Anti-lymphocytic Antibody Research Articles

SIGNIFICANCE OF MICROVASCULAR THROMBOSIS IN ALLOGRAFT KIDNEY: ROLE OF EX VIVO THROMBOLYTIC THERAPY

O326 Aims: The severe shortage of organs renders imperative the utilization of kidneys with microvascular thrombosis. This study examines the effects of ex vivo thrombolysis in such kidneys. Methods: Of 151 cadaveric kidney transplants performed between March 2000 and March 2002, 13 kidneys had histologic evidence of extensive disseminated intravascular thrombi. Donors averaged 25.5 years of age, with admission serum creatinine of 1.0 mg/dl, last 24 hour urine output 2100 cc. The causes of death were closed head trauma (2), open head injury (2) or intracerebral bleed (2). All kidneys wedge biopsies showed extensive intra glomerular thrombi but no cortical necrosis. Kidneys were flushed with 30-50 mg of activase, a recombinant plasminogen activator (t-PA) and stored in slush ice for 40 minutes. They were reflushed with Viaspan®. All but one were biopsied again prior to transplantation. CIT averaged 22 hours 14 minutes. Immunosuppression was obtained with simultaneous CSA, MMF and steroid. Antilymphocytic antibody was used in 7 patients. Methylprednisolone was used for rejection episodes. Results: All repeated biopsies showed complete resolution of fibrin thrombi. Neither post operative bleeding nor primary non function were noted. One kidney clotted on day 10. Three patients had ATN (25%). Current serum creatinine is 1.8 mg/dl after 24-48 months follow up. Graft survival is 92.2%. Conclusions: Back table t-PA thrombolysis can be used to open up partially thrombosed kidneys and makes them successfully transplantable with excellent long term survival and function.

Prognostic value of lymphocyte apoptosis in acute rejection of renal allografts.

Apoptosis is a cellular phenomenon generally found within rejecting transplants. It may play a role in physiological or therapy-associated deletion of infiltrating lymphocytes or in graft cell destruction. Our study focuses on apoptosis of infiltrating lymphocytes during acute kidney rejection after the initial steroid pulse therapy and on possible prognostic implications. Renal biopsy specimens of 23 transplant recipients with acute tubulo-interstitial rejection were examined for appearance of apoptosis and compared with 11 transplant biopsies with unspecific organ injury accompanied by lymphocyte infiltration. In all patients, biopsies were performed after steroid pulse therapy, and, after confirmation of rejection, antilymphocytic antibody treatment was carried out. Apoptosis was determined via terminal deoxynucleotidyltransferase-mediated dUTP-digoxigenin nick end labeling analysis and confirmed by electron microscopy. Apoptosis of lymphocytes or of tubular epithelium was detected in 11 cases of acute rejection (48%), respectively. Four biopsies showed lymphocytic as well as tubular apoptosis, whereas five sections showed no signs of programmed cell death. In biopsies revealing unspecific injury, tubular cell apoptosis was more frequently found (73%) compared with lymphocyte apoptosis (27%, P<0.05). Most interestingly, patients with a beneficial recovery from acute rejection had a higher proportion of lymphocyte apoptosis compared with patients with poor rejection outcome. The Bcl-2 oncoprotein was widely found within infiltrating lymphocytes without counter-regulating apoptosis. Lymphocyte apoptosis is found as frequently as tubular cell apoptosis in rejecting renal grafts after steroid pulse therapy and might have prognostic value for rejection outcome.

No B cell tolerance, but generation of antibody escape mutants in vivo in anti-lymphocytic choriomeningitis virus specific antibody transgenic mice

No B cell tolerance, but generation of antibody escape mutants in vivo in anti-lymphocytic choriomeningitis virus specific antibody transgenic mice

No B cell tolerance, but generation of antibody escape mutants in vivo in anti-lymphocytic choriomeningitis virus specific antibody transgenic mice

No B cell tolerance, but generation of antibody escape mutants in vivo in anti-lymphocytic choriomeningitis virus specific antibody transgenic mice

An improved assay for antibody dependent cellular cytotoxicity based on time resolved fluorometry

A new and faster assay for antibody dependent cellular cytotoxcity based on release of europium from target cells is described. This has a number of important advantages over the traditional assays based on release of chromium-51 ( 51Cr). The new method involves labelling of Wein 133 target cells (B cell non-Hodgkin's lymphoma cells) which express the antigen, CD w52, with the chelate europium diethylenetriaminopentaacetic acid (EuDTPA) according to the method of Blomberg et al. (1986). Labelled cells are sensitised (coated) with the anti-lymphocytic monoclonal antibody, Campath-1H. Human peripheral blood mononuclear cells are added to mediate lysis of EuDTPA labelled Wein 133 cells by ADCC. Release of EuDTPA from lysed cells is determined by mixing supernatants with enhancement solution containing 2-naphthoyl trifluoroacetone, 2-NTA, to form a highly fluorescent chelate which is measured using time resolved fluorometry. Results obtained with the new EuDPTA release assays were comparable to traditional assays based on the release of the radioisotope 51Cr. It is anticipated that this assay will have a widespread application among laboratories performing ADCC assays. The method is non-hazardous and has been used routinely for over 2 years to monitor production and purification of Campath-1H.

185.Specificity of anti-lymphocytic antibody and antigenicity of lymphoid cells

185.Specificity of anti-lymphocytic antibody and antigenicity of lymphoid cells

Surface membrane of lymphocytes. 1. Confirmation of antigenicity of lymphocytes.

Injection of serum or immunoglobulin, obtained after immunization of lymphocytes or thymocytes, caused lymphopenia either in normal rats or in RES‐blocked rats. Control rabbit serum, immunized with rat liver, caused leucocytosis instead of lymphopenia. The dose of administration was related to the degree of leucopenia.The presence of lymphocytic specific antigen determinants was confirmed with Ouchterlony agar plate method as well as other various heterologous antigens. Antilymphocytic antibody disappeared after absorption with lymphoid cells.Ferritin‐conjugated‐ALG were adsorbed along the plasma membrane of lymphocyte and did not infiltrate into the cytoplasm.

THE EFFECT OF ANTILYMPHOCYTIC ANTIBODY ON THE PRIMARY IMMUNE RESPONSE OF MICE TO SHEEP ERYTHROCYTES, BOVINE SERUM ALBUMIN, AND TYPE III PNEUMOCOCCUS POLYSACCHARIDE

The effect of antilymphocytic antibody on the primary immune response to sheep erythrocytes, alum-precipitated bovine serum albumin (BSA), and type III pneumococcus polysaccharide has been investigated in CBA/Ca, C3H/He, and A/HeJ strain mice. This material caused a highly significant suppression of the primary immune response to sheep erythrocytes and BSA in all three mouse strains. Its effect on the primary immune response to type III polysaccharide was less dramatic but, nevertheless, it significantly suppressed this response in CBA mice, indicating that our antilymphocytic antibody preparation did not readily discriminate between thymus-dependent and -independent humoral immune responses in this strain of mice.

Preparation of highly radioactive bovine anti-mouse lymphocyte antibody.

FOR use in humans the active components of anti-lymphocytic serum (ALS) must be sufficiently free of irrelevant substances, concentrated and non-toxic for it to be injected intravenously. With this in mind earlier investigations designed in the context of an experimental model1 were aimed at (1) preparing pure anti-lymphocytic antibody (ALSAb) from anti-lymphocytic globulin preparations (ALG) and (2) increasing the biological activity of such preparations by labelling the protein in them with a low energy gamma-emitting isotope (125I). It was hoped that the specifically localized irradiation would play a role in incapacitating the target lymphocytes1. ALS raised in rabbits is widely used, but many workers have reported considerable variation in the quality of ALS from individual rabbits. It would therefore be advantageous to raise ALS in larger animals such as cattle or pigs so that larger pools of material could be obtained. The earlier experiments with ALSAb prepared from rabbit ALS have been extended in the experiments described here, in which the effectiveness of purified bovine anti-mouse lymphocyte antibody (BALSAb) and 125I-BALSAb has been measured. A preliminary investigation has also been made of the possibility of using a column of thymocytes bound to ‘Sepharose’, for the preparation of BALSAb.

First Use of Antilymphocytic Serum

Excerpt To the editor: Last year the review Antilymphocytic Antibody Effects in Experimental Animals and Problems in Human Use by Stewart Sell appeared in the ANNALS(71:177-196, 1969). At the sam...

Effect of adjuvants on the immunosuppressive properties of antilymphocytic globulin.

IN recent years we have been primarily concerned with investigating factors affecting the immunosuppressive properties of antilymphocytic antibody. Detailed studies on humoral systems in rats have indicated that effective immunosuppression is dependent on the intactness of the antilymphocytic IgG molecule, the time of its administration in relation to the antigen, the strain of rats used and the antigen under test1. Because adjuvants are generally known to influence markedly the ontogeny of the humoral immune response, it seemed desirable to ascertain whether the ability of antilymphocytic antibody to suppress the humoral response (in this case, to bovine serum albumin) was also dependent on the adjuvant in which it was administered. These preliminary studies demonstrate that such a dependence exists.

In vitro studies on the heterogeneity of equine antibodies to human lymphoid tissue

The development of lymphocyte agglutinating, lysing and transforming activities in the serum of a horse undergoing prolonged immunization with human spleen have been investigated. The antibody activities of successive bleeds were assessed by standard in vitro immunological procedures and their molecular charaacteristeics were determined by G200 sephadex gel filtration. Anti-lymphocytic globulin was prepared from a selected serum sample and the distribution of the various activities in this sample, and the original serum, was extensively studied by a variety of physicochemical procedures. These included G200 sephadex gel filtration, gradient ultracentrifugation, DEAE cellulose chromatography and Porath column electrophoresis. The results indicate that both these samples contain a complex mixture of proteins with widely differing physiochemical properties and in vitro activities. Finally a preliminary assessement has also been made of the various methods currently used in preparing anti-lymphocytic antibody for therapeutic use.

Antilymphocytic antibody: effects in experimental animals and problems in human use.

The effects of antilymphocytic serum (ALS) and antilymphocytic globulin (ALG) in animals and in humans are reviewed. Antilymphocytic serum or ALG appear to affect: [1] the initial recognition of antigen by immunologically competent cells; [2] the expression of delayed hypersensitivity in previously sensitized individuals; [3] the induction of secondary responses to antigen (immunological memory); and [4] the production of circulating antibody, presumably by affecting precursors of antibody-forming cells. Antilymphocytic serum or ALG do not affect: plasma cells already producing antibody, the expression of immune reactions mediated by circulating antibody, or inflammation due to nonspecific initiation. Antilymphocyte globulin has been effective in suppressing delayed skin reactions and prolonging skin-allograft survival in humans. In view of the potential of ALG in prolonging allograft survival in human recipients, further controlled clinical trials and in vitro studies of antihuman lymphocyte sera are warranted. The experimental data now available can give considerable guidance to approach such trials.

Strain variations in the primary humoral response of rats and its susceptibility to inhibition by anti-lymphocytic antibody.

BECAUSE strain dependent differences in the primary humoral response of mice have been reported1–6, we have examined the primary humoral response of a number of inbred and random bred strains of rats and also determined the effect of anti-lymphocytic antibody on this process. The results of our preliminary studies indicate that, in the various groups of rats studied, marked variations occurred both in the primary immune response and in its susceptibility to anti-lymphocytic antibody inhibition.

Thein vivoAction of Anti-lymphocytic Antibody

It Is the general object of this paper to consider which of the many actions of anti-lymphocytic antibody contribute to its immunosuppressive action. However, it will be principally concerned with the kinetics of lymphopoiesis in mice receiving anti-lymphocytic antibody and the relevance of this activity to the resulting immunosuppression.

Large-scale Production of Anti-lymphocytic Antibody

The Wellcome Foundation is part of the M.R.C. Working Party on A.L.S. and the main role of the Foundation, as agreed with the Working Party as a whole, is to produce A.L.S. on a large scale for use in man.In Great Britain, the use of any new therapeutic agent is controlled by authority, in this case the Dunlop Committee. Before this committee could be approached for a licence to market such a product, it would be necessary for the pharmaceutical company concerned to have established conditions for large-scale production.

Purification of antilymphocytic antibody.

By elution from human lymphocytes it has been possible to obtain antilymphocytic antibody of much greater purity than before.

In vivo and in vitro effect of anti-lymphocytic IgG on sensitized spleen cells.

IT has been shown that treatment of a number of animals with species specific anti-lymphocytic antibody suppresses their primary immune response to a variety of antigens1. Cell transfer experiments have also shown that “non-sensitized” lymphoid tissue from mice treated with anti-lymphocytic antibody can exhibit a reduced immunological potential when transferred to suitably prepared recipients2–5. Furthermore, suppression of suspensions of sensitized mouse and rabbit lymphoid cells can also be achieved by incubating them in vitro with anti-lymphocytic antibody before transferring them to test recipients6,7. In order to confirm and extend these previous cell transfer results, preliminary experiments have been undertaken to determine the effect of anti-rat lymphocyte antibody treatment (in vivo and in vitro) on the humoral antibody producing potential of sensitized rat spleen cells on subsequent transfer to irradiated recipients.

Effect of F(ab′)2 from Rabbit Anti-mouse Lymphocyte IgG on the Graft versus Host Reaction in F1 Hybrid Mice

HETEROLOGOUS antisera raised against lymphoid cells (ALS) have been found to suppress a large variety of immune responses (see review by James1). In particular, lymphoid cells from mice which have been treated with ALS exhibit a diminished ability to produce graft versus host (GVH) reactions2–5. Such responses result from immunological interaction between lymphoid graft and the histocompatibility antigens of the host. One such reaction occurs when parental spleen cells are injected into adult F1 hybrid mice, and its occurrence and severity can be assayed by measurement of the resulting increase in size of spleen and liver6. As anti-lymphocytic antibody fragments seem incapable of suppressing humoral antibody formation7–10 and inhibiting allograft rejection11, we have investigated the effect of such preparations on the following GVH system.

Quantitative studies with antilymphocytic antibody.

Radioactive labelling has been used in measuring the amount of IgG taken up from antilymphocytic serum by lymphocytes, and the proportion of antilymphocytic IgG in various preparations of this immunoglobulin.