L MATERIALS AND METHODS P INTRODUCTION

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Summary :

Pneumocystis carinii pneumonia (PCP) is a highly frequent cause of morbidity and mortality in immunocompromised subjects,

particularly in HIV-infected ones. The biology of P. carinii is poorly understood because of the lack of reliable synthetic media or adequate cell lines to grow this opportunistic pathogen in continuous culture. W e reported the suitability of the M D C K (Madin Darby Canine Kidney, ATTC C C L 34) cell line to support the temporary microorganism's growth in vitro and the

experimental pharmacological trials, in comparison with the HEL 2 9 9 cell line, used as reference standard.

KEY WORDS : Pneumocystis carinii, in vitro culture, M DCK cell line.

Résumé : Madin Da r by Canine Kid n ey : unenouvelleligne CELLULAIRE POUR LA CULTURE IN VITRO d e PNEUMOCYSTIS CARINII La pneumonie à Pneumocystis carinii (PCP) est la cause la plus fréquente d e morbidité et mortalité ch ez les sujets

immunodéprimés, notamment les patients infectés par le VIH. La biologie d e P. carinii reste encore insuffisamment connue, du fait qu'on ne dispose pas encore de milieux synthétiques ou de lignées cellulaires permettant la culture continue d e c e micro­

organisme opportuniste. Dans cette étude les auteurs démontrent que la lignée cellulaire M D C K (M adin Darby Canine Kidney, A T C C CC L 34) peut supporter la culture temporaire de P. carinii et les tests d e pharmacosensibilité tout comme la lignée HEL 2 9 9 prise comme référence.

MOTS CLÉS : Pneumocystis carinii, culture in vitro, ligne cellulaire MDCK.

IN T R O D U C T IO N

P

nistic pneum onia (PCP) in im m unocom pro­

mised hosts. In particular PCP continues to be the most frequent cause o f morbidity and mortality in HIV-infected subjects (Glatt and Chirgwin, 1990).

The em ergence o f AIDS-related PCP has shed new light on the problems related with therapy. Both classic the­

rapeutic regimens, such as cotrim oxazole (TMP-SMZ) and pentamidine and new er ones, such as clinda- mycin-primaquine, trimetrexate and atovaquone (Sattler and Feinberg, 1992; Hughes et al., 1993), entail a high risk o f severe side effects. Research efforts to identify effective drugs have been at least partially hindered by the lack o f synthetic media or adequate cell lines to grow P. c a r in ii in continuous culture. Since the late 7 0 ’s, several cell lines have b een em ployed with acceptable results to maintain P. c a r in ii vegetative

* Laboratory o f Clinical Parasitology, Institute o f Infectious Diseases, University-IRCCS S.Matteo, 27100 Pavia, Italy.

Correspondence : Pr. Massimo Scaglia. Tel. : +(382) 502.698.

Fax : + (382) 42.33.20.

Parasite, 1996, 3, 183-185

forms in a short- or medium-term culture (Latorre et al., 1977; Pifer et al., 1977, 1978; Bartlett et al., 1979;

Cushion et al., 1985).

W e report on the satisfactory results w e obtained by culturing P. c a r in ii on the MDCK (Madin Darby Canine Kidney, ATTC CCL 34) cell line, which has already been em ployed successfully for the in vitro isolation and culture o f other microorganisms such as Crypto­

sp o rid iu m p a r v u m (Gut et al., 1991; Rosales et al., 1993) and Microsporidia (E n c ep h a lito z o o n spp.) (Can­

ning and Hollister, 1991; Curry and Canning, 1993; Hol­

lister et al., 1993). The HEL 299 line was used as a refe­

rence standard because o f its know n reliability in drug sensitivity tests (Bartlett, 1991; Atzori et al., 1993;

Mirovsky and Fishman, 1993; Q ueener et al., 1993).

MATERIALS A N D M ETH O D S

L

culum, and w ere prepared by extraction and purification from the lungs o f pharmacologically im munosuppressed rats w hich had been previously infected by the transtracheal instillation (Bartlett et al.,

N ote d e recherche 183

Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1996032183

ATZORI C., BRUNO A., AGOSTONÎ % Ï K $ A m , Û A f n S. & SCAGÍ.Ía’W . ¡

1988). The lungs o f the infected animals w ere hom o­

genized in a grinder with MEM medium and gently centrifuged at 500 rpm to remove the larger tissue frag­

ments. The num ber o f microorganisms was obtained by m icroscopical observation o f a sample o f superna­

tant and viability test perform ed at the same time by using the double staining fluorogenic method des­

cribed by Kaneshiro (1991).

MDCK and Hel 299 cells w ere cultured in 12- or 24- well plates (Flow Lab.) at 37 °C in 5 % CO 2 with minimal essential medium (MEM) to which 10 % fetal calf serum (FCS) was added. O nce a confluent m ono­

layer had developed, each well was inoculated with 50 μl o f lung hom ogenate to obtain a final con cen­

tration o f 5-6 x 105 trophozoites/ml.

The organisms developed in culture in the extracellular space; som e w ere adherent to the host cells, while others w ere free in the culture medium, at times for­

ming clusters.

O ne, 5 and 8 days after the inoculation, organism counts were performed after gentle pipetting the super­

natant in order to detach the adherent m icroorga­

nisms. Each point, plotted in the growth curves, repre­

sen ted the m ean tro p h o z o ites’ nu m ber per field (m agnification 1,000 x) obtained by blind count from two observers o f 30 fields o f a 10 μl drop o f super­

natant o f P. c a r in ii cell culture dried onto a 1 cm square slide and Giemsa stained: the m ean value has a mathem atical correlate, as multiplyied x 400,000 yields to the num ber o f trophozoites/ml. Viability test was perform ed at the m om ent o f the infection and repeated on day 8.

An experim ental trial with TMP-SMZ (9-45 μg/ml) was performed in parallel, by adding the drug to the wells together with the P. c a r in ii inoculum. Study control o f the direct effect o f the drug on m onolayer at the

same concentration used for P. c a r in ii infected cells was also done. Counts w ere perform ed by using the same approach described for growth curves.

Each experim ent was repeated at least three times before final evaluation, four well replications for each sample.

RESULTS A N D D ISC U SSIO N

R

rence HEL 299 cell line (Fig. 1). The increase in the num ber o f organisms was obviously related to the active m ultiplication o f trophozoites, w hich repre­

s e n te d th e a b so lu te ly p re v a le n t form o b se rv e d (approxim ately 99 % o f total) as com pared to the cysts, with a 95 % viability assessed on day 8. Accor­

ding to our observation, gentle pipetting the culture supernatant is very important in order to obtain detach­

m ent o f adherent forms, a phenom enon well dem ons­

trated also by different Authors (Aliouat et al., 1993;

Bartlett et al., 1994).

The suitability o f the in vitro culture technique was eva­

luated by double-blind counts and show ed a 10-fold increase in the num ber o f organisms/ml at the end o f the culture period (average initial count: 1.4 orga­

nisms/field; average final count, after 8 days o f culture:

over 13 organisms/field).

The results o f the pharmacological tests performed with TMP-SMZ w ere com parable in the two cell lines tested (Fig. 2). Less than 1 % o f TMP/SMZ treated m icroor­

ganisms w ere viable at day 8.

Fig. 1 - Growth curves o f rat-derived P. carin ii (Pc) in MDCK and HEL 299 cell lines.

Fig. 2. - In vitro culture o f P. carin ii (Pc) in MDCK and HEL 299 cells; response to addition o f cotrimoxazole (TMP-SMZ).

184- Parasite, 1996, 3, 183-185

P . CARINII IX v m o CULTURE

Given its already proven efficacy to support the deve­

lopm ent o f human strains o f protozoan pathogens (i.e . C. p a r v u m and Microsporidia) with good repro­

ducibility o f the culture (Gut et al., 1991; Rosales et al., 1993; Canning and Hollister, 1991; Curry and Can­

ning, 1993; Hollister et al., 1993), MDCK cell line may be considered a candidate for the role o f « all-purpose » cell line for the isolation, m aintenance and pharma­

cological testing o f opportunistic microorganisms with paramount importance in AIDS- and non-AIDS-related hum an infectious diseases.

ACKNOWLEDGEMENTS

T his w ork w as partially su pported by ECA;

contract * BMH CT94 PL 1118.

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Reçu le 22 septembre 1995 Accepté le 12 février 1996

Parasite, 1996, 3, 183-185

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