Lymphoma and methyldopa therapy.

I describe a patient who developed a drug associated lymphoma with methyldopa attributable to hypersensitive reaction. Several forms of immunologic changes have been observed with methyldopa therapy. In general, they have been considered to be hypersensitive changes from the common development of hemolytic anemia, lupus, retroperitoned fibrosis, thrombocytopenia, and hepatitis.

Immunogenicity studies of a synthetic antigen of alpha methyl dopa.

Since the idiosyncratic liver toxicity of methyl dopa (L-alpha-methyl-3,4- dihydroxy-phenylalanine) may be due to immune mediated mechanisms, immunologic tools are needed to detect methyl dopa induced antibody and antigen. Hapten (methyl dopa)--carrier (albumin) conjugates were synthesized to generate antibodies reactive with this drug. Studies were also conducted to test the immunogenicity of this hapten-carrier conjugate and its cross reactivity with methyl catechol and levodopa. Methyl dopa (MD), levodopa (LD) or methyl catechol (MC) were conjugated to rabbit serum albumin (RSA) under high pH (base) conditions or by a tyrosinase (tyr) catalyzed reaction. Under the base conjugation conditions, MD-RSA, LD-RSA and MC-RSA conjugates were produced at higher hapten: carrier ratios than conjugates produced by the enzyme catalyzed reaction. Rabbits were subsequently immunized with either MD-RSA(base) or MD-RSA(tyr). Antibodies elicited by MD-RSA(base) had marked reactivity to the carrier protein, albumin, whereas antibodies elicited by MD-RSA(tyr) did not. In addition, reactivity of anti-MD antibody was equal to or greater with MC-RSA than reactivity with either MD-RSA or LD-RSA. This work suggests that the conjugation method using the tyr catalyzed reaction produces the optimal immunogen with minimal modification of the carrier protein. In addition, the catechol moiety of MD, MC and LD appears to be the immunogenic epitope on these haptens.

Erythrocyte membrane proteins reactive with human (warm-reacting) anti-red cell autoantibodies.

Immunoglobulin G (IgG) autoantibodies of 20 patients with autoimmune hemolytic anemia (AHA) were used in immunoaffinity assays with surface-radioiodinated human red blood cells (RBCs), and detergent-solubilized products were analyzed by SDS-PAGE/autoradiography. Four membrane proteins were identified as candidate autoantigens: a nonglycosylated polypeptide with an apparent molecular mass of 34 kD (p34) that was expressed in all available RBC phenotypes except Rhnull but differed consistently in apparent molecular mass from the 32-kD Rh(D) polypeptide co-isolated by IgG allo-anti-D; a heterogenous 37-55-kD glycoprotein, also deficient in Rhnull RBCs, which disappeared after deglycosylation by N-glycanase, with the appearance of a sharp, new approximately 31-kD band distinct from p34 and from Rh(D) polypeptide; a approximately 100-kD major membrane glycoprotein identified by immunoblotting as the band 3 anion transporter; and glycophorin A (GPA), also confirmed by immunoblotting. GP37-55 was not seen in the absence of p34, and both proteins are likely to be members of the Rh family. Indeed, a 34-kD polypeptide band and 37-55-kD poly-disperse "smear," isolated concurrently from the same labeled RBCs by IgG allo-anti-e, were indistinguishable from their autoantibody-isolated counterparts and may well be the same protein identified at different epitopes by the auto- and allo-antibodies. Individual AHA patients' autoantibodies isolated p34 and gp37-55, alone or in combination with band 3 (nine cases); strong band 3 alone (five cases); and combinations of band 3 with GPA (six cases). The autoantibodies of three additional patients whose AHA had been induced by alpha-methyldopa also isolated p34 and gp37-55.

On the mechanisms of sensitization and attachment of antibodies to RBC in drug-induced immune hemolytic anemia.

The mechanisms of sensitization and attachment of drug-dependent antibodies to RBC in drug-induced immune hemolytic anemias are largely speculative. Nomifensine has been incriminated in causing immune hemolysis in a large number of patients. The hemolysis was usually of the so-called immune complex type, less commonly of the autoimmune type, and more surprisingly, few patients had developed both types of hemolysis. To determine whether nomifensine (metabolite)-dependent antibodies (ndab) exhibit specificity for antigenic structures of RBC membranes, 30 ndab were tested against large panels of RBC with common and rare antigens. We found that only 14 out of 30 ndab were invariably reactive with all cells tested. Nine antibodies were, similar to the majority of idiopathic or drug-induced autoantibodies, not or only weakly reactive with Rhnull RBC. Three antibodies did not react with cord RBC and could be inhibited by soluble I antigen. The remaining four antibodies gave inhomogeneous reaction patterns or were even negative with selected RBC; their specificity could not be identified. On a Scatchard plot analysis of one ndab, a maximum of 173,000 drug-dependent antibodies of the IgG class can specifically bind per RBC in the presence of the drug. Although nomifensine and its metabolites do not attach tightly onto RBC, our results clearly indicate that RBC do not act as "innocent bystanders," but rather serve as a surface for a loose attachment of drugs that possibly cause a subtle structural change in the cell antigens and, by this means, allow in vivo sensitization; and a specific binding of the resultant antibodies. This concept would explain why these antibodies can be directed against drug-cell complexes, against cell antigens alone (autoantibodies), or against both in the same patient.

Antibody mediated hepatocyte injury in methyl dopa induced hepatotoxicity.

To investigate the mechanisms underlying the hepatotoxicity induced by methyl dopa, we have examined sera from nine patients with liver damage following the use of the drug for evidence of sensitisation to drug altered liver cell membrane antigens using both immunofluorescence and antibody dependent cell mediated cytotoxicity. Five sera induced significant cytotoxicity to hepatocytes isolated from rabbits pretreated with methyl dopa after exposure to the mixed function oxidase inducer, Arachlor 1254. Sera from 10 patients on methyl dopa but with normal liver function and 32 patients with other drug and viral induced liver damage, gave normal cytotoxicity values. Two of the antibody positive sera gave a specific immunofluorescence pattern at the periphery of human hepatocytes when tested on liver biopsy specimens taken from patients taking methyl dopa. These findings are consistent with the view that immune mechanisms directed against drug associated antigens are involved in severe liver damage from methyl dopa administration and that metabolic activation of the drug is implicated in the generation of drug associated antigen. The need for a combination of immune and metabolic factors may explain the rarity of this condition.

Induction of direct Coombs positivity with alpha-methyldopa in chimpanzees.

Two of four chimpanzees developed a moderately strong positive direct Coombs test after injection of stroma of their autologous erythrocytes that had been treated with alpha-methyldopa in the presence of diethyldithiocarbamate. Similar experiments with rabbits were unsuccessful. The results are concluded to be a demonstration that the first step in the development of autoantibody in alpha-methyldopa therapy is interaction of the oxidized drug with certain, as yet unidentified, peptides of the erythrocyte membrane.

Autoimmune hemolytic anemia.

In autoimmune hemolytic anemia, individuals produce antibodies directed against one of their own erythrocyte membrane antigens. The hemolysis in autoimmune hemolytic anemia is most commonly extravascular rather than intravascular, and the liver and spleen play a major role in the clearance of the antibody-coated cells. The importance of complement in the destruction of IgM-coated cells has also been recognized.

Problems in pre-transfusion tests related to drugs and chemicals.

Ingestion of drugs can cause patients or blood donors to have a positive direct and sometimes indirect antiglobulin test. The most common cause of these positive reactions and immune hemolytic anemia due to drugs is the formation of red cell autoantibodies. These autoantibodies will react with the patient's own red cells and usually most other normal red cells in vitro without the drug being present. The prototype drug causing this type of reaction is alpha methyldopa (Aldomet). Other drugs cause positive antiglobulin tests by three different mechanisms, the drug antibodies reacting with red cells in vitro only in the presence of the drug. The first of these mechanisms causes positive reactions because the drug binds firmly to the red cell membrane, and antibody against the drug will combine with the drug on the membrane leading to IgG-sensitized red cells. The prototype drug for this mechanism is penicillin. The second mechanism involves chemical modification of the red cell membrane by the drug so that it takes up many proteins nonspecifically; the cephalosporins are the only group of drugs known to react in this fashion. The final mechanism involves the formation of an immune complex by the drug and its specific antibody. This immune complex will attach to cell membranes, usually activating complement in the process. Examples of drugs thought to operate by this mechanism are phenacetin, quinine, and quinidine. Some individuals have antibodies present in their serum that will react with chemical added to commercial blood bank reagents. Examples of these are antibodies to dyes added to ABO typing sera, antibodies to sodium caprylate in bovine albumin, and antibodies to chemicals added to red cell diluents, e.g., chloramphenicol, neomycin, and hydrocortisone. If these antibodies are present they can create problems in pretransfusion testing; in particular, they can present anomalies in ABO, Rh grouping, and antibody detection.

Retroperitoneal fibrosis during treatment with methydopa.

Retroperitoneeal fibrosis (R.P.F.) and a positive direct Coombs test developed in a patient who had received 1.4 kg. of alpha-methyldopa over a period of 5 years. Immunofluorescence microscopy demonstrated deposits of IgG, IgM, and IgA on the collagen fibres of the R.P.F. Biopsy speciments of the temporal artery, the right kidney, and the R.P.F. did not show signs of general arterial disease on light, immunofluorescence, and electron microscopy. Drugs which provoke autoimmunisation and interfere with nervous transmission are known to induce deposition of collagen or R.P.F. This suggests that R.P.F. in this patient was probably caused by alpha-methydopa.

Ultrastructural mapping of methyldopa and anti-D IgG erythrocyte antigen receptors.

The ultrastructural distribution pattern and site density of alpha-methyldopa immunoglobin G (alpha-MD IgG) on the red cell membrane was observed and compared with that of anti-D IgG, with ferritin-conjugated rabbit anti-human IgG and [125I]anti-D. alpha-MD IgG binds to all common types of human red cells, both Rho (D) positive and negative, to give a random, aperiodic distribution pattern grossly indistinguishable from the red cell D receptor site pattern. alpha-MD IgG inhibits the binding of [125I]anti-D to D-positive red cells when the reaction is controlled with respect to total reaction volume, ionic strength, and the appropriate concentrations of the two IgG reactants. To determine if a alpha-MD IgG binds to the D-antigen receptor, D-positive red cells were sensitized with alpha-MD and [125I]anti-D IgG spearately and with both IgG preparations. The cell-bound radioactivity served to identify what proportion of the total ferritin-labeled IgG sites were due to anti-D. With nonsaturating concentrations of anti-D the number of IgG sites observed was equal to the sum of the sites found when the red cell was sensitized separately with alpha-MD and anti-D IgG. With saturating concentrations of anti-D there was a reduction in the expected number of IgG sites, indicating that alpha-MD IgG was excluded from binding. There was no comparable interaction of alpha-MD IgG and anti-D IgG when D-negative red cells were used. The results obtained indicate that alpha-MD IgG does not bind to the D antigen. The interaction between alpha-MD IgG and anti-D IgG for binding sites on the red cell membrane may be due to the close physical proximity of the two receptors, so as to produce steric hindrance in binding of the two IgG preparations when both are present. The alpha-MD IgG receptor appears to be a part of the Rh antigen complex that occurs in both D-positive and D-negative red cells and probably contains receptors for other types of warm-antibody immune hemolytic anemias.