Intestinal and multivisceral transplantation / Transplante intestinal e multivisceral

Intestinal transplantation has shown exceptional growth over the past 10 years. At the end of the 1990’s, intestinal transplantation moved out of the experimental realm to become a routine practice in treating patients with severe complications related to total parenteral nutrition and intestinal failure. In the last years, several centers reported an increasing improvement in survival outcomes (about 80%), during the first 12 months after surgery, but long-term survival is still a challenge. Several advances led to clinical application of transplants. Immunosuppression involved in intestinal and multivisceral transplantation was the biggest gain for this procedure in the past decade due to tacrolimus, and new inducing drugs, mono- and polyclonal anti-lymphocyte antibodies. Despite the advancement of rigid immunosuppression protocols, rejection is still very frequent in the first 12 months, and can result in long-term graft loss. The future of intestinal transplantation and multivisceral transplantation appears promising. The major challenge is early recognition of acute rejection in order to prevent graft loss, opportunistic infections associated to complications, post-transplant lymphoproliferative disease and graft versus host disease; and consequently, improve results in the long run.

O transplante de intestino, ao redor do mundo, tem crescido de maneira sólida e consistente nos últimos 10 anos. No final da década de 1990, passou de um modelo experimental para uma prática clínica rotineira no tratamento dos pacientes com complicação severa da nutrição parenteral total com falência intestinal. Nos últimos anos, vários centros têm relatado uma crescente melhora nos resultados de sobrevida do transplante no primeiro ano (ao redor de 80%), porém, a longo prazo, ainda é desafiador. Diversos avanços permitiram sua aplicação clínica. O surgimento de novas drogas imunossupressoras, como o tacrolimus, além das drogas indutoras, os anticorpos antilinfocíticos mono e policlonal, nos últimos 10 anos, foi de suma importância para a melhora da sobrevida do transplante de intestino/multivisceral, mas, apesar dos protocolos bastante rígidos de imunossupressão, a rejeição é bastante frequente, podendo levar a altas taxas de perdas de enxerto a longo prazo. O futuro do transplante de intestino e multivisceral parece promissor. O grande desafio é reconhecer precocemente os casos de rejeição, prevenindo a perda do enxerto e melhorando os resultados a longo prazo, além das complicações causadas por infecções oportunistas, doenças linfoproliferativas pós-transplante e a doença do enxerto contra hospedeiro.

Establishment and application of flow cytometry in detecting human cells in human/goat chimera models / 中华检验医学杂志

Objective To establish the methodology of flow cytometry for detecting human cells in human/goat chimerisra.Methods Human hemopoietic stem/progenitor cells (CD+34 cells) or MIG-tranadueed-GFP CD+34 cells were transplanted into the peritoneal cavity of fetal goats in utero to obtain human/goat chimera modeL The peripheral blood cells from the chimeras were labeled with multiple mouse anti-human antibodies and the monoelonal antibodies that were specific for human but had not or only minimal cross-reaction with goat were screened as the primary antibodies for routine analysis in flow cytometry.Human cord blood was proportionally (25% ,50% ,75%,100%) added into the blood of the untransplanted goats and the cells were labeled with CD+34 monoclonal antibody.The region and size of the "gate" were chosen based on to the distribution of CD+34 cells or human cord blood.One human/goat chimera marked with GFP (MIG goat) was sacrificed and the substantial liver cells from its perfused liver were analyzed for the GFP+cells percentage and DNA contents by flow cytometry.Results CD7,CD15,CD38,CD45CD20CD34CD14and GPA monoclonal antibodies were chosen as the primary antibodies in rou tine detection by flow cytometry.The size and area of the "gate" were also defined.29.1% (29100/100 000 ) of the substantial liver cells from the MIG goat expressed GFP.DNA content analysis showed that the GFP+ cells obtained from the liver of MIG goat mainly manifested two peaks that were correspond to those of human.Conclusions Flow cytometry is rapid,simple and effective for the investigation of differentiation,homing and biological characteristics of stem cells in vivo.The selections of suitable surface antibodies and the "gate" are very important for detecting human cells accurately in the human/goat chimerism.

Expression of toll-like receptor on different cell surface in PMN recruiting in severe acute pancreatifis / 中华普通外科杂志

Objective To investigate the significance of expression of toll-like receptor on medullary system cell and endothelial cell surface in PMN recruiting in severe acute pancreatitis.Method In this study,26 C3H/He-J mice and 26 C3H/He-N mice were derided into 4 groups by bone marrow transplantation:group A, mosaic of medullary system cell TLR4-/- and endothelial cell TLR4+/+,group B, mosaic of medullary system cell TLR4+/+ and endothelial cell TLR4-/- ;group C, homozygote ofTLR4+/+ ;group D, homozygote of TLR4-/-. SAP was induced to all these mice. Amylase, TRIA of granular cell in peripheral blood,TRL4 expression in pancreas, Naphthol AS-D chloroacetate esterase, MPO activity were tested. Results Amylase level increased in all mice, there was no differences of the amylase level among groups, TRIA of granular cell in peripheral blood in group B and C was significant higher than A and D(P<0.05), TLR4 in pancreas were positive in group A and C, negative in group B and D, AS-D count and M PO activity in group A and C were higher than that in group B and D (P < 0. 05 ). Conclusions It was endothelial cell, not peripheral blood granulocyte, that plays a key role in recuiting neutrophils that triggers severe acute pancreatitis.

IL-2 Pathway Blocking in Combination with Anti-CD154 Synergistically Establishes Mixed Macrochimerism with Limited Dose of Bone Marrow Cells and Prolongs Skin Graft Survival in Mice

To facilitate the establishment of mixed chimerism with limited dose of bone marrow (BM) cells, and to achieve tolerance in skin graft model, combined blocking of costimulatory pathway and IL-2 pathway was used in minimally myeloablative model using busulfan. BM cells (2.5 x 10(7)) of BALB/c were injected into C57BL/6 mice at day 0 with full thickness skin graft after single dose injection of busulfan (25 mg/kg) on day-1. Recipients were grouped and injected the anti-CD154, CTLA4-Ig, anti-IL-2R at days 0, 2, 4, and 6 according to protocol. Mixed macrochimerism were induced in groups treated with anti-CD154+anti-CTLA4-Ig, anti-CD154+anti-IL-2R, and anti-CD154+anti-CTLA4 Ig+anti-IL-2R. Three groups having chimerism enjoyed prolonged graft survival more than 6 months. Superantigen deletion study revealed deletion of alloreactive T cells in combined blockade treated groups. In graft versus host disease model using CFSE staining, CD4+ T cell and CD8+ T cell proliferation were reduced in groups treated with CTLA4-Ig or anti-IL-2R or both in combination with anti-CD154. However, anti-IL-2R was not so strong as CTLA4-Ig in terms of inhibition of T cell proliferation. In conclusion, IL-2 pathway blocking combined with anti-CD154 can establish macrochimerism with limited dose of BM transplantation and induce specific tolerance to allograft.

IL-2 Pathway Blocking in Combination with Anti-CD154 Synergistically Establishes Mixed Macrochimerism with Limited Dose of Bone Marrow Cells and Prolongs Skin Graft Survival in Mice

To facilitate the establishment of mixed chimerism with limited dose of bone marrow (BM) cells, and to achieve tolerance in skin graft model, combined blocking of costimulatory pathway and IL-2 pathway was used in minimally myeloablative model using busulfan. BM cells (2.5 x 10(7)) of BALB/c were injected into C57BL/6 mice at day 0 with full thickness skin graft after single dose injection of busulfan (25 mg/kg) on day-1. Recipients were grouped and injected the anti-CD154, CTLA4-Ig, anti-IL-2R at days 0, 2, 4, and 6 according to protocol. Mixed macrochimerism were induced in groups treated with anti-CD154+anti-CTLA4-Ig, anti-CD154+anti-IL-2R, and anti-CD154+anti-CTLA4 Ig+anti-IL-2R. Three groups having chimerism enjoyed prolonged graft survival more than 6 months. Superantigen deletion study revealed deletion of alloreactive T cells in combined blockade treated groups. In graft versus host disease model using CFSE staining, CD4+ T cell and CD8+ T cell proliferation were reduced in groups treated with CTLA4-Ig or anti-IL-2R or both in combination with anti-CD154. However, anti-IL-2R was not so strong as CTLA4-Ig in terms of inhibition of T cell proliferation. In conclusion, IL-2 pathway blocking combined with anti-CD154 can establish macrochimerism with limited dose of BM transplantation and induce specific tolerance to allograft.

Donor specific tolerance for MHC mis-matched murine skin allograft induced by donor bone marrow transplantation with precondition of high dose immunosuppressants / 中华器官移植杂志

Objective To establish a non-irradiated precondition strategy for successful donor specific transplantation tolerance induced by bone marrow transplantation which may be introduced to clinical application by a murine model.Methods Male C57BL/6 and female BALB/c mice were used as skin transplant donors and recipients respectively. In all, 11 groups were studied: group 1, control mice with skin graft and without any other treatment; group 2, mice receiving only donor bone marrow transplantation (DBMT) before skin transplantation; groups 3～5, mice administrated by only high dose FK506, CsA or CTX respectively and then skin transplantation; groups 6～8, mice receiving DBMT preconditioned by high dose FK506, CsA or CTX respectively and followed by skin transplantation. Both skin and bone marrow donors were male C57BL/6 mice in above 8 groups. Mice in groups 9～11 were treated almost equally to groups 6～8 except that skin grafts were from the third party ICR donors to prove specificity of tolerance. Every group included 6 recipients. Survival time of skin graft was recorded. Macrochimerism were examined by PCR method.Results Neither standard dose DBMT nor high dose immnodepressants alone could prolong skin graft survival, and macrochimerism was not detected, either. But skin graft survival time was significantly prolonged and macrochimerism was also detected in mice of 6～8 groups. Survival time of skin graft from the third party mice was not prolonged. Conclusions Bone marrow transplantation preconditioned with high dose immunosuppressants and without irradiation can induce donor specific transplantation tolerance and prolong murine skin graft survival. It may work through the mechanism of establishment of macrochimerism.

Partial conditioning regimen for stable mixed chemerism after unrelated cord blood transplantation in adult patients with severe aplastic anemia / 中华器官移植杂志

Objective To study and observe the engraftment of donor cells from unrelated cord blood into adult patients with severe aplastic anemia (SAA) and the outcome of allo-CBSCT. Methods Six patients received cord blood provided by Guangzhou Cord Blood Bank, for three of which one unit of cord blood was given in a procedure, whereas for other 3 patients, 2 units of cord blood were infused at the same time in a transplant protocol. In all 9 units of umbilical cord blood (UCB) infused, UCB units contained 1.6 ～ 10.7 nucleated cells/kg body weight of the recipient after thawing. The patients were conditioned with decreased dosage of immunosuppressive agents of CTX (60?mg/kg) and ALG (120?mg/kg). The engraftment state of the donor cells into recipients was confirmed by microsatellite DNA fingerprinting and fluorescent quantitative PCR analysis. Results Engrafted evidence has been found in 5 patients by molecular biology analyses showing donor-recipient mixed chimerism post transplant which was stable and persistent. After a median follow-up of 21 months (range 7～50), 4 patients were alive and disease free. One patient died of severe infection in the 3rd month from transplant, though the evidence of engraftment was obvious. Another patient also died in the early stage posttransplant of serious aspergillus infection without the engrafted proof.Conclusion Durable donor-recipient stable mixed chimerism can be achieved by unrelated UCBT in patients with SAA. Umbilical cord blood could be employed as a source of hematopoietic stem cell for adult transplantation.

Studies on the microchimerism after kidney transplantation / 解放军医学杂志

Objective To investigate the state of microchimerism after kidney transplantation, and to evaluate the relationship between microchimerism and long term survival of transplanted kidney. Methods Leukocytes were collected from peripheral blood of 70 female recipients having received kidneys from males for the identification of microchimerism by means of amplifying the single copied sex determine region Y gene (SRY) by nested PCR. Results Half to 10 years after renal transplantation, the positive percentage of microchimerism in 70 female patients was 58.6%(41/70). These 70 patients were categorized into three groups according to the duration after the transplantation: Group 1(n=25), 0.5 to 2 years; Group 2 (n=27), 2 to 5 years; and group 3 (n=18), over 5 years. The positive rates of microchimerism for three groups were 68%(17/25), 44.4%(11/27) and 72%(13/18), respectively. The positive rates in both group 1 and group 3 were significantly different compared with that in group 2 (P

Infusion of donor specific bone marrow alleviates acute rejection reaction in liver allotransplantation / 中国病理生理杂志

100 d),and the histological grade of rejection was significantly lower than that in group Ⅱ(P

Clinical correlations of peripheral blood microchimerism after liver transplantation

The aim of this study was to evaluate microchimerism after human liver transplantation (LT). This study included 13 female recipients who received hepatic allograft from male donors at Asan Medical Center. A nested PCR specific for Y-chromosome gene (DYZ3) was used to analyze the small number of male cells in the peripheral blood mononuclear cells of the female recipients. Microchimerism was observed in 6 of 13 recipients and 16 out of 35 samples. Only 3 patients showed microchimerism 3 months after LT. There was no statistical difference between the presence of microchimerism and clinical findings such as type of donor, type of immunosuppression, episode of rejection and age of recipient. This study did not show any clinical relevance of microchimerism and further larger study are needed to confirm the results.

Clinical correlations of peripheral blood microchimerism after liver transplantation

The aim of this study was to evaluate microchimerism after human liver transplantation (LT). This study included 13 female recipients who received hepatic allograft from male donors at Asan Medical Center. A nested PCR specific for Y-chromosome gene (DYZ3) was used to analyze the small number of male cells in the peripheral blood mononuclear cells of the female recipients. Microchimerism was observed in 6 of 13 recipients and 16 out of 35 samples. Only 3 patients showed microchimerism 3 months after LT. There was no statistical difference between the presence of microchimerism and clinical findings such as type of donor, type of immunosuppression, episode of rejection and age of recipient. This study did not show any clinical relevance of microchimerism and further larger study are needed to confirm the results.

Studies on immune tolerance induced by the mixed infusion of mesenchymal stem cells and bone marrow cells after islet transplantation / 解放军医学杂志

Objective To examine the effects of mixed infusion of mesenchymal stem cells(MSCs) and bone marrow cells(BMCs) in the induction of chimerism and islet allograft tolerance.Methods BALB/C mouse was used as the recipient and C57BL/6 mouse was as the donor.BALB/C mice were rendered diabetic via injection of streptozotocin.The islet cells of donor mice were transplanted into the recipient mice under the capsule of kidney.Rat anti-mouse CD154 mAb was intraperitoneally injected to the recipient mice.All of recipient mice(n=25) were then randomly divided into five groups: A group(received nothing),B group(donor MSCs),C group(donor BMCs),D group(donor BMCs and MSCs) and E group(donor BMCs and the third strain-derived MSCs).The chimerism level of donor cells and the survival time of islet grafts were compared among these five groups on 7,30d and 60d after transplantation.Results On 30d and 60d after islet transplantation,the chimerism levels of donor cells in D and E groups,in which the recipient mice received the mixed infusion of MSCs and BMCs,were significantly higher than that in C group,in which the recipient mice received BMCs infusion only,and the survival time of islet graft prolonged from 53.0?16.4d to 77.0?7.7d and 61.0?2.2d,respectively(P