Dose-dependent effects of FK506 on neuroregeneration in a rat model

This study explored the effects of different doses of FK506 on peripheral nerve regeneration, to determine whether neuroregeneration could be enhanced without the toxicity of systemic immunosuppression. In the first part of the study, subimmunosuppressive doses of FK506 were determined by examining skin allograft survival in a rat model. Full-thickness skin grafts (2 cm2) from Wistar rats were grafted to recipient Lewis rats. The procedure was performed for six groups (n = 6). The control group received no FK506, and the other five groups received daily doses of FK506 of 0.125, 0.25, 0.5, 1.0, or 2.0 mg/kg. Animals that received 2.0 mg/kg FK506 per day exhibited complete skin graft take, whereas all other groups demonstrated complete rejection. After determination of the immunosuppressive dose of FK506, the neuroregenerative effects of different doses of FK506 were explored by assessing nerve regeneration in 80 rats after tibial nerve transection and repair. The control group received no FK506, whereas the other four groups were given daily doses of FK506 of 0.25, 0.5, 1.0, or 2.0 mg/kg. Rats were euthanized at three time points (25, 30, and 35 days), to fully investigate the effects of different FK506 dosing regimens on neuroregeneration. Histomorphometric analyses performed on postoperative days 30 and 35 demonstrated statistically significant improvements in neuroregeneration with subimmunosuppressive FK506 doses of 0.5 and 1.0 mg/kg per day. Therefore, the study demonstrated that neuroregeneration was enhanced at low doses of FK506 that were not sufficient to prevent skin allograft rejection.     

Anti-CD40 ligand antibody permits regeneration through peripheral nerve allografts in a nonhuman primate model

Systemic immunosuppression is typically required to prevent allograft rejection. Antibody-based therapies that induce immune unresponsiveness represent an appealing alternative to nonspecific immunosuppression, which is often associated with significant morbidity. In mice, successful prevention of nerve allograft rejection has been demonstrated through interference with the CD40/CD40 ligand interaction. This study investigated the effectiveness of anti-CD40 ligand monoclonal antibody as single-agent therapy in preventing rejection and supporting nerve regeneration across long nerve allografts in nonhuman primates. Twelve outbred cynomolgus macaques were arranged into six genetically mismatched pairs, with each animal receiving a 5-cm ulnar nerve allograft in the right arm and a 5-cm autograft in the left arm. Mixed lymphocyte reaction assays were used to assess resulting immune unresponsiveness. Treated animals (n = 10) received anti-CD40 ligand monoclonal antibody 10 mg/kg one time, locally applied, and 20 mg/kg systemically on postoperative days 0, 1, 3, 10, 18, and 28, and then monthly. Untreated animals (n = 2) served as the untreated controls. At 4 or 6 months after transplantation, nerves were harvested for histological analysis. Four treated animals underwent an additional challenge after cessation of anti-CD40 ligand monoclonal antibody therapy and nerve graft harvests. Autogenous and allogeneic skin and nerve inlay grafting was performed to assess the permanence of immune unresponsiveness induced by anti-CD40 ligand monoclonal antibody. Animals that received anti-CD40 ligand monoclonal antibody demonstrated robust regeneration across nerve allografts, similar to that seen in the autograft control in the contralateral arm. The histomorphometric analysis of allografts in the untreated animals demonstrated significantly worse measurements compared with their matched autograft controls. Animals that received anti-CD40 ligand monoclonal antibody with concomitant skin allografts had virtually no evidence of nerve regeneration through allografts. Allogeneic skin and nerve allografts applied 2 to 12 months after withdrawal of anti-CD40 ligand monoclonal antibody therapy were consistently rejected. This study demonstrates that anti-CD40 ligand monoclonal antibody prevents rejection and allows regeneration of peripheral nerve allografts in nonhuman primates. The effect of anti-CD40 ligand monoclonal antibody appears to be transient, however, with restoration of immunocompetence shortly after withdrawal of therapy.     

Effects of a dopaminergic stimulant,amfonelic acid,on anterior pituitary hormone release in conscious rats

The response of plasma LH, Prolactin, GH and TSH levels to systematic administration of a specific central dopaminergic stimulant, amfonelic acid (AFA), by intravenous pulse injection in ovariectomized (OVX) and OVX estrogen-progesterone primed conscious rats has been evaluated. Intravenous injection of 0.2 mg/kg of AFA had no influence on plasma LH concentration until 60 min after injection when it was significantly elevated. Increasing the dose to 1 mg/kg reduced LH titers at 15 and 30 min with a return to preinjection levels by 60 min. AFA produced a dose-dependent decrease in plasma prolactin levels; the decrease occurred as early as 5 min after injection. AFA, both at 0.2 and 1 mg/kg doses, was effective in producing a sharp, dose-related rise in plasma GH levels. By contrast, TSH levels were significantly suppressed by both doses of AFA. Injection of the 1 mg/kg dose of AFA did not modify plasma LH levels in OVX-steroid-primed animals, white producing a comparable effect on plasma prolactin, GH and TSH levels to that observed in OVX animals. The present results indicate that endogenously released DA can have profound effects on pituitary hormone release, inhibiting PRL and TSH discharge, stimulating GH release and either inhibiting or stimulating LH release.     

Selective effects of three herbicides on the fungus flora of egyptian soil

2,4-D (2,4-dichlorophenoxyacetio acid) when applied to the soil at three doses (1.9, 7.6 and 15.2 mg per kg dry soil) had a stimulating effect on the total count of soil fungi and on several fungal species especially between 5 and 20 d after treatment. When the herbicide was incorporated in the agar medium it had a stimulating effect on the counts of total fungi, Aspergillus sp., A. niger, A.fumigatus and Fusarium app. at the low dose (6.3 ppm), but wag toxic at this dose toward Humicola grisea and Myrothecium verrucaria at the medium and high doses (25.2 and 50.4 ppm), it was toxic to the total count of fungi and to the majority of fungal species. VCS-438 [2-(3,4-dichlorophenyl)-4-methyl-l,2,4-oxadiazolidine-3,5-dione] was beneficial to the total count of fungi 2 and 5 d after soil treatment with the medium dose (8.0 mg per kg dry soil). Some fungal species could benefit from the low and the high doses (2.0 and 16.0 mg per kg dry soil) after these experimental periods. In the agar medium the counts of total fungi, Aspergillus sp., A. niger and A.fumigatus were almost significantly reduced by the three doses (6.8, 27.2 and 54.8 ppm). Planavin (4-methylsulphonyl-2,6-dinitro-N,N-dipropylaniline) was stimulating for the total count of fungi, Aspergillus, A.niger and A.ochraceus 2 and 5 d after treatment with the medium dose (8.0 mg per kg dry soil), and was also stimulating to Fusarium population at the medium dose after 2 d and at the high dose (16 mg per kg dry soil) after 20 d. In the agar medium Planavin at the low dose (6.8 ppm) was stimulating to A.terreus and inhibitory to A.nidulans and A.fumigatus. The medium and high doses (27.2 and 54.8 ppm) were generally toxic to the total count of fungi.     

Action of cyclic adenosine monophosphate on the spleen antibody--forming cells of rabbits immunized with Escherichia coli]

Cyclic adenosine monophosphate (cAMP) parenterally injected to rabbits (immunized intraperitoneally with thymus-independent antigen of killed E. coli 0127/545) during January--April inhibited production of antibody-forming cells (AFC) in the spleen of these animals, and during May--June it increased the AFC count. Both the stimulating and inhibitory effect of cAMP was associated with the administration of the same doses of 25--250 microgram/kg. The nature of the cAMP effect on the production of the AFC depends on the initial immune response level. At the maximum immune response and in the absence of the dose-effect dependence cAMP inhibited the antibody formation, but when the immunological reaction was below the maximal level and in the presence of the dose-effect relationship cAMP increased the AFC production.     

Effect of various doses of cocaine on endurance capacity in rats

To determine the effects of a variety of doses of cocaine on endurance capacity, rats were injected intraperitoneally with either 0.1, 0.5, 2.5, 12.5, or 20 mg/kg body wt 20 min before running to exhaustion at 26 m/min up a 10% grade. Animals given saline ran 116 +/- 9 (SE) min. At doses of 12.5 and 20 mg/kg, cocaine reduced endurance time significantly (34 and 74%, respectively). At rest the drug had no effect on liver or fast-twitch muscle glycogen but significantly reduced (20-40%) soleus glycogen at the two highest doses. However, at exhaustion, the quantity of glycogen depleted in the fast-twitch red and white vastus muscles was similar in all groups despite the reduced run times of the animals receiving a higher dose implying a greater rate of glycogenolysis due to cocaine. Blood lactate in the 20 mg/kg group (9.9 +/- 1.2 mM) at exhaustion was nearly twice that of the saline controls at exhaustion (5.1 +/- 0.6). Before exercise plasma norepinephrine (at doses of 2.5, 12.5 and 20 mg/kg) was higher than saline controls and remained higher (20 mg/kg groups) at exhaustion. We conclude that high doses of cocaine cause rapid muscle glycogen depletion and early fatigue. The mechanism by which cocaine causes these effects is not clear.     

Peptide nucleic acid antisense prolongs skin allograft survival by means of blockade of CXCR3 expression directing T cells into graft

CXCR3, predominantly expressed on memory/activated T cells, is a receptor for both IFN-gamma-inducible protein 10/CXC chemokine ligand (CXCL)10 and monokine induced by IFN-gamma/CXCL9. It was reported that CXC chemokines IFN-gamma-inducible protein 10/CXCL10 and monokine induced by IFN-gamma/CXCL9 play a critical role in the allograft rejection. We report that CXCR3 is a dominant factor directing T cells into mouse skin allograft, and that peptide nucleic acid (PNA) CXCR3 antisense significantly prolongs skin allograft survival by means of blockade of CXCR3 expression directing T cells into allografts in mice. We found that CXCR3 is highly up-regulated in spleen T cells and allografts from BALB/c recipients by day 7 of receiving transplantation, whereas CCR5 expression is moderately increased. We designed PNA CCR5 and PNA CXCR3 antisenses, and i.v. treated mice that received skin allograft transplantations. The PNA CXCR3 at a dosage of 10 mg/kg/day significantly prolonged mouse skin allograft survival (17.1 +/- 2.4 days) compared with physiological saline treatment (7.5 +/- 0.7 days), whereas PNA CCR5 (10 mg/kg/day) marginally prolonged skin allograft survival (10.7 +/- 1.1 days). The mechanism of prolongation of skin allograft survival is that PNA CXCR3 directly blocks the CXCR3 expression in T cells, which is responsible for directing T cells into skin allograft to induce acute rejection, without interfering with other functions of the T cells. These results were obtained at mRNA and protein levels by flow cytometry and real-time quantitative RT-PCR technique, and confirmed by chemotaxis, Northern and Western blot assays, and histological evaluation of skin grafts. The present study indicates the therapeutic potential of PNA CXCR3 to prevent acute transplantation rejection.     

Critical, but conditional, role of OX40 in memory T cell-mediated rejection

Memory T cells can be a significant barrier to the induction of transplant tolerance. However, the molecular pathways that can regulate memory T cell-mediated rejection are poorly defined. In the present study we tested the hypothesis that the novel alternative costimulatory molecules (i.e., ICOS, 4-1BB, OX40, or CD30) may play a critical role in memory T cell activation and memory T cell-mediated rejection. We found that memory T cells, generated by either homeostatic proliferation or donor Ag priming, induced prompt skin allograft rejection regardless of CD28/CD154 blockade. Phenotypic analysis showed that, in contrast to naive T cells, such memory T cells expressed high levels of OX40, 4-1BB, and ICOS on the cell surface. In a skin transplant model in which rejection was mediated by memory T cells, blocking the OX40/OX40 ligand pathway alone did not prolong the skin allograft survival, but blocking OX40 costimulation in combination with CD28/CD154 blockade induced long-term skin allograft survival, and 40% of the recipients accepted their skin allograft for >100 days. In contrast, blocking the ICOS/ICOS ligand and the 4-1BB/4-1BBL pathways alone or combined with CD28/CD154 blockade had no effect in preventing skin allograft rejection. OX40 blockade did not affect the homeostatic proliferation of T cells in vivo, but markedly inhibited the effector functions of memory T cells. Our data demonstrate that memory T cells resisting to CD28/CD154 blockade in transplant rejection are sensitive to OX40 blockade and suggest that OX40 is a key therapeutic target in memory T cell-mediated rejection.     

Combined action of doxycycline and mytilan on the immune response to tularemia antigen]

Combined action of doxycycline and mytilan, a natural polysaccharide, on the primary immune response to the antigen of the tularemia vaccinal strain in CBA mice was studied. The polysaccharide was used to compensate the immunosuppressive effect of doxycycline high doses on the humoral immune response. The maximum stimulation of the antibody titers as compared to the controls (more than 250 per cent) was observed when mytilan was administered simultaneously with or prophylactically 3 days prior to the antibiotic in doses of 2.5 and 25 mg/kg. The use of mytilan in combination with doxycycline high doses made it possible to compensate the antibiotic-induced decrease of DTH and even to stimulate it as compared to the controls. The highest levels of DTH (150 per cent against the control) were observed when mytilan was administered prophylactically in doses of 2.5 and 11.25 mg/kg 3 days prior to immunization. Mytilan had the highest stimulating effect on antibody production. The combined use of doxycycline and mytilan was characterized by significant stimulation of antibody production and DTH when the dose/time regimens were rational.     

Effect of gentamycin in combination with prodigiozan on the immunological reactivity of the body

The effect of gentamicin sulphate and its combination will prodigiozan on antibody formation in experiments and the levels of the immunobiologic reactivity of patients with purulent inflammatory processes was studied with a purpose of developing rational schemes of antibiotic therapy of infectious diseases. A decrease in the titers of the antibodies to Aeromonas and the number of antibody-forming cells in the spleen was noted on repeated administration of gentamicin to albino mice in a dose of 20 mg/kg. This was prevented by the use of prodigiozan in a dose of 500 micrograms/kg once every 4 days. The use of gentamicin in patients with purulent inflammatory diseases in doses of 40 or 80 mg twice a day for 7--10 days had no significant effect on the titers of IgA, IgG, IgM, lysozyme blood serum levels, serum bactericidal activity and absorption activity of the peripheral blood neutrophils. Still, it induced a marked suppression of the neutrophil digestive capacity as compared to the initial levels, especially on administration of gentamicin in a dose of 40 mg twice a day. An increase in the level of IgM and no suppression of the neutrophil digestive capacity were noted after completion of the therapy in the patients treated with gentamicin administered in a dose of 40 mg twice a day and prodigiozan administered in a dose of 50 micrograms once every 4 days. It is recommended to use prodigiozan in combinaed therapy with gentamicin for correction of the changes in the specific and nonspecific protective forces of the host.     

I-TAC is a dominant chemokine in controlling skin intragraft inflammation via recruiting CXCR3 cells into the graft

Chemokines play a critical role in the acute transplant rejection. In order to provide an overview of the chemokine expression during the course of acute allograft rejection, the intragraft expression profile of 11 chemokines representative of all four chemokine subfamilies was analyzed in a murine skin transplantation model of acute rejection. It was found that RANTES/CCL5, TARC/CCL17 and FKN/CX₃CL1 were expressed at equivalent levels in iso- and allografts. However, the other eight chemokines expression was up-regulated to some extent in allograft compared with that in isograft. The levels of MIP-1α/CCL3, MIP-3α/CCL20 and CTACK/CCL27 were progressively increased from early stage (day 3 post-transplantation) to late stage (day 11). Mig/CXCL9, IP-10/CXCL10, I-TAC/CXCL11, CXCL16 and LTN/XCL1 expression was elevated at middle stage (day 7), and peaked at late stage. Among the up-regulated chemokines, I-TAC was the most obviously elevated chemokine. Therefore, the effect of I-TAC on the skin acute allograft rejection was evaluated. Block of I-TAC by the intradermal injection of anti-I-TAC monoclonal antibody (mAb) reduced the number of CXCR3⁺ cells in skin allograft and significantly prolonged the skin allograft survival. The mAb treatment did not influence the proliferation of the intragraft infiltrating cells in response to the allogeneic antigens, but significantly decreased the number of the infiltrating cells and consequently lowered the secretion of IFN-γ and TNF-α. These data indicate I-TAC might be a dominant chemokine involved in the intradermal infiltration and I-TAC-targeted intervening strategies would have potential application for the alleviation of acute transplant rejection.     

The mechanism of action of 3,6-bis(2-piperidinoethoxy)acridine trihydrochloride (CL 246,738) in the potentiation of natural killer cells

An effort has been made to determine the mechanism by which the immunomodulator 3,6-bis(2-piperidinoethoxy)acridine trihydrochloride (CL 246,738) enhances the cytotoxic activity of natural killer (NK) cells. Orally administered CL 246,738 produced augmentation of NK cell activity in mice in a dose-related fashion over a dose range of 10 to 160 mg/kg, with a peak stimulation occurring at 40 mg/kg. The stimulatory effect was short-lived and only persisted for 3 days after a single oral dose of the drug. However, it could be boosted by a subsequent treatment. With anti-asialo GM-1 (anti-ASGM-1) antibody used as an NK cell marker, it was determined that the compound increased the number of ASGM-1-positive cells in mice, as indicated by radioimmunoassay and immunofluorescence staining. NK cells of beige mice were also activated by CL 246,738. Furthermore, the compound at concentrations of 0.02 to 0.2 microgram/ml induced NK cell activity in vitro, with a minimum 3-day incubation being required for optimal activation. This effect was dependent on the presence of macrophages and was inhibited by anti-IFN-alpha + beta but not anti-IFN-beta antibody. Taken together, it is postulated that the compound functions by stimulating macrophages to release IFN-alpha, which subsequently activates NK cells. As an effective stimulator of IFN and NK cells, CL 246,738 may prove clinically useful in the immunotherapy of certain types of malignancy.     

Effects of Monoamine Oxidase Inhibitors on Methamphetamine-Induced Stereotypy in Mice and Rats

In male ICR mice, a single intraperitoneal administration of methamphetamine (METH) (10 mg/kg) induced stereotyped behavior such as continuous sniffing, circling, and nail biting, reaching a plateau level 20 min after the injection. Subcutaneous pretreatment with clorgyline, a monoamine oxidase (MAO)-A inhibitor, at a dose of 0.1 mg/kg 2 h prior to the drug challenge significantly decreased the initial (first 20 min) intensity of stereotypies and increased the latency to onset. The effect was not observed with either higher doses of clorgyline (1 and 10 mg/kg) or l-deprenyl, a MAO-B inhibitor, at doses of 0.1–10 mg/kg. In male Wistar rats, the inhibitory effect of clorgyline on METH-induced stereotypy was not observed. Pretreatment of the mice with clorgyline (0.1 mg/kg) had no effect on apparent serotonin and dopamine turnover in the striatum, although the higher doses of clorgyline (1 and 10 mg/kg) significantly decreased the turnover. These results suggest that a low dose of clorgyline tends to increase the latency and decrease the intensity of stereotypies induced by METH in a dopamine metabolism-independent manner in mice.     

Effect of acetylcholine on the mast cells of the dura mater]

The effects of various doses (100 mg/kg, 200 mg/kg and 500 mg/kg) of acetylcholine on the mast cells of the dura mater was studied in white rats by electron microscopy and the Falck fluorescent method. The qualitative and quantitative differences were found in the processes of accumulation and removal of monoamines depending on the drug dosage. The data obtained allow to suggest the stimulating effect of acetylcholine on monoamine secretion.     

Intravenous S-Ketamine Does Not Inhibit Alveolar Fluid Clearance in a Septic Rat Model

We previously demonstrated that intratracheally administered S-ketamine inhibits alveolar fluid clearance (AFC), whereas an intravenous (IV) bolus injection had no effect. The aim of the present study was to characterize whether continuous IV infusion of S-ketamine, yielding clinically relevant plasma concentrations, inhibits AFC and whether its effect is enhanced in acute lung injury (ALI) which might favor the appearance of IV S-ketamine at the alveolar surface. AFC was measured in fluid-instilled rat lungs. S-ketamine was administered IV over 6 h (loading dose: 20 mg/kg, followed by 20 mg/kg/h), or intratracheally by addition to the instillate (75 µg/ml). ALI was induced by IV lipopolysaccharide (LPS; 7 mg/kg). Interleukin (IL)-6 and cytokine-induced neutrophil chemoattractant (CINC)-3 were measured by ELISA in plasma and bronchoalveolar lavage fluid. Isolated rat alveolar type-II cells were exposed to S-ketamine (75 µg/ml) and/or LPS (1 mg/ml) for 6 h, and transepithelial ion transport was measured as short circuit current (ISC). AFC was 27±5% (mean±SD) over 60 min in control rats and was unaffected by IV S-ketamine. Tracheal S-ketamine reduced AFC to 18±9%. In LPS-treated rats, AFC decreased to 16±6%. This effect was not enhanced by IV S-ketamine. LPS increased IL-6 and CINC-3 in plasma and bronchoalveolar lavage fluid. In alveolar type-II cells, S-ketamine reduced ISC by 37% via a decrease in amiloride-inhibitable sodium transport. Continuous administration of IV S-ketamine does not affect rat AFC even in endotoxin-induced ALI. Tracheal application with direct exposure of alveolar epithelial cells to S-ketamine decreases AFC by inhibition of amiloride-inhibitable sodium transport.     

The effect of a peat-based preparation on mitogen-induced proliferation of thymocytes in non-treated and hydrocortisone-suppressed mice.

The studies were carried out on Balb/c mice (5-6 weeks of age) treated with a peat-based preparation (PBP), administered i.p. once or four times at 24 h intervals at doses of 0.01; 0.1 or 1 mg/kg. Additionally, hydrocortisone was injected i.p. to selected mice at a single dose of 125 mg/kg. The results show that PBP temporarily enhances the proliferative capability of murine thymocytes stimulated in vitro with concanavalin A (Con A) and phytohemagglutinin (PHA). The effect of PBP depends on the number of subsequent doses, but does not depend on the dose applied. A single PBP administration does not affect the proliferative response of thymocytes to Con A and PHA. A single injection of PBP (doses from 0.01 to 1 mg/kg) does not change the number of thymic cells and weight ratio of this organ. Increased doses of subsequent PBP injections (0.01 and 0.1 mg/kg) do not affect the number of thymocytes, but temporarily increase the weight ratio of the thymus two days after the last injection. Administration of PBP prior to hydrocortisone prevents the suppressive effect of the drug on proliferative response of thymocytes stimulated in vitro with Con A and PHA, at the same time increasing the proliferative response of thymic cells to the two mitogens in relation to the control group (hydrocortisone-free). The effect of a single dose of PBP depends on the dose applied--the weakest preventive effect was observed at a dose of 0.01 mg/kg. An increase in the number of subsequent PBP doses, irrespective of a dose applied, prolongs the protective action of the drug on proliferative activity of thymocytes stimulated in vitro with these mitogenes. Moreover, the results obtained in the studies show that PBP partially prevents the suppressive effect of hydrocortisone, as the number of thymic cells and weight ratio of this organ drastically decreased. PBP accelerates regeneration of the thymus, but this depends on a dose applied and the number of subsequent doses. The result was the strongest and the fastest when PBP was injected four times at a dose of 1 mg/kg. It seems quite likely that the thymic regeneration due to PBP is connected with the effect of this drug on maturation and differentiation of thymic cells.     

Effects of zomepirac and indomethacin on renal blood flow and electrolyte excretion in anesthetized monkeys

Zomepirac sodium is a new inhibitor of prostaglandin cyclooxygenase with an $\text{in vitro}$ potency equivalent to indomethacin. Since inhibitors of prostaglandin synthesis have marked effects on renal hemodynamics, zomepirac may be expected to reduce renal blood flow (RBF) in a manner similar to indomethacin. This study compares the effects of zomepirac and indomethacin on RBF and electrolyte excretion in anesthetized Rhesus monkeys. Each experiment consisted of a control period followed by 3 or 4 drug treatment periods in which increasing doses of zomepirac (0.5 to 20 mg/kg) or indomethacin (0.5 to 10 mg/kg were given. Indomethacin (5 mg/kg) reduced RBF by 22% and the higher dose (10 mg/kg) reduced RBF by an additional 13%. Zomepirac had little effect on RBF in doses as high as 20 mg/kg. At any given dose the mean plasma concentration of zomepirac was equal to or greater than indomethacin. Peak indomethacin concentration was 48 μg/ml after the 10 mg/kg dose while the peak zomepirac, after 20 mg/kg, was 158 μg/ml. Neither drug had a significant effect on either glomerular filtration rate or excretion rate of sodium or potassium. Thus, zomepirac had only minimal effects on RBF while indomethacin decreased RBF of anesthetized monkeys in a manner qualitatively similar to its effect in other species. The minimal renal effects caused by zomepirac relative to indomethacin in this primate may indicate a therapeutic advantage for zomepirac in man.     

The genotoxic and cytotoxic effects of ribavirin in rat bone marrow

The genotoxic and cytotoxic effects of the antiviral drug, ribavirin, was studied in rat bone marrow by employing the micronucleus assay. Ribavirin in doses of 10, 15, 20, 30, 50, 75, 100 and 200 mg/kg, and cyclophosphamide (CP) 40 mg/kg (only for sex-difference study) were injected intraperitoneally. Bone marrow was collected at 24 h and 48 h following the injection. To evaluate the recovery, the bone marrow was also sampled at 72 h from 20, 100 and 200 mg/kg treated rats. The micronucleus assay was conducted according to the standard procedure. Ribavirin elevated the incidence of micronuclei (except 10 mg/kg) in erythrocytes (P<0.01). The micronucleated polychromatic erythrocytes showed the initial steep increase at 15 and 20 mg/kg dose level, then with the gradual increase, possibly due to the limited metabolism and action of higher doses. The incidence of micronucleated normochromatic erythrocytes was not dose dependent. The effect was more at 48 h than 24 h due to prolonged toxicity of the drug or its metabolites, and by 72 h, recovery was observed eventhough the genotoxicity was significant. The PCE% decreased as the dose was increased up to 75 mg/kg, then without much difference between two higher doses. Only 100 mg/kg ribavirin and CP showed more toxicity on male rats. Cytotoxicity was seen due to hindered erythropoiesis or cell destruction. Our findings suggest that ribavirin is genotoxic and cytotoxic agent for rat bone marrow.     

Effect of toremifene on the activity of NK-cells in NZB/NZW mice

The effect of toremifene on NK-cells isolated from the spleen of NZB/NZW mice was studied in comparison to tamoxifen and estradiol. Unlike estradiol but like tamoxifen, toremifene did not influence the activity of NK-cells. Low doses (0.1 and 10.0 mg/kg) of toremifene did not suppress, but a high dose of toremifene and tamoxifen (50 mg/kg for 6 weeks) suppressed the stimulating effect of human interferon alpha on the cells.     

Cytogenetic effects of ribavirin on mouse bone marrow

The micronucleus test and mitotic chromosome analysis were used to study the in vivo mutagenic activity of ribavirin on bone marrow cells of Swiss albino mice. To determine the incidence of micronuclei, mice were injected i.p. twice, at an interval of 24 h. with the drug at doses of 20, 100 and 200 mg/kg. Animals were killed 6 h after the second dose and bone marrow was examined for the presence of micronuclei in developing erythrocytes. Ribavirin significantly (P less than 0.05) induced micronuclei in polychromatic erythrocytes at all doses. A study was conducted to investigate the cytogenetic effect of the drug on mitotic chromosomes. Ribavirin at 200 mg/kg/day was administered to mice for 3 and 5 days. Repeated treatment with the high dose of ribavirin produced a highly significant (P less than 0.02) increase in abnormal metaphase spreads. The results indicate that ribavirin is mutagenic to bone marrow cells of mice as evaluated by the micronucleus test and by chromosome analysis.