Biochemical Pharmacology, Vol. 35. No. 2, pp. 331-334, 1986. Printed in Great Britain. 000~295ys6 $3.00 + 0.00 @ 1986 Pergamon Press Lrd. NECESSITY OF ANTIBODY RESPONSE IN THE TREATMENT OF AFRICAN TRYPANOSOMIASIS WITH a-DIFLUOROMETHYLORNITHINE ALANJ. BITONTI,* PETERP. MCCANN and ALBERT~JOERDSMA Merrell Dow Research Institute, Cincinnati, OH 45215, U.S.A. (Receiued 19 May 1985; accepted 26 June 1985) Abstract-The role of the immune system in the clearance of Trypanosoma brucei brucei from the bloodstream during cu-difluoromethylomithine (DFMO) treatment was studied by determining the effects of dexamethasone on immune and therapeutic responses in rats infected with T. b. brucei. Normal DFMO-treated animals exhibited strong antibody responses to trypanosomes and were cured of T. b. brucei infection by a 7-day regimen of 2% DFMO in drinking water. Animals pretreated with dex- amethasone were not cured by the same level of DFMO treatment. Nonetheless, in rats pretreated with dexamethasone, trypanosomes were cleared from blood during treatment with DFMO, but all immunocompromised animals eventually succumbed to relapses of the infection. Athymic (nude) mice were cured of T. b. brucei infection by a 72-hr course of treatment with 2% DFMO in their drinking water. These findings suggest that relatively low levels of T-cell-independent, antitrypanosomal antibodies are adequate to clear the bloodstream of parasites during DFMO therapy but that an intact immune response is necessary for cures of the disease to be obtained. cu-Difluoromethylornithine (DFMO), a catalytic irre- versible inhibitor of ornithine decarboxylase [l, 21, has been shown to rapidly deplete the intracellular polyamines putrescine and spermidine in trypano- somes and to inhibit trypanosomal DNA synthesis and proliferation [3], as it does in a number of other cell types undergoing rapid proliferation [4]. Administration of DFMO effects cures of African trypanosomiasis in both experimental murine infec- tions [5] and natural human infections [6]. The effects of DFMO on trypanosomes are thought to be pri- marily cytostatic rather than cytolytic [5,7] and are clearly related to polyamine depletion since cures of murine trypanosomiasis by DFMO can be blocked by the coadministration of the polyamines putrescine or spermidine [8]. If DFMO is a cytostatic agent, there must be other factors involved in the clearance of trypanosome infections by the drug. One factor which could augment or supplement the effects of DFMO is an immune response to the trypanosome since antibody responses are known to be involved in the development of immunity to and clearance of trypanosomal infections [7,!9-111. In fact, it was shown earlier that cyclophosphamide-immunosup- pressed mice, which produced no trypanosome- specific antibodies, did not completely clear Try- panosoma rhodesiense from their blood during treat- ment with DFMO [7]. Treatment with glucocorticoids enhances the severity of infections with a variety of bacteria, viruses and parasites [12]. Dexamethasone treatment also inhibits the production of trypanocidal anti- bodies in rats infected with Trypanosoma lewisi, * Address all correspondence to Alan J. Bitonti, Ph.D., Merrell Dow Research Institute, 2110 East Galbraith Road, Cincinnati, OH 45215. resulting in a normally benign infection becoming lethal [13,14]. In the present work we have examined the production of antibodies by trypanosome- infected rats immunosuppressed with dexametha- sone, a synthetic glucocorticoid, which markedly depresses both humoral and cell-mediated immune response in susceptible species [15]. We have also studied the effects of DFMO on the long-term sur- vival of similarly immunocompromised rats and on congenitally athymic (nude) mice infected with TV- panosoma brucei brucei. MATERIALS AND METHODS Trypanosomes. Trypanosoma brucei brucei (EATRO llO), a monomorphic strain causing acute infections, was obtained from Dr. Cyrus Bacchi of the Haskins Laboratories, New York, NY. It was maintained in our laboratory by syringe passage in male Sprague-Dawley rats and was used in all experi- ments described herein. For survival experiments rats were injected intraperitoneally with 5 x 106 trypanosomes; the ensuing infection was lethal in untreated rats within 4-6 days. Drug treatment. Male Sprague-Dawley rats weigh- ing approximately 225 g at the beginning of the experiment were administered tetracycline (1 g/l) or tetracycline (1 g/l) plus dexamethasone (1 mg/l in I their drinking water. Dexamethasone at 1 mg 1 in drinking water has been shown previously to produce significant immunosuppression in rats [ 161. Tetra- cycline was present to protect the rats against bac- terial infections which could arise during immu- nosuppression. After 21 days of dexamethasone treatment the animals were infected with trypano- somes. After 24 hr of infection, one group of rats was given 2% DFMO in their drinking water in addition to the dexamethasone and tetracycline. 331332 A. J. BITONTI, P. P. MCCANN and A. SJOERDSMA DFMO was administered for 7 days and then removed. The animals were then observed daily for mortality and periodically for parasitemia. Since long-term dexamethasone treatment can also cause fatal Pneumocystis carinii infections in rats [16], we examined the lungs of a similarly-treated group of animals for the presence of P. carinii and found them to be uninfected even after 6 weeks of dex- amethasone treatment. Body, spleen and thymus weights as well as hema- tological variables were also determined after 21 days of treatment with dexamethasone. Erythro- cytes, leukocytes and platelets were all counted with an automated hematology analyzer. Lymphocyte percentages were counted in thin blood smears stained with Giemsa. In experiments designed to measure antibody responses,
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