Nicholas C. Palczuk

Preparation of a non-immunogenic arginase by the covalent attachment of polyethylene glycol.

Methoxypolyethylene glycol of 5000 daltons (PEG) was attached covalently to bovine liver arginase using 2,4,6-trichloro-s-triazine as the coupling agent. The conjugate (PEG-arginase), with PEG attached to 53% of the amino groups, retained 65% of its original enzymatic activity. Mice were injected intravenously with arginase or PEG-arginase for periods of one to three months. The blood-circulating life of PEG-arginase was greatly extended over that of arginase. The half-life of injected arginase at day 30 was less than 1 h, whereas that of the PEG-enzyme was 12 h. Antisera from mice injected with native arginase reacted against arginase but not against PEG-arginase when tested by immunodiffusion. Antisera from animals injected with PEG-arginase reacted neither with native arginase nor PEG-arginase. The data indicate that arginase modified by PEG has been rendered both non-immunogenic and non-antigenic when tested in mice. The injection of PEG-arginase into mice did not induce tolerance toward the native enzyme. Injected PEG-arginase, in the presence of precipitating antibody directed against native arginase, circulated at the same level as in virgin animals. The attachment of PEG to arginase altered its kinetic properties.

Properties of two urate oxidases modified by the covalent attachment of poly(ethylene glycol)

Poly(ethylene glycol) of 5 000 daltons has been attached covalently to preparations of urate oxidase (urate: oxygen oxidoreductase, EC 1.7.3.3) from hog liver and Candida utilis. Attachment of sufficient poly(ethylene glycol) to either urate oxidase renders the enzyme incapable of eliciting antibody production in mice, or of reacting with antibodies to the unmodified enzyme. The poly(ethylene glycol) : urate oxidase conjugates exhibit higher Km and lower V values than the unmodified urate oxidases. Optimal pH values are increased for the poly(ethylene glycol) : urate oxidases, and optimal temperatures are decreased. The blood circulating lives of the modified urate oxidases following intravenous injection are much longer than those of the unmodified urate oxidases: repetitive injections over a period of 90 days dd not alter the blood circulating lives of the poly(ethylene glycol) : urate oxidases. The unmodified enzymes, on the other hand, were cleared from the blood with extreme rapidity after a few intravenous injections.

Induction of tolerance in mice by uricase and monomethoxypolyethylene glycol-modified uricase

The ability to induce tolerance to uricase by the administration of native uricase, and uricase modified by the covalent attachment of monomethoxypolyethylene glycol (PEG) was examined. Uricase, and uricase with PEG attached to 35% (PEG-uricase 35%) and 70% (PEG-uricase 70%) of available amino groups were found to induce tolerance in mice not previously sensitized to uricase. There was a dampening of the IgG, IgE and IgM antibody response to uricase which persisted even after a second sensitizing dose of uricase was administered to these animals. PEG-uricases were found to have little or no immunogenicity when injected into mice and a reduced immunogenicity and antigenicity when tested in rabbits. Native uricase, however, was found to be immunogenic and antigenic in mice and rabbits. Mice sensitized to native uricase were injected with uricase, PEG-uricase 35% or 70% to induce tolerance. After a second sensitizing injection of uricase, circulating levels of IgE, IgG and IgM were measured. All three enzymes induced tolerance in the IgE class of antibody but there was no significant change in the hemagglutinating antibody levels of the mice. Mice injected with 1 mg of uricase died from anaphylaxis. PEG-uricase 35% was found to induce the most effective tolerance in both unsensitized and sensitized mice.

Enzyme-Polyethylene Glycol Adducts: Modified Enzymes with Unique Properties

There are many clinical disorders which, in theory might be treated by the use of appropriate enzymes. However, there are serious problems associated with enzyme therapy. First, inadequate amounts of human enzymes are available; and most foreign enzymes are immunogenic in humans. They can be administered only a few times. Second, the circulating life in the blood may be very short even on the first injection (often a matter of a few minutes). And third, the cost of highly purified enzymes may be too great to allow general use.

Genetic control of acquired resistance to visceral Leishmaniasis in mice

A series ofH-2 and non-H-2 congenic resistant (CR) strains on a C57BL/10Sn background were infected with 107 amastigotes ofLeishmania donovani. Non-H-2 congenic strains B10.LP-H-3b and B10.CE(30NX) and (B10.LP-H-3b × B10)F1 hybrids showed a very rapid decrease in liver-parasite burdens beyond day 21. Parasite counts for these strains at day 35 were significantly lower than for all other strains tested. The rapid decrease in parasite numbers, massive lymphocellular infiltration into the liver and strong delayed hypersensitivity reactions to parasite antigens in strains congenic for a portion of chromosome 2 indicated that acquired immunity toL. donovani was controlled by a dominant gene at or near theIr-2 locus. In addition, B10.129(10M) mice, which differ from C57BL/10Sn at theH-11 locus, showed highly significant increases in parasite numbers at day 35. Other observations supporting the absence of acquired immunity in B10.129(10M) included negative delayed hypersensitivity tests to parasite antigens and the absence of lymphocellular infiltrate into the liver. Although the differences were not as pronounced,H-2 CR strains withH-2b,H-2a, andH-2k haplotypes also showed significantly greater decreases in parasite numbers by day 35 as compared to otherH-2 CR strains.

Antibodies to DNA and a Synthetic Polydeoxyribonucleotide Produced by Oligodeoxyribonucleotides

Calf-thymus DNA was degraded into small fragments (oligodeoxyribonucleotides); the fragments were treated with methylated bovine serum albumin, and the complexes so formed were emulsified in complete Freunds adjuvant and injected into rabbits. The serums of the immunized rabbits contained antibodies that reacted with homologous and heterologous unfragmented heat-denatured DNA, and also with a synthetic polydeoxyadenylate-thymidylate. Relatively small DNA fragments (of the order of tetra-hexanucleotide) can thus serve as haptens for the production of DNA antibodies; this finding increases the probability of producing antibodies specific for unique sequences of nucleotides.

Leishmania donovani: Antibody response to chicken ovalbumin by infected golden hamsters☆

Abstract The individual antibody response to ovalbumin (OA) was studied in groups of hamsters infected with Leishmania donovani . Each group consisted of two infected and two uninfected hamsters. All the animals were immunized to OA at intervals, prior to or following the introduced infection. One week after the initial immunization, each hamster received a second injection making a total of 10 mg OA. This procedure was begun with Group 1, 12 days before infection and was continued in new groups of four hamsters every 3 days until there were 19 groups under study. Each hamster, 24 days after the initial sensitization to OA, was anesthetized, weighed, exsanguinated, and necropsied. Antibody titers to OA measured by tanned cell hemagglutination (HA) from the infected vs. the uninfected hamsters were similar until a moderately severe infection developed (900 “Total Spleen Lds”). Above this level of parasitization the anti-OA titers of the infected animals were lower despite increased concentrations of gamma globulin in their serum as shown by electrophoretic studies. The titers of the uninfected animals remained in the same range throughout the experiment. The possible explanations for the depression of the immune response are discussed.

Immunity to exoerythrocytic forms of malaria. III. Stage-specific immunization of turkeys against exoerythrocytic forms of Plasmodium fallax.

A method for immunization of turkeys against exoerythrocytic infection with Plasmodium fallax was devised using formalin-killed merozoites (FKM) from cultures of infected turkey embryo fibroblasts. A single intramuscular injection of FKM combined with complete Freunds adjuvant (CFA) followed at weekly intervals by 2 intravenous (i.v.) injections of FKM in Earles balanced salt solution (EBSS) (a total of 5.0 X 106 FKM) resulted in 100% survival of turkeys after challenge with infective merozoites when blood stages were drug-suppressed. Two weekly i.v. injections totaling 3.5 X 106 FKM without CFA resulted in 80% survival after challenge. Only 20% of untreated control birds, and about 40% of birds receiving normal cell culture medium overlay, with or without CFA prior to challenge, survived infection. When FKM-immunized birds were challenged with infective merozoites from culture and received no drug suppression of blood stages, parasitemias were similar to those in control turkeys. Also, challenge of FKM-immunized turkeys with parasitized erythrocytes resulted in parasitemias similar to those of control birds. The development of exoerythrocytic stages subsequent to blood-stage infection was suppressed in most FKM-immunized turkeys, while control turkeys eventually died with heavy exoerythrocytic infections in brain capillaries. Past efforts to immunize animals against infection with malaria parasites have been directed at the use of erythrocytic stages or sporozoites as immunogens. Varying degrees of success have been achieved using killed erythrocytic stages of avian malaria parasites (Redmond, 1939; Gingrich, 1941; Jacobs, 1943; Richards, 1966). The use of adjuvants in combination with killed erythrocytic parasites increased the degree of immunity to subsequent infection (Freund et al., 1945; Thomson et al., 1947). The use of killed sporozoites for immunization was effective against mosquito-transmitted avian malaria (Mulligan et al., 1941; Russell and Mohan, 1942), although birds immune to sporozoite challenge were susceptible to infection induced with erythrocytic stages (Russell et al., 1942). Richards (1966) achieved more effective immunization of chickens with killed sporozoites than with killed blood stages of Plasmodium gallinaceum. The development of a culture system for the growth and maintenance of cells infected with exoerythrocytic stages of avian malaria paraReceived for publication 18 September 1973. * Supported by Research Grant AI-08988, and in part by AI-00092, NIAID, U. S. Public Health Service. t Deceased, 27 March 1973. sites (review by Huff, 1969) presents an opportunity for specific immunization with that stage. The results of studies utilizing exoerythrocytic merozoites from infected cell cultures as immunogens against infection of turkeys with P. fallax are herein presented. MATERIALS AND METHODS

SPECTRAL MODEL OF LEISHMANIASIS IN CONGENIC STRAINS OF MICE

Nineteen congenic, resistant strains of mice on C57BL/10ScSn genetic background were infected with Leishmania donovani and the course of infection quantitated. Early in the infection, parasite burdens in the liver were similar for all strains, indicating that the parasite was able to establish, grow, and reproduce in the liver macrophages of each strain with equal facility. Differences in acquired resistance, indicated by decreases in parasite burden, among the strains were first noted at day 21 and became distinct by day 35 postinfection. The extremes were represented by B10.129(10M) mice in which the parasite burden continued to increase at day 35, and B10.LP-H-3b in which only 10% of the peak parasite population remained at this time. The other strains formed a complete continuum between the two extremes. Differences in hepatic pathology were noted among strains, but the severity was not related directly to the strength of the immune response as indicated by reduction in parasite burden; instead, it was more correlated with spleen-to-body weight ratios. Because of the range of responses observed, congenic strains of mice may be of use not only for immunization and chemotherapy studies of leishmaniasis, but also may yield fundamental information on spectral diseases in general.

Cardiac allografts in mice congenic at non-H-2 histocompatibility loci

Neonatal mouse heart fragments were grafted under the ear skin of adult recipients. Cardiac allograft survival was evaluated by visual observation of pulsation, electrocardiography, and histology. Employing a series of congenic resistant strains differing from C57BL/10Sn at theH-1, H-3,H-4, H-7, H-8, H-9, H-10, H-11, andH-12 loci, the median survival times of the heart grafts to and from C57BL/10Sn were obtained. The various interallelic combinations resulted in a wide variation of graft survival. Reciprocal transplants frequently showed different survival times.H-1c grafts were rejected by B10.129(5M)/nSn female mice with a median survival time of 90 days.H-1b grafts were not rejected by C57BL/10Sn mice for the experiments duration of 200 days. The weaker the histocompatibility barrier, the more variable the survival times and the smaller the ratio of rejected to total grafted heart fragments. Female recipients were observed to reject their grafts more rapidly and to reject a higher proportion than males of the same strain. Although the strength of the different non-H-2 barriers generally paralleled that determined by skin transplants, the rankings of the strongest minor barriers were not the same for both tissues.