Structural changes in rat Pneumocystis cariniisurface antigens after terbinafine administration in experimental P. carinii pneumonia

Carlo Continia,*, Elena Angelicib and Rita Caniparic

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Terbinafine is a synthetic antifungal agent which has recently been found to be highly effective against Pneumocystis carinii. This study evaluated the efficacy of terbinafine on rat P. carinii antigenic profile and the immune response by Western blot analysis, in comparison with atovaquone and co-trimoxazole in rats with pneumocystosis. Terbinafine was shown to target two specific major antigens, particularly those of 116 and 35-40 kDa. Antibodies reactive against these moieties were found in all rats treated with atovaquone and co-trimoxazole, but not in those treated with terbinafine. These surface antigen modifications could be related to disease severity and could provide additional information for monitoring the efficacy of this treatment.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
In recent years marked molecular and biological affinities have suggested that Pneumocystis carinii should be classified as a fungus. Antifungal agents could thus represent potential therapeutic and prophylactic drugs against this organism.1 A number of these compounds with different mechanisms of action have been evaluated extensively, both in vitro and in vivo, and seem to be promising. To date the most important agents are two classes of (1,3)-ß-D-glucan synthase inhibitors, the echinocandins and papulacandins.2 Because of the high incidence of side effects associated with standard anti-Pneumocystis regimens, research continues to identify other antifungal agents with anti-P. cariniipotential, though the sensitivity of the organism to these compounds alone or in combination is not completely established. Recently, an antifungal and antiprotozoal compound, the allylamine-derived terbinafine, has been shown to be very effective in vitro against P. cariniiand in the treatment of rat P. carinii pneumonia (PCP). This was first discovered by Contini et al.,3 and later confirmed by other investigators. 4

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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Rat immunosuppression and drug administration

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).


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
As reported previously, mean survival rates of the untreated control rats were found to be shorter but not significantly different from the treated groups. 6 Also, >50% of the control rats exhibited a heavy P. carinii burden and severe alveolar involvement, with marked weight loss (P < 0.01) after immunosuppression. The best inhibitory response was found in the terbinafine- and co-trimoxazole-treated groups in which PCP developed in a few animals with almost the same organism burden (mean infection score: 6 ± 0.7 and 8 ± 1.1, respectively, compared with 23 ± 2.1 in the atovaquone-treated group), whereas histological lung changes were absent or minimal.6 A significant relationship (P < 0.001, r = 0.472) was found when semiquantitative cyst counts between BALF and rat lungs were compared. This suggests that the P. carinii burden in BALF correctly reflects the organism burden in the lung.10

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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Figure 1. SDS-PAGE of silver-stained rat BALF preparations. N, BALF from non-steroid suppressed rats; C, BALF from steroid-suppressed rats with PCP; T and At, BALFs from rats treated with terbinafine or atovaquone, respectively. The arrows indicate the 116 and the 35-40 kDa bands. Molecular weights, in kDa, are shown on the left.

 
In preliminary experiments, sera collected before treatment have been shown to contain antibodies against some of the principal P. carinii proteins including those in the 35-40 kDa band. When the panel of each BALF preparation was probed with sera from P. carinii-infected rats, different patterns of immunoreactivity were detected by ECL Western blotting (Figure 2). Sera from control rats with PCP and from those treated with atovaquone recognized two or more antigens, particularly those of 116, 97, 66 and 35-40 kDa, but none of the terbinafine-treated rats had detectable antibodies against the 35-40 kDa protein, while a small number (two out of 30) showed a slight reactivity against the 116 kDa protein. Although no immunoreactivity towards the 116 or 35-40 kDa band was found by probing normal rat BALF with sera from infected rats or anti-P. carinii antiserum, the finding of faint bands on either silver staining preparations or blots is consistent with previous studies which confirm that animals as well as healthy humans have been exposed to P. carinii. 11



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Figure 2. Western blot patterns of BALFs probed with sera from steroid-suppressed rats with PCP (C), treated with terbinafine (T) or atovaquone (At). The arrows indicate the 116 and the 35-40 kDa bands. Molecular weights, in kDa, are shown on the left.

 
The purpose of these experiments was to investigate whether Western blotting would provide meaningful data about the immune response to P. cariniiantigens during drug administration. The 116 kDa protein is a component of the major surface glycoprotein (MSG), which participates in the attachment of P. cariniito alveolar epithelial cells. The immunoreactive moiety of molecular weight 35-40 kDa is the P. cariniiantigen most commonly found in the respiratory tract in rat and in humans and most frequently recognized by the host; it can thus serve as an important marker of P. cariniiinfection.11

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.


    Acknowledgments
 
This work was supported by the Italian Ministry of University and Scientific Research (Grant of 60% and 40%, 1997)


    Notes
 
* Tel and Fax: +39-532-291310; E-mail: cnc{at}ifeuniv.unife.it Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Stringer, J. R., Edman, J. C., Cushion, M. T., Richards F. F. & Watanabe, J. (1992). The fungal nature of Pneumocystis carinii. Journal of Medical and Veterinary Mycology 30 , Suppl. 1, 271–8.[ISI][Medline]

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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 , 727–35.[Abstract]

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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, 1935–42.[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, 649–54.[ISI][Medline]

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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, 4350–4.[Abstract]

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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, 2023–31.[Abstract/Free Full Text]

Received 23 June 1998; accepted 3 October 1998