This study examined the effects of Bangdeyun on the expressions of nuclear factor-kappaB (NF-kappaB), interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) in the endometrium of mice with embryo implantation dysfunction (EID) during the implantation time (namely on pregnancy day 5, 6, 7 and 8) and explored the local immune regulatory effects of Bangdeyun. The gestational mice were randomly divided into normal group, model group and Bangdeyun-treated group. EID models of mice were established by using indomethacin. The endometrial expression of NF-kappaB was detected by immunohistochemistry and Western blotting. IFN-gamma and IL-10 were measured by enzyme-linked immunosorbent assay (ELISA). The results showed that in the normal group, NF-kappaB and IFN-gamma were weakly expressed and IL-10 was strongly expressed in the endometrium during the whole implantation period. In the model group, the expressions of NF-kappaB and IFN-gamma were increased on pregnancy day 5, 6 and 7, and IL-10 expression decreased during the whole implantation time when compared with those in the normal group (P<0.01 for all). In the Bangdeyun-treated group, little amount of NF-kappaB and IFN-gamma was expressed and IL-10 expression was strong, much the way they were expressed in the normal group (P>0.05). The expressions of NF-kappaB and IFN-gamma were much lower in the Bangdeyun-treated group than those in the model group on pregnancy day 5, 6 and 7 (P<0.01 for all), while the expression of IL-10 was much higher than in the model group during the whole implantation time (P<0.01). It was suggested Bangderun may favor a shift from Th1- to Th2-type immune response, therefore inhibiting the maternal immune rejection, inducing the immune tolerance and improving the fetal implantation.
Drugs, Chinese Herbal/*pharmacology ; Embryo Implantation, Delayed/*drug effects ; Embryo Implantation, Delayed/immunology ; Endometrium/*immunology ; Endometrium/metabolism ; Interferon-gamma/genetics ; Interferon-gamma/metabolism ; Interleukin-10/genetics ; Interleukin-10/metabolism ; NF-kappa B/genetics ; NF-kappa B/metabolism

OBJECTIVETo investigate the expression of paired immunoglobin-like receptor B (PIR-B) on dendritic cells (DCs) and its relationship with tolerogenic DCs (T-DCs) in mouse.

METHODSDC2.4 cells, an immature dendritic cell line derived from C57BL/6 mouse, were stimulated by lipopolysaccharide (LPS) for 48 h to induce the mature dendritic cells (mDC) and cultured respectively with the recombined mouse interleukin-10 (rmIL-10) or recombined human transforming growth factor beta1 (rhTGF-beta1) to develop the tolerogenic dendritic cells (T-DC). Special small interference RNA (siRNA) molecular of PIR-B was chemically synthesized and transfected into DC2.4 cells (si-DC) by lip2000. The expression of PIR-B on DC2.4 cells was measured by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot. The allogeneic lymphocyte proliferative capacity of DCs was measured by mixed lymphocyte reaction (MLR) using 3H-thymidine incorporation test. The concentration of IFN-gamma in supernatants of MLR was analyzed by ELISA.

RESULTSSemi-quantitative RT-PCR and Western blot showed that PIR-B mRNA and protein were expressed on DC2.4 cells. RmIL-10 and rhTGF-beta1 induced the higher PIR-B mRNA and protein level on T-DCs (Relative values were 0.51 +/- 0.08 and 0.58 +/- 0.23; 0.85 +/- 0.07 and 0.87 +/- 0.14; 0.79 +/- 0.10 and 0.85 +/- 0.34, respectively) (P < 0.05). LPS down-regulated the PIR-B expression on mDC (0.35 +/- 0.10 and 0.32 +/- 0.04) (P < 0.05). The PIR-B mRNA and protein expression were inhibited by siRNA transfection (decreased by 78.9% and 74.2% respectively after 48 h interference) (P < 0.05). DC2.4 cells stimulated the proliferation of BALB/c mouse allo-genetic spleen cell. The mDC enhanced alloreactivity in MLR and the IFN-gamma secretion in supernatants. The T-DCs alleviated the allo-genetic spleen cell proliferation (P < 0.05) and IFN-gamma secretion in MLR (P < 0.05). Silence of the PIR-B expression (si-DC) also promoted of alloreactivity and enhanced the IFN-gamma secretion in MLR (P < 0.05).

CONCLUSIONHigh expression of immune inhibition receptor PIR-B is one of the general features and molecular mechanism of dendritic cells to acquire immune tolerance in mouse.

Animals ; Cell Line ; Dendritic Cells ; immunology ; metabolism ; Immune Tolerance ; Interleukin-10 ; pharmacology ; Membrane Glycoproteins ; genetics ; immunology ; metabolism ; Mice ; Mice, Inbred C57BL ; RNA, Small Interfering ; genetics ; Receptors, Immunologic ; genetics ; immunology ; metabolism ; Transfection ; Transforming Growth Factor beta1 ; pharmacology

The present study was designed to investigate the antimalarial activity of synthetic hepcidin and its effect on cytokine secretion in mice infected with Plasmodium berghei. The mice were infected with P. berghei intravenously and treated with hepcidin according to 4-day suppression test and Rane's test. The serum levels of interleukins (IL-1β, IL-2, IL-6, IL-10, IL-12p70, and IL-17A), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) in the experimental mice were determined using a cytometric bead array (CBA) kit. The survival rate of the infected mice was also registered. Additionally, the serum iron, alanine transaminase (ALT), aspartate transaminase (AST), and total bilirubin (BIL) were detected to evaluate liver functions. Hepcidin exerted direct anti-malarial function in vivo and increased survival rate in a dose-dependent manner. In addition, the secretion of T helper cell type 1 (Th1), Th2, and Th17 cytokines, TNF-α, and IFN-γ were inhibited by hepcidin. In conclusion, our results demonstrated that synthetic hepcidin exerts in vivo antimalarial activity and possesses anti-inflammatory function, which provides a basis for future design of new derivatives with ideal anti-malarial activity.
Animals ; Antimalarials ; chemical synthesis ; pharmacology ; Disease Models, Animal ; Drug Evaluation, Preclinical ; Hepcidins ; chemical synthesis ; pharmacology ; Humans ; Interleukin-10 ; immunology ; Interleukin-17 ; immunology ; Malaria ; drug therapy ; immunology ; mortality ; parasitology ; Male ; Mice ; Plasmodium berghei ; drug effects ; genetics ; metabolism

BACKGROUNDBAFF, the B cell activation factor, is a member of the tumor necrosis factor (TNF) ligand family that binds to BCMA, TACI, and BAFF-R. Previous studies have shown that members of the TNF family are detected in human placental trophoblast cells, but the expression patterns of BAFF involved in human decidua and the differential expression of BAFF between normal pregnancy and miscarriage are still incompletely documented or unknown. This study was designed to investigate the expression of BAFF and BAFF-R in the trophoblast and decidua of normal early pregnant women and recurrent spontaneous abortion (RSA) patients.

METHODSForty-five patients with RSA and 45 normal pregnant women were included in this study. By reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemical experiments, we explored the expression of BAFF and BAFF-R in the maternal-fetal interface of normal early pregnant women and RSA patients.

RESULTSAnalysis by RT-PCR and Western blotting revealed that BAFF was detected in both trophoblast and decidua of all the samples, and the expression level was higher in the tissues of normal early pregnant women (P<0.05) than that of recurrent spontaneous abortion patients under the same gestational weeks. Messages for BAFF-R were absent. Immunohistochemical experiments showed that expression of BAFF was cell-specific which was localized to villous cytotrophoblast and syncytiotrophoblast cells in trophoblast and to stromal cells in decidua. Whereas BAFF was prominent on the trophoblast and decidua of normal early pregnant women, it was decreased in the tissues of RSA patients.

CONCLUSIONSBAFF might steer maternal leukocytes away from a harmful immune response and toward a favorable one and play a potentially vital role for successful pregnancy.

Abortion, Habitual ; metabolism ; B-Cell Activating Factor ; analysis ; genetics ; physiology ; Decidua ; chemistry ; metabolism ; Female ; Humans ; Immunohistochemistry ; Interleukin-10 ; genetics ; Pregnancy ; RNA, Messenger ; analysis ; Th1 Cells ; immunology ; Th2 Cells ; immunology ; Trophoblasts ; chemistry ; metabolism

OBJECTIVETo prepare the live recombinant vaccine of attenuated Salmonella typhimurium SL3261 expressing Helicobacter pylori (H. pylori) B subunit (UreB) and to determine whether it could be used as an oral vaccine against H. pylori infection.

METHODSUsing genomic DNA of H. pylori Sydney strain (SSI) as template, the H. pylori UreB gene fragment was amplified by PCR and subcloned into the expression vector pTC01. The recombinant plasmid pTC01-UreB was then transferred into LB5000 to obtain modified forms, and further conversed into the attenuated Salmonella typhimurium SL3261 to obtain recombinant SL3261/pCT01-UreB as an oral immunization reagent, which was then used to orally immunize Balb/c mice twice at a three-week interval. Twelve weeks later, anti-UreB IgA antibodies in intestinal fluid and IgG antibodies in sera were determined by ELISA. The relating data in control groups (including body weight, gastric inflammation, etc.) were also collected.

RESULTSThe sequencing analysis showed that the UreB gene fragment amplified by PCR was consistent with the sequence of the H. pylori UreB gene. The restriction enzyme digestion revealed that the correct pTC01-UreB was obtained. SDS-PAGE and Western blot showed that a 61KD protein was expressed in SL3261/pTC01-UreB, which could be recognized by anti-H. pylori UreB antiserum and was absent in the control containing only Salmonella typhimurium SL3261 strain. The multiple oral immunization with SL3261/pTC01-UreB could significantly induce H. pylori specific mucosal IgA response as well as serum IgG responses. IFN-gamma and IL-10 levels were significantly increased in SL3261/pTC01-UreB group, and no obvious side effect and change in gastric inflammation were observed.

CONCLUSIONThe attenuated vaccine of Salmonella typhimurium expressing H. pylori UreB can be used as an oral vaccine against H. pylori infection.

Animals ; Antibodies, Bacterial ; blood ; Bacterial Vaccines ; administration & dosage ; immunology ; Female ; Gene Expression Regulation, Bacterial ; Gene Expression Regulation, Enzymologic ; Helicobacter Infections ; immunology ; prevention & control ; Helicobacter pylori ; enzymology ; genetics ; immunology ; Immunoglobulin G ; blood ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Mice ; Mice, Inbred BALB C ; Salmonella typhimurium ; genetics ; immunology ; metabolism ; Urease ; genetics ; immunology ; metabolism ; Vaccines, Attenuated ; genetics ; immunology ; Weight Loss

PURPOSE: High mobility group box 1 (HMGB1) plays a central role in the pathogenesis of sepsis and multiple organ dysfunction syndromes. We investigated the associations of a single nucleotide polymorphism (SNP; rs1045411) in HMGB1 with various clinical parameters, severity, and prognosis in patients with sepsis, severe sepsis, or septic shock. MATERIALS AND METHODS: We enrolled 212 adult patients followed for 28 days. All patients were genotyped for rs1045411, and the serum levels of HMGB1 and several cytokines were measured. RESULTS: The proportions of patients according to genotype were GG (71.2%), GA (26.4%), and AA (2.4%). Among patients with chronic lung disease comorbidity, patients with a variant A allele had higher positive blood culture rates and higher levels of various cytokines [interleukin (IL)-1beta, IL-6, IL-10, IL-17, and tumor necrosis factor-alpha] than those with the GG genotype. In the analysis of those with diabetes as a comorbidity, patients with a variant A allele had higher blood culture and Gram-negative culture rates than those with GG genotypes; these patients also had a higher levels of IL-17. In the analysis of those with sepsis caused by a respiratory tract infection, patients with a variant A allele had higher levels of IL-10 and IL-17 (all p<0.05). This polymorphism had no significant impact on patient survival. CONCLUSION: The variant A allele of rs1045411 appears to be associated with a more severe inflammatory response than the GG genotype under specific conditions.
Adult ; Aged ; Alleles ; Asian Continental Ancestry Group/genetics ; China/epidemiology ; Cytokines/*blood/*genetics ; Female ; Genotype ; HMGB1 Protein/blood/*genetics ; Humans ; Interleukin-10/genetics ; Interleukin-17/genetics ; Interleukin-6/blood ; Male ; Middle Aged ; Polymorphism, Genetic/*genetics ; Polymorphism, Single Nucleotide/*genetics ; Prognosis ; Republic of Korea ; Sepsis/immunology/*metabolism/mortality ; Shock, Septic/immunology/*metabolism/mortality ; Survival ; Tumor Necrosis Factor-alpha/genetics

OBJECTIVETo study the immunoregulatory effect of hepatitis B virus (HBV) e antigen (HBeAg) on peripheral blood monocytes (PBMCs).

METHODSPBMCs were isolated from patients with chronic hepatitis B (CHB; both HBeAg- and HBeAg+) and healthy controls, and cultured with recombinant HBeAg. The HBeAg-induced changes in expression of PD-1/PD-L1 were measured by flow cytometry of the cells and in secreted cytokines were measured by enzyme-linked immunosorbent assay of the supernatants. Comparisons between two groups were made by the independent-samples t-test; the relationship between PD-1/B7-H1 level and HBV DNA copy number was evaluated by Spearman's correlation analysis.

RESULTSExposure to HBeAg led to a significant decrease in CD3+CD4+ T lymphocyte-specific expression of IFNa for both the CHB patients' and healthy controls' samples (t = 2.382 and -4.190 respectively, P less than 0.01). For the HBeAg- CHB patients' and healthy controls' samples, the HBeAg exposure led to increased levels of secreted cytokines IL-6, IL-10 and TNFa (t = 2.504, 3.583 and 4.324, P less than 0.01 and t = 3.542, 6.246 and 5.273, P less than 0.01 respectively) and of CD14+ PBMC-specific expression of PD-L1 (t = 4.815 and 3.454, P less than 0.05 respectively). Compared to the HBeAg-negative CHB patients' and healthy controls' samples, the HBeAg+ CHB patients' samples had significantly lower CD3+CD4+ T cell-specific expression of IFNa (t = -3.177 and -4.541, P less than 0.01 respectively), but significantly higher levels of secreted IL-4 (t = 3.382 and 4.393, P less than 0.01 respectively), of CD3+ T cells-specific expression of PD-1/PD-L1 (t = 4.755, 2.942 and 4.518, 4.595, P less than 0.01 respectively), and of CD14+ T cells-specific expression of PD-L1 (t = 5.092 and 5.473, P less than 0.01 respectively). The CD3+ T cells-specific expression of PD-L1 was significantly higher in the samples from HBeAg- CHB patients than from the healthy controls (t = 3.214, P less than 0.01).

CONCLUSIONHBeAg was able to down-regulate the production of Th1-type cytokines (IFNgamma), and up-regulate the secretion of Th2-type cytokines (IL-6, IL-10) and the expression of PD-1/PD-L1on monocytes. These changes are conducive to the formation of immune tolerance to HBV. Therefore, HBeAg may play an important role in immune tolerance to chronic HBV infection.

Adult ; Case-Control Studies ; Cells, Cultured ; Female ; Hepatitis B e Antigens ; genetics ; immunology ; Hepatitis B, Chronic ; blood ; immunology ; Humans ; Interferon-gamma ; immunology ; Interleukin-10 ; immunology ; Interleukin-6 ; immunology ; Leukocytes, Mononuclear ; immunology ; metabolism ; Male ; Middle Aged ; Recombinant Proteins ; immunology ; Th1 Cells ; immunology ; Th1-Th2 Balance ; Th2 Cells ; immunology

OBJECTIVEIt is well known that vitamin A can improve mucosal immunity and anti-infection immunity. But the mechanisms thereof remain to be clarified. Previous studies on the role of vitamin A in immune regulation focused on lymphocytes, whereas little had been done about dendritic cells, which play very important roles in immune response. The objective of this study was to understand the effects of retinoic acid (RA), the metabolic product of vitamin A in vivo,on the differentiation, maturation and functions of dendritic cells from cord blood.

METHODSCord blood samples were collected from nine well-nourished full-term neonates. Mononuclear cells were isolated by Ficoll-Hypaque gradient centrifugation and cultured in the presence of 1000 u/ml GM-CSF, 500 u/ml IL-4 for 6 days, then TNF-alpha 20 ng/ml was added into the medium and cultured for another 3 days. The cells were incubated with or without 1 x 10(-6) MRA. Expression of surface molecules, CD1a, CD83, HLA-DR on DC was measured by flow cytometry. The ability of DC derived from the culture to induce proliferation of T cells in the mixed lymphocyte reaction (allo-MLR) was used for the evaluation of their function. IL-12, IFN-gamma, IL-4 and IL-10 were detected at mRNA levels by RT-PCR to understand the roles of DC treated with RA in regulation of Th1/Th2 balance.

RESULTSOn the sixth day of cell culture, the percentage of DC incubated with RA (57.28 +/- 9.22) was much lower than that without RA (79.57 +/- 11.85) (P < 0.001), but on the ninth day, there were no differences between the presence or absence of RA (76.18 +/- 10.27 vs. 73.72 +/- 15.58). When RA was added to the medium and the culture was continued for nine days, the percent of immature DC (CD1a + HLA-DR+) was much higher than that of the control (absence of RA) (58.93 +/- 4.70 vs. 45.80 +/- 7.88, t = 6.575, P < 0.001); whereas, mature DC (CD83 + HLA-DR+) percentage was markedly lower than that of the control (17.25 +/- 8.49 vs. 27.92 +/- 13.94, t = 4.435, P = 0.002). The T lymphocytes proliferation induced by the DC treated with RA was reduced from 16 857 +/- 3 643 to 11 924 +/- 2 576 cpm (t = 5.598, P < 0.001) in allo-MLR. Expression of mRNA for IL-12p35, IL-12p40, IFN-gamma in the cells that had been incubated with RA declined, but IL-10, IL-4 increased significantly.

CONCLUSIONVitamin A inhibited the differentiation and maturation of cord blood DC, reduced it's ability to stimulate allo-T lymphocytes proliferation, and down-regulated Th1 cytokines, up-regulated Th2 cytokines, consequently made immune response inclined to Th2.

Antigens, CD ; Antigens, CD1 ; analysis ; Cell Differentiation ; drug effects ; genetics ; immunology ; Cytokines ; genetics ; Dendritic Cells ; drug effects ; immunology ; metabolism ; Fetal Blood ; cytology ; immunology ; Flow Cytometry ; Gene Expression ; drug effects ; HLA-DR Antigens ; analysis ; Humans ; Immunoglobulins ; analysis ; Interferon-gamma ; genetics ; Interleukin-10 ; genetics ; Interleukin-12 ; genetics ; Interleukin-4 ; genetics ; Membrane Glycoproteins ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; Vitamin A ; pharmacology

OBJECTIVETo retrospectively analyze the quantity and status of the tumor infiltrating regulatory T lymphocytes in breast cancer and the draining lymph nodes, and to elucidate the clinical pathologic significance.

METHODSSeventy-four breast cancer samples with excised axillary lymph nodes were typed and staged histopathologically. The regulatory T lymphocytes were labeled by immunohistochemistry using EnVision method with the monoclonal antibodies against CD25 and Foxp3, and the immunophenotype was analyzed. In addition, the expression of IFN-γ, IL-10 and TGF-β1 mRNA in lymphocytes of lymph nodes draining the tumors was detected by in situ hybridization with the corresponding specific oligo nucleaic acid probes.

RESULTSThe number of CD25(+)Foxp3(+) T cells infiltrating the interstitium was much higher than that in the parenchymal tissue of the cancer. In the tumor draining lymph nodes, CD25(+) cells and Foxp3(+) cells were predominantly distributed in the paracortex with a proliferative pattern. TGF-β1, INF-γ and IL-10 mRNA positive cells showed a similar distribution pattern in the draining lymph nodes. Among the 39 cases with metastatic disease, the lymph nodes with metastases showed a much higher number of CD25(+)Foxp3(+) cells than that without metastases (23.5 vs 17.3 and 23.8 vs 15.5; P < 0.05). However, there was no difference in the density of Foxp3(+)CD25(+) cells in the draining lymph nodes between the death and survival groups (P > 0.05). Cytokine expression of TGF-β1, IL-10 and IFN-γ mRNA in the lymphocytes of draining lymph nodes in 24 cases showed that there were more IL-10 mRNA positive cells in the dead patients than that in the survived patients. A similar trend was observed for TGF-β1 mRNA positive cells but the difference was not statistically significant (P > 0.05). The expression rate of TGF-β1 and IL-10 mRNA in the draining lymph nodes was proportional to that of CD25(+) and Foxp3(+) cells (P < 0.05), and the expression of TGF-β1 positive cells was also proportional to that of IL-10 mRNA positive cells (P < 0.01). The expression of IFN-γ mRNA among these groups showed no significance (P > 0.05).

CONCLUSIONSRegulatory T cells may play important roles in inhibiting the host antitumor immunity, and the presence of increased regulatory T cells and Th2-secreting cells in paracortex with a proliferative pattern in the tumor draining lymph nodes implies that the paracortical proliferation of draining lymph nodes may not reflect positive antitumor effects.

Adult ; Aged ; Breast Neoplasms ; metabolism ; pathology ; surgery ; Female ; Follow-Up Studies ; Forkhead Transcription Factors ; metabolism ; Humans ; In Situ Hybridization ; Interferon-gamma ; genetics ; metabolism ; Interleukin-10 ; genetics ; metabolism ; Interleukin-2 Receptor alpha Subunit ; metabolism ; Lymph Nodes ; immunology ; metabolism ; Lymphatic Metastasis ; Middle Aged ; Neoplasm Staging ; RNA, Messenger ; metabolism ; Retrospective Studies ; Survival Rate ; T-Lymphocytes, Regulatory ; immunology ; metabolism ; Transforming Growth Factor beta1 ; genetics ; metabolism

OBJECTIVETo study the characteristics of host immune response against human gastric carcinoma of different histological types.

METHODSThe expression of 24 families of T cell receptor beta chain variable region (TCRVbeta) and cytokine profiles in isolated CD4(+) and CD8(+) subsets, as well as the cytokine profiles in purified epithelial cells from the tumor tissue and the residual benign tissue of patients with gastric carcinoma, was detected by a highly sensitive radioactivity labeled semi-quantitative RT-PCR technique.

RESULTSThe number of expanded T cell clones in CD8(+) subset from tumor tissue of the intestinal-type carcinoma was larger than that of diffuse-type (P = 0.046). The mRNA levels of IL-6, IL-8 in CD8(+) T subset, as well as the level of TNF-alpha in CD4(+) T subset from the tumor tissue of the diffuse type (0.61 +/- 0.29, 0.56 +/- 0.22, 0.09 +/- 0.03) were significantly higher than that from the residual benign tissue (0.14 +/- 0.05, 0.27 +/- 0.09, 0.04 +/- 0.02; P = 0.028, P = 0.043, P = 0.046). However, the mRNA level of IL-8 in CD8(+) subset and epithelial tumor cells of the intestinal-type (0.57 +/- 0.25, 0.27 +/- 0.07) was significantly higher than that from the residual benign tissue (0.21 +/- 0.07, 0.14 +/- 0.06; P = 0.028, P = 0.028).

CONCLUSIONSThe characteristics of host immune response against tumor are different between intestinal-type and diffuse-type gastric carcinoma. Both fewer expanded T cell clones and more suppressive cytokines suggest a more suppressive immune status in the local tumor lesion of diffuse-type than in the intestinal-type of the gastric carcinoma.

Adult ; Aged ; Aged, 80 and over ; CD4-Positive T-Lymphocytes ; metabolism ; pathology ; CD8-Positive T-Lymphocytes ; metabolism ; pathology ; Clone Cells ; Cytokines ; genetics ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Interferon-gamma ; genetics ; Interleukin-10 ; genetics ; Interleukin-2 ; genetics ; Interleukin-4 ; genetics ; Interleukin-6 ; genetics ; Interleukin-8 ; genetics ; Interleukins ; Male ; Middle Aged ; RNA, Messenger ; genetics ; metabolism ; Receptors, Antigen, T-Cell, alpha-beta ; genetics ; Stomach Neoplasms ; genetics ; immunology ; pathology ; T-Lymphocytes ; metabolism ; pathology ; Transforming Growth Factor beta ; genetics ; Tumor Necrosis Factor-alpha ; genetics