Total and active rabbit antithymocyte globulin (rATG;Thymoglobulin) pharmacokinetics in pediatric patients undergoing unrelated donor bone marrow transplantation.

Rabbit antithymocyte globulin (rATG; Thymoglobulin) is currently used to prevent or treat graft-versus-host disease (GVHD) during hematopoietic stem cell transplantation (HSCT). The dose and schedule of rATG as part of the preparative regimen for unrelated donor (URD) bone marrow transplantation (BMT) have not been optimized in pediatric patients. We conducted a prospective study of 13 pediatric patients with hematologic malignancies undergoing URD BMT at St. Jude Children's Research Hospital from October 2003 to March 2005, to determine the pharmacokinetics and toxicities of active and total rATG. The conditioning regimen comprised total body irradiation (TBI), thiotepa, and cyclophosphamide (Cy); cyclosporine (CsA) and methotrexate (MTX) were administered as GVHD prophylaxis. Patients received a total dose of 10 mg/kg rATG, and serial blood samples were assayed for total rATG by enzyme linked immunosorbent assay (ELISA) and active rATG by florescein activated cell sorting (FACS). We found that our weight-based dosing regimen for rATG was effective and well tolerated by patients. The half-lives of total and active rATG were comparable to those from previous studies, and despite high doses our patients had low maximum concentrations of active and total rATG. There were no occurrences of grade iii-iv GVHD even in patients having low peak rATG levels, and the overall incidence of grade II GVHD was only 15%. None of the patients had serious infections following transplantation. These data support the use of a 10 mg/kg dose of rATG in children with hematologic malignancies because it can be administered without increasing the risk of graft rejection, or serious infection in pediatric patients with a low rate of GVHD. These conclusions may not apply to patients with nonmalignant disorders.

Gene expression profile and overexpression of apoptosis-related genes (NGFI-B and Gadd 45 gamma) in early phase of Thy-1 nephritis model.

Mesangioproliferative glomerulonephritis (MPGN) is a disease of high incidence in humans. Rat Thy-1 nephritis (Thy-1 N), namely, anti-thymocyte serum (ATS)-induced nephritis, is considered to be an animal model for studying MPGN. Although previous studies have demonstrated that glomerular mesangial cell (GMCs) injury might be a feature of Thy-1 N, the mechanism of the disease (i.e., GMC apoptosis) remains unclear. We have examined the pathologic changes of GMCs and the gene expression profile of renal tissues in Thy-1 N. The pathologic changes of Thy-1 N include three phages: GMC apoptosis (40 min), necrosis (2 h), and proliferation (5 days). Many TUNEL-positive cells are found 40 min after administration of ATS. Concomitantly, 341 genes are up-regulated, whereas 392 genes are down-regulated, as shown by microarrays analysis. The mRNA and protein of two of the up-regulated genes (nerve growth factor induced protein I-B, NGFI-B; growth arrest- and DNA-damage-inducible protein 45 gamma, Gadd 45 gamma) in the GMC apoptotic phase of Thy-1 N are markedly elevated, as observed by real-time polymerase chain reaction and immunohistochemistry. Our data indicate that pathologic changes of Thy-1 N are involved in the abnormal gene profile. The overexpression of the NGFI-B and Gadd 45 gamma genes may be associated with GMC apoptosis of Thy-1 N.

Subcutaneous alemtuzumab vs ATG in adjusted conditioning for allogeneic transplantation: influence of Campath dose on lymphoid recovery, mixed chimerism and survival.

Sixty-nine consecutive patients (median age 54 years) were prospectively enrolled in a single-institution protocol for allogeneic transplantation with adjusted non-myeloablative fludarabine-melfalan-based conditioning including cyclosporin A and MMF, and one of three modes of serotherapy. Thirty-one donors (45%) were unrelated. The first cohort of 29 had ATG (Thymoglobulin 2 mg/kg x 3 days), the subsequent 26 had Campath 30 mg x 3 days subcutaneously, and the final cohort of 14 had 30 mg Campath once. The groups were similar as regards age, diagnosis and risk factors. Campath-patients had no acute toxicity, fewer days with fever and antibiotics, and required fewer transfusions than ATG-treated patients. 3-d-Campath patients showed lower lymphocyte counts from day +4, and CD4+, CD8+, CD19+ and NK cells recovered slower than in ATG-treated patients. More Campath patients developed mixed chimerism that required DLI. 3-d-Campath induced more serious and opportunistic infections than ATG, which resulted in a greater non-relapse mortality and an impaired overall survival despite a low tumor-related mortality. The change of the Campath dosing schedule to one dose abrogated the deleterious effect of 3-d-Campath on immune recovery, severe infections and survival. Subcutaneous Campath is simple and provides strong immune suppression with no early toxicity, but dose limitation to 30 mg once is recommended.

In vitro assessment of dose-dependent platelet activation by polyclonal antithymocyte globulins: a flow-cytometric analysis.

Polyclonal antithymocyte globulins (ATGs) are immunosuppressive drugs widely used in transplantation and hematologic disorders. Treatment with ATGs can induce side effects such as neutropenia and thrombocytopenia because of unspecific antibodies directed against nonmyeloid cells present in these preparations. Depletion, activation, and expression of adhesion molecules on platelets in vitro were studied in the whole blood of healthy volunteers by means of flow cytometry after incubation with different doses of three polyclonal ATGs. Our data show no ATG-mediated cytotoxic activity against platelets. ATGs are able to induce activation of platelets through increased expression of P-selectin and hLAMP-1 and higher percentages of gated thrombocytes expressing these molecules. Furthermore, increased expression of hLAMP-1 presented a dose-dependent pattern. ATGs induced activation and enhanced expression of adhesion molecules in unstimulated platelets. Increased adhesion may be responsible for undesirable side effects such as thrombocytopenia and reticulopenia.

The impact of antithymocyte globulin on short-term toxicity after allogeneic stem cell transplantation.

Antithymocyte globulin (ATG) is commonly used in allogeneic haematopoietic stem cell transplantation (HSCT). Little information is available, however, as to the optimal protocol for use and the side-effects occurring if ATG is administered in high daily doses (10-30 mg/kg). We report our experience with ATG Fresenius (ATG-F) in conditioning for allogeneic HSCT. During a period of 3 days, 47 patients received doses between 10 and 30 mg/kg either over 4 h preceded by 1-1.5 mg/kg prednisolone 30 min before the start of ATG-F (protocol A) or alternatively, over 12 h with 3-4 mg/kg prednisolone being administered before and 6 h after start of ATG (protocol B). During treatment with ATG-F, the side-effects observed included inflammation, disseminated intravascular coagulation, hyperdynamic circulation and renal dysfunction. Although these complications caused substantial morbidity, they were reversible within a few days. Side-effects were significantly more severe in patients treated according to protocol A than in those treated according to protocol B. As prolonged infusion of ATG-F does not reduce T cell clearance due to the long half-life of ATG-F, and since less cytokine release during conditioning might have beneficial long-term effects, we recommend administering ATG-F over 12 h preceded by high-dose steroid treatment.

Poor outcome in steroid-refractory graft-versus-host disease with antithymocyte globulin treatment.

Treatment of acute graft-versus-host disease (aGVHD) has relied on high-dose steroids, but less than 50% of patients show durable remission. Antithymocyte globulin (ATG) has become a standard salvage therapy. We now report our experience with ATG for the treatment of steroid-refractory GVHD in 69 patients treated from January 1, 1980, to May 1, 1999. Patients with GVHD were given an overall grade using standard criteria. Overall responses were similar to those in previously published literature. However, long-term survival for this group of patients was dismal. Of the 69 patients treated with ATG for steroid-refractory GVHD, only 3 (5%) are currently alive. The median survival of these patients by GVHD grade was 4.1 months for grade 2, 3.6 months for grade 3, and 2.7 months for grade 4. The age range of the surviving patients was 3 to 25 years. Only 5% of the deaths were due to relapse, with the remaining deaths due to GVHD, infection, and/or organ failure. In conclusion, ATG treatment can produce objective responses in patients with aGVHD, but these responses do not result in long-term survival. Given the poor survival rates of patients treated with ATG for steroid-refractory GVHD, treatment with ATG as standard therapy should be reconsidered. Patients with steroid-refractory GVHD should be enrolled in clinical study until there are data to support a standard salvage therapy.

Immune reconstitution following allogeneic stem cell transplantation in recipients conditioned by low intensity vs myeloablative regimen.

We have investigated the immune status of patients with hematologic malignancies treated with a low intensity conditioning in preparation for allogeneic stem cell transplantation. Conditioning consisted of fludarabine, anti-T lymphocyte globulin and low-dose busulfan, followed by infusion of allogeneic blood stem cells. This protocol resulted in rapid engraftment and complete replacement of host with donor hematopoietic cells. Immunological parameters of these patients were compared to those patients who were conditioned by an aggressive myeloablative regimen. Distribution of cell surface markers of lymphocyte subsets from both groups of patients was similar, but different from that of normal control cells. Reduced intensity or non-myeloablative conditioning prior to allogeneic stem cell transplantation (NST), hardly lowered the normal T cell-dependent mitogenic response even during the early period following transplant, while the myeloablative treatments resulted in a suppressed mitogenic reaction and in slow immune recovery. Reactivity of non-MHC restricted cytotoxic T cells was also at a normal level in patients who were treated with NST. We conclude that stem cell engraftment following reduced conditioning may result in early reconstitution of immune responses assessed in vitro. We hypothesize that clinical application of NST may lead to faster development of effective immune responses against residual host-type malignant and abnormal non-malignant hematopoietic cells, although the role of fludarabine on post-transplant infections remains to be investigated in a larger cohort of patients.

Intense immunosuppressive therapy followed by autologous peripheral blood selected progenitor cell reinfusion for severe autoimmune disease.

Autologous stem cell transplantation (HSCT) has been shown to be effective in curing a large spectrum of autoimmune disorders. Case reports are being collected in the EBMT/EULAR Autoimmune Disease Stem Cell Project registry, which reports transplant-related mortality (TRM) of 6%. In order to reduce TRM and preserve the anti-autoimmune effect we evaluated a more immunoablative as opposed to myeloablative conditioning regimen for the autotransplant of severe immunomediated diseases. We enrolled patients affected by systemic lupus erythematosus (SLE: 3 patients), by autoimmune thrombocytopenic purpura (AITP: one patient), by thrombotic thrombocytopenic purpura (TTP: one patient), by pure red cell aplasia (PRCA: one patient), and by a severe cryoglobulinemia (one patient). All patients were mobilized with cyclophosphamide (Cy) 4 g/m2 + G-csf. Conditioning regimen consisted of Cy 50 mg/kg/day (days -6 and -5); anti-T-globulin (ATG) 10 mg/kg/day and 6-methylprednisolone (PDN) 1 g/day (days -4, -3, and -2). Immunomagnetically selected CD34+ cells were re-infused on day 0. In three patients neutrophil count fell below 0.5 x 10(9)/l, while a PLT count below 20 x 10(9)/l was registered in two patients. Extrahematological toxicity was very low. Four patients (2 SLE, 1 TTP, 1 cryoglobulinemia) are in complete corticosteroid-free remission with a median follow up of 335 days. The third SLE patient improved considerably; however, he still needs low-dose corticosteroid maintenance. The AITP and PRCA patients achieved a CR but soon relapsed; nevertheless, the procedure restored a steroid-sensitive status. The use of this immunoablative conditioning regimen in auto-HSCT transplant was shown to be effective in controlling disease progression and could be a valuable strategy in reducing TRM.

Cyclophosphamide and antithymocyte globulin conditioning may be sufficient for Korean patients with early stage severe aplastic anemia transplanted with marrow from donors other than HLA-identical siblings.

We gave a regimen of cyclophosphamide and antithymocyte globulin (CY/ATG) to six patients with early stage severe aplastic anemia (SAA) transplanted with marrow from alternative donors. All patients engrafted and are alive with durable engraftment at a median follow-up of 406 days. The CY/ATG regimen may be sufficient in Korean patients with early stage SAA receiving marrow transplantation from alternative donors.

Treatment of steroid-resistant acute graft-versus-host disease with rabbit antithymocyte globulin.

Acute graft-versus-host disease (A-GVHD) is a life-threatening complication of allogeneic stem cell transplantation (SCT), and primary therapy consists of high-dose corticosteroids. Patients who fail to respond adequately to corticosteroids require salvage treatment, with anti-T cell antibodies being the most commonly utilized group of agents. We report our institution's experience treating steroid-resistant GVHD in 36 adult patients (median age 39 years, range 24-55) with a rabbit antithymocyte globulin product (thymoglobulin). Eleven patients had undergone sibling SCT (10 histocompatible, 1 one-antigen mismatched) and 25 patients had received unrelated donor bone marrow (17 matched, 8 one-antigen mismatched); 32 patients (89%) had grade III or IV A-GVHD. Thymoglobulin was administered in two different regimens; group 1 patients (n = 13) received 2.5 mg/kg/day x 4-6 consecutive days with maintenance of all other immunosuppressives. Group 2 patients (n = 21) were given the same dose of thymoglobulin on days 1, 3, 5, and 7 with discontinuation of cyclosporine for 14 days, during which the corticosteroid dose was held at 2-3 mg/kg/day. Two patients had severe adverse reactions to thymoglobulin (hypoxemia and hypotension) and could not complete treatment, however, in the other patients, aside from transient leukopenia (25%) and and hepatic dysfunction (25%), the antibody preparation was well tolerated. Of the 34 evaluable patients, 13 patients had a complete response (38%) and 7 patients (21%) had a partial response, for an overall response rate of 59%. Response rate was higher in group 1 patients (77%) compared to group 2 patients (48%), (p = 0.15); skin GVHD was more responsive (96% of patients) than gut GVHD (46% of patients) or hepatic GHVD (36% of patients). Opportunistic infections were a significant complication, with 11 patients developing systemic fungal infections and 9 patients serious viral infections; there were seven episodes of bacteremia following thymoglobulin treatment and one fatal protozoal infection. There were 9 patients (25%) who developed post-SCT lymphoproliferative disorder (PTLD) and 4 patients who had a relapse of underlying primary malignancy; none of these patients survived. Of the 36 patients entered on the study, only 2 patients (6%) survive, at 15+ and 34+ months post-unrelated donor SCT. Although thymoglobulin is associated with an impressive response rate when administered for advanced steroid-resistant GVHD, long-term survival is uncommon, even in responders, primarily due to the high risk of developing either an opportunistic infection or a PTLD.

Potency testing of anti-lymphocyte globulins: in vitro alternatives for the monkey skin graft assay.

Anti-lymphocyte globulins (ALG) are immunosuppressive agents of animal origin currently used in clinical organ transplantation and for the treatment of severe aplastic anaemia. The potency of each batch is tested in vivo using primates as recipients for allogeneic skin grafts. The two in vitro methods commonly used are (i) a cytotoxic assay and (ii) the rosette inhibition assay, both of which are evaluated by microscopy. In addition to animal welfare aspects, these methods require considerable experience, are difficult to validate, and the information as to the biological potency of the sera is questionable. The aim of our study is a better characterization of the biological properties of ALG in order to subsequently define an in vitro alternative for the potency test in monkeys. Several antibody specificities directed against functional molecules on T-cells, B-cells, NK-cells, macrophages as well as non-lineage specificities can be identified in competition assays with monoclonal antibodies. The cytotoxic capacity of ALG in the presence or absence of complement as well as DNA-fragmentation characteristic for apoptosis can be analysed by flow cytometry using propidiumiodide- (PI) incorporation. Immunoprecipitation of cell lysate with ALG and subsequent incubation with radioactive ATP (kinase-assay) shows specific bands which seem to be identical between different batches of one product.

Enhanced expression of membrane type-1 matrix metalloproteinase in mesangial proliferative glomerulonephritis.

Matrix metalloproteinase-2 (MMP-2, gelatinase A) is involved in the inflammatory and sclerotic events of glomerular diseases. Newly identified membrane-type matrix metalloproteinases (MT-MMP) have been shown to activate specifically proMMP-2. To date, several types of MT-MMP have been cloned; however, their expressions in glomerular diseases have not been evaluated. To investigate the role of MT-MMP in glomerular diseases, the glomerular gene expression and enzymatic activity of MT-MMP were examined during the time course of nephritis induced in rats by anti-Thy1.1 antibody injection. Both MT1-MMP and MMP-2 mRNA expression increased prominently 5 and 10 d after anti-Thy1.1 antibody injection and decreased thereafter, as assayed by semiquantitative reverse transcription-PCR. In contrast, there were no remarkable changes in the gene expression of MT2-MMP between normal and diseased tissue, and that of MT3-MMP was not detected in isolated glomeruli by reverse transcription-PCR analysis. The activation of proMMP-2 as analyzed by gelatin zymography correlated with the glomerular MT1-MMP gene expression, suggesting that proMMP-2 was activated by MT1-MMP. Protein and mRNA expression of fibronectin, one of the major mesangial matrix proteins and substrate of MMP-2, were also synchronized with MT1-MMP and MMP-2 expression. In situ hybridization revealed intense MT1-MMP mRNA expression in the proliferating mesangial cells. Interestingly, MT1-MMP gene expression exhibited a similar distribution as alpha-smooth muscle actin expression, which was closely associated with mesangial phenotypic change. These results suggest that among the newly identified MT-MMP, MT1-MMP may play the central role in activation of proMMP-2. Furthermore, the enhancement of MT1-MMP and MMP-2 expression associated with mesangial phenotypic change may contribute to the development of anti-Thy1.1 antibody-induced glomerulonephritis and remodeling of extracellular matrices.

Effect of anti-thymocyte serum on acquisition of resistance to infestation by Rhipicephalus appendiculatus larvae in rabbits.

Administration of specific goat anti-thymocyte serum (ATS) to rabbits, prior to a primary infestation by Rhipicephalus appendiculatus larvae, blocked the acquisition of resistance significantly only in the third infestation. The larvae which fed on these rabbits had higher engorgement masses than did those feeding on untreated control rabbits. Also, a higher percentage (92%) of larval ticks fed on these animals than on the controls (88%). ATS also induced a leucopenia due to a lymphopenia in the treated rabbits. It was concluded that a T-cell-dependent component might be involved in acquired resistance to infestation by R. appendiculatus.

Rapid cytotoxicity of human B lymphocytes induced by VH4-34 (VH4.21) gene-encoded monoclonal antibodies.

We have previously described two human cold agglutinin MoAbs 216 and A6(H4C5), that are derived from the VH4-34 (VH4.21) gene that bind specifically to a cell surface ligand on human B lymphocytes. In this study, we report that binding of 216 and A6(H4C5) leads to rapid killing of target B cells. This complement-independent cytotoxicity was measured by three independent assays, cell viability dye uptake on FACS, 3H-thymidine uptake, and the 3(4,5)-dimethylthiazol-2,5-diphenyl tetrazolium bromide (MTT) assay. Cytotoxicity was specific for CD20+ mononuclear cells in human spleen and peripheral blood. The MoAbs were also cytotoxic to human B cell lines Nalm-6, OCI-LY8, Arent and SUP-B8, but not to T cell lines HuT 78 and PEER. As observed by scanning electron microscopy, membrane pores were formed within 15 min of exposure to the MoAbs. Cytotoxic activity was dependent on MoAb concentration and temperature of exposure. Killing with greater at 4 degrees C than 37 degrees C. Sodium azide and EDTA did not block the cytotoxic activity. No DNA fragmentation typical of apoptosis was observed. This rapid cytotoxic activity, independent of physiologic cellular process and independent of complement, suggests a novel mechanism of all death via membrane perturbations.

Heparin decreases mesangial matrix accumulation after selective antibody-induced mesangial cell injury.

Anti-rat thymocyte antibody-induced injury of glomerular mesangial cells is characterized initially by lysis (1 h) and is followed by proliferation (beginning at 3 to 4 days), with resolution that can include a focal increase in mesangial matrix (by 28 days). Chronic administration (every 12 h) of heparin (anticoagulant or nonanticoagulant) resulted in a decrease in antibody-induced mesangial cell proliferation, which, in turn, was associated with a decrease in the size and number of areas of focal mesangial matrix increase. The effect could not be attributed to the effect of heparin on complement, to alterations in the small numbers of la-positive cells that characterize the lesion, or to binding of antibody to glomeruli. The beneficial effects of heparin in reducing mesangial cell proliferation, with a subsequent reduction in matrix increase, suggest that mesangial cell responses are a major element in the development of at least some forms of glomerulosclerosis. The possible mechanisms by which these effects of heparin may be achieved are discussed.

Beneficial and detrimental effects of RBC-adsorbed antilymphocyte globulin and prednisone on purified canine islet autograft and allograft function.

We have examined the effects of prednisone, cyclosporine, azathioprine, and RBC-adsorbed goat antidog antilymphocyte globulin on islet graft function in totally pancreatectomized canines with purified, quantitatively defined, autologous, or allogeneic islets transplanted to the liver. The objectives were twofold: (1) to determine the potential detrimental effects to islet autograft function of the aforementioned agents, and (2) to determine the relative efficacy of the "nontoxic" agents in prolonging purified islet allograft function administered in doses that would be considered tolerable in human. The islet autograft studies demonstrated that prednisone given in doses of 1-2 mg/kg/day had a detrimental effect on islet autograft function, and that the combinations of immunosuppression involving CsA, azathioprine, and ALG were not detrimental to islet autograft function to the extent that hyperglycemia would ensue. In the subsequent allograft studies, three groups of canines received islet transplants: (1) controls (n = 5; 7860 +/- 750 islets/kg/weight), (2) canines given CsA and azathioprine (n = 6; 6810 +/- 890 islets/kg/body weight), and (3) canines given CsA, azathioprine, and RBC-adsorbed goat antidog ALG (n = 8; 6540 +/- 710 islets/kg/body weight). The mean (+/- SE) day of rejection (serum glucose greater than or equal to 200 mg/dl) in the group of canine islet allograft recipients receiving CsA, azathioprine, and ALG was 11.8 +/- 1.4 days--significantly prolonged versus islet allograft recipients receiving no immunosuppression (mean survival 4.8 +/- 1.1 days, P less than 0.03), and versus allograft recipients receiving CsA/azathioprine without ALG (mean survival 4.4 +/- 1.4 days, P less than 0.05). Prednisone appears to be detrimental to islet graft function, even at low doses. ALG was not toxic, and significantly extended the survival of canine islet allografts. The inclusion of steroids as part of maintenance immunosuppression, or as treatment for acute rejection of islets, in human islet transplants should be reconsidered, whereas ALG or other antilymphocyte agents should continue to be used.