Effects of herbal cake separated moxibustion on Tim-4 and ubiquitination of PD-1 in rabbits with immunosuppression / 中国针灸

OBJECTIVE@#To observe the effects of herbal cake separated moxibustion on macrophage effector molecule T-cell immunoglobulin and mucin-domain containing-4 (Tim-4) and ubiquitination of programmed cell death protein 1 (PD-1) in rabbits with immunosuppression, and to explore the possible mechanism on herbal cake separated moxibustion in improving immunosuppression.@*METHODS@#Thirty-two big-ear white rabbits were randomly divided into a normal group, a model group, a moxa stick moxibustion group and a herbal cake separated moxibustion group, 8 rabbits in each group. Except the normal group, the immunosuppression model was established by intraperitoneal injection of cyclophosphamide of60 mg/kg in the other 3 groups. "Shenque" (CV 8), "Shenshu" (BL 23), "Zusanli" (ST 36), etc. were selected in both the moxa stick moxibustion group and the herbal cake separated moxibustion group. Moxa stick moxibustion was applied in the moxa stick moxibustion group, one cone at each acupoint; herbal cake separated moxibustion was applied in the herbal cake separated moxibustion group, 5 cones at each acupoint. The intervention was given once every other day for 10 times in both groups. Leukocyte content in peripheral blood was detected by blood cell analyzer; the positive expression of PD-1 in CD+4 T lymphocytes, CD+8T lymphocytes and CD+68 macrophages in peripheral blood was measured by flow cytometry, the serum levels of interleukin 2 (IL-2), CD8, CD68 and Tim-4 were detected by ELISA, and the expression of Tim-4 and F-box only protein 38 (FBXO38) in the liver and spleen tissues was measured by immunohistochemistry.@*RESULTS@#Compared with the normal group, in the model group, white blood cell count (WBC) and percentage of neutrophils (NEU%) were decreased while percentage of lymphocyte (LYM%) was increased (P<0.01) in peripheral blood; the positive expression rates of PD-1 in CD+4 T lymphocytes, CD+8T lymphocytes and CD+68 macrophages in peripheral blood were increased (P<0.01); the serum levels of IL-2, CD68 and Tim-4 were increased (P<0.01), the serum level of CD8 was decreased (P<0.01); the average optical density (AOD) of Tim-4 in the liver tissue and FBXO38 in the liver and spleen tissues was increased (P<0.01). Compared with the model group, in the moxa stick moxibustion group and the herbal cake separated moxibustion group, WBC and NEU% were increased (P<0.01); the positive expression rates of PD-1 in CD+4 T lymphocytes, CD+8T lymphocytes and CD+68 macrophages in peripheral blood were decreased (P<0.01); the serum levels of IL-2, CD68 and Tim-4 were decreased (P<0.01), the serum levels of CD8 were increased (P<0.01); the AOD of Tim-4 and FBXO38 in the liver tissue and FBXO38 in the spleen tissue was decreased (P<0.01, P<0.05). Compared with the moxa stick moxibustion group, in the herbal cake separated moxibustion group, the positive expression rate of PD-1 in CD+68 macrophages in peripheral blood was increased (P<0.05); serum level of Tim-4 was increased (P<0.01); AOD of Tim-4 in the liver tissue was decreased (P<0.05).@*CONCLUSION@#Herbal cake separated moxibustion can improve immunosuppression by regulating the expression of macrophage effector molecule Tim-4 and the FBXO38 mediated ubiquitination of PD-1, Tim-4 may be one of the specific indexes of immunomodulation involving with herbal cake separated moxibustion.

A Novel Recombinant BCG Vaccine Encoding Eimeria tenella Rhomboid and Chicken IL-2 Induces Protective Immunity Against Coccidiosis

A novel recombinant Bacille Calmette-Guerin (rBCG) vaccine co-expressed Eimeria tenella rhomboid and cytokine chicken IL-2 (chIL-2) was constructed, and its efficacy against E. tenella challenge was observed. The rhomboid gene of E. tenella and chIL-2 gene were subcloned into integrative expression vector pMV361, producing vaccines rBCG pMV361-rho and pMV361-rho-IL2. Animal experiment via intranasal and subcutaneous route in chickens was carried out to evaluate the immune efficacy of the vaccines. The results indicated that these rBCG vaccines could obviously alleviate cacal lesions and oocyst output. Intranasal immunization with pMV361-rho and pMV361-rho-IL2 elicited better protective immunity against E. tenella than subcutaneous immunization. Splenocytes from chickens immunized with either rBCG pMV361-rho and pMV361-rho-IL2 had increased CD4+ and CD8+ cell production. Our data indicate recombinant BCG is able to impart partial protection against E. tenella challenge and co-expression of cytokine with antigen was an effective strategy to improve vaccine immunity.

Positive effects of porcine IL-2 and IL-4 on virus-specific immune responses induced by the porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 DNA vaccine in swine

The purpose of this study was to investigate the effects of porcine interleukin (IL)-2 and IL-4 genes on enhancing the immunogenicity of a porcine reproductive and respiratory syndrome virus ORF5 DNA vaccine in piglets. Eukaryotic expression plasmids pcDNA-ORF5, pcDNA-IL-2, and pcDNA-IL-4 were constructed and then expressed in Marc-145 cells. The effects of these genes were detected using an indirect immunofluorescent assay and reverse transcription polymerase chain reaction (RT-PCR). Characteristic fluorescence was observed at different times after pcDNA-ORF5 was expressed in the Marc-145 cells, and PCR products corresponding to ORF5, IL-2, and IL-4 genes were detected at 48 h. Based on these data, healthy piglets were injected intramuscularly with different combinations of the purified plasmids: pcDNA-ORF5 alone, pcDNA-ORF5 + pcDNA-IL-2, pcDNA-ORF5 + pcDNA-IL-4, and pcDNA-ORF5 + pcDNAIL-4 + pcDNA-IL-2. The ensuing humoral immune responses, percentages of CD4+ and CD8+ T lymphocytes, proliferation indices, and interferon-gamma expression were analyzed. Results revealed that the piglets co-immunized with pcDNA-ORF5 + pcDNA-IL-4 + pcDNA-IL-2 plasmids developed significantly higher antibody titers and neutralizing antibody levels, had significantly increased levels of specific T lymphocyte proliferation, elevated percentages of CD4+ and CD8+ T lymphocytes, and significantly higher IFN-gamma production than the other inoculated pigs (p < 0.05).

Antitumor immunity induced by tumor cells engineered to express a membrane-bound form of IL-2

Transduction of cytokine gene into tumor cells is a promising method of tumor therapy, but the value is limited by accompanying side effects. To focus antitumor immune response to tumor antigen-specific CTL, we developed an antitumor vaccine by transfecting modified IL-2 gene in a membrane-bound form (mbIL-2) into B16F10 melanoma cells. The mbIL-2 clone showed reduced tumorigenicity and metastatic ability, and inhibited metastasis and prolonged the survival of mice against B16F10 cells. The inhibition of B16F10 metastasis by mbIL-2 was accompanied by the increment of CD8+ T cells. The metastasis of mbIL-2 clone was significantly increased in the CD8+ T cell-depleted mice, but not in CD4+ T cell depleted mice. Spleen cells immunized with the mbIL-2 clone showed higher CTL activity towards B16F10 cells than those immunized with control cells. The size of CD8+ T cell population in the lung of mice injected with the mbIL-2 clone was markedly greater than that of mice injected with B16F10 cells, but there was no detectible change in CD4+ and CD8+ T cell populations of lymph nodes and spleen. These results suggest that when the mbIL-2 clone is introduced into the blood stream, it migrates mainly to lung and activates CD8+ T cells in situ, possibly by direct priming. Such a tumor vaccine may ameliorate the toxic side effects encountered with conventional cytokine gene therapy.

Retroviral-mediated IL-2 gene transfer into murine neuroblastoma

We used retroviral-mediated gene transfer of the human interleukin (IL)-2 gene into murine neuroblastoma cells to investigate whether locally-secreted IL-2 is able to influence the generation of anti-tumor immune responses. Supernatant obtained from cultures of approximately 1 x 10(6) IL-2 gene-transduced, G-418 selected neuro-2a cells was assayed for human IL-2 production by ELISA kit. First, to estimate whether the local secretion of IL-2 from the genetically-modified tumor cells would affect their tumorigenicity in vivo, IL-2-secreting neuro-2a cells were s.c. injected into A/J mice and tumor growth was measured weekly. And to estimate whether IL-2 transfected neuroblastoma cells protect mice from tumor development after wild-type tumor cell challenge, IL-2-secreting neuro-2a cells were s.c. injected into A/J mice. Seven days after IL-2 gene-transfected neuroblastoma cell injection, unmodified neuro-2a cells were s.c. injected into the contralateral site of A/J mice and tumor growth was measured weekly. Finally, to estimate IL-2 effect on pre-established large tumor burdens, IL-2-secreting neuro-2a cells were s.c. injected into A/J mice with established tumor and its growth was measured weekly. The IL-2 gene-transduced neuro-2a clones secreted 120.25-177.3 IU of IL-2 per ml per 10(6) cells during 24 hr. None of the mice injected with IL-2-secreting neuro-2a cells developed tumors within 6 weeks, while all of the mice injected with wild-type neuro-2a cells developed tumors. Immunization of mice with IL-2 gene-transfected, irradiated neuro-2a cells protected these animals against a subsequent challenge with wild-type tumor cells. Finally, the size of large neuroblastomas decreased after IL-2-secreting neuro-2a cell injection into mice. Local secretion of IL-2 gene-transduced tumor cells abrogates their tumorigenicity and induces protective immunity and may inhibit the growth of neuroblastoma.