The Significance Of HLA-G And The Plasma Level Of Reg3α In Acute Graft-versus-host Disease Diagnosis And Treatment

This thesis will comprise two parts: Part I: Study on correlation between the plasmalevel of Regenerating islet-derived3-alpha and lower gastrointestinal acutegraft-versus-host disease; Part II: Part two Association of the expression of HLA-G withacute graft-versus-host disease after HLA-identical sibling hematopoietic stem celltransplantationPartⅠ Study on correlation between the plasma level of Regeneratingislet-derived3-alpha and lower gastrointestinal acute graft-versus-hostdisease【Objective】To explore the correlation of plasma Reg3α level with the lower gastrointestinal acutegraft-versus-host dease （LGI-aGVHD）. Expected to provide a reliable detectionbiomarkers for early diagnosis of LGI-aGVHD and severity of disease classification,suggesting that the effect of hormone therapy and prognosis.【Methods】1. The plasma was collected at multiple time points (d-9, d0, d14, d28,at the onset ofdiarrhea symptoms, at1and4weeks after GVHD therapy).2. The plasma concentrations of Reg3α were detected by enzyme-linkedimmunosorbent assay(ELISA).3. According to the severity of abdominal pain, diarrhea judges the occurrence ofaGVHD, and evaluate of anti-aGVHD treatment．4. Based on the occurrence of aGVHD and severity，the patients were divided into non-aGVHD group (17cases)greads, non-GVHD diarrhea group (27cases), Ⅰ-ⅡLGI-GVHD group (17cases), greads Ⅲ-Ⅳ LGI-GVHD group (32cases), simple skinaGVHD group (10cases).【Results】1. The plasma concentrations of Reg3α in patients with grades Ⅰ-Ⅱ, gradesⅢ-ⅣLGI-aGVHD group and non-aGVHD diarrhea group were52.9(40.6-90.2) ng/ml,124.1(87.1-211.6) ng/ml and23.9(14.5-89.5) ng/ml respectively with the difference beingstatistically significant (P=0.039and <0.001).2. The area under the ROC curve was0.902when plasma concentration of Reg3α wasset at87.73ng/ml. The sensitivity was81.48%and the specificity was82.86%when thecritical value was used in diagnosis of grades Ⅲ-Ⅳ LGI-aGVHD. The probability ofgrades Ⅲ-Ⅳ LGI-aGVHD had statistical difference above and below87.73ng/ml afterallo-HSCT(P<0.001).3. The plasma concentrations of Reg3α in17grades Ⅲ-Ⅳ LGI-aGVHD patients whohad no response to therapy at4weeks and7patients who experienced a complete or partialresponse were137.2(51.7-205.4) and679.4(122.3-896.8) ng/ml respectively with thedifference being statistically significant (P=0.028).【Conclusions】1.The high level of plasma Reg3α after transplantation could better predict gradesⅢ-Ⅳ LGI-aGVHD.2. Grades Ⅲ-Ⅳ LGI-aGVHD patients in the immunosuppressive treatment after fourweeks, the high level of plasma Reg3α protein indicates a poor prognosis.3. The plasma concentrations of Reg3α can be used as a specific biomarker ofLGI-aGVHD. PartⅡAssociation of the expression of HLA-G with acute graft-versus-hostdisease after HLA-identical sibling hematopoietic stem cell transplantation【Objective】To explore the correlation of HLA-G with acute graft-versus-host dease（aGVHD）after sibling bone marrow transplantation.Expected to HLA-G can be used as apre-transplant assessment of aGVHD risk factors.【Methods】1. The peripheral blood was collected at multiple time points (d-9, d14, d28).Collecting donor hematopoietic stem cells material2ml with heparin, extracting plasmaand mononuclear cells.2. The plasma sHLA-G concentrations of GVHD and non-aGVHD patients weredetected by enzyme-linked immunosorbent assay(ELISA).3. Detection mHLA-G expression of freshly peripheral blood by flow cytometry.4. HLA-G14bp polymorphisms were analyzed by PCR amplification of the entirecoding region followed by direct bidirectional DNA sequencing, defined as group del/del,group del/ins,group ins/ins. Real-time quantitative PCR was used to detect the relativeexpression level of HLA-G or between aGVHD and non-aGVHD patients.4. Clinical data were retrospectively analyzed in82patients received siblingmismatched HSCT. To analysis the relationship among the different genotypes andaGVHD.【Results】1. The plasma sHLA-G level of grades Ⅲ-Ⅳ LGI-aGVHD patients and no-aGVHDpatients were9.5(5.4-14.8)μg/L and19.5(9.4-21.5)μg/L respectively with thedifference being statistically significant,P=0.047. At the time of development ofaGVHD the plasma sHLA-G level of grades Ⅲ-Ⅳ LGI-aGVHD patientscontinued to decline compared with the28d. 2. The study failed to detect any HLA-G surface antigens in any freshly isolated celltested.3. The mRNA expression levels were significantly lower than the del/del and del/insgroup before and after transplantation.There were no differences between aGVHD andnon-GVHD.4. Analysis showed that there were no any association with aGVHD or grades Ⅲ-ⅣaGVHD among the14bp ins/ins, ins/del and del/del genotypes.【Conclusions】1. The plasma sHLA-G level of grades Ⅲ-Ⅳ LGI-aGVHD patients were lowercompared with no-aGVHD patients.2. The mRNA expression levels were significantly lower than the del/del and del/insgroup before and after transplantation. There were not statistically significant associationbetween the14bp ins/ins genotype and a lower risk of aGVHD compared to the14bpins/del or del/del genotypes.