JID 1992;166 (July)
Correspondence
primer pairs in highly conserved regions of viral genome. Third, the exquisite sensitivity of PCR is such that contamination of laboratory or nonspecific amplifications can lead to false-positive results. Our preliminary results do not support the existence of frequent HTLV-I and -II infection in French anti-HTLV-Ij II-negative IVDUs. Serologic tests used for diagnosis ofHTLV-I and -II infection do not underestimate the prevalence of infection.
4.
5.
6.
Francois Lefrere, Eric Wattel, Martine Mariotti, Frantz Agis, Emmanuel Gordien, and Jean-Jacques Lefrere 7.
8.
References I. Wattel E. Mariotti M. Agis F, et al. Human T Iymphotropic virus (HTLV) type I and II DNA amplification in HTLV-IfII-seropositive blood donors of the French West Indies. J Infect Dis 1992;165:36972. 2. D'Auriol L. Vernant JC, Ouka M, et al. Diagnosis ofHTLV-1 infected seronegative neurological patients by polymerase chain reaction amplification in Martinique. Nouv Rev Fr Hematol 1990;32: 113-6. 3. Haettich B. Desgranges C, Veillon L. Prier A. Kaplan G. Interet de I'amplification genique (polymerase chain reaction) dans l'identifi-
Identification of Toxoplasma gondii Bradyzoite-Specific Monoclonal Antibodies Colleagues- Toxoplasma gondii is a ubiquitous apicomplexan organism that can cause clinical syndromes in animals and humans, including one of the most common central nervous system infections in patients with AIDS. Despite recent progress in understanding the biology and antigenic structure of the rapidly replicating form (tachyzoites), very little is known about the cyst form (bradyzoites) of T. gondii. It is likely that bradyzoites and tachyzoites each possess unique stage-specific antigens [I]. The identification of such antigens and the development of reagents based on these antigens should greatly facilitate investigations on the mechanism of intraconversion between bradyzoites and tachyzoites and may aid in the development of diagnostic tests. We report the production and characterization of two bradyzoite-specific monoclonal antibodies. RH (Sabin isolate) or ME49 (a high-cyst-producing strain in mice) (provided by R. Mcl.eod, University of Chicago) tachy-
Grant support: National Institutes of Health (MH-45654. CA-13330). Reprints or correspondence: Dr. Louis M. Weiss. Albert Einstein College of Medicine, 1300 Morris Park Ave.. Room 504 Forchheimer, Bronx, NY 10461. The Journal of Infectious Diseases 1992;166:213-5 © 1992 by The University of Chicago. All rightsreserved. 0022-1899/92/6601-0040$01.00
9.
10.
cation des manifestations neurologiques associees au HTLV -I. Presse Med 1991;20:661. Ehrlich GO. Glaser JB. Lavigne K. et al. Prevalence of human 'f-cell leukemia/lymphoma virus (HTL V) type II infection among high-risk individuals: type specific identification of HTLVs by polymerase chain reaction. Blood 1989;74: 1658-64. Mariotti M. Lefrere JJ, Noel B, et al. DNA amplification of HIV-I in seropositive individuals and in seronegative at-risk individuals. AIDS 1990;4:633-7. Lemaire 1M. Coste J, Barin F. Courouce AM, et al. Etude epidemiologique des anticorps anti HTLV-lfII chez les donneurs de sang en France metropolitaine. Rev Fr Transfus Hemobiol 1991;34:21-34. Lee H. Swanson P. Shorty VS. Zack JA. Rosenblatt JD. Chen ISY. High rate of HTLV-II infection in seropositive IV drug abusers in New Orleans. Science 1989;244:471-5. Coste J. Lemaire 1M. Courouce AM. et al. Prevalence of HTLV-I/II antibodies among intravenous drug users in continental France [abstract WC3336].ln: Program and abstracts: VII International Conference on AIDS (Florence). 1991. Tuke PK. Garson JA, Loveday C, Mulcahy F. Tedder RS. The use of novel methodology for the determination of the prevalence of HTLV-I and HTLV-II infections in an HIV-I positive intravenous drug user cohort in Dublin [abstract MA 1282]. In: Program and abstracts: VII International Conference on AIDS (Florence), 1991. Ehrlich GO. GreenbergS, Abbott MA. etal. Detection of human T-cell lymphoma/leukemia virus. In: Erlich HA, ed. PCR technology: principles and applications for DNA amplification. New York: Stockton Press, 1990:325-36.
zoites of T. gondii were purified from L6E9 rat myoblasts by filtration through a 3-,.,.m polycarbonate membrane (Nucleopore, Pleasanton, CA) as previously described [2]. ME49 cysts (bradyzoites) were maintained by passage every 12 weeks in CDI mice (inoculated intraperitoneally [ip], --20 cysts/mouse). BALB/C-H-2 dm2 (dm2) and BIO.D2-H-2 dm ' (dml) mice (provided by R. Mcl.eod), which have defects in the H-2L locus, develop -- 104 cysts/mouse; these mice were used for the production oflarge numbers of cysts for immunologic studies [3]. The mice (dm 1and dm2) were inoculated ip with 20-40 ME49 cysts obtained from CD 1 mice and were treated with sulfamerazine (20 mg/l in drinking water) starting 3-5 days after inoculation. By this protocol, mortality at 4 weeks in dm2 mice was ~60% and in dm 1 mice, ~20%. At 4-5 weeks after ME49 infection, cysts were purified from brains removed from infected mice by isopycnic centrifugation in colloidal silica (Percoll; Pharmacia, Piscataway, NJ) [4]; --80% of the cysts were recovered. Bradyzoites for immunofluorescence were purified by digestion of the cyst wall with I%trypsin-saline followed by filtration through a cellulose column (CF-II; Whatman, Hillsboro, OR). Bradyzoites prepared in this manner were 99% viable by trypan blue exclusion. Rabbit antitachyzoite antiserum was prepared as previously described [2, 5]. Rabbit antibradyzoite antiserum was prepared by intradermal immunization of a 2.5-kg New Zealand White rabbit with a freeze-thawed preparation (10 cycles) of 104 ME49 cysts purified from mouse brain in complete Freund's adjuvant,
Downloaded from http://jid.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on June 17, 2015
Laboratoire de Biologie Moleculaire, Institut National de Transfusion Sanguine and CHU Saint-Antoine. Paris. France; Centre de Transfusion Sanguine. Guadeloupe. French West Indies
213
Correspondence
214
A
B
2
3
456
Figure 1. Western blots. A. Reactivity ofbradyzoite-specific monoclonal antibodies (MAbs) with tachyzoite and bradyzoite antigens . Lanes I and 4. mouse brain; 2 and 5. ME49 tachyzoites; 3 and 6. ME49 cysts or bradyzoites. Primary antiserum: 74.1.8 murine ascites (lanes 1-3) or 73.1.8 murine ascites (lanes 4-6). Secondary antiserum: anti-mouse IgG peroxidase [6). MAb 74.1.8 (IgG2b) reacts with 28-kDa bradyzoite-specific molecule and 73.1.8 (lgG3) with 116-kDa bradyzoite-specific molecule. B, Reactivity of murine serum after immunization with ME49 cysts. Dominant reactions are similar to those seen with MAbs 73.1.8 and 74.1.8 isolated after fusion for MAb production using spleen cells from this animal [6].
pies at once. Hybridoma supernatants were screened in pools of 5 wells: the clones present in positive pools were screened individually to select bradyzoite-reactive MAbs. Clones reactive to bradyzoite and not tachyzoite antigens were selected for further purification and analysis. Clones producing reactive MAbs were then further characterized with respect to the isotype [6] of the antibody produced, reactivity on Western blots [2, 5], and ability to immunoprecipitate radiolabeled T. gondii proteins [2]. Immunofluorescence was done on formaldehyde-fixed intact ME49 cysts, bradyzoites purified from ME49 cysts, and RH tachyzoites with polyclonal rabbit antisera or Mabs as the primary antibody. A I; 100 dilution of rabbit antiserum, 1:25-1 :50 dilution ofmurine antibradyzoite MAb ascites, I: 100 dilution of preimmunization rabbit antiserum, or I: 100 dilution of unrelated murine MAb ascites was used as the primary antiserum followed by a I:50 dilution of fluorescein anti-rabbit or antimouse IgG (Organon Teknika-Cappel, Malvern , PA) according to standard immunofluorescence methods [6]. Of the 360 hybridomas obtained from the initial fusions, 24 reacted on Western blot with bradyzoite antigens and not with tachyzoite antigens as assessed by ELISA or Western blot. Two patterns ofreactivity on Western blot were identified (figure lA) and representative MAbs were purified further. MAb 73.18 recognized a 116-kDa antigen on Western blotting (figure IA) and demonstrated weak fluorescence of the entire periphery of isolated bradyzoites and stained the wall of intact cysts on immunofluorescence studies (not shown). Isotyping demonstrated this antibody was IgG3. Clone 74.1.8 recognized a 28-kDa antigen on Western blotting (figure IA). Immunofluorescence studies demonstrated that this MAb reacted strongly with granular material within the cytoplasm of isolated bradyzoites and stained the bradyzoites within the cysts and amorphous material between the bradyzoites (not shown). Isotyping demonstrated that this antibody was an IgG2b . Neither 74.1.8 nor 73.1 .8 reacted with tachyzoites or murine brain on Western blot or with tachyzoites on ELISA. These two MAbs corresponded to the major bands seen in the Western blot of serum obtained from the immunized mouse used for fusion (figure I B). No immunofluorescence reactivity was seen when these MAbs were incubated with tachyzoites. Using polyclonal rabbit antibradyzoite antiserum absorbed with murine acetone, brain powder bands corresponding to 116 and 28 kDa were also seen. Anti-p30 MAb (7B8, gift ofL. Kasper, Dartmouth University, Hanover, NH) did not react on immunofluorescence with isolated ME49 bradyzoites or cysts. MAb 92-1OB [5], which we identified from a tachyzoite MAb library, reacted with both bradyzoites and tachyzoites by immunofluorescence and Western blot. It reacted at -22 kDa in bradyzoite and -29 kDa in tachyzoite Western blots. Several reports have noted stage-specific antigens in T. gondii [I. 7-10], but until recently there were only limited bradyzoitespecific MAbs available . Kasper [I] identified an IgM MAb that reacted only with bradyzoites, but the corresponding protein was not identified . Ornata et al. [9] described a MAb that reacted to a 20-kDa cytoplasmic bradyzoite antigen. Recently, Tomavo et al. [IO] described four bradyzoite MAbs (T84A 12-IgGI, T82C2-IgG I, T84G IO-IgGI, and T83B I-IgM) reacting to 36-,
Downloaded from http://jid.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on June 17, 2015
followed by booster immunization with a freeze-thawed preparation of 5000 ME49 cysts in incomplete Freund's adjuvant at 4, 8, and 16 weeks [6]. To produce the bradyzoite hybridomas, mice were immunized ip with solubilized (sonicated) ME49 cysts (5000 cysts/ mouse) that were emulsified with complete Freund's adjuvant [6]. Success of immunization was determined by measuring the animal's serologic response by Western blot with ME49 cysts as the antigen. Specificity of the reaction to bradyzoite antigens was confirmed by Western blot [2, 5] with mouse brain as antigen and by absorption of mouse sera with mouse brain acetone powder before use in the bradyzoite Western blot. Reaction to two distinct bradyzoite antigens was evident (figure IB). Crossreaction to tachyzoite antigens was measured by ELISA with RH and ME49 tachyzoites as the antigen as previously described [5]. At 19 weeks after primary immunization, mice were reimmunized intravenously with freeze-thawed ME49 cysts (5000 cysts/mouse). Hybridoma fusion was done using standard techniques [6] and the spleen repopulation method [6] previously reported for tachyzoite monoclonal antibodies (MAbs) [5]. The supernatant from each well in which a hybridoma cell colony was growing was tested for antitoxoplasma antibody by ELISA with ME49 tachyzoites as antigen and Western blot with ME49 cysts as antigen [5]. Western blots were done by overnight incubation of supernatant using a manifold device (Miniblotter 45; Immunetics, Cambridge, MA) that allowed screening of45 sam-
JID 1992; 166 (July)
Correspondence
JID 1992;166 (July)
References
Louis M. Weiss, Denise LaPlace, Herbert B. Tanowitz, and Murray Wittner
I. Kasper LH. Identification of stage specific antigens of Toxoplasma gondii. Infect Immun 1989;57:668-72. 2. Weiss LM. Udem SA, Tanowitz HB. Wittner M. Western blot analysis of the antibody response of patients with AIDS and toxoplasma encephalitis: antigenic diversity among toxoplasma strains. J Infect Dis 1988; 157:7-13. 3. Brown CR. McLeod R. Class I MHC genes and CD8+ T cells determine cyst number in T. gondii infection. J Immunol1990; 145:343841. 4. Cornelissen AWCA. Overdalve JP. Haenderboom JM. Separation of Isospora (Toxoplasma) gondii cyst and cystozoites from mouse brain by continuous density-gradient centrifugation. Parasitology 1981; 83: 103-8. 5. Weiss LM. Udem SA, Salgo M. Tanowitz HB. Wittner M. Sensitive and specific detection of toxoplasma DNA in an experimental murine model: use of Toxoplasma gondii-specific cDNA and the polymerase chain reaction. J Infect Dis 1991; 163: 180-6. 6. Coligan JE. Kruisbeek AM. Margulies DH. Shevach EM, Strober W, eds. Current protocols in immunology. New York: John Wiley & Sons. 1991. 7. Lunde MN. Jacobs L. Antigenic differences between endozoites and cystozoites of Toxoplasma gondii. J Parasitol 1983;69:806-8. 8. Woodison G. Smith JE. Identification of the dominant cyst antigens of Toxoplasma gondii. Parasitology 1990; 100:389-92. 9. Ornata Y. Igarashi M. Ramos MI. Nakabayashi T. Toxoplasma gondii: antigenic differences between endozoites and cystozoites defined by monoclonal antibodies. Parasitol Res 1989;75: 189-93.
Departments ofMedicine. Division of Infectious Diseases. and Pathology. Division ofTropical Medicine and Parasitology. Albert Einstein College of Medicine. Bronx. New York
10. Tomavo S. Fortier B. Soete M. Ansel C. Camus D. Dubremetz JF. Characterization of bradyzoite-specific antigens of Toxoplasma gondii. Infect Immun 1991;59:3750-3.
Ketoconazole in the Treatment of Antimony- and Pentamidine-Resistant Kala-Azar
treating kala-azar [5]. Here we describe our findings with oral ketoconazole in 9 cases of drug-resistant kala-azar. Nine patients who failed to respond to antimonials, pentamidine, or both were studied. The diagnosis ofkala-azar was based on the demonstration of Leishmania donovani bodies in splenic and bone marrow aspirates. The parasitic load in the splenic aspirate was graded as 0, 1+, 2+, 3+, 4+, 5+, and 6+ as described earlier [1]. Symptoms had lasted 3 months to 2 years. All 9 patients had received sodium antimony gluconate in the recommended dosage (20 rug/kg/day) for 20-50 days without any response. Four were subsequently treated with pentamidine isethionate for 15 days but with no response. (An interesting observation was that 2 of the 4 patients treated with pentamidine developed diabetes mellitus and required insulin for its control. ) All patients were treated with ketoconazole (6DO mg/day in three divided doses) for 4 weeks. Clinical evaluation every third day noted improvement in symptoms and signs of the disease and presence of any side effects (anorexia, nausea, vomiting, epigastric pain, dizziness, paraesthesias, headache, or gynecomastia). Splenic aspiration for parasite grading was done weekly. For cases in which splenic aspiration was not possible due to regression of the spleen with treatment, bone marrow aspiration
Colleagues-Kala-azar, a protozoal disease, is endemic in several parts of the world, including India. The drugs of choice for treatment are pentavalent antimonials, such as sodium antimony gluconate and meglumine. Patients who do not respond to these are treated with pentamidine isethionate and amphotericin B. Because of potentially serious side effects of pentamidine and amphotericin B [1, 2] and because these drugs are given parenterally, there is a need for an orally administered drug that is effective in kala-azar patients who do not respond to antimonials. Some previous studies have indicated a possible role for allopurinol, metronidazole, and rifampicin in the treatment of kala-azar [3], but a recent report doubts the utility of these drugs [4]. Recently, we have shown the efficacy of ketoconazole in
Reprints or correspondence: Dr. Jyoti P. Wali, Department of Medicine. All India Institute of Medical Sciences. New Delhi-Ll 0029. India.
The Journal of Infectious Diseases
1992;166:215-6
© 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6601-0041 $0 1.00
Downloaded from http://jid.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on June 17, 2015
34-, 21-, and 18-kDa antigens, respectively, by Western blot. These stained the periphery of the bradyzoite on immunofluorescence. Trypsin treatment of bradyzoites suggested that most of these MAbs recognized surface proteins [10]. The MAbs we have identified differ from those previously described. MAb 74.1.8 recognized a 28-kDa antigen that by immunofluorescence appeared to be present within the cytoplasm of bradyzoites and between parasites in the intact cyst. Parasitic antigens lying between parasites in intact cysts have been identified previously by electron microscopy. MAb 73.1.8 recognized a 116-kDa antigen and is ofIgG3 class. IgG3 antibodies are often associated with carbohydrate epitopes, and it is possible this Mab is recognizing such an epitope on a bradyzoite glycoprotein in the cyst wall. The availability of MAbs to bradyzoite-specific antigens should allow one to follow the expression of such antigens and thus the differentiation of bradyzoites in tissue culture. In fact, using T. gondii 76K, bradyzoite-like organisms have been demonstrated in tissue culture [10] with MAb-confirming studies that demonstrate the existence of bradyzoites in tissue cultures by use of a bioassay in cats. These bradyzoite MAb probes allow in vitro studies of modulation of stage transition. In addition, the availability of bradyzoite-specific MAbs may make the screening of DNA libraries for these antigens feasible.
215
Correspondence
primer pairs in highly conserved regions of viral genome. Third, the exquisite sensitivity of PCR is such that contamination of laboratory or nonspecific amplifications can lead to false-positive results. Our preliminary results do not support the existence of frequent HTLV-I and -II infection in French anti-HTLV-Ij II-negative IVDUs. Serologic tests used for diagnosis ofHTLV-I and -II infection do not underestimate the prevalence of infection.
4.
5.
6.
Francois Lefrere, Eric Wattel, Martine Mariotti, Frantz Agis, Emmanuel Gordien, and Jean-Jacques Lefrere 7.
8.
References I. Wattel E. Mariotti M. Agis F, et al. Human T Iymphotropic virus (HTLV) type I and II DNA amplification in HTLV-IfII-seropositive blood donors of the French West Indies. J Infect Dis 1992;165:36972. 2. D'Auriol L. Vernant JC, Ouka M, et al. Diagnosis ofHTLV-1 infected seronegative neurological patients by polymerase chain reaction amplification in Martinique. Nouv Rev Fr Hematol 1990;32: 113-6. 3. Haettich B. Desgranges C, Veillon L. Prier A. Kaplan G. Interet de I'amplification genique (polymerase chain reaction) dans l'identifi-
Identification of Toxoplasma gondii Bradyzoite-Specific Monoclonal Antibodies Colleagues- Toxoplasma gondii is a ubiquitous apicomplexan organism that can cause clinical syndromes in animals and humans, including one of the most common central nervous system infections in patients with AIDS. Despite recent progress in understanding the biology and antigenic structure of the rapidly replicating form (tachyzoites), very little is known about the cyst form (bradyzoites) of T. gondii. It is likely that bradyzoites and tachyzoites each possess unique stage-specific antigens [I]. The identification of such antigens and the development of reagents based on these antigens should greatly facilitate investigations on the mechanism of intraconversion between bradyzoites and tachyzoites and may aid in the development of diagnostic tests. We report the production and characterization of two bradyzoite-specific monoclonal antibodies. RH (Sabin isolate) or ME49 (a high-cyst-producing strain in mice) (provided by R. Mcl.eod, University of Chicago) tachy-
Grant support: National Institutes of Health (MH-45654. CA-13330). Reprints or correspondence: Dr. Louis M. Weiss. Albert Einstein College of Medicine, 1300 Morris Park Ave.. Room 504 Forchheimer, Bronx, NY 10461. The Journal of Infectious Diseases 1992;166:213-5 © 1992 by The University of Chicago. All rightsreserved. 0022-1899/92/6601-0040$01.00
9.
10.
cation des manifestations neurologiques associees au HTLV -I. Presse Med 1991;20:661. Ehrlich GO. Glaser JB. Lavigne K. et al. Prevalence of human 'f-cell leukemia/lymphoma virus (HTL V) type II infection among high-risk individuals: type specific identification of HTLVs by polymerase chain reaction. Blood 1989;74: 1658-64. Mariotti M. Lefrere JJ, Noel B, et al. DNA amplification of HIV-I in seropositive individuals and in seronegative at-risk individuals. AIDS 1990;4:633-7. Lemaire 1M. Coste J, Barin F. Courouce AM, et al. Etude epidemiologique des anticorps anti HTLV-lfII chez les donneurs de sang en France metropolitaine. Rev Fr Transfus Hemobiol 1991;34:21-34. Lee H. Swanson P. Shorty VS. Zack JA. Rosenblatt JD. Chen ISY. High rate of HTLV-II infection in seropositive IV drug abusers in New Orleans. Science 1989;244:471-5. Coste J. Lemaire 1M. Courouce AM. et al. Prevalence of HTLV-I/II antibodies among intravenous drug users in continental France [abstract WC3336].ln: Program and abstracts: VII International Conference on AIDS (Florence). 1991. Tuke PK. Garson JA, Loveday C, Mulcahy F. Tedder RS. The use of novel methodology for the determination of the prevalence of HTLV-I and HTLV-II infections in an HIV-I positive intravenous drug user cohort in Dublin [abstract MA 1282]. In: Program and abstracts: VII International Conference on AIDS (Florence), 1991. Ehrlich GO. GreenbergS, Abbott MA. etal. Detection of human T-cell lymphoma/leukemia virus. In: Erlich HA, ed. PCR technology: principles and applications for DNA amplification. New York: Stockton Press, 1990:325-36.
zoites of T. gondii were purified from L6E9 rat myoblasts by filtration through a 3-,.,.m polycarbonate membrane (Nucleopore, Pleasanton, CA) as previously described [2]. ME49 cysts (bradyzoites) were maintained by passage every 12 weeks in CDI mice (inoculated intraperitoneally [ip], --20 cysts/mouse). BALB/C-H-2 dm2 (dm2) and BIO.D2-H-2 dm ' (dml) mice (provided by R. Mcl.eod), which have defects in the H-2L locus, develop -- 104 cysts/mouse; these mice were used for the production oflarge numbers of cysts for immunologic studies [3]. The mice (dm 1and dm2) were inoculated ip with 20-40 ME49 cysts obtained from CD 1 mice and were treated with sulfamerazine (20 mg/l in drinking water) starting 3-5 days after inoculation. By this protocol, mortality at 4 weeks in dm2 mice was ~60% and in dm 1 mice, ~20%. At 4-5 weeks after ME49 infection, cysts were purified from brains removed from infected mice by isopycnic centrifugation in colloidal silica (Percoll; Pharmacia, Piscataway, NJ) [4]; --80% of the cysts were recovered. Bradyzoites for immunofluorescence were purified by digestion of the cyst wall with I%trypsin-saline followed by filtration through a cellulose column (CF-II; Whatman, Hillsboro, OR). Bradyzoites prepared in this manner were 99% viable by trypan blue exclusion. Rabbit antitachyzoite antiserum was prepared as previously described [2, 5]. Rabbit antibradyzoite antiserum was prepared by intradermal immunization of a 2.5-kg New Zealand White rabbit with a freeze-thawed preparation (10 cycles) of 104 ME49 cysts purified from mouse brain in complete Freund's adjuvant,
Downloaded from http://jid.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on June 17, 2015
Laboratoire de Biologie Moleculaire, Institut National de Transfusion Sanguine and CHU Saint-Antoine. Paris. France; Centre de Transfusion Sanguine. Guadeloupe. French West Indies
213
Correspondence
214
A
B
2
3
456
Figure 1. Western blots. A. Reactivity ofbradyzoite-specific monoclonal antibodies (MAbs) with tachyzoite and bradyzoite antigens . Lanes I and 4. mouse brain; 2 and 5. ME49 tachyzoites; 3 and 6. ME49 cysts or bradyzoites. Primary antiserum: 74.1.8 murine ascites (lanes 1-3) or 73.1.8 murine ascites (lanes 4-6). Secondary antiserum: anti-mouse IgG peroxidase [6). MAb 74.1.8 (IgG2b) reacts with 28-kDa bradyzoite-specific molecule and 73.1.8 (lgG3) with 116-kDa bradyzoite-specific molecule. B, Reactivity of murine serum after immunization with ME49 cysts. Dominant reactions are similar to those seen with MAbs 73.1.8 and 74.1.8 isolated after fusion for MAb production using spleen cells from this animal [6].
pies at once. Hybridoma supernatants were screened in pools of 5 wells: the clones present in positive pools were screened individually to select bradyzoite-reactive MAbs. Clones reactive to bradyzoite and not tachyzoite antigens were selected for further purification and analysis. Clones producing reactive MAbs were then further characterized with respect to the isotype [6] of the antibody produced, reactivity on Western blots [2, 5], and ability to immunoprecipitate radiolabeled T. gondii proteins [2]. Immunofluorescence was done on formaldehyde-fixed intact ME49 cysts, bradyzoites purified from ME49 cysts, and RH tachyzoites with polyclonal rabbit antisera or Mabs as the primary antibody. A I; 100 dilution of rabbit antiserum, 1:25-1 :50 dilution ofmurine antibradyzoite MAb ascites, I: 100 dilution of preimmunization rabbit antiserum, or I: 100 dilution of unrelated murine MAb ascites was used as the primary antiserum followed by a I:50 dilution of fluorescein anti-rabbit or antimouse IgG (Organon Teknika-Cappel, Malvern , PA) according to standard immunofluorescence methods [6]. Of the 360 hybridomas obtained from the initial fusions, 24 reacted on Western blot with bradyzoite antigens and not with tachyzoite antigens as assessed by ELISA or Western blot. Two patterns ofreactivity on Western blot were identified (figure lA) and representative MAbs were purified further. MAb 73.18 recognized a 116-kDa antigen on Western blotting (figure IA) and demonstrated weak fluorescence of the entire periphery of isolated bradyzoites and stained the wall of intact cysts on immunofluorescence studies (not shown). Isotyping demonstrated this antibody was IgG3. Clone 74.1.8 recognized a 28-kDa antigen on Western blotting (figure IA). Immunofluorescence studies demonstrated that this MAb reacted strongly with granular material within the cytoplasm of isolated bradyzoites and stained the bradyzoites within the cysts and amorphous material between the bradyzoites (not shown). Isotyping demonstrated that this antibody was an IgG2b . Neither 74.1.8 nor 73.1 .8 reacted with tachyzoites or murine brain on Western blot or with tachyzoites on ELISA. These two MAbs corresponded to the major bands seen in the Western blot of serum obtained from the immunized mouse used for fusion (figure I B). No immunofluorescence reactivity was seen when these MAbs were incubated with tachyzoites. Using polyclonal rabbit antibradyzoite antiserum absorbed with murine acetone, brain powder bands corresponding to 116 and 28 kDa were also seen. Anti-p30 MAb (7B8, gift ofL. Kasper, Dartmouth University, Hanover, NH) did not react on immunofluorescence with isolated ME49 bradyzoites or cysts. MAb 92-1OB [5], which we identified from a tachyzoite MAb library, reacted with both bradyzoites and tachyzoites by immunofluorescence and Western blot. It reacted at -22 kDa in bradyzoite and -29 kDa in tachyzoite Western blots. Several reports have noted stage-specific antigens in T. gondii [I. 7-10], but until recently there were only limited bradyzoitespecific MAbs available . Kasper [I] identified an IgM MAb that reacted only with bradyzoites, but the corresponding protein was not identified . Ornata et al. [9] described a MAb that reacted to a 20-kDa cytoplasmic bradyzoite antigen. Recently, Tomavo et al. [IO] described four bradyzoite MAbs (T84A 12-IgGI, T82C2-IgG I, T84G IO-IgGI, and T83B I-IgM) reacting to 36-,
Downloaded from http://jid.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on June 17, 2015
followed by booster immunization with a freeze-thawed preparation of 5000 ME49 cysts in incomplete Freund's adjuvant at 4, 8, and 16 weeks [6]. To produce the bradyzoite hybridomas, mice were immunized ip with solubilized (sonicated) ME49 cysts (5000 cysts/ mouse) that were emulsified with complete Freund's adjuvant [6]. Success of immunization was determined by measuring the animal's serologic response by Western blot with ME49 cysts as the antigen. Specificity of the reaction to bradyzoite antigens was confirmed by Western blot [2, 5] with mouse brain as antigen and by absorption of mouse sera with mouse brain acetone powder before use in the bradyzoite Western blot. Reaction to two distinct bradyzoite antigens was evident (figure IB). Crossreaction to tachyzoite antigens was measured by ELISA with RH and ME49 tachyzoites as the antigen as previously described [5]. At 19 weeks after primary immunization, mice were reimmunized intravenously with freeze-thawed ME49 cysts (5000 cysts/mouse). Hybridoma fusion was done using standard techniques [6] and the spleen repopulation method [6] previously reported for tachyzoite monoclonal antibodies (MAbs) [5]. The supernatant from each well in which a hybridoma cell colony was growing was tested for antitoxoplasma antibody by ELISA with ME49 tachyzoites as antigen and Western blot with ME49 cysts as antigen [5]. Western blots were done by overnight incubation of supernatant using a manifold device (Miniblotter 45; Immunetics, Cambridge, MA) that allowed screening of45 sam-
JID 1992; 166 (July)
Correspondence
JID 1992;166 (July)
References
Louis M. Weiss, Denise LaPlace, Herbert B. Tanowitz, and Murray Wittner
I. Kasper LH. Identification of stage specific antigens of Toxoplasma gondii. Infect Immun 1989;57:668-72. 2. Weiss LM. Udem SA, Tanowitz HB. Wittner M. Western blot analysis of the antibody response of patients with AIDS and toxoplasma encephalitis: antigenic diversity among toxoplasma strains. J Infect Dis 1988; 157:7-13. 3. Brown CR. McLeod R. Class I MHC genes and CD8+ T cells determine cyst number in T. gondii infection. J Immunol1990; 145:343841. 4. Cornelissen AWCA. Overdalve JP. Haenderboom JM. Separation of Isospora (Toxoplasma) gondii cyst and cystozoites from mouse brain by continuous density-gradient centrifugation. Parasitology 1981; 83: 103-8. 5. Weiss LM. Udem SA, Salgo M. Tanowitz HB. Wittner M. Sensitive and specific detection of toxoplasma DNA in an experimental murine model: use of Toxoplasma gondii-specific cDNA and the polymerase chain reaction. J Infect Dis 1991; 163: 180-6. 6. Coligan JE. Kruisbeek AM. Margulies DH. Shevach EM, Strober W, eds. Current protocols in immunology. New York: John Wiley & Sons. 1991. 7. Lunde MN. Jacobs L. Antigenic differences between endozoites and cystozoites of Toxoplasma gondii. J Parasitol 1983;69:806-8. 8. Woodison G. Smith JE. Identification of the dominant cyst antigens of Toxoplasma gondii. Parasitology 1990; 100:389-92. 9. Ornata Y. Igarashi M. Ramos MI. Nakabayashi T. Toxoplasma gondii: antigenic differences between endozoites and cystozoites defined by monoclonal antibodies. Parasitol Res 1989;75: 189-93.
Departments ofMedicine. Division of Infectious Diseases. and Pathology. Division ofTropical Medicine and Parasitology. Albert Einstein College of Medicine. Bronx. New York
10. Tomavo S. Fortier B. Soete M. Ansel C. Camus D. Dubremetz JF. Characterization of bradyzoite-specific antigens of Toxoplasma gondii. Infect Immun 1991;59:3750-3.
Ketoconazole in the Treatment of Antimony- and Pentamidine-Resistant Kala-Azar
treating kala-azar [5]. Here we describe our findings with oral ketoconazole in 9 cases of drug-resistant kala-azar. Nine patients who failed to respond to antimonials, pentamidine, or both were studied. The diagnosis ofkala-azar was based on the demonstration of Leishmania donovani bodies in splenic and bone marrow aspirates. The parasitic load in the splenic aspirate was graded as 0, 1+, 2+, 3+, 4+, 5+, and 6+ as described earlier [1]. Symptoms had lasted 3 months to 2 years. All 9 patients had received sodium antimony gluconate in the recommended dosage (20 rug/kg/day) for 20-50 days without any response. Four were subsequently treated with pentamidine isethionate for 15 days but with no response. (An interesting observation was that 2 of the 4 patients treated with pentamidine developed diabetes mellitus and required insulin for its control. ) All patients were treated with ketoconazole (6DO mg/day in three divided doses) for 4 weeks. Clinical evaluation every third day noted improvement in symptoms and signs of the disease and presence of any side effects (anorexia, nausea, vomiting, epigastric pain, dizziness, paraesthesias, headache, or gynecomastia). Splenic aspiration for parasite grading was done weekly. For cases in which splenic aspiration was not possible due to regression of the spleen with treatment, bone marrow aspiration
Colleagues-Kala-azar, a protozoal disease, is endemic in several parts of the world, including India. The drugs of choice for treatment are pentavalent antimonials, such as sodium antimony gluconate and meglumine. Patients who do not respond to these are treated with pentamidine isethionate and amphotericin B. Because of potentially serious side effects of pentamidine and amphotericin B [1, 2] and because these drugs are given parenterally, there is a need for an orally administered drug that is effective in kala-azar patients who do not respond to antimonials. Some previous studies have indicated a possible role for allopurinol, metronidazole, and rifampicin in the treatment of kala-azar [3], but a recent report doubts the utility of these drugs [4]. Recently, we have shown the efficacy of ketoconazole in
Reprints or correspondence: Dr. Jyoti P. Wali, Department of Medicine. All India Institute of Medical Sciences. New Delhi-Ll 0029. India.
The Journal of Infectious Diseases
1992;166:215-6
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34-, 21-, and 18-kDa antigens, respectively, by Western blot. These stained the periphery of the bradyzoite on immunofluorescence. Trypsin treatment of bradyzoites suggested that most of these MAbs recognized surface proteins [10]. The MAbs we have identified differ from those previously described. MAb 74.1.8 recognized a 28-kDa antigen that by immunofluorescence appeared to be present within the cytoplasm of bradyzoites and between parasites in the intact cyst. Parasitic antigens lying between parasites in intact cysts have been identified previously by electron microscopy. MAb 73.1.8 recognized a 116-kDa antigen and is ofIgG3 class. IgG3 antibodies are often associated with carbohydrate epitopes, and it is possible this Mab is recognizing such an epitope on a bradyzoite glycoprotein in the cyst wall. The availability of MAbs to bradyzoite-specific antigens should allow one to follow the expression of such antigens and thus the differentiation of bradyzoites in tissue culture. In fact, using T. gondii 76K, bradyzoite-like organisms have been demonstrated in tissue culture [10] with MAb-confirming studies that demonstrate the existence of bradyzoites in tissue cultures by use of a bioassay in cats. These bradyzoite MAb probes allow in vitro studies of modulation of stage transition. In addition, the availability of bradyzoite-specific MAbs may make the screening of DNA libraries for these antigens feasible.
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