Using Tree Shrews (Tupaia belangeri) as a Novel Animal Model of Liver Transplantation.

Liver transplantation (LT) is most effective and promising approach for end-stage liver disease. However, there remains room for further improvement and innovation, for example, to reduce ischemic reperfusion injury, transplant rejection and immune tolerance. A good animal model of LT is essential for such innovation in transplant research. Although rat LT model has been used since the last century, it has never been an ideal model because the results observed in rat may not be applied to human because these two species are genetically distinct from each other. In this study, we for the first time performed LT using the tree shrew (Tupaia belangeri), a species in the Order Scandentia which is closely related with primates, and evaluated the possibility to adopt this species as a new model of LT. We performed LT on 30 animals using the two-cuff technique, examining the success rate, the survival rate and the immunological reaction. The recipient operation time was 60 min averagely, and we limited the time of the anhepatic phase within 20 min. Twenty-seven (90%) of the animals survived for at least 3 days after the transplantation. Thirteen animals that did not receive any immunosuppressive drug died in 8 days mostly because of acute rejection effect (n=9), similar to the reaction in human but not in experimental rat. The rest 14 animals that were given rapamycin survived significantly longer (38 days) and half of them survived for 60 days until the end of the study. Our results suggest that performing LT in tree shrews can yield high success rate and high survival rate. More importantly, the tree shrews share similar immunological reaction with human. In addition, previous genomics study found that the tree shrews share more proteins with human. In sum, the tree shrews may outperform the experimental rats and could be used as a better and cost-effective animal model for LT.

Upregulation of B7-H4 promotes tumor progression of intrahepatic cholangiocarcinoma.

Recent reports show that B7-H4 is highly expressed in a variety of tumor cells, functions as a negative regulator of T cells and then promotes tumor progression. However, its expression and role in intrahepatic cholangiocarcinoma (ICC) remain unclear. In present study, B7-H4 expression in ICC and peritumoral tissues was determined at the level of mRNA and protein, and its bioactivity in ICC cells was studied after modification of B7-H4 expression. Then, the mechanism related to tumor progression induced by B7-H4 expression in ICC cells was explored. Finally, clinical significance of B7-H4 expression in ICC patients was further analyzed. The results showed that B7-H4 expression in ICC was much higher than that in peritumoral tissues at the level of both mRNA and protein. The high level of B7-H4 in ICC cells induced epithelial-to-mesenchymal transitions and promoted invasion and metastasis of tumor cells through activation of ERK1/2 signaling. The elevated B7-H4 expression was associated with the downregulated Bax, upregulated Bcl-2 expression, and activation of caspase-3. Clinically, high B7-H4 expression in tumor samples was significantly related to malignant phenotype, such as lymph node metastasis, high tumor stage, and poor differentiation. ICC patients with high expression of B7-H4 had shorter overall survival (OS) and disease-free survival. Moreover, the B7-H4 expression was an independent prognostic factor for predicting OS and tumor recurrence of ICC patients after operation. In conclusion, high expression of B7-H4 promotes tumor progression of ICC and may be a novel therapeutic target for ICC patients.

A tolerogenic semimature dendritic cells induce effector T-cell hyporesponsiveness by activation of antigen-specific CD4+CD25+ T regulatory cells that promotes skin allograft survival in mice.

Semimature dendritic cells (smDCs) can induce autoimmune tolerance by activation of host antigen-specific CD4(+)CD25(+) regulatory T (Treg) cells. We hypothesized that donor smDCs injected into recipients would induce effector T-cell hyporesponsiveness by activating CD4(+)CD25(+)Treg cells, and promote skin allograft survival. Myeloid smDCs were derived from C57BL/6J mice (donors) in vitro. BALB/c mice (recipients) were injected with smDCs to generate antigen-specific CD4(+)CD25(+)Treg cells in vivo. Allograft survival was prolonged when BALB/c recipients received either C57BL/6J smDCs prior to grafting or C57BL/6J smDC-derived CD4(+)CD25(+)Treg cells post-grafting, and skin flaps from these grafts showed the highest IL-10 production regardless of rapamycin treatments. Our findings confirm that smDCs constitute an independent subgroup of DCs that play a key role for inducing CD4(+)CD25(+)Treg cells to express high IL-10 levels, which induce hyporesponsiveness of effector T cells. Pre-treating recipients with donor smDCs may have potential for transplant tolerance induction.

Tolerogenic semimature dendritic cells induce effector T-cell hyporesponsiveness by the activation of antigen-specific CD4+ CD25+ T-regulatory cells.

OBJECTIVES: Researchers recently discovered a group of semimature dendritic cells that induce autoimmune tolerance by activating host antigen-specific CD4+CD25+ T-regulatory cells. We hypothesized that donor semimature dendritic cells injected into recipients would induce effector T-cell hyporesponsiveness by activating CD4+CD25+ T-regulatory cells. MATERIALS AND METHODS: Donor myeloid semimature dendritic cells were cultivated for 6 days and were then stimulated with tumor necrosis factor a for 24 hours. BALB/c mice were pretreated with semimature dendritic cells to generate antigen-specific CD4+CD25+ T-regulatory cells in vivo. The role of CD4+CD25+ T-regulatory cells in transplant immunity was studied via mixed lymphocyte culture in vitro. RESULTS: Surface markers and cytokines secreted by semimature dendritic cells differed from those secreted by immature myeloid dendritic cells or mature dendritic cells. Semimature dendritic cells and immature myeloid dendritic cells did not activate allogenic lymphocyte responses in coculture studies. CD4+CD25+ T-regulatory cells of recipients challenged by donor semimature dendritic cells, which expressed a high level of interleukin-10, induced hyporesponsiveness in host effector T cells that were stimulated by donor splenocytes. In contrast, CD4+CD25+ T-regulatory cells did not induce hyporesponsiveness in effector T cells when the host T cells were stimulated by third-party antigen from DBA2 mice splenocytes. CONCLUSIONS: Our findings confirm that semimature dendritic cells are an independent subgroup of dendritic cells in both immune function and morphologic profile. It may be the cytokine secretion profile of semimature dendritic cells (rather than that of surface markers) that has a key role in inducing CD4+CD25+ T-regulatory cells to express a high level of interleukin-10. Immunization with donor semimature dendritic cells may be an effective method of inducing transplant tolerance, but further evidencebased studies of that topic are necessary.

[Culture and identification of mouse myeloid semimature dendritic cells].

OBJECTIVE: To investigate the methods of culturing and identifying mouse myeloid semimature dendritic cell (smDC) in vitro. METHODS: Myeloid monocytes derived from 6-week-old C57 BL/6 mice were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 2 ng/ml recombinant murine granulocyte macrophage-colony stimulating factor (GM-CSF), and 20 ng/ml recombinant murine interleukin (IL)-4 for 9 days. Then cells were incubated with 40 ng/ml tumor necrosis factor-alpha (TNF-alpha) for 24 hours to obtain smDC. Meanwhile, smDC was differentiated into mature dendritic cell (mDC) or immature dendritic cell (iDC) by treatment with 1 micro/m1 lipopolysaccharide (LPS) or without LPS. The morphological features of smDC were assayed by inverted microscopy and scanning electron microscopy. Surface markers such as CD11c, CD4O, CD8O, CD86, and MHC-II were tested by flow cytometry. IL-1beta, IL-6, IL-12, and IL-10 in the supernatant were tested by ELISA. The activation of allogene lymphocyte (BALB/c mice) stimulated by C57BL/6 myeloid smDC in mixed lymphocyte reaction was examined by Cell Counting Kit-8 in vitro. RESULTS: The shape of smDC was round or oval-shaped, and the diameter of smDC was about 15 microm. The length of smDC dendrite was between 5 to 10 microm. smDC, iDC, and mDC all expressed high level of CD11 c. The expressions of MHC-II, CD40, CD80, and CD86 on smDC were higher than those of iDC and lower than those of mDC. IL-1beta, IL-6, and IL-12 secretion of smDC was significantly lower than that of mDC (P < 0.01), and IL-12 was significantly lower than that of iDC (P < 0.05), while no significant difference of IL-1beta and IL-6 secretion was found between smDC and iDC (P > 0.05). Furthermore, IL-10 secretion was not significantly different among these three kinds of DCs (P > 0.05). The effect of allogene lymphocytes activation on smDC was significantly lower than that of mDC and positive control (P < 0.01), but had no significant difference when compared with that of iDC and negative control (P > 0.05). CONCLUSIONS: smDC may be a relatively independent dendritic cell sub-population in terms of function and morphology. It is a feasible way to induce myeloid monocytes to differentiate into smDC using GM-CSF, IL-4, and TNF-alpha in vitro.

[Rapamycin combined with donor bone marrow-derived immature dendritic cells induces mouse skin allograft tolerance].

OBJECTIVE: To investigate the synergic effects of rapamycin and donor bone marrow-derived immature dendritic cells (DCs) in inducing skin allograft tolerance in mice. METHODS: The recipient BALB/c mice receiving transplantation of skin allograft from C57BL/6 mice were divided into control group (without perioperative treatments), rapamycin group (receiving rapamycin at 1 mg.kg(-1).d(-1) by gavage for 7 consecutive 7 days after skin transplantation), immature DC group (receiving an injection of donor bone marrow-derived immature DCs of 2 x 10(6) via tail vein before skin transplantation), combined group (receiving an injection of the DCs of 2 x 10(6) before transplantation and rapamycin at 1 mg.kg(-1).d(-1) for 7 consecutive days after transplantation). The survival time of the skin allograft was observed in each group. RESULTS: The survival time of the skin allograft in the control, rapamycin, immature DC and immature DC +rapamycin groups were 6.9-/+1.9, 12.3-/+3.0, 17.0-/+3.4 and 20.8-/+3.6 days, respectively, showing significant differences among the groups (P<0.05), and SNK test also indicated significant differences between every two groups. CONCLUSIONS: Rapamycin and donor bone marrow-derived immature DCs have synergic effects in inducing skin allograft tolerance in mice.