a Section of Infectious Diseases, Department of Clinical and Experimental Medicine, University of Ferrara, via Fossato di Mortara 23, I-44100 Ferrara; b Department of Clinical Medicine, via Regina Elena 331; c Department of Histology and Medical Embryology, `La Sapienza' University, via Antonio Scarpa 14, I-00161 Rome, Italy
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Abstract |
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Introduction |
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Although various mechanisms of action of terbinafine have been suggested, it is not known how P. carinii is cleared from rat lung after treatment. In this paper we examined the anti-P. cariniiefficacy of terbinafine by investigating the antigenic pattern of P. cariniiisolated from the bronchoalveolar lavage fluid (BALF) of immunosuppressed rats and by assessing the specific serological response by Western blotting in comparison with two reference anti-P. cariniidrugs, atovaquone and co-trimoxazole.
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Materials and methods |
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PCP was induced by immunosuppressing 180-250 g male Sprague-Dawley rats (Charles River, Como, Italy) with 25 mg/L of subcutaneous methylprednisolone acetate (Solu-medrol; Upjohn, Milan, Italy) twice weekly for 10-12 weeks, and giving a low-protein diet to enhance the immunosuppression. Tetracycline was given by gavage to minimize other opportunistic infections. The percent survival was monitored. At intervals, randomly selected sample animals were killed and parasite burdens in the lungs were determined. The intensity of PCP was graded by scanning the impression smears and assigning an infection score of 0 (no infection evident) to 4+ (heavy infection).5,6 PCP progressed consistently over time for each of the inoculation groups. By week 4, rats began to show signs of wasting, with marked weight loss; P. carinii infection became intense with massive organism burden in 6-8 weeks. Rats were then randomly selected and divided into three groups of about 30 animals each of which received, by oral gavage, either co-trimoxazole (Sigma-Aldrich, Milan, Italy) (at a dose of 12.5 mg/kg/day of trimethoprim and 62.5 mg/kg/day of sulfamethoxazole) or atovaquone (Wellcome Italia, Rome, Italy) at 100 mg/kg/day, both found to have a significant dose-related effect in the treatment of pneumocystosis. Terbinafine (Sandoz, Basel, Switzerland) was given at 80 mg/kg/day, which has been shown to lead to significant clearance of P. carinii without significant histological changes.6 After 4 weeks of drug administration, rats were killed and lungs were removed aseptically and weighed. Evaluation of drug efficacy was studied by comparing the P. cariniiburden (mean infection score ± S.D.) and histological changes in the treatment groups with those in the controls in the same experiment. Control groups consisted of steroid-suppressed rats with PCP and uninfected and untreated rats. The in-vivo study was approved by the Institutional Animal Care and Use Committee.
Specimen collection and parasite load determination
BALF specimens were collected at the end of treatment at post-mortem. Broncheolar lavage of the right lung was performed with approximately 10 mL of 0.15 M NaCl, instilled in 3 mL fractions. Aliquots of BALF were subjected to microbiological tests, and any BALF samples containing bacteria or fungi that could potentially interfere with the detection of P. carinii were discarded. For quantification of P. carinii, BALF samples were centrifuged at 3500g for 10 min and the cellular pellet was resuspended in Hank's solution. Ten-microlitre drops of cell pellet were stained by methenamine silver stain. The number of cysts was scored on a semiquantitative scale (0 to 4+) and the score was correlated with the cyst burden recorded in each rat lung (Spearman's rank correlation coefficient; statistical significance was at P < 0.05). Infected sera were obtained by cardiac puncture when rats were bled. Sera were found to contain a high specific IgG antibody titre as measured by ELISA.7
Electrophoresis and Western blot analysis
After BALF protein determination (Bio-Rad Laboratories, Richmond, CA, USA), BALF pellets were solubilized by boiling for 10 min in 2% sodium dodecyl sulphate (SDS) lysis buffer containing 0.06 M Tris, 1% glycerol and 1.25% ß-mercaptoethanol. 8 Samples were centrifuged and then loaded at a concentration of 10 µg of protein per lane (approximately 106 P. carinii cysts) to discontinuous SDS-polyacrylamide 4% stacking gel and 10% running gels at 10°C under 60 mA constant current using bromophenol blue as a marker. After completion of the run, gels containing separated antigens were either fixed in 40% methanol/10% acetic acid and stained with silver stain or were transferred to nitrocellulose in a Trans-blot cell (Bio-Rad) for 1 h at 60 V according to Towbin et al. 9 Nitrocellulose blots were probed overnight with infection-derived rat serum (1:200 dilution) and then washed in 0.5% Tween 20- Tris-buffered saline (TTBS). After incubation with alkaline phosphatase-conjugated goat anti-rat IgG (Dako, Copenhagen, Denmark) diluted 1:2000, strips were finally washed in TTBS and developed with the ECL chemiluminescence substrate (Amersham plc, Amersham, UK).
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Results and discussion |
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The electrophoretic pattern of BALF preparations analysed by SDS-PAGE is presented in Figure 1. Strong bands corresponding to major P. cariniiantigens and ranging in size from 30 to 116 kDa were demonstrated in BALFs from control rats with PCP and, similarly, in atovaquone preparations. Neither the electrophoretic profile nor the antigen recognition pattern of BALFs from co-trimoxazole-treated rats differed substantially from that of atovaquone-treated animals (data not shown). In BALF preparations from terbinafine-treated animals, the 116 kDa protein was not stained but the bands at molecular masses of 35-40 kDa were almost absent. Bands at 97, 66 and 45 kDa were occasionally seen in normal BALF samples.
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Among the squalene-epoxydase inhibitors, terbinafine is the most potent agent against pathogenic fungi and is also active against protozoa. Although its mechanism of activity against P. cariniiis not fully established, there is experimental evidence that terbinafine may target two important surface P. carinii antigens, the 116 and 35-40 kDa proteins. These changes may impair the organism's structural integrity and consequently either the mechanism of host-parasite interaction or the host immune response, thus providing additional information for monitoring the efficacy of this treatment.
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Acknowledgments |
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Notes |
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References |
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2 . Schmatz, D. M., Romancheck, M. A., Pittarelli, L. A., Schwartz, R. E., Fromtling, R. A., Nollstadt, K. H. et al. (1990). Treatment of Pneumocystis carinii pneumonia with 1,3-beta-glucan synthesis inhibitors. Proceedings of the National Academy of Sciences of the USA 87, 59504.[Abstract]
3 . Contini, C., Manganaro, M., Romani, R., Tzantzoglou, S., Poggesi, I., Vullo, V. et al. (1994). Activity of terbinafine against Pneumocystis carinii in vitro and its efficacy in the treatment of experimental pneumonia. Journal of Antimicrobial Chemotherapy34 , 72735.[Abstract]
4 . Cirioni, O., Giacometti, A., Balducci, M., Burzacchini, F. & Scalise, G. (1995). In-vitro activity of terbinafine, atovaquone and co-trimoxazole against Pneumocystis carinii. Journal of Antimicrobial Chemotherapy 36, 7402.[ISI][Medline]
5 . Walzer, P. D., Foy, J., Steele, P., Kim, C. K., White, M., Klein, R. S. et al. (1992). Activities of antifolate, antiviral and other drugs in an immunosuppressed rat model of Pneumocystis carinii pneumonia. Antimicrobial Agents and Chemotherapy 36, 193542.[Abstract]
6 . Contini, C., Colombo, D., Cultrera, R., Prini, E., Sechi, T., Angelici, E. et al. (1996). Employment of terbinafine against Pneumocystis carinii infection in rat models. British Journal of Dermatology 134, Suppl. 46,30 2.[ISI][Medline]
7 . Nielsen, P. B. & Mojon, M. (1988). Enzyme linked immunosorbent assay compared with indirect immunofluorescence test for detection of Pneumocystis carinii specific immunoglobulins G, M, and A. APMIS 96, 64954.[ISI][Medline]
8 . Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 6805.[ISI][Medline]
9 . Towbin, H., Staehelin, T. & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the USA 76, 43504.[Abstract]
10 . Smulian, A. G., Linke, M. J., Cushion, M. T., Baughman, R. P., Frame, P. T., Dohn, M. N. et al. (1994). Analysis of Pneumocystis carinii organism burden, viability and antigens in bronchoalveolar lavage fluid in AIDS patients with pneumocystosis: correlation with disease severity. AIDS 8, 155562.[ISI][Medline]
11
.
Kovacs, J. A, Halpern, J. L, Swan, J. C., Moss, J.,
Parrillo, E. & Masur, H. (1988). Identification of antigens and antibodies specific
for Pneumocystis carinii. Journal of Immunology 140, 202331.
Received 23 June 1998; accepted 3 October 1998