alpha-glucan induces monocytes to differentiate into DC (Glu-MoDC) with the same altered phenotype and functional behaviour of DC derived from Mtb-infected monocytes (Mt-MoDC). In fact, Glu-MoDC lack CD1 molecule expression, fail to upregulate CD80 and produce IL-10 but not IL-12. We also showed that Glu-MoDC are not able to prime effector T cells or present lipid antigens to CD1-restricted T-cell clones. Thus, we propose a mechanism of Mtb-monocyte interaction mediated by CW-associated alpha-glucan, which allows the bacterium to evade both innate and acquired immune responses.

BCG-infected adherent mononuclear cells release cytokines that regulate group 1 CD1 molecule expression

Increasing evidence is now available showing that CD1-restricted T cell responses against non-peptide mycobacterial antigens could play a role in the immune resistance against tuberculosis. BCG, widely used in anti-tubercular vaccination, shares various constituents with Mycobacterium tuberculosis, but does not provide full protection. In the present study we have investigated the pattern of group 1 CD1 molecule expression in adherent mononuclear cells (AMNC) of human peripheral blood, infected in vitro with BCG. Shortly after exposure to BCG, both BCG-positive and BCG-negative AMNC showed a moderate CD1 expression elicited by BCG-induced release of GM-CSF presumably acting through an autocrine and a paracrine mechanism. This was demonstrated using two-color flow cytometry with green fluorescent BCG and anti-CD1 PE-labeled antibodies. However, high CD1 expression induced by exogenously added GM-CSF in AMNC was reduced if target cells were cocultivated with BCG. Monoclonal antibodies against IL-10 partially restored CD1 expression, thus showing that IL-10, released from infected AMNC, is involved, at least in part, in CD1 negative modulation. Therefore, through a complex cytokine network, including not yet identified factor(s), BCG triggers but does not allow full expression of CD1 on AMNC. It cannot be excluded that this mechanism could play a role in the limited efficiency of BCG vaccination.

Down-regulation of expression of survivin and induction of apoptosis in the human Burkitt lymphomas cells by ligation of CD40-CD40L

To study the biological effects of ligation of CD40 mediated by soluble recombinant human CD40 ligand (srhCD40L) on the human malignant hematogenous cells and to explore the molecular mechanism thereof. Human Burkitt lymphoma cells of the line CA46 were cultured. Flow cytometry was used to detect the expression of CD40 molecule on the cell surface. CA46 cells were put into 96-well plate and added with solutions of srhCD40L of the terminal concentrations of 0.04 microg/ml, 0.2 microg/ml, 1.0 microg/ml, and 5.0 microg/ml respectively, and 24, 28, 72, 96, and 120 hours later cell growth curve was drawn. MTT assay was used to other CA46 cells co-incubated with srhCD40L of the terminal concentrations of 0.04 microg/ml, 0.2 microg/ml, 1.0 microg/ml, and 5.0 microg/ml respectively so as to calculate the proliferation inhibition rate. CA46 cells were treated with srhCD40L of the concentrations of 1.0 microg/ml for 24, 48, and 72 hours respectively, FCM was used to analyze the DNA cycle and TUNEL was used to calculate the apoptotic rate. Annexin-V labeling method was used to detect the positive rate of apoptotic cells. CA46 cells were treated with srhCD40L of the concentration of 1 microg/ml for 24, 48, 72, or 96 hours, semi-quantitative RT-PCR was used to detect the mRNA expression of survivin, an anti-apoptosis protein, and the protein expression of survivin was detected by Western blotting. The expression rate of CD40 in the human Burkitt CA46 cells was 99%. srhCD40L dose-dependently inhibited the proliferation of the CD46 cells. Treated by srhCD40L, the progress of cells at S stage into G(2)/M stage was inhibited. TUNEL showed that treated by srhCD40L (1.0 microg/ml) for 24, 48, and 72 hours the apoptotic rates of the cells were 9%, 18%, and 35% respectively. Annexin-V showed that after incubation with srhCD40L (1.0 microg/ml) for 24 h the apoptotic rate was 10.04%. Two apoptotic peaks appeared 48 and 72 hours later. Semi-quantitative RT-PCR and Western blotting showed that the survivin mRNA expression and protein expression were both down-regulated. Ligation of CD40 by srhCD40L inhibits the proliferation of malignant hematogenous cells and induces their apoptosis. Expression of survivin mRNA and protein may be related to cell growth inhibition and to the apoptosis mediated by Ligation of CD40 by srhCD40L.

Phenotypic and Functional Characterization of Vaginal Dendritic Cells in a Rat Model ofCandida albicansVaginitis

This study analyzes the phenotype of vaginal dendritic cells (VDCs), their antigenic presentation and activation of T-cell cytokine secretion, and their protective role in a rat model of Candida vaginitis. Histological observation demonstrated a significant accumulation of OX62(+) VDCs in the mucosal epithelium of Candida albicans-infected rats at the third round of infection. We identified two subsets of OX62(+) VDCs differing in the expression of CD4 molecule in both noninfected and Candida-infected rats. The OX62(+) CD4(+) subset of VDCs displayed a lymphoid cell-like morphology and expressed the T-cell antigen CD5, whereas the OX62(+) CD4(-) VDC subset exhibited a myeloid morphology and was CD5 negative. Candida infection resulted in VDC maturation with enhanced expression of CD80 and CD134L on both CD4(+) and CD4(-) VDC subsets at 2 and 6 weeks after Candida infection. CD5(-) CD4(-) CD86(-) CD80(-) CD134L(+) VDCs from infected, but not noninfected, rats spontaneously released large amounts of interleukin-12 (IL-12) and tumor necrosis factor alpha, whereas all VDC subsets released comparable levels of IL-10 and IL-2 cytokines. Furthermore, OX62(+) VDCs from infected rats primed naïve CD4(+) T-cell proliferation and release of cytokines, including gamma interferon, IL-2, IL-6, and IL-10, in response to staphylococcal enterotoxin B stimulation in vitro. Adoptive transfer of highly purified OX62(+) VDCs from infected rats induced a significant acceleration of fungal clearance compared with that in rats receiving naive VDCs, suggesting a protective role of VDCs in the anti-Candida mucosal immunity. Finally, VDC-mediated protection was associated with their ability to rapidly migrate to the vaginal mucosa and lymph nodes, as assessed by adoptive transfer of OX62(+) VDCs labeled with 5 (and 6-)-carboxyfluorescein diacetate succinimidyl ester.