Intestinal antigen-presenting cells in mucosal immune homeostasis:Crosstalk between dendritic cells,macrophages and B-cells

The intestinal immune system maintains a delicate balance between immunogenicity against invading pathogens and tolerance of the commensal microbiota. Inflammatory bowel disease (IBD) involves a breakdown in tolerance towards the microbiota. Dendritic cells (DC), macrophages (MΦ) and B-cells are known as professional antigen-presenting cells (APC) due to their specialization in presenting processed antigen to T-cells, and in turn shaping types of T-cell responses generated. Intestinal DC are migratory cells, unique in their ability to generate primary T-cell responses in mesenteric lymph nodes or Peyer’s patches, whilst MΦ and B-cells contribute to polarization and differentiation of secondary T-cell responses in the gut lamina propria. The antigen-sampling function of gut DC and MΦ enables them to sample bacterial antigens from the gut lumen to determine types of T-cell responses generated. The primary function of intestinal B-cells involves their secretion of large amounts of immunoglobulin A, which in turn contributes to epithelial barrier function and limits immune responses towards to microbiota. Here, we review the role of all three types of APC in intestinal immunity, both in the steady state and in inflammation, and how these cells interact with one another, as well as with the intestinal microenvironment, to shape mucosal immune responses. We describe mechanisms of maintaining intestinal immune tolerance in the steady state but also inappropriate responses of APC to components of the gut microbiota that contribute to pathology in IBD.

The current role of dendritic cells in the progression and treatment of colorectal cancer

Colorectal cancer(CRC)is the third most common cancer and the second leading cause of cancer-related deaths worldwide.Dendritic cells(DCs)constitute a heterogeneous group of antigen-presenting cells that are important for initiating and regulating both innate and adaptive immune responses.As a crucial component of the immune system,DCs have a pivotal role in the pathogenesis and clinical treatment of CRC.DCs cross-present tumor-related antigens to activate T cells and trigger an antitumor immune response.However,the antitumor immune function of DCs is impaired and immune tolerance is promoted due to the presence of the tumor microenvironment.This review systematically elucidates the specific characteristics and functions of different DC subsets,as well as the role that DCs play in the immune response and tolerance within the CRC microenvironment.Moreover,how DCs contribute to the progression of CRC and potential therapies to enhance antitumor immunity on the basis of existing data are also discussed,which will provide new perspectives and approaches for immunotherapy in patients with CRC.

Effect of vitamin D3 on maturation and antigen-presenting function of dendritic cells treated with Mycobacterium tuberculosis

Objective:To investigate the phenotypic characteristics and functional capability differences of mouse bone marrow-derived dendritic cells after stimulation with Mycobacterium tuberculosis in the presence or absence of vitamin 03.Methods:Mouse bone marrow-derived cells were cultured with GM-CSF(20 ng/mL).Then,one was added with 100 nmol/L of 25(OH)D3,while the other did not.On day 6.5 μg/mL of BCG was added to stimulate the cells for 24 h.On day 7,suspension cells were harvested for phenotypic and functional analyses.Results:The percentages of CD86 dendritic cells(DCs) in the control group and 25(OH)D3 group were 66.97%± 8.29%and 52.18%± 8.52%.respectively;the mean fluorescence intensities of MHC- Ⅱ in the control group and 25(OH)D3 group were 1 102.16 ± 371.02 and 681.62± 292.71.The expression levels of MHC- Ⅱ and CD86 on the surface of the DCs in 25(OH)D3 group were significantly lower than those of the control group.The ability of the DCs to stimulate proliferation of T-lymphocytes was also significantly lower than that of the control group.Conclusions:These findings suggest that 25(OH)D3 modulates the immune response by affecting the maturation and function of DCs in Mycobacterium tuberculosis period.

Changes of the Transcriptional Levels of Molecules Associated with Endogenous Antigen Processing and Presentation in Porcine Skin-derived Dendritic Cells Infected with PCV2 in vivo

[Objective] This study aimed to investigate the changes of the transcriptional levels of molecules associated with endogenous antigen processing and presenta- tion in porcine skin-derived dendritic cells infected with PCV2 in vivo. [Method] Healthy 40-day-old Landrace piglets were infected with porcine circovirus type 2 （PCV2） and euthanized on the 34, 7rd, 14th, 21st and 35th d post inoculation （DPI）. The porcine skin-derived dendritic cells （DCs） were collected to analyze the transcrip- tional levels of molecules （LMP7, UBP, MHC-I, calreticulin） associated with endogenous antigen processing and presentation by using real-time fluorescent quantitative PCR （real-time FQ-PCR）. [Result] The results showed that the level of LMP7 mR- NAs was reduced significantly on the 3DPI （P〈0.05）; the level of UBP mRNAs was consistently up-regulated, which increased significantly on the 21DPI and 35DPI （P〈 0.05）; the level of MHC-I mRNAs was significantly down-regulated on the 7DPI （P〈 0.05）; the level of calreticulin mRNAs was up-regulated slightly without significant dif- ference. [Conclusion] PCV2 can inhibit the endogenous antigen processing and presentation ability of porcine skin-derived DCs at early stages of infection.

Characterization of colonic dendritic cells in normal and colitic mice

AIM： Recent studies demonstrating the direct involvement of dendritic cells （DC） in the activation of pathogenic T cells in animal models of inflammatory bowel disease identify DC as important antigen presenting cells in the colon. However, very little is known about the properties of colonic DC. METHODS： Using immunohistochemistry, electron microscopy and flow cytometry we have characterized and compared colonic DC in the colon of healthy animals and interleukin-2-deficient （IL2-/-） mice that develop colitis. RESULTS： In the healthy colon, DC resided within the lamina propria and in close association with the basement membrane of colonic villi. Type i myeloid （CD11c^＋, CD11b^＋, B220, CD8） DC made up the largest （40-45%） population and all DC expressed low levels of CD80, CD86, and CD40, and had high endooltic activity consistent with an immature phenotype. In colitic IL2-/- mice, colonic DC numbers increased four- to five-fold and were localized within the epithelial layer and within aggregates of T and B cells. They were also many more DC in mesenteric lymph nodes （MLN）. The majority （〉85%） of DC in the colon and MLN of IL2./ mice were type 1 myeloid, and expressed high levels of MHC class II, CD80, CD86, CD40, DEC 205, and CCR5 molecules and were of low endocytic activity consistent with mature DC. CONCLUSION： These findings demonstrate striking changes in the number, distribution and phenotype of DC in the inflamed colon. Their intimate association with lymphocytes in the colon and draining lymph nodes suggest that they may contribute directly to the ongoing inflammation in the colon.

Flow cytometry assay of myeloid dendritic cells (mDCs)in peripheral blood during acute hepatitis C:Possible pathogenetic mechanisms

AIM： To asses the expression of myeloid dendritic cells （CD11c＋） subset during acute HCV hepatitis and its possible involvement in natural history of the infection.METHODS： We enrolled 11 patients with acute hepatitis C （AHC） （Group A）, 10 patients with acute hepatitis A （AHA） （as infective control-Group B） and 10 healthy donors （group C） in this study. All patients underwent selective flow cytometry gating strategies to assess the peripheral number of the myeloid dendritic cells （mDCs） to understand the possible role and differences during acute hepatitis.RESULTS： Eight of 11 patients with acute HCV hepatitis did not show any increase of mDCs compared to healthy individuals, while a significant decrease of mDCs was found in absolute cell count （z=-2.37, P〈0.05） and percentage （z=-2.30; P〈 0.05） as compared with AHA. On the contrary, The remaining three patients of the group A had a higher mDCs number and percentage as occur in group B. Interestingly, after six months, those patients did not show any increase of mDCs subset were chronically infected, while the three subjects with an increase of peripheral mDCs, as in HAV acute infection, resolved the illness.CONCLUSION： The lack of increase of mDCs during acute hepatitis C might be an important factor involved in chronicization of the infection.

Over-expression of programmed death-ligand 1 and programmed death-1 on antigen-presenting cells as a predictor of organ dysfunction and mortality during early sepsis: a prospective cohort study

BACKGROUND:This study aimed to explore the changes of programmed death-ligand 1(PDL1)and programmed death-1(PD-1)expression on antigen-presenting cells(APCs)and evaluate their association with organ failure and mortality during early sepsis.METHODS:In total,40 healthy controls and 198 patients with sepsis were included in this study.Peripheral blood was collected within the first 24 h after the diagnosis of sepsis.The expression of PDL1 and PD-1 was determined on APCs,such as B cells,monocytes,and dendritic cells(DCs),by flow cytometry.Cytokines in plasma,such as interferon-γ(IFN-γ),tumor necrosis factor-α(TNF-α),interleukin-4(IL-4),IL-6,IL-10,and IL-17A were determined by Luminex assay.RESULTS:PD-1 expression decreased significantly on B cells,monocytes,myeloid DCs(mDCs),and plasmacytoid DCs(pDCs)as the severity of sepsis increased.PD-1 expression was also markedly decreased in non-survivors compared with survivors.In contrast,PD-L1 expression was markedly higher on mDCs,pDCs,and monocytes in patients with sepsis than in healthy controls and in non-survivors than in survivors.The PD-L1 expression on APCs(monocytes and DCs)was weakly related to organ dysfunction and infl ammation.The area under the receiver operating characteristic curve(AUC)of the PD-1 percentage of monocytes(monocyte PD-1%)+APACHE II model(0.823)and monocyte PD-1%+SOFA model(0.816)had higher prognostic value than other parameters alone.Monocyte PD-1%was an independent risk factor for 28-day mortality.CONCLUSION:The severity of sepsis was correlated with PD-L1 or PD-1 over-expression on APCs.PD-L1 in monocytes and DCs was weakly correlated with infl ammation and organ dysfunction during early sepsis.The combination of SOFA or APACHE II scores with monocyte PD-1%could improve the prediction ability for mortality.

Study on mechanism of increased allergenicity induced by Ara h 3 from roasted peanut using bone marrow-derived dendritic cells

Little information was so far available about allergenic mechanism of the roasted peanut allergens during initial stages of allergy.The purpose of this study was to determine the influence of roasting(150℃,20 min)on biochemical and biological properties of Ara h 3,a major peanut allergen.Allergenicity of roasted peanut emulsion to mice,differences in uptakes between Ara h 3 purified from raw peanuts(named as Ara h 3-Raw)and that purified from roasted peanuts(named as Ara h 3-Roasted)by bone marrow-derived dendritic cells(BMDCs)and the implication of cell surface receptors involving in uptake,and changes in glycosylation and structure of Ara h 3 after roasting were analyzed in this study.This study suggested that roasting increased allergenicity of peanut to BALB/c mice.Maillard reaction and structural changes of Ara h 3 induced by roasting significantly altered the uptake of Ara h 3-Roasted by BMDCs,and modified Ara h 3 fate in processes involved in immunogenicity and hyper allergenicity,indicating that food processing pattern can change food allergenicity.

Lethal effect of mononuclear cells derived from human umbilical cord blood differentiating into dendritic cells after in vitro induction of cytokines on neuroblastoma cells

BACKGROUND： Dendritic cell is the most major antigen presenting cell of organism. It is proved in recent studies that human umbilical cord blood mononuclear cells induced and cultured in vitro by recombinant human granulocyte-macrophage colony stimulating factor （rhG-MCSF） and recombinant human interleukin-4 （rhlL-4） can generate a great many dendritic cells and promote the lethal effect of T cells on human neuroblastoma, but it is unclear that whether the lethal effect is associated with the most proper concentration of dendritic cells. OBJEETIVE： To investigate the lethal effect of human umbilical cord blood mononuclear cells induced in vitro by cytokines differentiating into dendritic cells on human neuroblastoma, and its best concentration range. DESIGN ： Open experiment SEI-FING： Department of Pediatrics, the Medical School Hospital of Qingdao University MATERIALS ： The study was carried out in the Shandong Provincial Key Laboratory （Laboratory for the Department of Pediatrics of the Medical School Hospital of Qingdao University） during September 2005 to May 2006. Human umbilical cord blood samples were taken from the healthy newborn infants of full-term normal delivery during October to November 2005 in the Medical School Hospital of Qingdao University, and were voluntarily donated by the puerperas. Main instruments： type 3111 CO2 incubator （Forma Scientific, USA）, type 550 ELISA Reader （Bio-Rad, USA）. Main reagents： neuroblastoma cell line SK-N-SH （Shanghai Institute of Life Science, Chinese Academy of Sciences）, RPMI-1640 culture fluid and fetal bovine serum （Hyclone）, rhlL-4 （Promega, USA）, rhG-MCSF （Harbin Pharmaceutic Group Bioengineering Co.Ltd）, rat anti-human CDla monoclonal antibody and FITC-labeled rabbit anti-rat IgG （Xiehe Stem cell Gene Engineering Co.Ltd）. METHODS： ① Human umbilical cord blood mononuclear cells obtained with attachment methods differentiated into human umbilical cord blood dendritic cells, presenting typical morphology of dendritic cells after in vitro induction by rhG-MCSF and rhlL-4. ② Different concentrations of dendritic cells[ dendritic cells： neuroblastoma cells=20：1,50：1,100：1 （2×10^8 L^-1,5×10^8 L^-1,1×10^9 L^-1）], 1×10^9 L^-1 T cells and 1×10^7 L^-1 neuroblastoma cells were added in the experimental group. 1 ×10^9 L^-1 T cells and 1 ×10^7 L^-1 neuroblastoma cells were added in the control group. ③ Main surface marker CDla molecules of dendritic cells were detected with indirect immunofluorescence, and the percent rate of dendritic cells was counted with ultraviolet light and expressed as the expression rate of CD1a^＋ cells. ④Single effector cells and target cells were respectively set in the experimental group and control group to obtain the lethal effect. The lethal effect of dendritic cells on neuroblastoma cells was indirectly evaluated by detecting cellular survival with MTT assay. The lethal effect（%）= （1-A experimentat well-A effector cell /A target cell well）×100%.⑤The expenmental data were presented as Mean ±SD, and paired t test was used. MAIN OUTCOME MEASURES： ① Morphological characters of dendritic cells in the process of induction and differentiation. ②CD1a^＋ cellular expression rate. ③Lethal effect of dendntic cells on neuroblastoma cells. RESULTS： ①Morphological characters of dendritic cells in the process of induction and differentiation： On the 15^th day after human umbilical cord blood mononuclear cells were induced by rhG-MCSF and rhlL-4, typical morphology of dendritic cells could be seen under an inverted microscope. ②Expression rate of CD1a^＋ cells was （43.12±5.83）%. ③Lethal effect of dendritic cells on neuroblastoma cells： Lethal effect of dendritic cells stimulated T cells in each experimental group （ dendritic cells： neuroblastoma cells=100：1,50：1, 20：1 respectively） on neuroblastoma cells was significantly higher than that in control group[（31.00 ±4.41 ）%, （30.92±5.27）%,（33.57±5.35）%,（26.23±5.20）%, t=3.51,2.98,4.24, P〈 0.01 ）; But the lethal effect of dendntic cells on neuroblastoma was significantly lower when their ratio was 100：1 and 50：1 in comparison with 20：1 （t=2.01,2.36, P 〈 0.05）, and no significant difference in lethal effect existed between the ratio at 100：1 and 50：1 （t=0.06,P 〉 0.05）. CONCLUSION： Dendritic cells differentiated from human umbilical cord blood mononuclear cells after in vitro induction of cytokines can promote the lethal effect of T cells on neuroblastoma cells. The lethal effect is associated with the concentration of dendritic cells within some range.

Novel heat shock protein Hsp70L1 activates dendritic cells and acts as a Th1 polarizing adjuvant

Heat shock proteins (HSPs) are reported to act as effective adjuvants to elicit anti-tumor and anti-infection immunity. Here, we report that Hsp70-like protein 1 (Hsp70L1), a novel HSP derived from human dendritic cells (DCs), has potent adjuvant effects that polarize responses toward Th1. With a calculated molecular weight of 54.8 kDa, Hsp70L1 is smaller in size than Hsp70 but resembles it both structurally and functionally. Hsp70L1 shares common receptors on DCs with Hsp70 and can interact with DCs, promoting DC maturation and stimulating secretion of the proinflammatory cytokines interleukin 12p70 (IL-12p70), IL-1beta, tumor necrosis factor-alpha (TNF-alpha), and the chemokines IP-10, macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and normal T cell expressed and secreted (RANTES). The induction of interferon-gamma-inducible protein 10 (IP-10) secretion by Hsp70L1 is not shared by Hsp70, and other functional differences include more potent stimulation of DC IL-12p70, CC-chemokine, and CCR7 and CXCR4 expression by Hsp70L1. Immunization of mice with the hybrid peptide Hsp70L1-ovalbumin(OVA)(257-264) induces an OVA(257-264)-specific Th1 response and cytotoxic T lymphocyte (CTL) that results in significant inhibition of E.G7-OVA tumor growth. The ability of Hsp70L1 to activate DCs indicates its potential as a novel adjuvant for use with peptide immunizations; the Hsp70L1 antigen peptide hybrid may serve as a more effective vaccine for the control of cancer and infectious diseases.

Culture of Porcine Peripheral Blood Monocyte-derived Dendritic Cells in vitro

[Objective] This study aimed to establish an in vitro culture model for porcine peripheral blood monocyte-derived dendritic cells （MoDCs）. [Method] Fresh peripheral blood mononuclear cells （PBMCs） were separated from pig, and precursor dendritic cells were obtained by adherence method. The dendritic cells were treated by recombinant porcine granulocyte-monocyte colony stimulating factor （rpGM-CSF） and recombinant porcine interleukin-4 （rplL-4） together, and lipopolysaccharide （LPS） respectively. The cells in different time periods were collected. The morphology of the collected cells was observed by scanning electron microscopy; the expression of surface molecules and phagocytic ability to FITC-dextran were detected by flow cy- tometry; and the stimulating ability for allogeneic T cells was detected by mixed lymphocyte reaction. [Result] The DCs suffering maturation induction in vitro showed typical dendritic morphology; compared with those of DCs untreated by LPS, the cell surface expression of CDla, CD80, CD86, SLAII and CD172a of DCs treated by LPS was significantly increased, the phagocytic ability was reduced slightly, and the stimulating ability for allogeneic T cells was enhanced to some extent. [Conclusion] An in vitro culture method was successfully established for porcine MoDCs in this study, laying a foundation for further study on the role of porcine MoDCs in immunoregulation and anti-virus infection.

Induction of cytotoxic T lymphocyte respones in vivo after immunotherapy with dendritic cells in patients with nasopharyngeal carcinoma

The aim of the present study was to determine the efficacy of immunotherapy with dendritic cells to elicit EBV-specific CTL-immunity in advanced cases of EBV-positive patients with nasopharyngeal carcinoma （NPC） and to determine the safety and toxicity of this preparation. Nine cases of histologically confirmed patients with NPC undergoing treatment with radiological therapy were enrolled in this study. Dendritic cells, generated in vitro from blood monocytes of patients were cultured and matured with cytokines and then infected with recombinant adenovims vaccine containing EBV-latent membrane protein-2 （Ad-LMP2）. On 9 days＇ cultivation of cells, the matured DCs were harvested, irradiated with 60^Co and then injected intradermally to patients with NPC. The injections were performed 3 times totally. After immunization, the CTL responses were assayed by means of cytotoxicity and epitope-specific IFN-γ production. The results of this trial showed that all patients could tolerate this kind of treatment without any side effect, during which marked increase of LMP2-specific CIL-responses could be demonstrated in 5 patients of this group. And the level of IgA/VCA antibody decreased in 8 of 9 patients, thus accounting for a better prognosis for these patients. All patients will be followed up for another one year. At least, the present work shows that intradermal vaccination with autologons DCs infected with recombinant Ad-LMP2 adenovirus is a safe procedure in NPC patients, in which this procedure can enhance the LMP2-specific CTL responses in patients. These data are encouraging to develop more effective vaccine strategies for the treatment of nasopharyngeal carcinoma.

Sinomenine promotes differentiation of induced pluripotent stem cells into immature dendritic cells with high induction of immune tolerance

BACKGROUND Immature dendritic cells(imDCs)play an important role in the induction of donor-specific transplant immunotolerance.However,these cells have limitations,such as rapid maturation and a short lifespan in vivo.In previous studies,induced pluripotent stem cells(iPSCs)differentiated into imDCs,and sinomenine(SN)was used to inhibit the maturation of imDCs.AIM To study the capacity of SN to maintain iPSC-derived imDCs(SN-iPSCs-imDCs)in an immature state and the mechanism by which SN-iPSCs-imDCs induce immunotolerance.METHODS In this study,mouse iPSCs were induced to differentiate into imDCs in culture medium without or with SN(iPSCs-imDCs and SN-iPSCs-imDCs).The imDCrelated surface markers,endocytotic capacity of fluorescein isothiocyanate Dextran and apoptosis were analyzed by flow cytometry.The effects of iPSCs-imDCs and SNiPSCs-imDCs on T-cell stimulatory function,and regulatory T(Treg)cell proliferative function in vitro were analyzed by mixed lymphocyte reaction.Cytokine expression was detected by ELISA.The apoptosis-related proteins of iPSCs-DCs and SN-iPSCs-DCs were analyzed by western blotting.The induced immunotolerance of SN-iPSCs-DCs was evaluated by treating recipient Balb/c skin graft mice.Statistical evaluation of graft survival was performed using Kaplan–Meier curves.RESULTS Both iPSCs-imDCs and SN-iPSCs-imDCs were successfully obtained,and their biological characteristics and ability to induce immunotolerance were compared.SN-iPSCs-imDCs exhibited higher CD11c levels and lower CD80 and CD86 levels compared with iPSCs-imDCs.Reduced major histocompatibility complex II expression,worse T-cell stimulatory function,higher Treg cell proliferative function and stronger endocytotic capacity were observed with SN-iPSCs-imDCs(P<0.05).The levels of interleukin(IL)-2,IL-12,interferon-γin SN-iPSCs-imDCs were lower than those in iPSCs-imDCs,whereas IL-10 and transforming growth factor-βlevels were higher(P<0.05).The apoptosis rate of these cells was significantly higher(P<0.05),and the expression levels of cleaved caspase3,Bax and cleaved poly(ADP-ribose)polymerase were higher after treatment with lipopolysaccharides,but Bcl-2 was reduced.In Balb/c mice recipients immunized with iPSCsimDCs or SN-iPSCs-imDCs 7 d before skin grafting,the SN-iPSCs-imDCs group showed lower ability to inhibit donor-specific CD4+T-cell proliferation(P<0.05)and a higher capacity to induce CD4+CD25+FoxP3+Treg cell proliferation in the spleen(P<0.05).The survival span of C57bl/6 skin grafts was significantly prolonged in immunized Balb/c recipients with a donor-specific pattern.CONCLUSION This study demonstrated that SN-iPSCs-imDCs have potential applications in vitro and in vivo for induction of immunotolerance following organ transplantation.

IMMUNOHISTOCHEMICAL STUDY ON S100 PROTEIN-POSITIVE DENDRITIC CELLS IN PATIENTS WITH GASTRIC CARCINOMA

The density of dendritic cells (DC) and macro-phages (Mφ) in tissue specimens of gastric carcinoma (GC n=65) was investigated by ABC im-munohistochemical method using anti-S100 protein and anti-lysozyme antibodies, and was compared with that in gastric ulcer (GU n=19), chronic atrophic gastritis (CAG n=28) and normal gastric mucosa (NGM n=15). The mean density if DC (cells/mm2) in GC (15.0 was significantly higher than that in NGM (3.8) and GU (8.3), but was remarkably lower when compared to that in CAG (29.5) (P<0.01). Statistically significant difference in the population density of DC was observed between well- and poorly-differentiated GC (P<0.01). With their unique dendritic processes, DC were mainly concentrated within dense lymphoid infiltrates or in the T-area of reactive lymphoid follicles and were interspersed among the tumor cells. In contrast, Mφ were present around the necrotic foci and were rarely seen within the non-necrotic neoplastic tissues. These data suggest that DC, which differ in morphology, distribution, number and function form Mφ may be more directly involved in the host immune reaction against tumor by acting as antigen presenting cells.

Experimental study on dendritic cells pulsed with brain tumor stem cells for immunotherapy of glioma

Objective To investigate the effect of dendritic cells pulsed with brain tumor stem cells which are used to treat on intracranial glioma. Methods We obtained murine brain tumor stem cells by grow ing C6 cells in epidermal grow th factor/basic fibroblast grow th factor w ithout serum.Dendritic cells isolated from rat bone marrow w ere pulsed w ith BTSCs. Rat brain

Dysfunction of peripheral blood dendritic cells from patients with chronic hepatitis B virus infection

AIM: To identify the property of dendritic cells (DCs) of peripheral blood monocytes (PBMC) in patients with chronic HBV infection. METHODS: Twenty patients with persistent HBV infection were included in this study, 10 healthy subjects being used as a control group. The peripheral blood mononuclear cells (PBMC) of T cell-depleted populations were incubated and induced into mature dendritic cells in the RPMI-1640 medium in the presence of cytokines GM-CSF, IL-4, FLt-3,TNF-alpha and 100mL.L(-1 )of fetal calf serum for a total of 10-12 days. The expressions of surface markers on DCs were evaluated using flow cytometric analysis. ELISA method was used to determine the cytokine levels of interleukin-12 (IL-12) and IL-10 in the supernatant produced by DCs. For detection of the stimulatory capacity of DCs to T cell proliferation, mytomycin C-treated DC were incubated with allogenic T cells. RESULTS: A typical morphology of mature DCs from healthy subjects and HBV-infected patients was induced in in vitro incubation, but the proliferation ability and cellular number of DCs from HBV-infected patients significantly decreased compared with healthy individuals. In particular, the expression levels of HLA-DR, CD80 (B7-1) and CD86 (B7-2) on DC surface from patients were also lower than that from healthy individuals (0.46 vs 0.92 for HLA-DR, 0.44 vs 0.88 for CD80 and 0.44 vs 0.84 for CD86,P【0.05). The stimulatory capacity and production of IL-12 of DCs from patients in allogenic mixed lymphocyte reaction (AMLR) significantly decreased, but the production level of nitric oxide (NO) by DCs simultaneously increased compared with healthy subjects (86 +/- 15 vs 170 +/- 22 micromol.L(-1), P 【0.05). CONCLUSION: The patients with chronic HBV infection have the defective function and immature phenotype of dendritic cells, which may be associated with the inability of efficient presentation of HBV antigens to host immune system for the clearance of HBV.

Clostridium butyricum alleviates intestinal low-grade inflammation in TNBS-induced irritable bowel syndrome in mice by regulating functional status of lamina propria dendritic cells

BACKGROUND Irritable bowel syndrome (IBS) is one of the most common functional gastroenterological diseases characterized by abnormal visceral sensitivity and lowgrade inflammation. The role of Clostridium butyricum (C. butyricum) in reducing intestinal low-grade inflammation via immune pathways has been well defined. However, the detailed mechanisms of the effects of C. butyricum on intestinal mucosal immunity, especially on immune cells of the lamina propria, remain unclear. Dendritic cells (DCs), which are important immune cells, secrete proinflammatory cytokines (IL-1β, IL-6, and others) and express T cell immunoglobulin and mucin domain-3 (TIM3), promoting proliferation and activation of DCs, and mediating Th1 and Th17 inflammatory responses. AIM To investigate the role of DCs in the development of IBS in a rat model and to understand the regulation of DCs after C. butyricum intervention. METHODS An IBS animal model was established using C57BL/6 mice, and C. butyricum was continuously administered via the intragastric route to simulate different intestinal immune states. Intestinal visceral hypersensitivity and histopathology were assessed using the abdominal withdrawal reflex (AWR) test and hematoxylin & eosin (H&E) staining, respectively. The expression of proinflammatory cytokines (IL-1β and IL-6) and TIM3 was analyzed by Western blot analysis and real-time PCR. Flow cytometry was applied to analyze the quantity, function, and membrane molecule TIM3 of the lamina propria dendritic cells (LPDCs). The regulatory effect of C. butyricum was verified in bone marrowderived dendritic cells by in vitro experiments. RESULTS The secretion of proinflammatory cytokines (IL-1β and IL-6) in mice with IBS was significantly increased compared with that of the control group, which suggested that the intestinal mucosa in mice with IBS was in a low-grade inflammatory state. The expression of CD11C+CD80+ and CD11c+TIM3+ in intestinal LPDCs in mice with IBS increased significantly. Meanwhile, the cytokines (IL-1β and IL-6) were significantly reduced after the intervention with probiotic C. butyricum. The amount and function of LPDCs and the TIM3 on the surface of the LPDCs were decreased with the alleviation of the intestinal inflammatory response. CONCLUSION The results suggest that C. butyricum regulates the amount and functional status of LPDCs in the intestinal mucosa of mice with IBS, and therefore modulates the local immune response in the intestine.

Effect of a cancer vaccine prepared by fusions of hepatocarcinoma cells with dendritic cells

AIM: To prepare a cancer vaccine (H(22)-DC) expressing high levels of costimulatory molecules based on fusions of hepatocarcinoma cells (H(22)) with dendritic cells (DC) of mice and to analyze the biological characteristics and induction of specific CTL activity of H(22)-DC. METHODS: DCs were isolated from murine spleen by metrizamide density gradient centrifugation, purified based on its characteristics of semi-adhesion to culture plates and FcR-,and were cultured in the medium containing GM-CSF and IL-4. A large number of DC were harvested. DCs were then fused with H(22) cells by PEG and the fusion cells were marked with CD11c MicroBeads. The H(22)-DC was sorted with Mimi MACS sorter. The techniques of cell culture, immunocytochemistry and light microscopy were also used to test the characteristics of growth and morphology of H(22)-DC in vitro. As the immunogen, H(22)-DC was inoculated subcutaneously into the right armpit of BALB/C mice, and their tumorigenicity in vivo was observed. MTT was used to test the CTL activity of murine spleen in vivo. RESULTS: DC cells isolated and generated were CD11c+ cells with irregular shape, and highly expressed CD80, CD86 and CD54 molecules. H22 cells were CD11c- cells with spherical shape and bigger volume, and did not express CD80, CD86 and CD54 molecules.H(22)-DC was CD11c+ cells with bigger volume, being spherical, flat or irregular in shape, and highly expressed CD80, CD86 and CD54 molecules, too. H(22)-DC was able to divide and proliferate in vitro, but its activity of proliferation was significantly decreased as compared with H(22) cells and its growth curve was flatter than H(22) cells. After subcutaneous inoculation over 60 days, H(22)-DC showed no tumorigenecity in mice, which was significantly different from control groups (P【0.01). The spleen CTL activity against H(22) cells in mice implanted with fresh H(22)-DC was significantly higher than control groups (P 【 0.01). CONCLUSION: H(22)-DC could significantly stimulate the specific CTL activity of murine spleen, which suggests that the fusion cells have already obtained the function of antigen presenting of parental DC and could present H(22)specific antigen which has not been identified yet, and H(22)-DC could induce antitumor immune response; although simply mixed H(22) cells with DC could stimulate the specific CTL activity which could inhibit the growth of tumor in some degree, it could not prevent the generation of tumor. It shows that the DC vaccine is likely to become a helpful approach in immunotherapy of hepatocarcinoma.