Anti-human T-lymphocyte Immunoglobulin Research Articles

In Vitro Effects of ATG-Fresenius on Immune Cell Adhesion

Introduction: ATG-Fresenius, a highly purified rabbit polyclonal antihuman T-lymphocyte immunoglobulin resulting from immunisation of rabbits with the Jurkat T-Lymphoblast cell line is currently used for the prevention of acute rejection in patients receiving solid organ-transplants. The aim of this study was to investigate the in vitro activity of ATG-Fresenius that underlies its activity in prevention of ischemia-reperfusion injury in solid organ transplantation. Material and methods: Human vascular endothelial cells (HUVEC) and peripheral blood mononuclear cells (PBMCs) were isolated from umbilical vein or peripheral blood, incubated 20 to 24 h before analysis of adhesion molecule expression and use in the ATG Fresenius-binding or immune cell adhesion assays. The cells were cultured alone or activated with TNF-alpha (HUVECs) or phytohaemagglutinin (PBMCs) for 20 h. HUVEC were incubated for 30 min at 2-8°C with 10 and 100 mg/mL ATG-Fresenius (Fresenius Biotech GmbH) or reference rabbit IgG (Fresenius Biotech GmbH). Analysis of adhesion of immune cells to endothelial cells used co-cultures of peripheral blood mononuclear cells (PBMC) and adherent HUVEC. Endothelial cell expression of the adhesion molecules CD62E and CD54 was determined by flow-cytometry. The relative amounts of T-, B- and NK-cells attached to HUVEC were also determined by flow cytometry (FACSCalibur). T cells were identified by CD3 expression, B-cells by CD19 expression, NK-cells by CD16 expression, and HUVEC by CD31 expression. Groups were compared using one-way analysis of variance (ANOVA) and Tukey-Kramer multiple comparison test Results: We show that ATG-Fresenius binds to endothelial cells, with a higher level of binding to activated endothelial cells that express increased levels of E-selectin and ICAM-1. The increased binding of ATG-Fresenius to cytokine-activated endothelial cells is consistent with its known binding to ICAM-1 and selectins. We also show that ATG-Fresenius inhibits adhesion of prestimulated immune cells to activated endothelium. Conclusion: Our in vitro results provide a rationale supporting of pre-reperfusion administration of ATG-Fresenius in solid organ transplantation.

Feasibility of tacrolimus, methotrexate, and prednisolone as a graft-versus-host disease prophylaxis in non-T-cell-depleted haploidentical hematopoietic stem cell transplantation for children

In this study, we evaluated the feasibility of our graft-versus-host disease (GVHD) prophylaxis with tacrolimus, methotrexate, and prednisolone in non-T-cell-depleted haploidentical hematopoietic stem cell transplantation (HSCT) for children. Twenty-one consecutive patients including those with hematological malignancies (n = 11), solid tumors (n = 7), and non-malignancies (n = 3) were analyzed. Myeloablative and reduced intensity conditionings were carried out in 5 and 16 patients, respectively, and both of the regimens contained anti-human T-lymphocyte immunoglobulin. Twenty (95%) of the 21 patients achieved primary engraftment. Acute GVHD of grades II-IV and III-IV were observed in nine (47%) and one (5%) patient, respectively, all of which were controllable by steroids. Chronic GVHD was observed in eight (51%) of the 17 evaluable patients, and one of them developed steroid refractory chronic GVHD. Treatment-related mortality occurred in three patients (15%), as a result of acute pancreatitis, chronic GVHD, and EB virus associated lymphoproliferative disease. The median follow-up of the 13 survivors was 24 months, and the two-yr probability of overall survival was 68%. The Karnofsky performance scale score of the 13 survivors was 100%. These results indicated the feasibility of our GVHD prophylaxis in non-T-cell-depleted haploidentical HSCT for children.

Renal Complications after Busulfan-Based Reduced-Intensity Stem Cell Transplantation in 286 Patients with Hematological Disorders.

Abstract 3353Poster Board III-241 [Background]Renal dysfunction is a life-threatening complication after hematopoietic stem cell transplantation, and the incidence of acute and chronic renal dysfunction after non-myeloablative transplantation is reportedly, 40 to 50% and 16 to 35%, respectively. In this study, we evaluated this complication after reduced-intensity stem cell transplantation (RIST) at a single institute. [Patients and Methods]We retrospectively reviewed the medical records of 286 patients (median age, 54 years; range, 21-68) with various hematological disorders who underwent RIST between 1999 and 2007, using a conditioning regimen that consisted of busulfan (oral 8 mg/kg or iv 6.4 mg/kg) and fludarabine (180 mg/m2, n=214) or cladribine (0.66 mg/kg, n=72). Sixty-seven patients also received 2-4 Gy of total body irradiation (TBI). The diagnosis included AML (n=77), ALL (n=9), MDS (n=56), malignant lymphoma (n=116), CML (n=14), and other (n=14). Whereas 188 patients were transplanted from a related donor (BM n=8, PB n=180), 98 were transplanted from an unrelated donor (BM n=80, PB n=1, CB n=17). GVHD prophylaxis consisted of cyclosporine (CSP, starting dose 3 mg/kg/day civ, target whole blood conc. 250-350 ng/ml, n=235) or tacrolimus (starting dose 0.03mg/kg/day civ, target whole blood conc. 10-20 ng/ml, n=51), with (n=131) or without (n=155) methotrexate, and 71 patients also received anti-human T-lymphocyte immunoglobulin (ATG, Fresenius, 5-10 mg/kg). Renal function was assessed in terms of the serum creatinine concentration and the estimated glomerular filtration rate (eGFR) as calculated by the Modification of Diet in Renal Disease equation (MDRD) for our population (eGFR=0.741*175*Age-0.203*Cr-1.154, *0.742, if female). Acute renal failure (ARF) within 100 days was categorized as grade 0 (decrease in eGFR of <25% of the baseline value), grade 1 (less than two-fold rise in the serum creatinine concentration with a decrease in eGFR of >25% but <50% of the baseline value), grade 2 (greater than two-fold rise in the serum creatinine concentration compared to the baseline value but not requiring dialysis), and grade 3 (same as grade 2 but requiring dialysis). Chronic kidney disease (CKD) was defined as two or more estimates of eGFR <60 ml/min/1.73 m2 within a minimum 30-day interval after day 100. Patients with eGFR <60 at baseline and/or less than 131 days of follow-up (n=121) were excluded from the CKD study cohort, which left 165 patients eligible for analysis. [Results]The median follow-up in surviving patients was 746 days (87-3291). The numbers of patients who developed grade 1, 2 and 3 ARF were 139, 72 and 9, respectively. The cumulative incidence of grade 2-3 ARF was 29 % at 100 days after RIST. The overall survival was significantly better in patients with grade 0-1 ARF than in those with grade 2-3 ARF (62% vs 42% at 2 years, p<0.0001, Figure 1). There was no significant difference in the occurrence of ARF between the cladribine and fludarabine groups (p=0.28). Multivariate analyses for ARF showed that TBI and CSP were significantly associated with a higher incidence of grade 2-3 ARF. CKD developed in 15 of the 165 evaluable patients (9.1%), and all had received PBSCT and CSP for GVHD prophylaxis. The cumulative incidence of CKD was 3.8% at 1 year, 9.0% at 2 years and 11.7% at 3 years after RIST. Grade 2-3 ARF (p=0.014) and the development of chronic GVHD (p=0.048) were significantly associated with a higher incidence of CKD. There was no significant difference in survival between patients with and without CKD (p=0.16). [Conslusion]We found that ARF was significantly associated with a higher mortality after busulfan-based RIST, and that preceding grade 2-3 ARF led to the occurrence of CKD in surviving patients, which highlights the importance of preventing ARF. This study also indicated that the incidence of acute and chronic renal complications was lower in our population compared to previously reported data from Western countries. Therefore, ethnicity should be taken into account when designing future international clinical trials. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Impact of T Cell Chimerism on Clinical Outcome in 117 Patients Who Underwent Allogeneic Stem Cell Transplantation with a Busulfan-Containing Reduced-Intensity Conditioning Regimen

Within the concept of reduced-intensity stem cell transplantation (RIST) there is a wide range of different regimens used, and little information is available on the clinical impact of chimerism status in patients conditioned with a busulfan-containing regimen. Therefore, we retrospectively reviewed lineage-specific chimerism and the subsequent clinical outcome in 117 patients (median age, 55 years; range: 29-68) who underwent busulfan-containing RIST. The conditioning regimen consisted of busulfan (oral 8 mg/kg or i.v. 6.4 mg/kg) and fludarabine (180 mg/m2, n = 64) or cladribine (0.66 mg/kg, n = 53), with or without 2-4 Gy total-body irridiation (TBI) (n = 26) or antihuman T-lymphocyte immunoglobulin (ATG; 5-10 mg/kg; n = 31). Chimerism was evaluated with peripheral blood samples taken on days 30, 60, and 90 after transplantation by polymerase chain reaction (PCR)-based amplification of polymorphic short tandem repeat regions. The median follow-up of surviving patients was 1039 days (153-2535). The percent donor-chimerism was significantly higher in granulocyte than T cell fraction throughout the entire course, and the median (mean) values were, respectively, 100% (96%) versus 95% (83%), 100% (98%) versus 100% (89%), and 100% (98%) versus 100% (91%) at days 30, 60, and 90 after RIST. In a multivariate analysis, having received <2 types of chemotherapy regimens before RIST was the only factor that was significantly associated with low donor T cell chimerism (<60%) at day 30 (hazard ratio [HR]: 6.1; 95% confidence interval [CI], 2.1-18.4; P < .01). The median percentage of donor T cell chimerism at day 30 was 9% (0%-63%) in 5 patients who experienced graft failure, which was significantly lower than that (97%; 15%-100%) in the rest of the patients (P < .01). No correlation was found between the kinetics of T cell chimerism and the occurrence of acute or chronic GVHD (aGVHD, cGVHD). The stem cell source and the addition of TBI or ATG were not associated with the degree of T cell chimerism, overall survival (OS) or event-free survival (EFS). In a Cox proportional hazard model, low donor T cell chimerism of <60% at day 30 was associated with both poor OS (HR: 2.2; 95% CI, 1.1-4.5; P = .02) and EFS (HR: 2.0; 95% CI, 1.1-3.8; P = .02). In conclusion, we found that 43% of the patients retained mixed donor T cell chimerism (<90% donor) at day 30, whereas 92% achieved complete chimerism in granulocyte fraction. Low donor T cell chimerism of <60% at day 30 may predict a poor outcome, and a prospective study to examine the value of early intervention based on chimerism data is warranted.