Rabbit antithymocyte globulin treatment in childhood acquired severe aplastic anemia.

Acquired severe aplastic anemia (SAA) is a life threatening bone marrow failure characterized by pancytopenia and hypocellular bone marrow. Matched sibling donor is not available for majority of the patients and many children receive immunosuppressive therapy (IST). Although horse antithymocyte globuline (ATG) is the preferred option, our patients received rabbit ATG; since horse ATG is not available in Turkey. We reviewed the medical records of children with SAA who were treated with rabbit ATG, cyclosporine, and granulocyte colony stimulating factor (GCSF) between 2006 and 2012. Fifteen children with SAA aged between 1.5 and 17 years received rabbit ATG as first line treatment. Only two of them showed partial response and the others did not give any response at 3rd, 6th, and 12th months after the first course of IST. The second course of ATG was given to 8 of the patients; Rabbit ATG at the same dosage was used for 3 of them, and others were given horse ATG. None of the patients responded to the second course of ATG. Invasive fungal infection (IFI) which was seen in 80% of the patients was the most significant problem. Overall survival rate was 60%. The median time between the diagnosis and initiation of IST was 57 (range; 29-144) days. This delay might be significantly contributed to unresponsiveness. In our series, the use of rabbit ATG was not effective for these patients as first line treatment modality. Response rate was very low and the incidence of fungal infections was very high in the SAA patients who received rabbit ATG.

Optimal detection of serum antipaternal antileukocytic antibodies after injection of allogenic lymphocytes in women with habitual abortions.

The level of antipaternal antileukocytic antibodies detected by flow cytometry is a parameter reflecting the efficiency of alloimmunization of women with reproductive disorders during preparation to pregnancy. The results of evaluation of antipaternal antileukocytic antibodies by two modifications of the method are presented. The optimal method for detection of antipaternal antileukocytic antibodies after immunocytotherapy is selected.

The mechanism of action of anti-lymphocyte serum. Studies of antibody eluate.

Studies designed to gain insight into the mechanism of action of the active component of antilymphocyte serum were carried out using an antibody eluate prepared from the IgG fraction of anti-lymphocyte serum by absorption and subsequent elution from thymocyte membrane. The resulting antibody eluate was labeled with radionuclide tracer to determine the fate of the antibody in vivo. The result indicated that anti-lymphocytic antibodies are eliminated from recipients extremely rapidly. The mechanism for this rapid clearance appears to depend upon the absorption of antibody molecules onto lymphocyte surfaces and the subsequent clearing and degradation of the antibody-lymphocyte complexes by the reticuloendothelial system. Distribution studies confirm that the major site of antibody-lymphocyte interaction occurs in the periphery with relatively little penetration of antibody within lymphoid organs. Radioautographic studies showed that the pattern of localization within lymphoid and other organs is confined to rather specific areas. These observations are believed to offer strong support for the notion that anti-lymphocyte serum achieves its immunosuppressive effect by bringing about a selective ablation of the population of recirculating lymphocytes.

Common epitope in human immunodeficiency virus (HIV) I-GP41 and HLA class II elicits immunosuppressive autoantibodies capable of contributing to immune dysfunction in HIV I-infected individuals.

We previously reported the identification of highly conserved homologous regions located in the carboxy terminus of the HIV I gp41-envelope (aa 837-844), and the amino-terminal of the beta chain of all human HLA class II antigens (aa 19-25). Murine monoclonal antibodies, raised against synthetic peptides from these homologous regions, bound not only to the isolated peptides, but also to the native gp160 and class II molecules. In this study one-third of sera from HIV I-infected individuals, at different disease stages, were found to react with both the gp41 and class II-derived peptides. These sera also reacted with "native" HLA class II molecules. The potential affects of such autoantibodies on normal immune functions were examined. It was found that in the presence of class II-cross-reactive (but not control) sera, the proliferative responses of normal CD4+ T cells to tetanus toxoid and allogeneic stimuli were markedly decreased. In addition, these sera could eliminate class II-bearing cells by antibody dependent cellular cytotoxicity. Similar affects were seen with affinity-purified IgG antibodies from patients' sera. Thus, the "molecular mimicry" between HIV I and HLA class II antigens, may lead to the generation of autoantibodies in HIV I-infected individuals that may contribute to the early functional impairment of CD4+ T cell observed in many HIV I-infected individuals.

Collaborative study to establish human immunoglobulin BRP batch 3 and human immunoglobulin (molecular size) BRP batch 1.

A study was carried out by the European Directorate for the Quality of Medicines (EDQM) as part of the joint Biological Standardisation Programme of the Council of Europe and the European Commission with the aim to establish replacement batches of the European Pharmacopoeia (Ph. Eur.) human immunoglobulin Biological Reference Preparation (BRP) batch 2. Twenty-eight laboratories participated in this study. The suitability of the candidate reference preparations to serve as working references in the tests for distribution of the molecular size, anticomplementary activity and Fc function, in accordance with the specifications of the Ph. Eur. monographs Human normal immunoglobulin for intravenous administration (0918), Human normal immunoglobulin (0338) and Anti-T lymphocyte immunoglobulin for human use, animal (1928) was demonstrated. The candidates were therefore established as human immunoglobulin BRP batch 3 and Human immunoglobulin (molecular size) BRP batch 1. The prescribed use of the latter BRP is limited to the test for distribution of molecular size.

Effect of thimerosal in leukemia, in leukemic cell lines, and on normal hematopoiesis.

Anti-thymocyte globulin (ATG), a horse antiserum to human thymus tissue, has been shown to induce granulocytic differentiation of the HL-60 human leukemia cell line. In this paper we describe the effect of ATG on leukemic blasts and its effect on other human leukemia cell lines in vitro. The in vitro differentiation effect of ATG was observed in blasts from two patients with leukemia and the human leukemia cell line K562. The differentiation effect of ATG was attributable to its preservative, thimerosal, separable from ATG by high pressure liquid chromatography or dialysis. Subsequent studies with thimerosal alone showed it to induce differentiation in leukemic blasts from three patients and the human leukemia cell lines U937, K562, and KG-1. The differentiation effect of thimerosal is blocked by a sulfhydryl-protective agent, dithiothreitol, suggesting that the mechanism of differentiation may be mediated via a sulfhydryl group-dependent process.

Removal of therapeutic anti-lymphocyte antibodies from human sera prior to anti-human leukocyte antibody testing.

Both monoclonal (e.g. Orthoclone (OKT3), rituximab) and polyclonal (e.g. ATGAM, Thymoglobulin (Thymo)) anti-lymphocyte Abs (ALAs) are used extensively in organ transplantation for immunosuppression induction, desensitization, and treatment of acute rejection. ALAs often interfere with post transplant immunologic monitoring. We describe a method that uses magnetic beads to selectively remove ALAs from patient serum. Rabbit anti-mouse Fc-specific (180 mug), or rabbit anti-mouse Fab-specific (180 microg), or rabbit anti-horse heavy and light chain-specific and rabbit anti-horse F(ab')2 (200 microg) (Jackson Immunoresearch) was adsorbed to 6.7 x 10(8) Dynabeads M-280 conjugated with sheep anti-rabbit IgG (Dynal Biotech). Fifty microliters of normal human serum (NHS) with 2 microg/ml of OKT3 or 100 microg/ml ATGAM, Thymo, or rituximab were incubated with conjugated beads for several incubations. NHS containing ALAs before and after treatment by the protocol were incubated with human lymphocytes and labeled with FITC-antibody to immunoglobulin of the species used to produce the particular ALA. Residual ALA was determined using flow cytometry. Average median channel for serum with or without ALA was 11.1 and 0.120, respectively for OKT3; 64.4 and 0.344 for ATGAM; 108.5 and 0.200 for Thymo; and 1022.5 and 11.4 for rituximab. Treatment lowered the median channel for serum with OKT3 to 0.103, 0.309 for ATGAM, 0.199 for Thymo, and 12.1 for rituximab. ALAs can be effectively removed from serum by the use of magnetic beads conjugated with Ab specific for ALA thereby permitting immunologic monitoring without interference.

Failure to demonstrate anti-lymphocytic antibody in serum of patients with AIDS.

Several studies have produced evidence for anti-lymphocytic antibodies (ALA) in AIDS. We attempted to demonstrate ALA by immunofluorescent flow cytometry. Normal human peripheral blood lymphocytes (PBL) and the T-cell line, CEM, were incubated with sera from patients with AIDS, patients with chronic HIV infection and HIV-seronegative blood donors. ALA were not detected in the AIDS sera with fluorescein isothiocyanate (FITC)-labelled rabbit anti-mu, anti-alpha or the F(ab)2 fragment of anti-human gamma. A small number of CEM cells (2%) fluoresced with either AIDS or normal serum. A larger proportion of PBL were immunofluorescent after serum treatment but there was no difference between normal and AIDS serum. We were able to detect ALA in the serum of patients with systemic lupus erythematosus with both CEM and PBL. In contrast, incubation of either CEM or PBL with some AIDS sera, and to a lesser degree normal sera, enhanced the binding of intact FITC-rabbit anti-gamma. Anti-gamma was not bound by CEM cells unexposed to human serum. The binding was blocked by rabbit immunoglobulin, demonstrable with CEM fixed in 1% formalin, and unrelated to the density of CD4 on CEM cells.

E-rosette inhibitory factor in sera from patients with mycosis fungoides.

Peripheral blood lymphocytes from some of patients with mycosis fungoides disease showed decreased ability to form rosettes with sheep erythrocytes. This decreased percentage of E-rosette forming cells could be normalized when those cells were incubated in culture for 20 hr. Since these data led us to considering a possible inhibitory factor present in patients' sera, we tested their ability to inhibit E-rosetting by T lymphocytes from normal donors, and found that sera from mycosis fungoides patients with low levels of E-rosetting blood lymphocytes showed greater inhibitory effect on E-rosette formation by normal T cells when compared to those either from normal donors or from mycosis patients who had almost normal levels of E-rosetting blood lymphocyte number. The E-rosette inhibitory factor was sensitive to 2-mercaptoethanol treatment and was copurified with serum IgM by ammonium sulfate precipitation and by sequential gel filtrations, suggesting that it might be an anti-T lymphocyte antibody naturally occurring during the disease process.

A simple and convenient method for producing anti-activated lymphocyte alloantisera which does not require prior absorption.

A BALB/c B cell lymphoma, 2PK-3, was found to be activation lymphocyte alloantigen (Ala-1)-positive by virtue of its ability to absorb out the cytotoxic anti-Ala-1.1-like antibody activity of an antiserum against activated BALB/c (Ala-1.1) lymphocytes. Injection of 2PK-3 cells from the spleens or ascites of lymphomatous BALB/c mice into H-2d compatible DBA/2 mice led to the rapid development of an antiserum with properties identical to anti-Ala-1.1 alloantisera. Thus, it did not significantly kill normal spleen cells from any one of 7 different strains tested, but killed over 85% of Con A- or LPS-stimulated spleen cells of 4 Ala-1.1 positive strains; activated lymphocytes of Ala-1.2 were not affected. Furthermore, these properties were seen with unabsorbed antisera. The results indicate that the Ala-1 phenotype may, in some cases, be retained on neoplastic lymphocytes and that such cells, being rapidly and easily obtained in vivo in large quantities, can serve as a convenient source of immunogen for the development of anti-Ala-1 alloantisera in certain donor-recipient combinations.

Methods for producing T- and B-lymphocyte receptor-specific antisera.

Spent tissue culture medium from two continuous lymphoblastoid cell lines, FL-74 and CT45-S, expressing the T-lymphocyte receptor for guinea pig E and the B-lymphocyte receptor for EAC respectively were used to produce receptor-specific antisera. Anti-E receptor sera blocked E rosette formation on FL-74 cells, canine and feline lymphocytes and canine and feline thymocytes but not EAC rosette formation by CT45-S cells or canine and feline lymphocytes. Anti-EAC receptor sera blocked EAC rosette formation on CT45-S cells and canine or feline lymphocytes. Absorption of antisera will the appropriate lymphoblastoid cell line removed E or EAC-blocking activity. The results of this study suggest that similar methods may be used to produce lymphocyte subpopulation-specific antisera in other species including man.

Induction of specific anti-guinea pig T cell sera in rabbits.

In an attempt to increase the specificity of antisera raised in rabbits against strain 2 guinea pig thymocytes and brain, the rabbits were screened for titres of natural antibodies to thymocytes and other lymphocytes. Although unimmunized rabbits commonly had moderate titres of cytotoxic antibodies to guinea pig thymocytes, occasional animals had low titres to thymocytes and moderate titres to bone marrow cells. Intravenous immunization of this latter group of rabbits with thymocytes led to the production of high titred anti-thymocyte sera which were easily made specific for thymus-derived lymphocytes (T cells) by absorption with L2 C lymphoma, a bone marrow-derived lymphoma of strain 2 guinea pigs. Sera raised against guinea pig brain in complete Freund's adjuvant which had high titres of antibodies to both thymocytes and bone marrow cells could be made specific for T cells only with great difficulty. The cytotoxic activity of the anti-T cell serum could be absorbed by strain 2 thymocytes and brain homogenates, while high dilutions of this serum inhibited the formation of spontaneous rosettes between guinea pig lymphoid cells and normal rabbit erythrocytes.

A simple method of labelling mouse Thy-1 antibodies with FITC.

Unlike antibodies produced in rabbits and other animals, mouse antibodies have proved to be difficult to covalently label with fluorochromes. By careful control of the labelling conditions, a method has been established for labelling mouse Thy-1 antibodies obtained by immunisation with normal thymus cells. The resultant FITC Thy-1 antibodies suffer little loss in cytotoxic properties, react with the correct specificity and can be used to label Thy-1 positive cells.

Preparation and characterization of antithymocyte serum and globulin without stem cell activity.

Immunosuppressive antimouse and antihuman thymocyte sera and antihuman T lymphocyte sera, raised in rabbits, inhibited colony formation in vitro by bone marrow stem cells. Absorption of the antisera with fetal liver, or isolation of the gamma-globulin from the antisera, removed the inhibitory effect on bone marrow stem cells but left the anti T lymphocyte activity unimpaired. Absorption with cultured lymphoblasts (B cell lines) failed to remove the affinity of the globulins for B lymphocytes.

The preparation and testing of antihuman lymphoblast globulin for clinical use.

Antibodies to cultured human lymphoblasts were raised in horses using a schedule employing both subcutaneous and intravenous routes of injection. Plasma from groups of horses was pooled and the IgG prepared from each pool was tested extensively for safety and immunosuppressive efficacy in vitro and in vivo. On the basis of the results of skin grafting in monkeys, only globulins derived from the first main bleeds were blended to produce a bulk for clinical use. One early pool of globulin was discarded because when undiluted, it was lethal in monkeys by the intravenous route, and another pool was discarded because it contained antibodies which bound to the glomerular basement membrane of rats. No signs of toxicity as judged by haematology, blood biochemistry, and histology were detected in monkeys receiving the clinical blend of globulin either subcutaneously or intravenously.

Cytotoxic endothelial cell antibodies in mixed connective tissue disease.

We examined the presence of anti-endothelial cell antibodies in the sera of 44 patients with mixed connective tissue disease (MCTD). Warm antibodies against endothelial cells were found in 45.4% of patients; the presence of these antibodies was positively correlated with anti-monocyte antibodies (P less than 0.01) but not with anti-lymphocytic antibodies. Strong correlations were found between the presence of these antibodies and abnormalities of pulmonary ventilatory capacity, neurophysiologic and myocardial function. Anti-endothelial antibodies were related to the high rate of spontaneous abortion noted in female MCTD patients. The identification of the antigen identified by MCTD sera in endothelial cells would help in understanding disease manifestations.

Attempts to isolation and characterization of autolymphocytotoxins from syphilitic sera.

To solve problem if autolymphocytotoxins (AL) present in syphilitic sera are not circulating immune complexes (CIC), sera containing strong AL activity and weak CIC were precipitated with polyethylene glycol (PEG). Received PEG-precipitates were filtrated on Bio-Gel A-1.5 column to separate CIC from AL. The Al were found in the first three after void volume fractions of the column. The fractions were examined on content of protein, sugar and sialic acid. The only correlation seen refers to the sugar and protein. The fraction which display the AL activity have the ratios of sugar to protein higher than non active fractions. The data indicated besides that AL is not CIC because it is not immunoglobulin nor Treponema pallidum antigen. For further biochemical characterization of these factors more material is needed.

Donor-specific antibodies. Clinical relevance of antibodies detected in lymphocyte crossmatches.

Positive donor crossmatches due to lymphocytotoxic antibodies generally have an adverse effect on allograft survival, but with numerous variations. Different laboratory techniques used to characterize lymphocytotoxic antibody, new antibody systems, such as those incited by endothelial/monocyte and epithelial antigens, the variable tempos and histopathologic changes of early rejections caused by these antibodies, and the specific features imposed by each organ, all combine to make the once simple crossmatch test a complex, clinical and laboratory crossroads, that directs the initial and perhaps ultimate course of the allograft. An integrated assessment of these factors is provided in this article.

Treatment of aplastic anemia with an investigational antilymphocyte serum prepared in rabbits.

The authors evaluated antilymphocyte serum prepared in rabbits (ALS-R) as an alternative to antilymphocyte serum prepared in horses (ALG-H) in the therapy of aplastic anemia. Between 1980 and 1993, 57 evaluable patients received ALS-R and prednisone +/- cyclosporine +/- androgens. Standard response criteria were used and patients were evaluated at 3 months from the start of therapy. Median age was 43 years. Disease was present for up to 2 months in 24 patients, 2-5 months in 14 patients, and 6 months or more in 19 patients. Disease was severe in 30 patients and moderate in 27. Responses occurred in 16 (28%) of 57 patients. Responses were more frequent in females, in patients treated within 6 months of diagnosis, and in patients with severe disease. Among patients receiving ALS-R and cyclosporine within 2 months of diagnosis, 46% responded. After ALS-R therapy, 20 patients received ALG-H; 8 (40%) of 20 responded. Eight patients receiving ALS-R previously had received ALG-H; 2 (25%) of these 8 patients responded. Toxicity of ALS-R was minimal. Antilymphocyte serum prepared in rabbits, in conjunction with other immunosuppressive agents, represents an effective alternative to ALG-H in aplastic anemia, especially in patients previously treated with ALG-H.

Characterization of IgG-containing DEAE fractions of canine serum.

A high-titered canine antilymphocyte serum was chromatographed on DE-52 cellulose and the fractions were assayed for cytotoxic activity. Although cytotoxicity was associated with the first 63% of the column effluent corresponding to fractions 1 through 7, significant activity (256) was demonstrable only in fractions 2, 3, 4, and 5. An immunoelectrophoretic study of fractions 1 through 5 showed only IgGa in fraction 1, IgGa and IgGb as electrophoretically separate arcs in fraction 2, and as electrophoretically inseparable arcs in fractions 3 through 5. Cytotoxicity is postulated to be associated with the IgGb subclass, since titers correspond with the distribution of IgGb in column fractions.