| Background?-thalassemia major is a blood system disease, which known as hemolytic anemia, characterized by special thalassemia face, hepatosplenomegaly and increased hemoglobin. P-thalassemia major patients required blood transfusion and application to iron medication regularly. Allogeneic hematopoietic stem cell transplantation is the only way to cure thalassemia at present. The bone marrow transplantation of ?-thalassemia major compared with other disease is more difficult because?-thalassemia major patients effect of bone marrow cell rich characteristics of itself and repeated blood transfusions, as graft versus host disease is important factor in graft failure and transplantation related mortality. cytokine storm is one of the important reasons of various pathological graft target organ damage because of graft-versus-host disease,which caused by Condition and infection lead to gastrointestinal mucosal epithelial cell injury, the degradation products of bacteria such as lipopolysaccharide into the blood. Some research indicates that cytokines plays a critical role in the occurrence of graft versus host diseasedevelopment. IL-2 and TNF-? is the most studied cytokines. Miyamoto was first reported in third days after transplantation, sIL-2R level can predict acute graft-versus-host disease, Mathias reported that sIL-2R was associated with the pathogenesis and severity of acute graft-versus-host disease. Choi was measured in 438 patients with myeloablative pretreatment seventh days after transplantation of soluble TNFR1 levels increased more than 2.5 times the original, which increasing the risk of transplant related mortality,and related to ?-? degree of acute graft versus host disease. Berger.reported that higher tumor necrosis factor indicated increasing risk when graft-versus-host disease appear. Many studies suggest that elevated IL-10 may reduce the occurrence of graft-versus-host disease. Although researchs show that the relationship between cytokines and graft versus host disease closely, but there are a lot of cytokines and graft versus host diseaserelationship is not clear, at the same time, thalassemia patients with graft versus host disease may exist its particularity which is a non neoplastic diseases of Mediterranean anemia. Therefore, to understand the change of cytokines when graft versus host disease appears of?-thalassemia major after hematopoietic stem cell transplantation has the vital significance,for the prevention and treatment of graft-versus-host disease after hematopoietic stem cell transplantation and improve ?-thalassemia major patient's quality of lif. This research adopted Liquid chip technology to measure the changes of IL-6, IL-8, IL-12, TNF-?, MIF before and after graft-versus-host disease of ?-thalassemia major patients after hematopoietic stem cell transplantation,assessing whether it can be used as indicators for monitoring the occurrence and curative effect ofgraft-versus-host disease.Objects and Method:Objects:The study included 52 patients received hematopoietic stem cell transplantation in the Department of Pediatrics in Nanfang Hospital of Southern Medical University Between 2011 July to 2015 March, including 47 cases of thalassemia major,5 cases of not thalassemia developed GVHD.The patients were made to check the thalassemia gene, genotype for beta thalassemia gene homozygote or double heterozygote, included 27 male cases,20 female cases, the median age was 6.28 years (age range 2-14 years old). Before transplantation,47 cases of severe thalassemia children in domestic thalassemia clinical grading (based on age, liver and ferritin level is divided into three degrees), similar to the Pesaro index, including 4 cases of grade I,42 cases of second degree,1 case of third degree.24 cases were relative stem cell transplantation,23 cases were not relative stem cell transplantation,2 cases of bone marrow transplantation,29 cases of peripheral stem cell transplantation,7 cases of cord blood,4 cases of bone marrow plus cord blood,1 cases peripheral stem plus bone marrow,4 case of peripheral stem cells plus cord blood.41 cases were HLA identically matched (6/6,8/8 or 10/10), 3 patients who met HLA 8/10 matched,1 patients who met HLA 7/8 matched,2 cases of haploidentical matched. In 16 cases of acute GVHD,2 case of first degree, second degree in 4 cases,8 cases of grade III, IV in 2 cases.Conditioning Regimen:Mostly give myeloablative regimen based on cyclophosphamide/busulfan:Cyclophosphamide(Cy,50-60mg/kg/d,-10d?-9d); Busulfan(BU,2.8-4.4mg/kg/d,-8d?-6d);Fludarabine(Flu,40mg/m2/d,-8d-4d);Thiote pa(TT,10mg/kg/d,-5d);Rabbit anti-human T lymphocyte immunoglobulin (ATG, 5-10 mg/kg/d,-3d?-1d), or Thymoglobuline (2.5mg/kg/d,-3d?-1d)Prevention of GVHD:Use of cyclosporine A, mycophenolate mofetil combined with methotrexate in conventional prevented GVHD. GVHD diagnosis of Seattle in accordance with the diagnostic criteria. The treatment of GVHD:For those who have aGVHD of limit skin rash, ignore it or treated with topical cortisone ointments. But if there are such as rash area expanded rapidly, the severity of skin lesions, fever, flu like symptoms, or suspected of intestine or liver GVHD happens to give systemic treatment. The first-line therapy of acute GVHD:methylprednisolone 2mg/kg/d graded give or dexamethasone; Second-line therapy:Tacrolimus, mycophenolate mofetil, human T-lymphocyte rabbit immunoglobulin, CD25 antibody, anti lymphocyte globulin. Treatment of chronic GVHD line:methylprednisolone and cyclosporine use alternately;second-line treatment:azathioprine, tacrolimus, mycophenolate mofetil, cyclophosphamide,methotrexate therapy.Hematopoietic stem cell collection and transfusion:After completion of pretreatment, hematopoietic stem cell graft infusion in next day. For bone marrow stem cells plus cord blood stem cell transplantation,bone marrow stem cells transplantation input central vein as soon as possible in the day of transplantation as bone marrow hematopoietic stem vivo collected, the morning of the next day of infusion of umbilical cord blood stem cells. Infusion of mononuclear cell number for an average of 7.41 x 108/kg, infusion of CD34+ cell number for an average of 6.33 x 106/kg.Prevention of Complications:(1)GVHD prophylaxis:cyclosporine A was started at 1.5mg/kg/day i.v. from day -10 to day -2, increased to 3mg/kg/day on day-1 up to day 25, and subsequently administered orally at targeted concentrations of 200 ±50 ng/ml.The dose of CsA was tapered from day 60. Mycophenolate mofetil was administered on day 1 at 15mg/kg bid and was discontinued on day 30 if there were no signs of acute GVHD. Short-term methotrexate was administered on days 1,3, and 6 at 15,10, and 10 mg/m2, respectively. When GVHD occurred, we add methylprednisolone or immune inhibitors according to GVHD grading. (2) Hepatic veno-occlusive disease prophylaxis:Heparin and ursodiol were used for the prophylaxis of Hepatic veno-occlusive disease. The dose of heparin was gradually increased from 100 to 200 U/kg to maintain the levels of activated partial thromboplastin time that were slightly higher than normal. When Hepatic veno-occlusive disease occurred, diuresis treatment was used. (3) Hemorrhagic cystitis prophylaxis:hydration was applied before 6 hours preconditioning to maintain a higher 60-100 ml/m2,and mesna was applied when Cy was intravenous infused.Use bladder irrigation treatment if necessary. (4) Prevention of infection: clear dental necrosis and other focus of infection before transplantation. All patients were given preemptive prophylactic treatment for cytomegalovirus infection before entering the laminar flow ward. Itraconazole was used form the fifth day until the third day after granulocyte rose up to 0.5×109/L on three consecutive days. when infection occurred,we choose sufficient antibiotics based on experience.Observation index and definitions:(1) hematopoietic reconstitution:Myeloid engraftment was defined to occur on the first of 3 consecutive days in which the absolute neutrophil count (ANC) was >0.5×109/L. Platelet engraftment was defined as seven consecutive days of a platelet count of >20×109/L maintained without transfusion. (2) The GVHD was defined in accordance with the classification system of Glucksberg and Przepiorka. VOD was diagnosed with the Baltimore criteria. Engraftment of donor cells was assessed through the use of implanted evidence sex chromosome fluorescence in situ hybridization method (donor and recipient of the same sex) or short tandem repeat polymerase chain reaction (different donor recipient gender)regularly;Primary graft failure was defined as the absence of donor-originated hematopoietic reconstitution on day 28 after the allograft infusion. Graft rejection was defined to encompass thalassemia recurrence at any time after engraftment.Cytokine detection:Take 3 mL blood sample from peripheral vein for fasting in the morning(EDTA anticoagulant), standing at room temperature for 1 h, put to fridge at 4 "Cfor 1 h,3000 r/min, centrifuge for 10 min 3000 r/min use Ordinary centrifuge,and took 0.5 mL supernatan after centrifugation, keep in frozen 80? before measured. Measure cytokines by liquid chip technology: Operate strictly in accordance with the requirements of American company Bio.Padexperiments, using the company's detecting instrument and Bio-Plex cytokine reagent kit to purchase detection, and measure IL-6?IL-8?IL-12? TNF-??MIF level per 200 ?L serum samples. Data was analyzed using the Bio-Plex management software, can detect the levels of cytokines in range 0001-32 00 ng/L, the sensitivity of<10 ng/L.Statistical methods:The SPSS 19.0 software package was used for the analyses, data were expressed as x±s inter group compared with the Friedman M test, the comparison between the two indexes by using Spearman correlation analysis. P<0.05 is a significant test standard.ResultsThe study included 52 patients received hematopoietic stem cell transplantation, including 47 cases of thalassemia major,of which 16 cases developted GVHD,5 cases of not thalassemia developted GVHD, take specimen before the GVHD cytokine levels of basic value (TO), GVHD (Tl)appears, GVHD appeared fourth days (T2), GVHD appeared in seventh days (T3), GVHD symptoms completely disappeared (T4) changes in different phase IL-6, IL-8, IL-12, TNF-a, MIF level, The remaining 26 patients specimens take before transplantation specimen.1?The cytokines variation tendency in different time point before and after graft versus host disease for ?-thalassemia major There was significant in each phase of interleukin 6 in comparison to the level difference(P=0.001), the highest level appears when graft versus host disease occurred. There was significant in each phase of interleukin 8 in comparison to the level difference(P=0.001), the highest level appears when GVHD occurred fourth days. There was significant in each phase of interleukin 12 in comparison to the level difference(P=0.003), the highest level appears when GVHD occurre, followed by the GVHD appeared in seventh days. There was significant in each phase of TNF-a in comparison to the level difference(P=0.005), the highest level appears when GVHD occurre, followed by the GVHD appeared in seventh days. There was significant in each phase of MIF in comparison to the level difference(P=0.006), the highest level appears when GVHD occurre, followed by the GVHD appeared in seventh days.2?The cytokines variation tendency in different time point without graft versus host disease for ?-thalassemia major.Each phase of IL-6, IL-8, IL-12, TNF-, MIF level in comparison was not statistically significant (P> 0.05).3?The cytokines variation tendency in different time point before and after graft versus host disease for not ?-thalassemia major. There was significant in each phase of interleukin 6 in comparison to the level difference(P=0.005), the highest level appears when graft versus host disease occurred. There was significant in each phase of interleukin 8 in comparison to the level difference(P=0.017), the highest level appears when GVHD occurred fourth days. There was significant in each phase of interleukin 12 in comparison to the level difference(P=0.005), the highest level appears when GVHD occurre, followed by the GVHD appeared in seventh days. There was significant in each phase of TNF-a in comparison to the level difference(P=0.01), the highest level appears when GVHD occurre, followed by the GVHD appeared in seventh days. There was significant in each phase of MIF in comparison to the level difference(P=0.01), the highest level appears when GVHD occurre, followed by the GVHD appeared in seventh days.4. Correlation analysis:4.1?T0 phase:IL-6, IL-8, IL-12, TNF-alpha, MIF between the two groups had no significant correlation (P>0.05),4.2, T1 phase:there is a significant positive correlation between IL-6 and IL-12 (r=0.962,P=0.001), there is a significant positive correlation between IL-12 and TNF-alpha (r=0.73,P=0.01), there is a significant positive correlation between IL-12 and MIF (r=0.724,P=0.012), there is a significant positive correlation between TNF-alpha and MIF (r=0.804,P=0.003),4.3, T2 phase:there is a significant positive correlation between IL-12 and TNF-alpha(r=0.661, P=0.027),4.4, T3 phase:there is a significant positive correlation between IL-12 and TNF-alpha(r=0.981,P=0.001), there is a significant positive correlation between IL-12 and MIF(r=0.835, P=0.001), there is a significant positive correlation between TNF-alpha and MIF(r=0.782, P=0.004),4.5, T4 phase:there is a significant positive correlation between IL-6 and IL-12 (r=0.895,P=0.001), there is a significant positive correlation between IL-12 and TNF-alpha(r=0.816,P=0.002), there is a significant positive correlation between IL-12 and MIF (r=0.608,P=0.047), there is a significant positive correlation between TNF-a and MIF (r=0.734,P=0.01).Conclusion:l.The dynamic expression of IL-6?IL-8?IL-12?TNF-a, MIF can estimate the immune function of P-thalassemia major patients who develops graft-versus-host disease after allogeneic hematopoietic stem cell transplantation, and can be used as the immunobiology indicators for the early diagnosis of graft-versus-host disease.2. There was a serious aGVHD may lead to cGVHD more easily For related cytokines, The highest value of the number, the slower will them decline, prompting graft-versus-host disease, target organ damage more serious, and poor prognosis.3. Cytokines production and secretion have mutual regulation role, and IL-6, IL-8, IL-12, TNF-a, and MIF correlation with each other in different time of graft versus host disease. |
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