optimization-of-the-cuff-technique-for-murine-heart-transplantation

Optimization of the Cuff Technique for Murine Heart Transplantation

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to ...

Xiamen University

Fujian Key Laboratory of Organ and Tissue Regeneration

Donor-reactive memory T (Tm) cells undermine transplanted organs more readily than naive T cells. Rapamycin (RAPA) and tacrolimus (FK-506) are current mainstay immunosuppressants used for preventing acute allograft rejection. Although their efficacy in suppressing naive T cell is established, their suppressing effect on memory T cells is undefined. This study was conducted to investigate the inhibiting capability of RAPA or FK-506 against transferred alloreactive CD4(+) Tm cells in a mouse cardiac transplant model. We found that these drugs alone prolonged the median survival time (MST) of allograft from 5days to 9days in recipient mice with CD4(+) Tm infusion (P<0.01), which however was not significantly longer than that (8days) in untreated recipient mice without CD4(+) Tm infusion (naive control). Mean histologic rank of rejection activity in section of cardiac allograft on day 5 postgrafting was Grade 4 in the Tm control recipients versus Grade 3A in both of the immunosuppressant treatment recipients with CD4(+) Tm infusion. RAPA or FK-506 alone failed to completely suppress proliferation and differentiation of the alloreactive CD4(+) Tm, which was confirmed by in vitro mixed lymphocyte reaction (MLR) and by flow cytometry (FCM) of the splenocytes for detecting CD44(high)CD62L(-) effector/memory as well as CD69(+)/CD25(+) activation phenotype cells from the respective recipients. Furthermore, the agent alone didn't completely inhibit the activation of CD4(+) Tm, for serum level of IFN-gamma and its gene expression at the cardiac allograft from the immunosuppressant-treated recipients were as still high as the untreated naive control. Thus, RAPA or FK-506 alone couldn't completely suppress the proliferation and activation of the alloantigen-primed CD4(+) Tm cells responding to the alloantigen, indicating that alloreactive CD4(+) Tm was insensitive to these immunosuppressants. The characteristics of alloreactive CD4(+) Tm to resist immunosuppressants and its potency to initiate quick and vigorous rejection despite treatment with the immunosuppressant make it to be a critical barrier to prolongation of allograft survival and induction of transplant tolerance.

Rapamycin or tacrolimus alone fails to resist cardiac allograft accelerated rejection mediated by alloreactive CD4(+) memory T cells in mice.

Although immunosuppressive treatments are available for acute cardiac rejection no viable treatment exists for long-term cardiac graft failure. Moreover, the extended use of calcineurin inhibitor immunosuppressants, the mainstay of current treatment for cardiac transplantation, leads to significant side effects such as nephrotoxicity and an increased risk of cardiac disease. Because some agents used in Traditional Chinese Medicine (TCM) have strong immunosuppressive effects coupled with low toxicity, we investigated the effect of Compound K (K), the synthesized analogue of highly unsaturated fatty acids from Isatis tinctoria L., either as a single treatment or combined with tacrolimus (FK-506) on acute cardiac allograft rejection. We compared the ability of K alone, or in combination with FK-506, to inhibit acute heart transplant rejection both in vitro and in vivo. We found that the inhibition of lymphocyte proliferation was positively correlated with K concentration. K significantly reduced IL-2 and IFN-gamma expression levels and significantly inhibited lymphocyte proliferation in both a lymphocyte transformation test and a mixed lymphocyte reaction (MLR). We also found that the inhibitory effect of a combination of K and a sub-therapeutic dose of FK-506 (SubFK-506) was stronger than that of full-dose FK-506 alone. Oral administration of K reduced acute cardiac allograft rejection in mice and had no apparent toxicity. In vivo, the immunosuppressive effect of K combined with a half-dose of FK-506 was equivalent to that of a full-dose of FK-506 alone. K combined with a half-dose of FK-506 reduced the expression levels of IL-2 and IFN-gamma (both within the graft and in the recipients' serum) more effectively than a full-dose of FK-506. These results show that K has significant immunosuppressive effects both in vitro and in vivo. When used as a combination therapy with FK-506 we see a powerful inhibition of rejection with no obvious toxic side effects. The mechanism of action is postulated to involve the inhibition of IL-2 and IFN-gamma expressions by lymphocytes, rather than the activation of Tr1 cells via the production of IL-10.

Synergistic effects of Isatis tinctoria L. and tacrolimus in the prevention of acute heart rejection in mice.

Donor-reactive memory T cells threaten the survival of transplanted organs via multiple pathways. This study was undertaken to induce tolerance of cardiac allografts in mice, in which alloreactive memory T cells were adoptively transferred, by combined costimulatory blockade of both effector and memory T cells. We found that the median survival time (MST) of the grafts was 5.17 days in the untreated group, 10.33 days in the CTLA4Ig- and anti-CD40L-treated (2-combined) group, and more than 100 days in the CTLA4Ig-, anti-CD40L-, anti-LFA-1-, and anti-OX40L-treated (4-combined) group. Histological analysis revealed that the mean rejection level was Grade 4 in the untreated group, Grade 3 in the 2-combined treatment group, and Grade 0 in the 4-combined treatment group. CD44(high) T cells were detected only in the untreated group. The in vitro proliferation of lymphocytes of both untreated and 2-combined group was higher than that of the 4-combined treatment group (p < 0.01). Compared with the untreated group, the expression levels of IL-2, IFN-gamma, and Foxp3 were lower in the 2-combined treatment group; the expression levels of these genes were the lowest in the 4-combined treatment group. IL-10 expression was significantly higher in the 4-combined treatment group than in the other groups. These results demonstrate the inhibition efficacy of combined costimulation blockade in accelerated-rejection models and the possible mechanisms underlying the suppression of cellular immunity in mice receiving grafts as well as in inducing the activation of IL-10-producing Tr1 cells in grafts.

Combined costimulation blockade inhibits accelerated rejection mediated by alloantigen-primed memory T cells in mice.

The presence of alloreactive memory T cells in recipient is a critical handicap to achieving transplantation tolerance. To make a mouse model that can as closely as possible mimic the presensitized transplant patient is important for research on this subject. Thus, we developed a novel retransplant model and compared the alloresponse in this model with that in the memory T cells-transfer model (transfer control). Mean survival time of allograft was compared between 3 groups, including blank transplant control, memory transfer control and retransplant groups. Cellular rejection activity in allografts was evaluated via HE staining of cardiac graft section. Proliferation and differentiation of the alloreactive effector T cells were assayed by in vitro mixed lymphocyte reaction and flow cytometry, respectively. Real-time quantitive RT-PCR was used to assess gene expression of cytokines and surum IFN-gamma was measured via ELISA. It showed that the median survival time of allograft in retransplant recipients was significantly shortened compared to that of transfer control, and it was the same in rejection score of graft. Moreover, proliferation and differentiation of the alloreactive effector T cells were more intensive in retransplant recipients than that in transfer control, which was confirmed by in vitro mixed lymphocyte reaction and by flow cytometry of the splenocytes for detecting CD44highCD62L- memory/effector phenotype cells. Furthermore, activation of CD4+ memory T cells is reflected by high level of surum IFN-gamma and the intensive gene expression of IFN-gamma and IL-2 at cardiac allograft in retransplant recipients. Collectively, the recall alloresponse in retransplantation is more intensive than that in a memory-transfer setting, and this retransplant model is closer to the clinic situation than the memory-transfer model in rodents.

The recall alloresponse following retransplantation is more intense compared with the T cell memory-transfer model.

Memory T cells are known to play a key role in prevention of allograft tolerance in alloantigen-primed mice. Here, we used an adoptively transferred memory T cell model and an alloantigen-primed model to evaluate the abilities of different combinations of monoclonal antibodies (mAb) to block key signaling pathways involved in activation of effector and memory T cells. In the adoptively transferred model, the use of anti-CD134L mAb effectively prevented activation of CD4(+) memory T cells and significantly prolonged islet survival, similar to the action of anti-CD122 mAb to CD8(+) memory T cells. In the alloantigen-primed model, use of anti-CD134L and anti-CD122 mAbs in addition to co-stimulatory blockade with anti-CD154 and anti-LFA-1 prolonged secondary allograft survival and significantly reduced the proportion of memory T cells; meanwhile, this combination therapy increased the proportion of regulatory T cells (Tregs) in the spleen, inhibited lymphocyte infiltration in the graft, and suppressed alloresponse of recipient splenic T cells. However, we also detected high levels of alloantibodies in the serum which caused high levels of damage to the allogeneic spleen cells. Our results suggest that combination of four mAbs can significantly suppress the function of memory T cells and prolong allograft survival in alloantigen primed animals.

Suppressing memory T cell activation induces islet allograft tolerance in alloantigen-primed mice.

Donor-reactive CD4(+)memory T cells threaten the survival of transplanted organs. In this study, we used anti-CD44 monoclonal antibody (mAb) to inhibit adoptively transferred B6-reactive CD4(+)memory T cells (BALB/c origin) and to induce tolerance of B6 hearts in nude mice. The median survival time (MST) of the grafts was 6 days in the isotype group, and more than 100 days in the group treated with 8 doses of anti-CD44 at four-day intervals. Histological analysis revealed that the mean rejection level was Grade 3 in the isotype group, and Grade 0 or 1 in the multi-dose anti-CD44 treatment group. Compared with the isotype group, the multiply treated anti-CD44 group had significantly decreased IL-2 and IFN-γ expressions, while IL-10 and TGF-β were increased in the serum and the graft. Foxp3 in the graft was also increased. These data demonstrate that alloreactive CD4(+) memory T cells mediate the destruction of allografts, and the adhesion molecule CD44 plays an important role in this course. Anti-CD44 mAb may promote the reduction of CD4(+)memory T cells and the production of regulatory T cells (Tregs). Furthermore, Tregs are maintained at a certain level while suppressing cellular immunity and inducing the grafts long-term survival in transplant recipients.

Anti-CD44 monoclonal antibody inhibits heart transplant rejection mediated by alloantigen-primed CD4(+) memory T cells in nude mice.

To evaluate the Lung Operation Three-dimensional Imaging System software by Xiamen TRONG Technology Co. Ltd in detecting the precise position of solitary pulmonary nodules or ground-glass opacity nodules before surgery.

Thoracoscopic anatomic pulmonary segmentectomy: a 3-dimensional guided imaging system for lung operations.

Xenotransplantation is an effective way to solve the problem of donor shortage in clinical transplantation. However, clinical use of xenotransplantation is currently limited due to immunological challenges such as acute vascular rejection and cell-mediated rejection. To finally surpass this immunological barrier, more preclinical research is needed into the molecular mechanisms of rejection and the possible effects of new immunosuppressants. Our aim was to create a refined, highly reproducible protocol to establish the most suitable rat-to-mouse heterotopic heart transplantation model using the cuff technique.

A highly reproducible cervical cuff technique for rat-to-mouse heterotopic heart xenotransplantation.

 Cardiac extracellular matrix (cECM) scaffolds are promising biomaterials for clinical applications. Our aim is to determine the immunogenicity of decellularized scaffolds from different sources for use as artificial organs during organ transplantation.  We transplanted Lewis rats with syngeneic (Lewis rat cECM), allogeneic (BN rat cECM) or xenogeneic (hamster cECM) decellularized cardiac scaffolds. Acute vascular and cellular rejection was quantified by immunohistochemistry and immune cell infiltration.  BN rat and hamster hearts were rejected following transplantation. BN and hamster cECMs had similarly low immunogenicity compared with Lewis rat cECMs and did not lead to increased rejection.  We found that scaffolds from all sources did not induce vascular or cellular rejection and exhibited low immunogenicity.

Immunogenicity analysis of decellularized cardiac scaffolds after transplantation into rats.

Patients with end-stage heart failure must receive treatment to recover cardiac function, and the current primary therapy, heart transplantation, is plagued by the limited supply of donor hearts. Bioengineered artificial hearts generated by seeding of cells on decellularized scaffolds have been suggested as an alternative source for transplantation. This study aimed to develop a tissue-engineered heart with lower immunogenicity and functional similarity to a physiological heart that can be used for heart transplantation.

Generation of bioartificial hearts using decellularized scaffolds and mixed cells.

Accelerated transplant rejection mediated by donor reactive memory T cells is another barrier to the induction of graft tolerance. The aim of this study was to investigate the immunosuppressing effects of vitamin D (1,25(OH)2D3), administered alone or in combination with a costimulatory blockade treatment, on rejection of secondary heart allografts in a mouse model.

Combined treatment with vitamin D3 and antibody agents suppresses secondary heart transplant rejection in the early postoperative period.

An ideal animal model is a prerequisite for the basic research of uterus transplantation. This study aimed to develop a new cervical ectopic uterus transplantation mice model, which was established by vascular anastomosis of the right common iliac artery and vein of the donor with the right common carotid artery and external jugular vein of the recipient, respectively, using the cuff method. The survival status of the transplanted uterus was assessed by macroscopic observation and histological examination after surgery, and the function of the graft uterus was tested by verifying whether the pregnancy is possible. A total of 40 transplants were performed, of which only 1 failed due to donor hemorrhage. After 26 transplants, the total operation time reduced to 52.4 ± 3.8 minutes, of which the total ischemia time took 6.6 ± 1.1 minutes. Sixty days after transplantation, all the graft uteri had a good blood supply and spontaneous contraction. The histology showed no significant difference between the transplanted uterus and the native. Embryo transfer experiments have proven that the transplanted uterus has uterine function. In conclusion, this new model is an effective and simple mice model for the studies of the scientific issues related to uterus transplantation.

Article	Year
Rapamycin or tacrolimus alone fails to resist cardiac allograft accelerated rejection mediated by alloreactive CD4(+) memory T cells in mice. Transplant immunology\| PubMed ID: 19755159	2010
Synergistic effects of Isatis tinctoria L. and tacrolimus in the prevention of acute heart rejection in mice. Transplant immunology\| PubMed ID: 19766187	2009
Combined costimulation blockade inhibits accelerated rejection mediated by alloantigen-primed memory T cells in mice. Immunological investigations\| PubMed ID: 19811427	2009
The recall alloresponse following retransplantation is more intense compared with the T cell memory-transfer model. Immunological investigations\| PubMed ID: 20064084	2010
Suppressing memory T cell activation induces islet allograft tolerance in alloantigen-primed mice. Transplant international : official journal of the European Society for Organ Transplantation\| PubMed ID: 20536791	2010
Anti-CD44 monoclonal antibody inhibits heart transplant rejection mediated by alloantigen-primed CD4(+) memory T cells in nude mice. Immunological investigations\| PubMed ID: 20718664	2010
Thoracoscopic anatomic pulmonary segmentectomy: a 3-dimensional guided imaging system for lung operations. Interactive cardiovascular and thoracic surgery\| PubMed ID: 27099268	2016
A highly reproducible cervical cuff technique for rat-to-mouse heterotopic heart xenotransplantation. Xenotransplantation\| PubMed ID: 28833558	2017
Immunogenicity analysis of decellularized cardiac scaffolds after transplantation into rats. Regenerative medicine\| PubMed ID: 31070505	2019
Generation of bioartificial hearts using decellularized scaffolds and mixed cells. Biomedical engineering online\| PubMed ID: 31164131	2019
Combined treatment with vitamin D3 and antibody agents suppresses secondary heart transplant rejection in the early postoperative period. Transplant immunology\| PubMed ID: 31953155	2020
Live birth after cervical ectopic uterus transplantation in mice. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons\| PubMed ID: 32092213	2020

Baiyi Xie

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