Development of an algorithm of satellite markers for monitoring chimerism status in post‐allogeneic haematopoietic stem cell transplantation patients

Introduction: Analysis of variable number tandem repeats (VNTRs) by polymerase chain reaction (PCR) is a common method used to predict engraftment status in post‐allogeneic haematopoeitic stem cell transplantation (HSCT) patients. Different populations have different copies of repeated DNA sequence and hence, different percentage of informativeness between patient and donor. Methods: PCR amplification of four highly polymorphic VNTR markers (YNZ‐22, D1S80, D1S111 and ApoB) was conducted on 60 patient‐donor pairs. The informativeness of the markers was analysed using 3% agarose gel electrophoresis. Results: We developed an algorithm for identification of informative VNTR markers in 60 post‐allogeneic HSCT patients. YNZ‐22 was the most informative (72%), followed by D1S80 (63%) and D1S111 (60%), while the least informative was ApoB (47%). The degree of informativeness achieved was 95%, which could discriminate 57 patient‐donor pairs, when all four markers were combined. Conclusion: Since population genetic studies on VNTR loci are not well established in Southeast Asia, the present study is useful to determine reliable markers during the initial screening step of chimerism analysis. By following this algorithm, we are able to reduce time and cost of finding a suitable VNTR marker in our cohort.     

异基因外周血干细胞移植后淋巴细胞和粒细胞嵌合体的动态改变

目的：探讨异基因外周血干细胞移植(allo-PBSCT)后T细胞、粒细胞嵌合体的动态改变及临床价值。方法：将多重PCR扩增短串联重复序列(STR—PCR)的法医试剂进行条件优化，间隔短时间抽取9例恶性血液病患者(5例清髓性PBSCT，4例非清髓性PBSCT)的外周血样，STR—PCR定量分析T细胞和粒细胞的嵌合体，并观察其对移植后应用免疫抑制剂的指导作用。结果：STR—PCR定量分析嵌合体的敏感性为5％，并具高度可重复性。清髓性PBSCT后10d( 10d)，5／5例患者的粒细胞迅速演变为完全供者嵌合体(CDC)， 14d，4，／5例患者的T细胞获得CDC。非清髓性PBSCT， 7～ 14d，供者T细胞信号的植入速度快于粒细胞；随后，供者粒细胞的比例突然增加，并迅速获得CDC，T细胞的植入却渐缓慢，最后，T细胞取得CDC的时间迟于粒细胞。依据供者T细胞信号的植入程度，及时调整非清髓性PBSCT后环孢素A(CsA)的用量，移植早期T细胞即获CDC，随访2～16个月，T细胞和粒细胞均呈稳定的供者植入状态。结论：供者T细胞的完全植入迟于粒细胞．动态监测T细胞嵌合体，可能有助于免疫抑制剂的调整。     

Hematopoietic donor chimerism and graft-versus-myeloma effect in relapse of multiple myeloma after allogeneic bone marrow transplantation

 A large group of patients relapsing after allogeneic bone marrow transplantation (BMT) have obtained remission after infusion of leukocytes from their original donor, suggesting a graft-versus-myeloma effect. However, side effects such as graft-versus-host disease and myelosuppression are severe, and sometimes fatal, complications of this therapeutic approach. Previously we demonstrated that patients with leukemia who lack donor hematopoiesis in relapse after BMT experience severe and lasting aplasia after infusion of donor leukocytes. In two patients – one with extramedullary and one with marrow relapse after a sex-mismatched transplantation – we analyzed hematopoietic chimerism by cell sorting and bone marrow cultures. CD34-positive cells, CD4-CD8-positive cells, committed progenitors, and LTC-IC were of donor origin, as demonstrated by two-color fluorescence in situ hybridization (FISH). Additionally, in relapse complete donor T-cell chimerism was seen. In contrast, plasma cells were of recipient origin in the patient who had a relapse in the bone marrow. Both patients were treated with infusions of donor leukocytes from their original donor. Neither patient suffered myelosuppression, and one achieved a stable complete remission. Received: February 26, 1999 / Accepted: April 14, 1999     

Graft-versus-host disease after liver transplantation: A comprehensive literature review

AIM:To determine the factors affecting mortality in patients who developed graft-versus-host disease (GvH) after liver transplantation (LT) METHODS:We performed a review of studies of GvH following LT published in the English literature and accessed the PubMed, Medline, EBSCO, EMBASE, and Google Scholar databases Using relevant search phrases, 88 articles were identified. Of these, 61 articles containing most of the study parameters were considered eligible for the study. Risk factors were first examined using a univariate Kaplan-Meier model, and variables with a significant association (P 0 05) were then subjected to multivariate analyses using a Cox proportional-hazards model RESULTS:The 61 articles reported 87 patients, 58 male and 29 female, mean age, 40.4 ± 15.5 years (range:8 mo to 74 years), who met the inclusion criteria for the present study. Deaths occurred in 59 (67.8%) patients, whereas 28 (32.2%) survived after a mean follow-up period of 280.8 ± 316.2 d (range:27-2285 d). Among the most frequent symptoms were rash (94.2%), fever (66.6%), diarrhea (54%), and pancytopenia (54%). Theaverage time period between LT and first symptom onset was 60.6 ± 190.1 d (range: 2-1865 d). The Kaplan-Meier analysis revealed that pancytopenia (42.8% vs 59.3%,P = 0.03), diarrhea (39.2%vs 61.0%,P = 0 04), age difference between the recipient and the donor (14.6 ± 3.1 yearsvs 22.6 ± 2.7 years,P 0.0001), and time from first symptom occurrence to diagnosis or treatment (13.3 ± 2.6 mo vs 15.0 ± 2.3 mo, P 0.0001) were significant factors affecting mortality, whereas age, sex, presence of rash and fever, use of immunosuppressive agents, acute rejection before GvH , etiological causes, time of onset, and donor type were not associated with mortality risk The Cox proportional-hazards model, determined that an age difference between the recipient and donor was an independent risk factor (P = 0 03; hazard ratio, 7.395, 95% confidence interval, 1.2-46.7). CONCLUSION:This study showed that an age difference between the recipient and donor is an independent risk factor for mortality in patients who develop GvH after LT.     

Cellular repopulation of myocardial infarction in patients with sex-mismatched heart transplantation.

AIMS: Recent studies have suggested that human extracardiac progenitor cells are capable of differentiating into cardiomyocytes. In animal studies, myocardial infarction attracted bone marrow stem cells and enhanced their differentiation into cardiomyocytes. Based on these findings, we hypothesised that myocardial infarction stimulates the invasion of progenitor cells and their differentiation into endothelial and cardiac cells in the human heart. METHODS AND RESULTS: We compared autopsy samples from male control patients who had received a female donor heart with samples from such patients who developed myocardial infarction after transplantation. Fluorescence in situ hybridisation (FISH) for detection of the Y-chromosome was combined with immunofluorescence staining for CD45 and CD68 to distinguish host-derived inflammatory cells. Additionally, we used a 3D-confocal imaging technique to indisputably assign Y-chromosome-positive nuclei to their cytoplasm. In patients with myocardial infarction after heart transplantation (n=5), host-derived non-inflammatory progenitor and endothelial cells were significantly increased compared to non-infarcted patients (n=9). Yet, by using this novel multi-step approach, only 0.02% of all cells were estimated to be male cardiomyocytes and their increase in infarcted regions to 0.07% was not significant. CONCLUSION: Myocardial infarction enhances the invasion of extracardiac progenitor cells and their regeneration of endothelial cells. However, a significant differentiation into cardiomyocytes as a physiological mechanism of postischaemic regeneration does not occur in transplanted patients.     

Macrochimerism and clinical transplant tolerance

Current theory holds that macrochimerism is essential to the development of transplant tolerance. Hematopoietic cell transplantation from the solid organ donor is necessary to achieve macrochimerism. Over the last 10–20?years, trials of tolerance induction with combined kidney and hematopoietic cell transplantation have moved from the preclinical to the clinical arena. The achievement of macrochimerism in the clinical setting is challenging, and potentially toxic due to the conditioning regimen necessary to hematopoietic cell transplantation and due to the risk of graft-versus-host disease. There are differences in chimerism goals and methods of the three major clinical stage tolerance induction strategies in both HLA-matched and HLA-mismatched living donor kidney transplantation, with consequent differences in efficacy and safety. The Stanford protocol has proven efficacious in the induction of tolerance in HLA-matched kidney transplantation, allowing cessation of immunosuppressive drug therapy in 80% of study participants, with the safety profile of conventional transplantation. In HLA-mismatched transplantation, multi-lineage macrochimerism of over a year’s duration can now be consistently achieved with the Stanford protocol, with complete withdrawal of immunosuppressive drug therapy during the second post-transplant year as the next experimental step and test of tolerance.     

Preclinical and clinical studies for transplant tolerance via the mixed chimerism approach

Based upon observations in murine models, we have developed protocols to induce renal allograft tolerance by combined kidney and bone marrow transplantation (CKBMT) in non-human primates (NHP) and in humans. Induction of persistent mixed chimerism has proved to be extremely difficult in major histocompatibility complex (MHC)-mismatched primates, with detectable chimerism typically disappearing within 30–60?days. Nevertheless, in MHC mismatched NHP, long-term immunosuppression-free renal allograft survival has been achieved reproducibly, using a non-myeloablative conditioning approach that has also been successfully extended to human kidney transplant recipients. CKBMT has also been applied to the patients with end stage renal disease with hematologic malignancies. Renal allograft tolerance and long-term remission of myeloma have been achieved by transient mixed or persistent full chimerism. This review summarizes the current status of preclinical and clinical studies for renal and non-renal allograft tolerance induction by CKBMT. Improving the consistency of tolerance induction with less morbidity, extending this approach to deceased donor transplantation and inducing tolerance of non-renal transplants, are critical next steps for bringing this strategy to a wider range of clinical applications.