Improved antitumor efficacy of neutrophils stimulated by bacillus Calmette­Guérin.

Bacillus Calmette­Guérin (BCG) has become a significant treatment for bladder cancer, and neutrophils are reported to be associated with the antitumor effect of BCG. The aim of the present study was to clarify the antitumor function of neutrophils stimulated by BCG. Initially, the killing effect and cytotoxic activity of neutrophils treated with BCG was detected. Subsequently, the effectiveness of BCG­treated neutrophils extracted from tumor­bearing mice was analyzed. The results revealed that the cytotoxic effect of neutrophils was stronger in the BCG­treated group compared with that in the normal saline (NS)­treated and control groups (P<0.05). A significantly higher concentration of cytokines tumor necrosis factor (TNF)­α, interleukin (IL)­1ß, IL­6 and TNF­related apoptosis­inducing ligand occurred in the BCG­treated neutrophil group compared with the NS and control groups (P<0.01), which was also associated with the BCG dose (P<0.01). The gross tumor volume percentage in BCG­treated neutrophils from tumor­bearing mice (BCGT group) was significantly lower in comparison with that in the NS­treated neutrophils from tumor­bearing mice (NST group; P<0.05). In addition, the survival rate of tumor­bearing mice was higher in the BCGT group compared with the NST group (P<0.05), while more BCG­treated neutrophils from tumor­bearing mice were infiltrated in the MethA tumor (P<0.01). In conclusion, BCG­treated neutrophils were observed to enhance the antitumor efficacy and extend the life span of mice.

Pioglitazone Improves Mitochondrial Function in the Remnant Kidney and Protects against Renal Fibrosis in 5/6 Nephrectomized Rats.

Pioglitazone is a type of peroxisome proliferator-activated receptor γ (PPARγ) agonist and has been demonstrated to be effective in chronic kidney diseases (CKD) treatment. However, the underlying mechanism involved in the renoprotection of pioglitazone has not been fully revealed. In the present study, the renoprotective mechanism of pioglitazone was investigated in 5/6 nephrectomized (Nx) rats and TGF-ß1-exposed HK-2 cells. Pioglitazone attenuated renal injury and improved renal function, as examined by 24 h urinary protein, blood urea nitrogen and plasma creatinine in Nx rats. Renal fibrosis and enhanced expressions of profibrotic proteins TGF-ß1, fibronectin and collagen I caused by Nx were significantly alleviated by pioglitazone. In addition, pioglitazone protected mitochondrial functions by stabilizing the mitochondrial membrane potential, inhibiting ROS generation, maintaining ATP production and the activities of complexes I and III, and preventing cytochrome C leakage from mitochondria. Pioglitazone also upregulated the expression levels of ATP synthase ß, COX I and NDUFB8, which were downregulated in the kidney of Nx rats and TGF-ß1-exposed HK-2 cells. Furthermore, pioglitazone increased fusion proteins Opa-1 and Mfn2 expressions and decreased fission protein Drp1 expression. The results imply that pioglitazone may exert the renoprotective effects through modulating mitochondrial electron transport chain and mitochondrial dynamics in CKD. Finally, these recoveries were completely or partly inhibited by GW9662, which suggests that these effects at least partly PPARγ dependent. This study provides evidence for the pharmacological mechanism of pioglitazone in the treatment of CKD.

Analysis of murine and human Treg subsets in inflammatory bowel disease.

Previous studies have indicated that regulatory T cells serve essential roles in maintaining intestinal homeostasis, however, the role of different Treg subsets in modulating inflammatory bowel disease has still not been addressed clearly. In the present study, the authors measured the percentage of Foxp3+ IL­10+ TGF­ß+ natural Tregs, Foxp3­ IL­10+ TGF­ß­ induced Tregs, CD127­ induced Tregs and CD8+ Tregs at different time points in DSS­induced experimental colitis model in murine lamina propria lymphocytes, mesenteric lymph node and peripheral blood. In addition, the authors compared the frequency of four Treg subsets in patients diagnosed of ulcerative colitis at different stages with enrolled healthy controls. The percentage of Foxp3+ IL­10+ TGF­ß+ natural Tregs decreased in acute stage of both human and mice was observed, but proliferated significantly during remittent stage. Foxp3­ IL­10+ TGF­ß­ inducible (i) Treg and CD127­ iTreg was observed as being significantly decreased percentage in LPL at 4 and 7 days, the frequency of Foxp3­ IL­10+ TGF­ß­ iTreg cells became decreased and CD127­ iTreg only slightly increased at the chronic stage following DSS induction. However, the proportion of both Foxp3­ IL­10+ TGF­ß­ iTreg and CD127­ iTreg was nearly unchanged in human IBD. Although intestinal inflammation decreased the percentage of CD8+ Tregs, it remained lower in the remittent stage of human IBD. Only enhanced proliferation of lamina propria lymphocytes­derived CD8+ Treg was reported at 7 days in dextran sodium sulfate­induced murine colitis. The results demonstrated that Foxp3+ IL­10+ TGF­ß+ natural Tregs may serve an essential role in exhibiting suppressive and protecting from immune­related mucosal injury during chronic stage in inflammatory bowel disease.

Sodium selenite ameliorates dextran sulfate sodium-induced chronic colitis in mice by decreasing Th1, Th17, and γδT and increasing CD4(+)CD25(+) regulatory T-cell responses.

AIM: To assess the effect of sodium selenite on the severity of dextran sulfate sodium (DSS)-induced colitis in C57BL/6 mice. METHODS: Mice were randomly divided into four groups (n = 10/group): normal group, selenium (Se) group, chronic colitis group, and Se + chronic colitis group. The mice were sacrificed on day 26. Survival rates, clinical symptoms, colon length, and histological changes were determined. The percentages and absolute numbers of immune system cells in the lamina propria lymphocytes (LPL) of the colon, the expression of mRNA in colon tissue, and the concentrations of Th1, Th17, and Treg cytokines in LPL from the large intestine, were measured. RESULTS: Se significantly ameliorated the symptoms of colitis and histological injury (P < 0.05 each), increasing the proportions of neutrophils and CD4+ CD25+ T cells (P < 0.05 each) and decreasing the proportions of γδT cells, CD4+, CD4+CD44+, and CD4+ CD69+ T cells in LPL (P < 0.05 each). Moreover, Se reduced the expression of IL-6, IFN-γ, IL-17A, IL-21, T-bet, and RORγt (P < 0.05 each), but enhanced the expression of IL-10 and Foxp3 (P < 0.05 each). CONCLUSION: These results suggest that Se protects against DSS-induced chronic colitis perhaps by increasing the number of CD4(+)CD25(+) Tregs that suppress the secretion of proinflammatory cytokines and populations of Th1, Th17, and γδT cells.

Th1/Th2 Balance and Th17/Treg-Mediated Immunity in relation to Murine Resistance to Dextran Sulfate-Induced Colitis.

BACKGROUND: The role of the Th17/Treg balance in the development of experimental colitis remains poorly understood. METHODS: We exploited the differential response of BALB/c mice and C57BL/6 mice towards drinking water mediated by dextran sulfate sodium (DSS) challenge. RESULTS: DSS-resistant BALB/c mice were characterized by low levels of IFN-γ and TNF-α but high levels of IL-4, IL-6, IL-10, IL-17A, IL-17F, and colon lamina propria and mesenteric lymph node (MLN) CD4+CD25+FoxP3+ T cells when compared to C57BL/6 mice. Collectively, these data indicate the propensity of BALB/c mice towards a Th2/Th17/Treg-polarized immunity protecting these animals against DSS challenge, whereas Th1-polarization of C57BL/6 mice confers sensitivity to DSS-induced colitis. CONCLUSIONS: The intrinsic congenital capacity of mouse strains with respect to T cell proliferation determines sensitivity to experimental colitis.

Novel adjuvant for immunization against tuberculosis: DNA vaccine expressing Mycobacterium tuberculosis antigen 85A and interleukin-15 fusion product elicits strong immune responses in mice.

OBJECTIVES: To investigate the potential of interleukin (IL)-15 as a novel adjuvant for Mycobacterium tuberculosis (Mtb) antigen 85A (Ag85A) vaccine. RESULTS: C57BL/6 mice were intramuscularly immunized three times with a plasmid expressing the Ag85A-IL-15 fusion protein (pcDNA3.1-Ag85A-IL-15), with the empty pcDNA3.1 vector and the pcDNA3.1-Ag85A as control. Mice vaccinated with pcDNA3.1-Ag85A-IL-15 generated more secretory IgA (sIgA) into their lung (209 ± 21 µg/ml) and acquired an enhanced serum IgG response to Ag85A. IgG2a/IgG1 ratios were upregulated, natural killer cell activity was augmented and Ag85A-specific splenic T cell proliferation was enhanced in these mice as well. Vaccination with pcDNA3.1-Ag85A-IL-15 promoted the polarization of CD4+ T cells towards a Th1 type in the spleen, and significantly upregulated the serum level of interferon (IFN)-γ (458 ± 98 pg/ml), a typical Th1 cytokine. IFN-γ-expressing CD8+ cells were also increased in the spleen after pcDNA3.1-Ag85A-IL-15 immunization. CONCLUSIONS: A superior immune type I response in mice vaccinated with plasmid Ag85A-IL-15 has been achieved.

Dextran sulfate sodium-induced acute experimental colitis in C57BL/6 mice is mitigated by selenium.

BACKGROUND: Sodium selenite has been shown to have a protective role in experimental colitis. Th1 and Th17 responses are involved in the pathogenesis of dextran sulfate sodium (DSS)-induced colitis and inflammatory bowel disease. This study investigated whether sodium selenite can suppress Th1/Th17-mediated experimental colitis. METHODS: Mice were administered sodium selenite (2µg/g body weight) by gavage daily for 30days. Beginning on day 21, mice were administered 2.5% oral DSS for 9days. The mice were sacrificed on day 31. Survival rates, clinical symptoms, colon lengths, and histological changes were determined. RESULTS: Pretreatment with sodium selenite (2µg/g body weight) improved survival rates, colon shortening, body weight loss, disease activity index, and histopathological score in mice with DSS-induced colitis. Pretreatment with sodium selenite restored interleukin-10 and Foxp3 excretion, as well as reducing the levels of interferon-γ and interleukin-17A. CONCLUSIONS: Pretreatment with sodium selenite showed therapeutic potential for preventing colitis in mice. This effect may be mediated by the immunomodulation of regulatory T cells, expressing anti-inflammatory genes that suppress Th1 and Th17 responses.

Pioglitazone, a Peroxisome Proliferator-Activated Receptor x03B3; Agonist, Ameliorates Chronic Kidney Disease by Enhancing Antioxidative Capacity and Attenuating Angiogenesis in the Kidney of a 5/6 Nephrectomized Rat Model.

BACKGROUND/AIMS: Pioglitazone is a type of peroxisome proliferator-activated receptor x03B3; agonist and is capable of alleviating renal ischemia-reperfusion injury. METHODS: A5/6 nephrectomized rat model was established to induce renal impairments mimicking chronic kidney diseases (CKDs). The effect of pioglitazone on renal structure, function, antioxidative capacity, and angiogenesis in the nephrectomized rats was assessed. Moreover, the expression of HIF-1α, eNOS, VEGF, Flt-1 and Flk-1 was determined to reveal the possible pathways through which pioglitazone exerted its beneficial effect on CKDs. RESULTS: Subtotal nephrectomy caused severe damages to rat renal tissues, and administration of pioglitazone dramatically restored the structure and function of the kidney, which was evidenced by Periodic acid- Schiff staining and the reduced levels of urinary proteins, blood urea nitrogen, and creatinine. Furthermore, pioglitazone decreased the level of malondialdehyde and increased the level of superoxide dismutase in the injured renal tissues, suggesting that the antioxidative capacity in the injured kidney was augmented by pioglitazone. Additionally, pioglitazone inhibited HIF-1α-dependent angiogenesis by down-regulating the expression of a panel of angiogenic factors. CONCLUSION: The current study demonstrates that pioglitazone benefits renal failure through activation of the antioxidative system and inhibition of angiogenesis in the injured kidney. Our study provides preliminary evidences for the potential application of this agent in the treatment of CKDs.

IL-33 alleviates DSS-induced chronic colitis in C57BL/6 mice colon lamina propria by suppressing Th17 cell response as well as Th1 cell response.

Interleukin (IL)-33, a member of the IL-1 cytokine family, is associated with autoimmune diseases including inflammatory bowel diseases (IBD). A few studies on animal models have shown that IL-33 can suppress Th1 cell response and improve Th2 cell response in mesenteric lymph nodes (MLN) and sera. However, there is little data published about the effect of IL-33 on Th17 cell in and Th1/Th2 cell in colon lamina propria. The aim of this study was to investigate the effect of IL-33 on Th17 cell in colon lamina propria of mice with dextran sulfate sodium (DSS) induced chronic colitis. We studied the influence of IL-33 on colonic tissue injury and clinical symptoms of colitis. The T cell subsets were measured by flow cytometry and the production of cytokines secreted by lamina propria lymphocytes (LPL) was measured by Enzyme-Linked Immunosorbent Assay (ELISA) and quantitative real-time PCR. We have found that rIL-33 treatment led to a significant alleviation of DSS induced chronic colitis as evidenced by 1) alleviation of weight loss, DAI, macroscopic changes and histological score; 2) down-regulating the rates and absolute cell numbers of Th17 and Th1 cell in LPL; 3) inducing secretion of lower levels of IFN-γ and IL-17A. It is therefore concluded that IL-33 may play a therapeutic role in DSS-induced chronic colitis in mice by suppressing Th17 response and switching Th1 to Th2 response.

Enhanced therapeutic effects against murine colon carcinoma induced by a Colon 26/Ag85A-CD226 tumor cell vaccine.

Genetically modified tumor cells represent one of the most effective cancer vaccine strategies. In the present study, we describe our approach for inducing an immune response against a colon carcinoma in BALB/c mice, using a Colon 26 tumor cell line expressing Ag85A and CD226. We investigated whether CD226 plays a promotive role for Ag85A against Colon 26 colon carcinoma. The therapeutic efficacy was investigated. The cytotoxic T lymphocyte (CTL) and natural killer (NK) cell cytotoxicity were assessed. Dynamic changes in interferon (IFN)-γ levels in the spleen and the number of IFN-γ-producing CD4+ or CD8+ T cells in the spleen or mesenteric lymph nodes were detected by enzyme-linked immunoabsorbent assay or flow cytometry. Extended survival times, delayed appearances of tumors, and reduced tumor volumes were achieved by preventive vaccination with the Colon 26/Ag85A-CD226 tumor cell vaccine. NK cell or CTL cytotoxicity in the spleens of mice immunized with the Colon 26/Ag85A-CD226 tumor cell vaccine was significantly higher than that in the other treatment groups. The numbers of CD4+ IFN-γ+ and CD8+ IFN-γ+ T cells were both significantly increased in mice immunized with the Colon 26/Ag85A-CD226 tumor cell vaccine in both the spleen and mesenteric lymph nodes. Our results indicated that the tumor vaccine expressing Ag85A and CD226 induced more intensive antitumor immunity than tumor vaccine expressing Ag85A or CD226 only. Moreover, the results suggest that Ag85A and CD226 play a synergistic antitumor effect and CD226 could be used as a genetic adjuvant to enhance the effects of Ag85A vaccine against murine colon carcinoma.

CD226 as a genetic adjuvant to enhance immune efficacy induced by Ag85A DNA vaccination.

Antigen-85A (Ag85A) is one of the major proteins secreted by Mycobacterium tuberculosis. Many studies on animal models have shown that vaccination with the recombinant Ag85A-DNA or Ag85A protein induces powerful immune response. However, these vaccines cannot generate sufficient protective immunity in the systemic compartment. CD226, a member of the immunoglobulin superfamily, is expressed in the majority of NK cells, T cells, monocytes, and platelets, and can be served as a co-stimulator that contributes to multiple innate and adaptive responses. However, there has been no study where either CD226 protein or DNA has been used as an adjuvant for vaccine development. The aim of this study was to develop a novel Ag85A DNA vaccine with CD226 as the genetic adjuvant to increase the immune efficacy induced by Ag85A. Oral vaccination with pcDNA3.1-Ag85A-CD226 DNA induced potent immune responses in mice. CD226 was an effective genetic adjuvant that improved the immune efficacy induced by Ag85A and enhanced the activity of cytotoxic T lymphocytes (CTL) and NK cells in mice. Th1 dominant cytokines (i.e. IL-2, IFN-γ and TNF-α), cellular immunity (i.e. CD4(+)IFN-γ(+)T cells and CD8(+)IFN-γ(+)T cells in splenocytes) and MLNs were also significantly elevated by pcDNA3.1-Ag85A-CD226 DNA vaccination. Our results suggest that CD226 is an effective adjuvant to enhance the immune efficacy induced by Ag85A. Our findings provide a new strategy for the development of a DNA vaccine co-expressing Ag85A and CD226.

Dendritic cell vaccine modified by Ag85A gene enhances anti-tumor immunity against bladder cancer.

The ability of dendritic cells to provide all the signals required for T-cell activation makes them an ideal cancer vaccine platform. With the use of established DC2.4 cell line, originated from C57BL/6 mice and developed by superinfecting GM-CSF transduced bone marrow cells with myc and raf oncogenes, we investigated whether the DC 2.4 cell line transfected with Ag85A gene could enhance immunity against bladder cancer. Both phenotypic and functional analyses of Ag85A-DCs were done with use of FCM and T cell proliferation test. The cytotoxicity of Ag85A-DCs loaded with tumor cell lysate was verified by LDH. Finally, the production of interferon gamma was assayed by both ELISA and FCM. The immunotherapeutic effect of DC vaccine on murine bladder cancer was assessed pharmacologically and pathologically. Our results showed that Ag85A gene transfected DCs expressed high levels of key surface markers such as CD80, CD86 and MHC-II. The CTL primed with MB49 lysate-pulsed Ag85A-DCs elicits higher activity against MB49 tumor cells and upregulated level of IFN-γ production. Furthermore, the significant inhibitive effect on tumor growth in mice was found in the group of Ag85A-DC vaccine. The infiltration of CD4(+) or CD8(+) T cell within established tumor treated by Ag85A-DC vaccine significantly increased as compared with control groups. It is therefore concluded that DCs engineered by Ag85A gene exerts enhanced anti-tumor immunity against bladder cancer and this study might provide a meaningful mode of action with the use of Ag85A engineered DC vaccination in anti-cancer immunotherapy.

Comparison of stimulating effect on subpopulations of lymphocytes in human peripheral blood by methionine enkephalin with IL-2 and IFN-γ.

The aim of this study was to investigate the effects of mechanisms of methionine enkephalin (MENK) on lymphocytes in human peripheral blood. We detected CD4+T cells, CD8+T cells, CD4+CD25+ regulatory T cells (Treg), dendritic cells (DCs), natural killer cells (NK), NKT cells and γδT cells before and after treatment with 10 (-12) M MENK, in cell culture by FCM and RT-PCR. Our findings show that MENK stimulating expansion of lymphocyte subpopulationns by inhibiting CD4+CD25+ regulatory T cells (Treg), which is unique discovery of our study. We may use MENK as a drug to treat cancer patients, whose immune systems are damaged by chemotherapy or radiotherapy.

Identification of the risk for liver fibrosis on CHB patients using an artificial neural network based on routine and serum markers.

BACKGROUND: Liver fibrosis progression is commonly found in patients with CHB. Liver biopsy is a gold standard for identifying the extent of liver fibrosis, but has many draw-backs. It is essential to construct a noninvasive model to predict the levels of risk for liver fibrosis. It would provide very useful information to help reduce the number of liver biopsies of CHB patients. METHODS: 339 chronic hepatitis B patients with HBsAg-positive were investigated retrospectively, and divided at random into 2 subsets with twice as many patients in the training set as in the validation set; 116 additional patients were consequently enrolled in the study as the testing set. A three-layer artificial neural network was developed using a Bayesian learning algorithm. Sensitivity and ROC analysis were performed to explain the importance of input variables and the performance of the neural network. RESULTS: There were 329 patients without significant fibrosis and 126 with significant fibrosis in the study. All markers except gender, HB, ALP and TP were found to be statistically significant factors associated with significant fibrosis. The sensitivity analysis showed that the most important factors in the predictive model were age, AST, platelet, and GGT, and the influence on the output variable among coal miners were 22.3-24.6%. The AUROC in 3 sets was 0.883, 0.884, and 0.920. In the testing set, for a decision threshold of 0.33, sensitivity and negative predictive values were 100% and all CHB patients with significant fibrosis would be identified. CONCLUSIONS: The artificial neural network model based on routine and serum markers would predict the risk for liver fibrosis with a high accuracy. 47.4% of CHB patients at a decision threshold of 0.33 would be free of liver biopsy and wouldn't be missed.

Liposomal oral DNA vaccine (mycobacterium DNA) elicits immune response.

Tuberculosis is one of the leading causes of mortality from an infectious disease worldwide, however, the efficacy of BCG vaccine against adult pulmonary tuberculosis still remains instability. Therefore, it is an urgent work to develop both safe and effective vaccine to TB. To clarify the liposome encapsulated DNA vaccine was effectively working as a vaccine delivery system to evoke mucosal intestinal immune responses. A Mycobacterium pcDNA3.1(+)/Ag85A DNA was constructed and encapsulated into liposome. Ag85A protein antigen was observed to substantially express in the epithelium, microfold cells (M cells), dendritic cells (DCs) and Peyer's patches (pp) of the small intestine, respectively, after oral administration into C57BL/6 mice 3 times each 14 days interval. Furthermore, levels of IL-2 and IFN-gamma in the IELs isolated from the small intestine were markedly increased, IL-4 level was not significantly changed as compared to those in control group after oral administration of Ag85A DNA encapsulated in liposome, together with the augmented Ag85A-specific cytotoxicity of IELs, indicating a local Th1 dominant cellular immune response was elicited, and thus enhanced cytotoxicity of IELs as compared to those in control mice. Furthermore, sIgA level was also elevated in liposomal encapsulated Ag85A DNA immunized mice. These data indicated that oral vaccination with the liposomal-pcDNA 3.1(+)/Ag85A DNA is able to induce antigen specific mucosal cellular and humoral immune responses. Especially, cellular compartment in the epithelium of small intestine plays a key role on the regulation of immune response to eliminate TB. These findings have important understanding and possible implications for the design of new strategies based on oral DNA vaccine on regulation of immune response in protection against TB. Further study is clearly necessary to improve the effectiveness of Ag85A DNA vaccines against TB as compared with BCG.

Inhibitory effect of recombinant IL-25 on the development of dextran sulfate sodium-induced experimental colitis in mice.

The role of interleukin 25 (IL-25) in a number of human diseases still has not been extensively studied, here we attempt to evaluate the role of recombinant IL-25 (rIL-25) in the development of dextran sulfate sodium (DSS)-induced experimental colitis. Acute colitis was induced in female C57BL/6 mice by oral administration of 2.5% DSS in drinking water ad libitum. At the same time as the start of DSS exposure, mice were injected intraperitoneally with 0.4 microg of rIL-25 or PBS. Then disease activity index (DAI), histological changes and survival rate were observed. The levels of IL-17, IL-23, and TGF-beta1 in colon tissues were determined by ELISA, and the production of IL-17 by CD4(+)/CD8(+) T cells was detected by intracellular flow cytometry. In contrast to the DSS treated mice, DSS + rIL-25 treated mice displayed a lower DAI, limited histological changes and prolonged survival. The levels of IL-23 and TGF-beta1 were significantly elevated in the DSS + rIL-25 treated mice compared to the DSS treated mice. There was no significant difference in the production of IL-17 in colon tissues and CD4(+)/CD8(+) T cells between the DSS + rIL-25 treated mice and DSS treated mice. Our findings suggest the role of IL-25 in inhibiting development and progression of acute colitis in DSS-induced mouse colitis model.