Immunosuppressive Effects of the S-Adenosylhomocysteine Hydrolase Inhibitor, 3-Deazaadenosine

Abstract

Immunosuppressive effects of 3-deazaadenosine (3-DAA), an inhibitor of S-adenosylhomocysteine hydrolase, were tested in vivo in immune assays against sheep red blood cells (SRBC), involving serum titrations for hemagglutinins and hemolysins, cellular cytotoxicity tests and the direct plaque-forming cell assay. At daily doses up to 100 mg/kg, the compound was suppressive when injected before antigen and the effect appeared to be dose-dependent (ED₅₀ = 52.6 ± 4.9 mg/kg). When doses of 25 mg/kg of 3-DAA were given before antigen, co-injections of 250 mg/kg of L-homocysteine (L-HC) potentiated the suppressive effect, although L-HC alone was inactive. Daily administration of 100 mg/kg of 3-DAA or 250 mg/kg of L-HC alone was not suppressive when given after the antigen; however, in combination they were able to induce suppression. The possible biochemical mechanisms of the suppression, particularly those involving the inhibition of S-adenosylmethionine-dependent methylation reactions, are discussed.     

Optimal Conditions for Antitumor Activity of C. Parvum Against the Ascites Form of Sarcoma 180

Single and multiple doses of Corynebacterium parvum (C. parvum) ranging from 0.1-60 mg/kg were tested for antitumor activity against 10⁶ sarcoma 180 cells in male CD1 mice. Determinations were made of the optimal dose and time of treatment needed to produce maximum suppression of the tumor using both median survival time and percent survival to day 90 as endpoints. A dose of 1 mg/kg given 3 days before sarcoma 180 transplant produced complete protection (100% survival). All other treatment regimens produced less of an effect. Single doses of 1, 10 and 60 mglkg had significant antitumor activity when administered either on day 3, 2 or 1 before tumor implant, 0.1 mg/kg protected only when given on day 3. All single doses given 5 or 8 days before and anytime after tumor were ineffective.

Multiple doses were only of advantage over single doses when treatments were after tumor cell inoculation. In vitro cytotoxicity studies demonstrated that both 1 and 60 mg/kg enhanced tumor cell killing by cells isolated from the peritoneal cavity, with the 1 mg/kg dose producing a greater effect. It was concluded that dose and time of administration of C. parvum, in relation to tumor implant, were important in determining optimal antitumor activity against sarcoma 180.     

The Dose-Dependent Influence of Mechloretamine on the Response to Sheep Erythrocytes in Mice. Comparison of Immunostimulating Properties of Mechloretamine with Levamisole

The effect of the following doses of mechloretamine: 1, 5, 10, 25, 50, 100, 250 and 500 μg^/kg on the immunological response in mice immunized with sheep red blood cells (SRBC) was investigated. the number of plaque forming cells (PFC) to SRBC, the serum hemagglutinins level and the number of lymphocytes forming E or EAC-rosettes were determined. Depending on mechloretamine dose the following effects on the tested parameters were obtained : (i) only stimulating -1 and 5 pg/kg, (ii) stimulating or suppressive according to the test -10-100 pg/kg, (iii) only suppressive -250 and 500 μg/kg. Mechloretamine (5 μg/kg) induced the increase in PFC in comparison with levamisole (2 mg/kg). the difference between the action of mechloretamine and levamisole used in immunostimulating doses on the increased anti-SKBC antibodies or on the E-rosette forming lymphocytes was revealed.     

Suppression of Experimental Allergic Encephalomyelitis by En3638: Dependence

Oral administration of 2, 3 or 4 doses of 100 or 250 mg/kg of EN3638 during the incubation period of experimental allergic encephalomyelitis delayed the onset and reduced the incidence and severity of clinical signs and histological lesions. Five doses of 50 or 100 mg/kg effected virtually complete and permanent suppression of clinical signs even after cessation of therapy, and five doses of 250 mg/kg eliminated histological lesions as well. Optimum results required coverage of the entire incubation period regardless of dose level. These results were obtained only when carbonyl iron was used as the adjuvant for production of EAE. When complete Freund's adjuvant was used, EN3638 delayed the onset but had little or no influence on late-developing clinical signs and histologic lesions after cessation of therapy. The permanence of suppression when carbonyl iron was used is related to the absence of an oil depot. Carbonyl iron is a superior adjuvant for drug suppression studies.     

Skin test suppression by antihistamines and the development of subsensitivity

The suppression of skin test reactivity by single doses of six antihistamines was measured before and after a period of daily antihistamine ingestion in 18 subjects. Single doses of hydroxyzine, 50 mg; chlorpheniramine, 16 mg; and promethazine, 50 mg; induced significant suppression of skin test reactivity at 2 hr, whereas the suppression produced by tripelennamine, 100 mg; diphenhydramine, 50 mg; and cyproheptadine, 16 mg; did not differ significantly from that produced by placebo. After 3 wk of treatment with hydroxyzine, 75 mg per day, the suppressive effect of hydroxyzine as well as the five clinically unrelated antihistamines was significantly reduced. Although the response to chlorpheniramine was also reduced after chronic treatment with chlorpheniramine, 24 mg per day, the difference was not statistically significant. We conclude that antihistamines in the doses used differ greatly in their suppressive effect on skin test reactivity. The antihistamine producing the most skin test suppression, hydroxyzine, when it was taken daily for 3 wk, caused the development of partial tolerance not only to its own effect but to those of clinically unrelated antihistamines.     

Effects of cocaine on the immune system of Balb/C mice

Cocaine was given to Balb/C mice by intramuscular injection to assess the effects of the drug on their immune system. An injection of 5 mg/kg of cocaine 24 hr before assay suppressed phagocytic activity of peritoneal macrophages and decreased the numbers of thymocytes and white blood cells in a dose-dependent manner. The suppression appeared to be reversible. Tumor-implanted mice received 10 consecutive days of injections of smaller doses of cocaine (0.05, 0.25, or 5 mg/kg), also resulting in a dose-dependent suppression of phagocytosis 24 hr after the last injection. Cocaine decreased the number of plaque-forming cells when 5 mg/kg of cocaine was injected on the day of immunization but no inhibition was detected if the drug was given later. The size of tumors appeared to be increased in mice injected with cocaine for 10 consecutive days in comparison to the control mice. The effects were concentration dependent. Our study showed that cocaine had general suppressive effects on the mouse immune system.     

Antigenic competition in IgE antibody production. II. Effect of cyclophosphamide.

The effects of cyclophosphamide (CY) on antigenic competition in IgE antibody production were studied in mice treated with the drug on different days and immunized with a mixture of two non-related antigens. Injection of 100 mg of CY/kg of body weight 3 days before or 6 days after immunization resulted in a partial or total recovery of the IgE, but not of the IgGl antibody response to the test antigen. In contrast, when the same dose was given together or 3 days after immunization both responses were much more suppressed than in untreated animals. This same effect was obtained when a higher concentration (200 mg/kg) of cyclophosphamide was injected on day--3. When a different antigenic system was tested, the suppressive effects of competition in IgGl antibody production were also abolished after CY treatment. These results seem to provide further evidence for an important role of suppressor T cells in the mechanism of antigenic competition.     

Inhibition of postpyrogenic increase of phagocytic and killing activity of neutrophils by nonsteroid antiinflammatory drugs

Acetylsalicylic acid (300 mg/kg), mefenamic acid (30 mg/kg) or indomethacin (20 mg/kg) given orally in the doses preventing the postpyrogenic fever, inhibited the stimulatory effect of LPS on phagocytic and killing activity of neutrophils. The dose of acetylsalicylic acid that did not eliminate fever in rabbits (100 mg/kg), had no suppressive effect upon fever-stimulated killing activity of neutrophils. The drugs administered twice a day to normothermic animals did not evoke any suppressive changes in the activity of neutrophils.     

Comparison of the suppressive effect of thymus cells and the suppression by neonatal application of antigen

The suppressive effect of thymus cells on the agglutinating antibody response in chickens is shown to be antigen-specific. Experiments reveal that this effect can be seen several weeks after thymus cell transfer, if the respective antigen (A) is given 3 times weekly after transfer. Repeatedly applied high doses of the antigen (A) result in a stronger effect than low antigen doses, and mercaptoethanol-resistant antibody (IgG) is usually more affected than the total titer (of which IgM is the major contributor). Finally, several weeks after thymus cell transfer, when the response to antigen A is still lower than that of the normal controls, the antibody response to an unrelated antigen B is normal. By these three criteria, as well as by the degree of suppression, the suppressive effect of thymus cells strikingly resembles the phenomenon of antigen-specific suppression that is seen after antigenic stimulus in the prenatal or early postnatal period. The suppressive effect is already given by thymus cells from 10-day old donors. As in previous studies, the suppressive activity was significantly diminished in thymus cells from neonatally bursectomized donors. The data indicate that a specifically acting suppressor cell compartment exists which is different from B cells and cooperative or graft-vs-host-active T cells. The results are discussed in light of current concepts of “self-tolerance”.     

Radioprotective efficacy and acute toxicity of 5-androstenediol after subcutaneous or oral administration in mice

We previously showed that one subcutaneous (sc) injection of 5-androstene-3beta,17beta-diol (AED) stimulated the innate immune system in mice and prevented mortality due to hemopoietic suppression after whole-body ionizing irradiation with gamma rays. In the present study, we tested whether there was any significant toxicity in mice that might hinder development of this steroid for human use. There were no indications of toxicity in chemical analyses of serum after sc doses as high as 4000 mg/kg. At this dose, 2 of 54 mice died when given AED alone. When 4800 mg/kg was given orally, no deaths resulted. The only adverse findings attributed to AED administration were 1) a moderate elevation of granulocytes in abdominal organs and fat after sc injections of 320 mg/kg; and 2) occasional wasting of skin over the injection site in female B6D2F1 but not male C3H/HeN mice. Significant weight loss (6%) was observed after sc injections of 320 mg/kg but not 160 or 80 mg/kg. When male C3H/HeN mice were injected sc with AED at doses of 0-200 mg/kg 24 h before whole body gamma-irradiation (9 Gy), a significant improvement in survival was observed at doses as low as 5 mg/kg. Oral administration of AED produced significant survival enhancement at a dose of 1600 mg/kg. We conclude that the radioprotective efficacy of AED is accompanied by low toxicity.Androst-5-ene-3 beta, 17 beta-diol; Ionizing radiation; Experimental radiation injuries; Toxicity; Clinical chemistry; Histopathology     

RADIOPROTECTIVE EFFICACY AND ACUTE TOXICITY OF 5-ANDROSTENEDIOL AFTER SUBCUTANEOUS OR ORAL ADMINISTRATION IN MICE

ABSTRACT

We previously showed that one subcutaneous (sc) injection of 5-androstene-3beta,17beta-diol (AED) stimulated the innate immune system in mice and prevented mortality due to hemopoietic suppression after whole-body ionizing irradiation with gamma rays. In the present study, we tested whether there was any significant toxicity in mice that might hinder development of this steroid for human use. There were no indications of toxicity in chemical analyses of serum after sc doses as high as 4000 mg/kg. At this dose, 2 of 54 mice died when given AED alone. When 4800 mg/kg was given orally, no deaths resulted. The only adverse findings attributed to AED administration were 1) a moderate elevation of granulocytes in abdominal organs and fat after sc injections of 320 mg/kg; and 2) occasional wasting of skin over the injection site in female B6D2F1 but not male C3H/HeN mice. Significant weight loss (6%) was observed after sc injections of 320 mg/kg but not 160 or 80 mg/kg. When male C3H/HeN mice were injected sc with AED at doses of 0–200 mg/kg 24 h before whole body gamma-irradiation (9 Gy), a significant improvement in survival was observed at doses as low as 5 mg/kg. Oral administration of AED produced significant survival enhancement at a dose of 1600 mg/kg. We conclude that the radioprotective efficacy of AED is accompanied by low toxicity.Androst-5-ene-3 beta, 17 beta-diol; Ionizing radiation; Experimental radiation injuries; Toxicity; Clinical chemistry; Histopathology     

Effects of apomorphine and haloperidol on response suppression learning of young chicks

In four experiments, the effects of augmenting or blocking dopamine receptor activity on response suppression learning of Colburn X Colburn chicks were determined. In each experiment, 4-day-old chicks were trained to key peck for heat reward and then tested for response suppression learning by using either a response-contingent punishment or an extinction-punishment task. Before response suppression testing, different groups of chicks were injected ip with apomorphine (1.0, 2.0, or 4.0 mg/kg) either alone or after pretreatment with haloperidol (0.5 or 1.0 mg/kg). Regardless of the response suppression task used, chicks injected with apomorphine had difficulty inhibiting their responding; whereas, chicks injected with haloperidol, either alone or before apomorphine treatment, responded on fewer trials than saline-treated chicks. During extinction testing, 4-day-old chicks given only apomorphine showed the typical suppressive effect of punishment on responding rather than the paradoxical punishment-induced increase in responding found in normal 1-day-old chicks. These results indicate that activation of dopamine receptors retards response suppression learning of the 4-day-old chick, but functional changes in central dopaminergic mechanisms are not primarily responsible for the normal age-dependent improvement in response suppression learning of the young chick.     

Intestinal nutrients elicit satiation through concomitant activation of CCK(1) and 5-HT(3) receptors

Previous studies demonstrate that cholecystokinin type-1 (CCK(1)) and serotonin type-3 (5-HT(3)) dependent pathways are independently involved in intestinal nutrient-induced meal termination. In the current study, we employed selective antagonists to investigate the relative contribution of CCK(1) and 5-HT(3) receptors in mediating the anorexia produced by duodenal infusion of Polycose or Intralipid in rats. Combined administration of 1 mg/kg ondansetron (Ond) and 1 mg/kg devazepide (Dev) reversed 132 mM Polycose-induced suppression to the level of control intake and significantly attenuated 263 mM Polycose-induced suppression greater than either antagonist alone. Similar results were observed when subthreshold doses of Ond (500 microg/kg) and Dev (5 microg/kg) were co-administered prior to 263 mM Polycose infusion. Suppression of intake resulting from 130 mM Intralipid was reversed to the level of control when Ond and Dev were co-administered at both independent effective doses (1 mg/kg each) and subthreshold doses (500 microg/kg and 5 microg/kg, respectively). Finally, combined administration of the antagonists increased sucrose intakes beyond intakes following control or treatment with either antagonist alone when rats were infused with saline. These data demonstrate that intestinal carbohydrates and lipids inhibit food intake through simultaneous CCK(1) and 5-HT(3) receptor activation and that these receptors appear to completely mediate the Intralipid-induced suppression of intake.     

RM-11, a new izoxasole derivative, is a potent stimulator of the humoral and cellular immune responses in mice

In this report we describe immunostimulatory properties of RM-11 in several in vivo and in vitro tests in the murine model. We found that RM-11 significantly stimulated the humoral immune response to sheep erythrocytes (SRBC) when given intraperitoneally (i.p.) at doses of 10 and 100 microg or per os (doses of 20 and 200 microg) 3 h before immunization. The compound was also stimulatory with regard to generation of delayed type hypersensitivity (DTH) to SRBC when given i.p. or per os (doses of 10, 100 and 500 microg/mouse). The described immunostimulatory activities of RM-11 were higher compared to that of the reference drug, levamisole. RM-11 stimulated, in addition, concanavalin A (ConA)-induced splenocyte proliferation. Lastly, we showed that RM-11 was not toxic when given to mice per os at doses 250 mg/kg body weight. Taken together, RM-11 appeared to be a universal stimulator of the immune response in mice. Lack of toxicity and the ability to stimulate the immune response, when administered per os, predispose the compound for further preclinical studies.     

Environment modulates naloxone's suppressive effect on feeding in diabetic and non-diabetic rats

We evaluated the effect of environment on naloxone-induced suppression of feeding in streptozotocin rats and sham injected controls. Naloxone was administered to animals fasted for 24 hours and food intake was measured at 30, 60 and 120 minutes. Diabetic rats, in their home cages, were insensitive to naloxone's suppressive effect for the first 30 minutes and the 5 mg/kg dose suppressed feeding only at 120 minutes. In control rats, feeding was suppressed at 1 and 5 mg/kg naloxone during the first 30 minutes. In contrast, when animals were placed in novel plastic cages, control animals were insensitive to naloxone at all time points at doses as high as 5 mg/kg. In novel cages, diabetic rats responded to doses of 1 and 5 mg/kg during the first 30 minute period by lowering food intake. It should also be noted that basal food intake was suppressed (40-53%) when animals were placed in novel cages. These data suggest that stress of a novel environment alters the neuroregulatory system involved in inducing feeding. Lack of response of normal rats to naloxone's suppressive effect in a novel environment suggests that (1) a non-opioid feeding system operates under these conditions, or (2) opioid receptors are occupied as a result of the release of endogenous opioids due to stress. The opposite result observed in the diabetics indicates that glucose has a modulating effect on opioid effects.     

Beneficial effects of ascorbic acid on preimplantation mouse embryos after exposure to cyclophosphamide in vivo

To study mechanisms of embryotoxicity in early pregnancy, we have evaluated the genotoxic and embryolethal effects of ascorbic acid (AA) alone or in combination with cyclophosphamide (CPA). Female mice were exposed on day 3 of pregnancy. Embryotoxicity was investigated at term and genotoxicity shortly after treatment using the chromosomal aberration test and the sister chromatid exchange (SCE) assay as sensitive end points. Additionally, cytotoxic effects were determined by a proliferation test. AA was not found to be embryotoxic, cytotoxic, or genotoxic when given alone. In combination with 10 mg/kg CPA, however, which induced 50% aberrant metaphases, 100% increase SCE frequency, and a strong inhibition of cell proliferation, AA in a dose range of 25-1,600 mg/kg did not change SCE and proliferation, but reduced the rate of aberrant metaphases significantly. This anticlastogenic effect was clearly correlated to a beneficial effect on embryolethality at term when 200 mg/kg ascorbic acid was given in combination with 40 mg/kg CPA. The results suggest that during early pregnancy AA is not genotoxic even at so-called megadoses doses, but it seems to protect early embryos against damage induced by genotoxic agents like CPA.     

Combined treatment with Ren-shen-yang-rong-tang (Japanese name: Ninjin-youei-to) plus prednisolone on adjuvant-induced arthritis in Lewis rat

The effects of combined treatment with 250 mg/kg Ren-shen-yang-rong-tang (Japanese name: Ninjinyouei-to, NYT) plus 4 mg/kg, an average dosage, or 0.2 mg/kg, a suboptimal dosage, of prednisolone (PSL) on adjuvant-induced arthritis in Lewis rats were investigated using two treatment schedules. The agents were administered orally every day from day 0 to 21 (schedule A), or from day -7 to 21 (schedule B) after adjuvant injection. PSL treatment (4 mg/kg) obviously inhibited paw swelling due to non-immune inflammation, diminished the weights of the thymus, spleen, adrenals and iliac lymph nodes, and suppressed the increment of serum interleukin (IL)-6 concentration in both schedules compared to controls. NYT treatment alone inhibited paw swelling due to immune inflammation, diminished the weight of the adrenals and decreased IL-6 concentration only in schedule A. Combined treatment with NYT plus PSL (4 mg/kg) showed: (1) a superior effect to that of PSL on paw swelling in the uninjected hind foot, especially in schedule B, (2) a tendency to diminish adrenal weight in schedule A and the weights of all four organs in schedule B compared with PSL treatment alone, and (3) a suppressive effect on IL-6 concentration weaker than that of PSL alone in schedule B. The suppressive effect of combined treatment with NYT plus PSL (0.2 mg/kg) on paw swelling was significantly stronger compared with either NYT or PSL treatment alone in schedule B. Although this dose of PSL had no influence upon the IL-6 concentration, the combined treatment or NYT alone increased the IL-6 concentration. Mechanisms underlying the synergistic suppressive effects on paw swelling are discussed.     

Glomerular thrombosis and cortical infarction in cyclosporin-treated rabbits with acute serum sickness.

Rabbits given acute serum sickness (ASS) and treated with cyclosporin A (CyA) developed glomerular capillary thrombosis and cortical infarction, lesions not seen in unmodified ASS. Thirty-three NZW rabbits received a single intravenous injection of 250 mg/kg bovine serum albumen (BSA) with or without endotoxin (5 micrograms/kg) on day 0. Groups of rabbits were given intramuscular CyA as follows: 15 mg/kg from day -2 to +8, or 25 mg/kg/day from day -20 to +3 or day 0 to 5. Signs of this renal injury were haematuria, transient proteinuria, glycosuria and oliguria and they occurred during the rapid phase of antigen elimination when immune complexes were being formed. Seventeen of the 33 rabbits developed glomerular capillary thrombi and 11 of 17 also had glomerular and tubular infarction. Electron microscopic examination showed that these lesions were associated with severe endothelial injury and platelet-fibrin-leucocyte thrombi. These changes were more severe in the groups given 25 mg/kg. The lesions were not seen in untreated rabbits and ASS, nor in normal rabbits given equivalent doses of CyA alone. A strikingly similar renal lesion has been seen in patients receiving CyA following bone marrow transplantation, and also in the haemolytic uraemic syndrome. The model we describe may be valuable for the study of the mechanisms of endothelial injury and thrombosis in the kidney.     

Analysis of carrageenan-induced suppression of antibody response.

This paper describes the induction of immunosuppression by multiple injections of carrageenan-lambda (cgn) in BALB/c mice. While cgn alone induced non-specific suppression that persisted for up to 25 days, priming with sheep red blood cells (SRBC) of mice within 10 days of cgn treatment prolonged the suppressive state. Also, antigen (SRBC)-specific suppression was observed when these mice were given a secondary challenge 25-30 days afer priming. Thus multiple cgn treatment along with priming SRBC generated non-specific and specific suppressive states which were transferable by Thy-1+ Lyt-2+ splenocytes.     

Pharmacological studies of antigen-induced arthritis in BALB/c mice I. Characterization of the arthritis and the effects of steroidal and non-steroidal anti-inflammatory agents

Chronic monoarticular allergic arthritis was induced in BALB/c mice using methylated BSA as antigen and Freund's complete adjuvant, together with Bordetella pertussis as a secondary adjuvant. The optimum conditions for induction of chronic persistent arthritis and the histological characteristics of the arthritic lesion are described.Both the synovitis and erosive progression of the arthritis could be suppressed by daily treatment with prednisolone (1–10 mg/kg) or dexamethasone (0.5–2.5 mg/kg) for 4 weeks commencing 2 weeks after the induction of arthritis. In contrast, daily treatment with the non-steroidal anti-inflammatory agents ibuprofen (50–100 mg/kg), flurbiprofen (1–9 mg/kg) or indomethacin (0.1–3 mg/kg) had no significant effect on either the synovitis or erosions as judged histologically. Synovial fluid differential leukocyte counts were altered by treatment with ibuprofen and indomethacin but not by flurbiprofen or the corticosteroids.The suppressive effect of the corticosteroids was not due to either suppression of antibody synthesis or alteration of the number of leukocytes in the peripheral circulation.