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Antimicrobial Agents and Chemotherapy, September 1999, p. 2305-2306, Vol. 43, No. 9
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Lack of In Vitro Antimicrosporidian Activity
of Thalidomide
Olivier
Ridoux and
Michel
Drancourt*
Unité des Rickettsies, CNRS UPRES-A
6020, Faculté de Médecine, Université de la
Méditerranée, Marseille, France
Received 26 April 1999/Returned for modification 27 May
1999/Accepted 24 June 1999
 |
ABSTRACT |
Thalidomide was evaluated for its in vitro activity against
Encephalitozoon species by using the MRC-5 cell system. A
cytotoxic effect was observed for concentrations of 101
µg/ml (P < 105) and 5 µg/ml
(P < 105). Thalidomide did not
significantly inhibit the growth of any of the microsporidia under
study. These data suggest that thalidomide is not an etiologic
treatment in microsporidial enteritis.
 |
TEXT |
Microsporidia are spore-forming,
obligate intracellular microorganisms known to parasitize almost every
group of animals (23). In humans, Enterocytozoon
bieneusi, Encephalitozoon intestinalis, and
Encephalitozoon cuniculi are emerging pathogens increasingly recognized as major causes of diarrhea, weight loss, and wasting in
human immunodeficiency virus (HIV)-infected patients (5) and
in non-HIV-infected patients (2, 11, 20, 22). Enteric infection is localized to the small intestine and is associated with
villus damage and functional alterations (14). E. bieneusi infection is limited to the intestine, whereas
Encephalitozoon species are also responsible for
antimicrobial-resistant, life-threatening systemic infections
(5, 7, 10).
Thalidomide, first developed as a sedative in the late 1950s, was
withdrawn from widespread use because of teratogenicity (3).
However, recently, thalidomide was approved for the treatment of
leprosy (4). It has been shown to prevent HIV replication in
monocytes in vitro (9) but has no known antimicrobial
activity. It has also been successfully used as a treatment for
microsporidian enteritis in HIV-infected persons (18), with
improvement in clinical symptoms and histological parameters (16,
18). Microsporidial alterations were noted at all stages of the
life cycle (18). These observations suggested a direct
antimicrosporidian effect of thalidomide, but no experimental model has
been developed to assess this hypothesis. We therefore tested the
potential antimicrosporidian activity of thalidomide against E. intestinalis, E. cuniculi, and Encephalitozoon
hellem using a cell culture system.
Thalidomide (LAPHAL, Paris, France) was dissolved in sterile dimethyl
sulfoxide (DMSO) and the potential toxicity of thalidomide and DMSO on
MRC-5 cells was examined by microplaque colorimetric assay
(12). Untreated control cells, DMSO-treated cells, and cells
treated with one of 10 to 10
6 mg/ml (final concentration)
serial dilutions of thalidomide were tested per plate. The optical
density at 492 nm (OD492) of treated cells was compared
with that of control cells after incubation in the presence of 0.15%
neutral red dye (pH 5.5) (Sigma Chemical, St. Louis, Mo.) and
extraction by 10% phosphate ethanol buffer (pH 4.2). A thalidomide
concentration was considered toxic when the mean OD492 of
the treated cells was lower than the mean OD492 of the
control cells.
Microsporidia (kindly provided by T. van Gool, University of Amsterdam,
Amsterdam, The Netherlands) were cocultivated with MRC-5 embryonic lung
fibroblasts (BioMérieux, Lyon, France) on coverslips in 24-well
plates. For the thalidomide activity assay, 1 ml of 1% decomplemented
fetal calf serum culture medium, with or without thalidomide, was added
and the plate was incubated at 37°C in a 5% CO2
incubator for 6 days. For each plate, line 1 was a positive control
without drug, and nine serial dilutions of thalidomide from 10 to
10
6 µg/ml were tested in lines 2 to 6 of the plate.
After incubation, fixed cells were stained by quick-hot
gram-chromotrope staining, and coverslips were mounted on microscope
slides and observed with a light microscope using a 100× oil immersion
lens objective. The mean and standard deviation of the mean number of
microsporidia per field were determined after 20 fields had been
observed. The percentage of microsporidian growth inhibition was
calculated as [1
(mean number of infected cells in replicate
cultures with thalidomide/mean number of infected cells in control
cultures)] × 100 (± standard error).
Analysis of variance was used to compare the means and standard
deviations of optical density in the toxicity test and percentages of
growth inhibition for each microsporidian species and for each concentration of thalidomide.
A toxic effect was observed for thalidomide concentrations of 5 to 10 µg/ml (P < 105) (Table
1). DMSO at a final concentration of
10
3 µg/ml had no toxic effect on MRC-5 cells. The
percentage of growth inhibition varied from 0 to 8.2% for E. hellem, from 0 to 5.6% for E. cuniculi, and from 0 to
8.4% for E. intestinalis, with thalidomide concentrations
of 101, 5, 100, 10
1,
10
2, 103, 10
4, and
10
5 µg/ml (Table 1). Statistical analysis indicated
that no thalidomide concentration significantly reduced the growth of
any of the three Encephalitozoon species under
investigation. We did not observe morphological modifications in any of
the three species under investigation.
View this table:
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TABLE 1.
Intracellular activity of thalidomide on microsporidian
growth and estimation of MRC-5 host cell viability
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|
Thalidomide was reported to relieve the clinical symptoms of
microsporidian enteritis in HIV-1-infected patients as assessed by
lower stool frequency and body weight gain (16, 18).
Clinical improvement correlated with the normalization of the villus
height/crypt depth ratio, along with morphological alterations of
microsporidia (16). Electron microscopy analysis of pre- and
post-thalidomide treatment intestinal biopsies disclosed no reduction
in the microsporidian load but a significant increase in the number of
ultrastructurally abnormal forms. These included membrane damage,
vacuolated nuclei and cytoplasm, megaspores, and meganuclei for
E. bieneusi. In the case of E. intestinalis,
plasmodia and spores detected in the stool after treatment were
disrupted (16). Data herein reported demonstrate that
thalidomide had no direct antimicrosporidian effect resulting in such
abnormalities and no significant microsporidian growth inhibition
effect over the concentration range we studied. The model used in this
study has been previously validated for antimicrosporidian screening of
various drugs (1, 12). There is no pharmacokinetic study of
thalidomide in HIV-1-infected patients, but a 5-µg/ml level in plasma
was achieved in patients presenting with chronic graft-versus-host
disease (21). One may assume that the concentration range we
tested comprises expected levels in serum during the treatment of
microsporidian enteritis. Elevated levels of fecal tumor necrosis
factor alpha (TNF-
) were found in HIV-infected patients with
microsporidial enteritis (17), and a marked, albeit
nonsignificant, decrease of fecal TNF-
level from 17.9 to 8.9 U/ml
was observed after thalidomide treatment (16, 18). TNF-
may play a role in the mucosal abnormalities in microsporidial
enteritis (14). Thalidomide has been proved to selectively
inhibit TNF-
from a monocytic cell line (13). In a
tuberculosis meningitis rabbit model with a 50% death rate, the
combination of thalidomide with appropriate antibiotics resulted in a
100% survival rate. Increased survival of animals correlated with
decreased TNF-
levels in the cerebrospinal fluid and plasma of
infected animals (19). Also, thalidomide has been shown to inhibit immunoglobulin M synthesis (15), to inhibit
interleukin 12 production (8), and to increase cytotoxic
responses in the CD8+ human T lymphocytes subset
(6). It is therefore likely that thalidomide-induced
immunomodulation is the principal mechanism of action of thalidomide in
microsporidian enteritis, and this hypothesis is under investigation in
the laboratory in an original coculture model.
 |
ACKNOWLEDGMENTS |
We acknowledge the support of Programme Hospitalier de Recherche
Clinique 1997, Assistance Publique, Hôpitaux de Marseille.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Unité des
Rickettsies, Faculté de Médecine, 27 Boulevard Jean Moulin,
13385 Marseille cedex 5, France. Phone: 33 (0) 4.91.32.43.75. Fax. 33 (0) 4.91.83.03.90. E-mail:
Michel.Drancourt{at}medecine.univ-mrs.fr.
 |
REFERENCES |
| 1.
|
Beauvais, B.,
C. Sarfati,
S. Challier, and F. Derouin.
1994.
In vitro model to assess effect of antimicrobial agents on Encephalitozoon cuniculi.
Antimicrob. Agents Chemother.
38:2440-2448[Abstract/Free Full Text].
|
| 2.
|
Cegielski, J. P.,
Y. R. Ortega,
S. McKnee,
J. F. Madden,
L. Gaido,
D. A. Schwartz,
K. Manji,
A. F. Jorgensen,
S. E. Miller,
U. P. Pulipaka,
A. E. Msengi,
D. H. Mwakyusa,
C. R. Sterling, and L. B. Reller.
1999.
Cryptosporidium, Enterocytozoon, and Cyclospora infections in pediatric and adult patients with diarrhea in Tanzania.
Clin. Infect. Dis.
28:314-321[Medline].
|
| 3.
|
Dally, A.
1998.
Thalidomide: was the tragedy preventable?
Lancet
351:1197-1199[Medline].
|
| 4.
|
Fox, J. L.
1998.
Thalidomide approved for treating leprosy.
ASM News
64:557-558.
|
| 5.
|
Franzen, C.,
D. A. Schwartz,
G. S. Visvesvara,
A. Muller,
A. Schwenk,
B. Salzberger,
G. Fatkenheuer,
P. Hartmann,
G. Mahrle,
V. Diehl, and M. Schrappe.
1995.
Immunologically confirmed disseminated, asymptomatic Encephalitozoon cuniculi infection of the gastrointestinal tract in a patient with AIDS.
Clin. Infect. Dis.
21:1480-1484[Medline].
|
| 6.
|
Haslett, P. A.,
L. G. Corral,
M. Albert, and G. Kaplan.
1998.
Thalidomide costimulates primary human T lymphocytes, preferentially inducing proliferation, cytokine production, and cytotoxic responses in the CD8+ subset.
J. Exp. Med.
187:1885-1892[Abstract/Free Full Text].
|
| 7.
|
Molina, J.-M.,
E. Oksenhendler,
B. Beauvais,
C. Sarfati,
A. Jaccard,
F. Derouin, and J. Modaï.
1995.
Disseminated microsporidiosis due to Septata intestinalis in patients with AIDS: clinical features and response to albendazole therapy.
J. Infect. Dis.
171:245-249[Medline].
|
| 8.
|
Moller, D. R.,
M. Wysocka,
B. M. Greenlee,
X. Ma,
L. Wahl,
D. A. Flockhart,
G. Trinchieri, and C. L. Karp.
1997.
Inhibition of IL-12 production by thalidomide.
J. Immunol.
159:5157-5161[Abstract].
|
| 9.
|
Moreira, A. L.,
L. G. Corral,
W. Ye,
B. Johnson,
D. Stirling,
G. W. Muller,
V. H. Freedman, and G. Kaplan.
1997.
Thalidomide and thalidomide analogs reduce HIV type 1 replication in human macrophages in vitro.
AIDS Res. Hum. Retroviruses
13:857-863[Medline].
|
| 10.
|
Orenstein, J. M.,
D. T. Dieterich,
A. E. Lew, and D. Kotler.
1993.
Albendazole as a treatment for intestinalis and disseminated microsporidiosis due to Septata intestinalis in AIDS patients: a report of four patients.
AIDS
7:S40-S42.
|
| 11.
|
Rabodonirina, M.,
M. Bertocchi,
I. Desportes-Livage,
L. Cotte,
H. Levrey,
M. A. Piens,
G. Monneret,
M. Célard,
J. F. Mornex, and M. Mojon.
1996.
Enterocytozoon bieneusi as a cause of chronic diarrhea in a heart-lung transplant recipient who was seronegative for human immunodeficiency virus.
Clin. Infect. Dis.
23:114-117[Medline].
|
| 12.
|
Ridoux, O., and M. Drancourt.
1998.
In vitro susceptibility of the microsporidia Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis to albendazole and its sulfoxide and sulfone metabolites.
Antimicrob. Agents Chemother.
42:3301-3303[Abstract/Free Full Text].
|
| 13.
|
Sampaio, E. P.,
E. N. Sarno,
R. Galilly,
Z. A. Cohn, and G. Kaplan.
1991.
Thalidomide selectively inhibits tumor-necrosis-factor- production by stimulated human monocytes.
J. Exp. Med.
173:699-703[Abstract/Free Full Text].
|
| 14.
|
Schmidt, W.,
T. Schneider,
W. Heise,
J. D. Schulzke,
T. Weinke,
R. Ignatus,
R. L. Owen,
M. Zeitz,
R. Riecken, and R. Ullrich.
1997.
Mucosal abnormalities in microsporidiosis.
AIDS
11:1589-1594[Medline].
|
| 15.
|
Shannon, E. J.,
R. O. Miranda,
M. J. Morales, and R. C. Hastings.
1981.
Inhibition of de novo IgM antibody synthesis by thalidomide as a relevant mechanism of action in leprosy.
Scand. J. Immunol.
13:553-562[Medline].
|
| 16.
|
Sharpstone, D.,
A. Rowbottom,
N. Francis,
G. Tovey,
D. Ellis,
M. Barret, and B. Gazzard.
1997.
Thalidomide: a novel therapy for microsporidiosis.
Gastroenterology
112:1823-1829[Medline].
|
| 17.
|
Sharpstone, D.,
A. Rowbottom,
M. Lepper,
M. Nelson, and B. Gazzard.
1996.
Faecal tumor necrosis factor-alpha in HIV-related diarrhea.
AIDS
10:989-994[Medline].
|
| 18.
|
Sharpstone, D.,
A. Rowbottom,
M. Nelson, and B. Gazzard.
1995.
The treatment of microsporidial diarrhoea with thalidomide.
AIDS
9:658-659[Medline].
|
| 19.
| Tsenova, L., K. Sokol, V. H. Freedman, and G. Kaplan. A combination of thalidomide plus antibiotics protects
rabbits from mycobacterial meningitis-associated death. J. Infect.
Dis. 177:1563-1572.
|
| 20.
|
Van Gool, T.,
J. C. M. Vetter,
B. Weinmayr,
A. Van Dam,
F. Derouin, and J. Dankert.
1997.
High seroprevalence of Encephalitozoon species in immunocompetent subjects.
J. Infect. Dis.
175:1020-1024[Medline].
|
| 21.
|
Vogelsang, G. B.,
E. R. Farmer,
A. D. Hess,
V. Altamante,
W. E. Beschorner,
D. A. Jabs,
R. L. Cario,
L. S. Levin,
O. R. Colvin,
J. R. Waigard, and G. W. Santos.
1992.
Thalidomide for the treatment of chronic graft-versus-host disease.
N. Engl. J. Med.
326:1055-1058[Abstract].
|
| 22.
|
Wankle, C. A.,
P. DeGirolami, and M. Federman.
1996.
Enterocytozoon bieneusi infection and diarrheal disease in patients who were not infected with human immunodeficiency virus: case report and review.
Clin. Infect. Dis.
23:816-818[Medline].
|
| 23.
|
Weber, R.,
R. T. Bryan,
D. A. Schwartz, and R. L. Owen.
1994.
Human microsporidial infections.
Clin. Microbiol. Rev.
7:426-461[Abstract/Free Full Text].
|
Antimicrobial Agents and Chemotherapy, September 1999, p. 2305-2306, Vol. 43, No. 9
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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