HLA配型新策略:融测序与构象于一体的RSCA系统

造血干细胞移植后可发生一系列的并发症 ,如移植物抗宿主病 (ＧＶＨＤ)和感染 ,约有 90 %的患者在移植后发生ＧＶＨＤ。其中对诱发ＧＶＨＤ及其程度影响最大的是供、受者的ＨＬＡ相合程度。目前 ,已成熟的 8～ 9种ＨＬＡ分型方法 ,基本上可分为两种类型。第一类是以鉴别ＨＬＡ基因序列不同为基础的ＨＬＡ分型技术 ,如血清学分型、序列特异性引物 (ＳＳＰ)ＰＣＲ、序列特异性寡核苷酸探针 (ＳＳＯＰ)分型技术以及单核苷酸序列分析 (ＳＢＴ)等。由于ＨＬＡ各等位基因之间存在序列互换现象 ,即同一序列可以出现在不同的等位基因中 ,这使得上述…     

他克莫司在重度急性移植物抗宿主病治疗中的应用

异基因造血干细胞移植是治疗恶性血液病及再生障碍性贫血等疾病的有效方法[1] ,但这种移植受到供、受者ＨＬＡ配型的限制 ,即使在ＨＬＡ完全相合的供、受者之间移植 ,仍然有 5 0 %左右的受者术后发生急性移植物抗宿主病(ＡＧＶＨＤ) ,而其中又有约一半的受者最终死于本病或本病的免疫抑制治疗。因此 ,ＡＧＶＨＤ是异基因造血干细胞移植的主要并发症 ,它的预防及治疗效果是移植成功与否的关键。近 1年来 ,我们将免疫抑制剂他克莫司 (ＦＫ5 0 6 )用于 5例难治性重度 (Ⅳ度 )ＡＧＶＨＤ的治疗中 ,取得较满意的结果。报告如下。一、资料与方法1.…     

异种移植中的移植物抗宿主病

目的 了解异种移植物抗宿主病（ＧＶＨＤ）的组织学特点,与同种ＧＶＨＤ的区别及异种脾脏移植的关系。方法 对近十年来有关异种移植中ＧＶＨＤ的特点及其与同种ＧＶＨＤ区别的文献进行检索、综述。结果异种ＧＶＨＤ由于共刺激信号、粘附分子的不匹配及所识别抗原的差异、其发生发展速度,组织学特点等均与同种ＧＶＨＤ不同。受体脾切除加异种脾移植有利于异种ＧＶＨＤ的发生。结论 可利用异种ＧＶＨＤ模型来观测异种移植排斥反应     

HLA-DR基因相容对肾移植长期存活的影响

目的 研究人类白细胞Ⅱ类抗原（ＨＬＡ－ＤＲ）基因相容对肾移植长期存活的影响。方法 采用基因分型技术,回顾性分析５１８例首次肾移植ＨＬＡ－ＤＲ基因相容性情况。结果 单个移植中心达到基因水平ＤＲ相配的受者超过１０Ａ％,半数以上可达１个ＤＲ相配。ＨＬＡ－ＤＲ相容的受者急性排斥反应显著减少,早期肾功能恢复顺利,１－５年人存活率提高１０％－２１．７％,肾存活率提高１７％－３７．７％,差异有显著性。     

慢性肾小球肾炎与HLA免疫遗传强关联

目的 评估人类组织相容性抗原（ＨＬＡ）在慢性肾小球肾炎所致肾功能衰竭病人中的免疫遗传学本质。方法 用单抗免疫磁珠ＨＬＡ－Ｉ类分型和ＰＣＲ－ＳＳＰ－ＨＬＡ－Ⅱ类分型技术，对广东地区２１９例汉族慢性肾小球肾炎病人和４０６例健康人进行ＨＬＡ－Ｉ，Ⅱ类抗原频率，基因频率，单倍型频率，风险系数等分析，结果 疾病组ＨＬＡ－Ｂ１３，Ｂ３５，Ｂ５５，ＤＲ７，ＤＲ１０，ＤＲ１２，ＤＱ７基因频率和Ａｌｌ－Ｂ６０，Ａｌ     

快速PCR-SSP-HLA-DRB分型与血清学分型的比较

为适应临床器官移植组织配型的要求，采用酚氯仿快速ＤＮＡ提取技术，建立起２．５小时完成的ＰＣＲＳＳＰＨＬＡＤＲＢ基因分型方法，并与血清学实验进行对照实验。结果显示，８５０份标本ＤＮＡ提取数量平均为１４．９μｇ，ＤＮＡ平均纯度为１．６；静脉血和腹腔血之间的ＨＬＡＤＲＢ１抗原频率无显著性差异，ＤＮＡ质量有显著性差异。结果表明此法具有快速、准确和适应腹腔血ＨＬＡＤＮＡ分型的优点     

同种脾移植治疗血友病甲五例报告

为５例重症血友病甲患者施行了同种全脾移植，３例为尸体供脾，２例为母亲供脾。１例因脾蒂扭转于手术当天丧失移植脾，余４例术后第Ⅷ因子（ＡＨＧ）显著升高。１例术后３４天因严重移植物抗宿主反应（ＧＶＨＲ）而切脾，另３例移植物有功能超过１年，其中１例至今已超过３年，生活正常。本组病例在数量和疗效上均居国际领先水平。结果提示，脾是产生ＡＨＧ的重要器官之一，脾移植（特别是亲属脾移植）有可能使血友病甲获根治，尸脾移植的排斥和ＧＶＨＲ强烈，是导致失败的主要原因，供受体术前预处理可预防和减轻排斥及ＧＶＨＲ。     

MHC基因胸腺转移诱导异基因小鼠皮肤移植免疫耐受

目的通过胸腺内注射质粒ＰＸＮ（Ｎ２Ｂ１９Ｈ２Ｋｂ）,表达外源性主要组织相容性抗原复合物（ＭＨＣ）抗原,以小鼠皮肤移植为模型,诱导移植免疫耐受,为免疫耐受提供理论和实验依据。方法应用逆转录病毒载体介导的基因转移技术,通过ＢＡＬＡ／Ｃ胸腺内注射质粒ＰＸＮ（Ｎ２Ｂ１９Ｈ２Ｋｂ）,将外源性ＭＨＣ基因转移到胸腺细胞,１４天后行异基因小鼠皮肤移植。应用聚合酶链反应（ＰＣＲ）,反转录聚合酶链反应（ＲＴＰＣＲ）,单克隆抗体免疫荧光染色流式细胞仪检测胸腺细胞ＤＮＡ,ｍＲＮＡ和ＭＨＣ蛋白质表达。结果外源性ＭＨＣ基因已整合到靶细胞染色体ＤＮＡ并有效地转录,胸腺细胞表面有外源性ＭＨＣ分子表达,转染效率为５％,胸腺内注射质粒ＰＸＮ（Ｎ２Ｂ１９Ｈ２Ｋｂ）能明显延长异基因小鼠的皮肤移植时间,平均２７天,对照为１０天（Ｐ＜０００１）。受体鼠脾细胞对ＣｏｎＡ的增殖反应在正常范围。结论供体鼠ＭＨＣ基因转移至受体鼠胸腺诱导了对供体皮肤移植的免疫耐受。     

表皮生长因子和维生素A对高浓度葡萄糖抑制腹膜间皮细胞增殖和分化的影响

目的　研究表皮生长因子（ Ｅ Ｇ Ｆ） 和维生素 Ａ（ Ｖｉｔ Ａ） 对高浓度葡萄糖抑制腹膜间皮细胞增殖和分化的影响，以探讨 Ｅ Ｇ Ｆ和 Ｖｉｔ Ａ 在腹膜修复中的作用。方法　采用胰蛋白酶消化法，从人腹膜组织中分离间皮细胞，并建立体外人腹膜间皮细胞（ Ｈ Ｐ Ｍ Ｃ） 培养模型；以３ Ｈ胸腺嘧啶核苷（３ Ｈ Ｔｄ Ｒ） 掺入法测定细胞内 Ｄ Ｎ Ａ 含量示细胞增殖程度；以免疫组织化学染色法，用图像分析仪测细胞表面角蛋白和波形蛋白（ｖｉｍｅｎｔｉｎ） 表达量示细胞分化程度。结果　随着葡萄糖浓度增高和培养时间的延长， Ｈ Ｐ Ｍ Ｃ３ Ｈ Ｔｄ Ｒ 掺入降低； Ｅ Ｇ Ｆ 和 Ｖｉｔ Ａ 可增加 Ｈ Ｐ Ｍ Ｃ 对３ Ｈ Ｔｄ Ｒ 的掺入，但两者ｃｐｍ 值比较无显著性差异（ Ｐ＞ ００５） ；在含高浓度葡萄糖的培养液中分别加入 Ｅ Ｇ Ｆ或 Ｖｉｔ Ａ 后， Ｅ Ｇ Ｆ 可明显促进 Ｈ Ｐ Ｍ Ｃ 的增殖，其３ Ｈ Ｔｄ Ｒ 掺入量明显增加，与 Ｖｉｔ Ａ 组比较有显著性差异（ Ｐ＜ ００５） 。 Ｅ Ｇ Ｆ 和 Ｖｉｔ Ａ 不影响细胞表面角蛋白和波形蛋白的表达。结论　 Ｅ Ｇ Ｆ和 Ｖｉｔ Ａ 对腹膜间皮细胞的分化未显示出明显作用，但两者均可刺激细胞的增殖，特别是 Ｅ Ｇ Ｆ可改善     

异基因外周血干细胞移植后后叶脑白质病性综合征一例

在我们的异基因外周血干细胞移植(Ａｌｌｏ ＰＢＳＣＴ)患者中 ,有 1例发生较为少见的可逆性神经系统并发症 ,报道如下。患者为女性 ,36岁 ,确诊为慢性髓性白血病 (慢性期 ) ,入院行Ａｌｌｏ ＰＢＳＣＴ。供者为其兄长。供、受者ＨＬＡ配型为不全相合 (一个Ｂ位点不合 )。预处理方案为白消安加环磷酰胺 ,以环孢素Ａ(ＣｓＡ)及短疗程甲氨蝶呤预防移植物抗宿主病 (ＧＶＨＤ)。ＣｓＡ术前 1ｄ开始使用 ,剂量为 3～ 4ｍｇ·ｋｇ-1·ｄ-1,血中ＣｓＡ的浓度维持于 15 0～ 2 30 μｇ/Ｌ。术后第 14ｄ发生Ⅳ度ＧＶＨＤ ,加用甲泼尼龙 (40～ 4 80ｍｇ…     

HLA不全相合骨髓移植治疗白血病五例

为探讨应用亲属中ＨＬＡ不全相合供体骨髓移植治疗白血病的疗效，选择性应用ＨＬＡ１～２个位点不合的血缘供体骨髓移植治疗白血病５例，并长期随访观察其远期疗效。结果５例患者中有３例无病存活１３４０、１１６０和９００天以上，恢复了正常的生活、工作和学习，１例慢性髓性白血病患者无病存活３年，于嵌合造血状态下复发，正在供体淋巴细胞输注治疗中，１例表兄弟间２个位点不合移植患者因Ⅳ度移植物抗宿主病和配型不合综合征致死。认为ＨＬＡ不全相合的亲属供体骨髓移植治疗白血病是有应用前景的，然而对移植物抗宿主病的发生应予以足够的重视。     

Relevance of HLA compatibility in living donor liver transplantation: the double‐edged sword associated with the patient outcome

HLA compatibility in living donor liver transplantation (LDLT) seems relevant to the acceptability of graft livers because LDLT recipients often share most or some part of HLAs with the respective donors. This study retrospectively investigated whether HLA compatibility affected the outcome of LDLT. Three hundred ninety LDLTs were performed in this hospital, and 346 pairs of HLAs (HLA‐A, B, DR) were retrieved from the medical record between October 1996 and March 2011. The dates of the deaths were censored when a recipient apparently died of or was retransplanted by other causes than graft failure because of host‐versus‐graft (HVG) response to purely analyze the outcomes of LDLT in view of HVG response. The relationship between HLA compatibility and graft‐versus‐host disease (GVHD) was also analyzed. No recipients with recipient‐against‐donor HLA mismatch (R→D MM) 0 experienced graft failure by HVG response. On the other hand, three of five recipients with “R→D MM 0” together with “donor‐against‐recipient MM 3” died of fatal GVHD. HLA compatibility in LDLT not only affected the long‐term acceptance of graft livers but also the risk of fatal GVHD.     

One-way donor-recipient HLA-matching as a risk factor for graft-versus-host disease in living-related liver transplantation

Abstract Although one-way matching between an HLA-homozygous donor and a haploidentical recipient is a recognized risk factor in transfusion-associated graft-versus-host disease (GVHD), its impact in living-related liver transplantation (LRLT) has so far not been investigated. We present a case of fatal acute GVHD in our LRLT program that was attributed to one-way HLA matching between donor and recipient. Although the disappearance of donor cells in peripheral blood was suggested by genetic analysis, severe septicemia led to a fatal outcome. We further reviewed 280 LRLT cases and correlated one-way HLA matching with outcome. A total of 8 out of 280 donors (2.9%) and 11 out of 278 recipients (4.0%) were completely HLA homozygous in our LRLT program. Complete one-way HLA matching linked to GVHD was observed in four cases, including the present case. Although other contributing factors also need to be clarified, one-way HLA matching is a definite risk factor for GVHD in LRLT. We advocate caution before proceeding with one-way HLA donor-recipient combinations.     

Evans' syndrome complicating chronic graft versus host disease after cadaveric liver transplantation

Acute graft versus host disease (GVHD) occurred in a patient after cadaveric liver transplantation from an HLA disparate donor. Immunosuppression resulted in a remission, but chronic GVHD with a scleroderma-like syndrome ensued. This was further complicated by immune hemolytic anemia and thrombocytopenia (Evan's syndrome). Semi-quantitative microsatellite analysis of circulating lymphoid cells showed that T cells were predominantly of donor origin, thereby explaining the chronic GVHD. The marrow hematopoietic cells remained recipient, so that the immune cytopenias were expected to be alloimmune in nature. However, the red cell antibodies were shown to have anti-C and anti-e specificity, with both the donor (R1R1) and recipient (R1r) possessing the C and e antigens. Therefore, the immune hemolysis might be considered both alloimmune and autoimmune. The patient finally died of sepsis. This case illustrates that chronic GVHD due to stable donor T cell engraftment may rarely occur in liver transplantation despite HLA disparity. Immunosuppression may result in dysregulation of T cell functions, leading to alloimmune and autoimmune problems.     

Neutrophils and lymphoid chimerism after adult living-related liver transplantation from a homozygous donor

Chimerism and graft-versus-host disease (GVHD) pose significant risks to liver transplant patients. The risk of chimerism and GVHD is higher among cases of living-related liver transplant (LRLT). Donors homozygous at all HLA loci carry a higher risk for GVHD. Herein we present a case of LRLT. The recipient suffered from end-stage liver disease and received a right lobe graft from his son. After 8 months posttransplant, the patient developed profound bone marrow depression. The patient was negative for CMV, Brucella, HHV6, HHV8, HBV, HCV, and parvovirus. No skin or GI signs of GVHD were noted. The patient and donor were HLA typed by SSP. The donor was homozygous for all HLA loci while the patient shared the class II homozygosity and was class I heterozygous. Chimerism studies were prompted after noting that the neutrophil compartment of the patient was homozygous for all HLA loci. This initiated further studies of the PMN and lymphocytes by microsatellite analysis. A total 15 microsatellites were analyzed. The results suggest that the majority (75%) of the PMNs and 45% of the lymphocytes were of donor origin. The patient was treated with G-CSF; his WBC counts returned to normal. At 2.5 years posttransplant the patient had not developed GVHD, despite the large number of donor lymphocytes circulating in his bloodstream. The only complaint he had was severe arthritis, which was treated with steroids. It must be investigated whether this was the result of GVHD.     

Fatal graft-versus-host disease after living donor liver transplantation: differential impact of donor-dominant one-way HLA matching.

Graft-versus-host disease (GVHD) is an uncommon but potentially devastating complication following liver transplantation. Recently, it was shown that use of a human leukocyte antigen (HLA)-homozygous donor leading to one-way HLA matching significantly increases the risk of GVHD after living donor liver transplantation (LDLT). However, the precise impact of HLA matching between donor and recipient on the risk of GVHD is not yet clear. We surveyed instances of fatal GVHD following LDLT in Japan and reviewed all 8 cases in detail, especially with respect to HLA matching. Serological typing showed that 7 of those cases had donor-dominant one-way HLA matching in the 3 loci of HLA-A, -B, and -DR, while one had donor-dominant one-way HLA matching in the 2 loci of HLA-A and -DR and identical alleles in the B locus. However, DNA typing revealed that the latter case had 1-way HLA matching in the 3 loci. Further, we analyzed HLA typing of 906 donor-recipient pairs who underwent LDLT. There were 5 cases with donor-dominant one-way matching in 2 loci and 2 with donor-dominant one-way matching in 1 locus. All of those cases except 1, who died from an unrelated cause, are alive without an obvious presentation of GVHD. In conclusion, our results suggest that the total number of loci with donor-dominant one-way HLA matching is important for determining the risk of fatal GVHD following LDLT, and that DNA typing of HLA alleles is indispensable in some cases to identify the true risk of donor-dominant 1-way HLA matching.     

Impact of amino acid substitution at residue 9 of HLA‐A2 on the development of acute GVHD in Korean pediatric patients receiving unrelated hematopoietic stem cell transplantation

Incompatibility of human leukocyte antigen (HLA) alleles between donors and recipients of unrelated hematopoietic stem cell transplantation (UHSCT) increases the risk of acute graft‐versus‐host disease (GVHD). We evaluated the positional effect of amino acid substitutions in HLA molecules on severe acute GVHD in Korean pediatric recipients of UHSCT. All of 64 donor–recipient pairs were serologically matched for HLA‐A, ‐B, and ‐DR loci. Only substitution at residue 9 resulting from an HLA‐A*02 polymorphism was significantly associated with the risk of severe acute GVHD in patients (OR = 7.0, P = 0.033) on multivariate analysis. Recipients of this mismatched HLA also showed shortened overall survival (HR = 9.7, P < 0.001) and increased risk for transplant‐related mortality (HR = 9.1, P = 0.027). Structural modeling showed that the amino acid substitution could alter the peptide preference of the ligand‐binding pocket. A single amino acid substitution at position 9 was a major predictor of severe acute GVHD in Korean pediatric patients.     

Unusual onset of chronic graft-versus-host disease after adult living-related liver transplantation from a homozygous donor

Graft-versus-host disease (GVHD) is a rare complication that occurs after living-related liver transplantation (LRLT). This condition usually occurs early after transplantation and is fatal. Although one-way matching between a human leukocyte antigen (HLA)-homozygous donor and a haploidentical recipient has been shown to be a significant risk factor for the development of acute GVHD, chronic GVHD has not been clarified. A 50-year-old woman underwent LRLT for primary biliary cirrhosis with her 32-year-old HLA-homozygous son as the donor. The patient developed chronic GVHD 114 days posttransplant, presenting with a skin rash only. Her atypical onset included neither fever nor gastrointestinal symptoms. The patient responded to corticosteroid therapy and is now doing well at home. Careful donor selection and HLA matching before LRLT should be performed to prevent GVHD. However, the risks associated with grafts from homozygous donors may be unavoidable at present because of the shortage of cadaveric donors in Japan.     

Recipient and donor factors influence the incidence of graft-vs.-host disease in liver transplant patients.

Acute cellular graft-vs.-host disease (GVHD) following liver transplantation has an incidence of 1 to 2% and a mortality rate of 85%. Our aim was to identify a patient population at high risk for developing GVHD using a large clinical database to study both recipient and donor factors. We compared our liver transplant patients who developed GVHD to those that did not for recipient and donor factors and combinations of factors. For 2003-2004 we had 205 first-time liver transplant patients surviving >30 days. From this group, 4 (1.9%) developed GVHD. Compared to the control group, there were no significant differences in recipient age, recipient gender, donor age, donor gender, total ischemia time, donor-recipient human leukocyte antigen (HLA) mismatch, or donor-recipient age difference. Percentages of liver disease etiologies among the patients who developed GVHD were as follows: 16% (1/6) autoimmune hepatitis (AIH) (P = 0.003), 5.6% (3/54) alcoholic liver disease (ALD) (P = 0.057), and 7.1% (3/42) hepatocellular carcinoma (HCC) (P = 0.026). The incidence of GVHD in patients with glucose intolerance (either Type I or Type II diabetes mellitus [DM]) was significant (P = 0.022). Focusing on patients only with high-risk factors for GVHD during the years 2003-2005, we had 19 such patients. Four of these high-risk patients developed GVHD. Three of these 4 patients had received a donor liver with steatosis of degree >or=mild compared to only 2 of the 15 high-risk patients who did not develop GVHD (P = 0.037). In conclusion, we have identified liver transplant patients with AIH or the combination of ALD, HCC, and glucose intolerance who receive a steatotic donor liver as being at high risk for developing GVHD.     

Prediction of graft versus host disease by frequency analysis of cytotoxic T cells after unrelated donor bone marrow transplantation

HLA "matched" unrelated donor bone marrow transplants are associated with an increased incidence and severity of graft-versus-host disease in comparison with HLA-identical sibling transplants. This is presumably due to HLA and non-HLA histocompatibility differences between donor and recipient. Using a limiting dilution assay, we have previously demonstrated a relationship between cytotoxic T lymphocyte precursor frequency and HLA disparity. In this study we have compared CTL-p frequencies with clinical GVHD, and demonstrate for the first time a significant correlation (P less than 0.005) between high CTL precursor frequency prior to BMT and severity of acute GVHD after HLA A, B, DR "matched" unrelated donor transplants using T cell depleted marrow. This assay system may be of value in the final selection of HLA "matched" unrelated donors for BMT.