Immunological Response to Cell-Free of Paracoccidioides ...IMMUNOLOGICAL RESPONSE TOP. BRASILIENSIS...

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JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1993, p. 671-676 0095-1137/93/030671-06$02.00/0 Copyright © 1993, American Society for Microbiology Immunological Response to Cell-Free Antigens of Paracoccidioides brasiliensis: Relationship with Clinical Forms of Paracoccidioidomycosis M. H. S. L. BLOlTA' AND Z. P. CAMARGO2* Departamento de Patologia Clinica, Faculdade de Ciencias Medicas, UNICAMP,' and Disciplina de Biologia Celular, Escola Paulista de Medicina, 04023-062 Rua Botucatu 862,2 Sdo Paulo, Brazil Received 24 August 1992/Accepted 15 December 1992 Sera from patients with the acute (AF) and chronic (CF) forms of paracoccidioidomycosis (PCM) were tested against Paracoccidioides brasiiensis cell-free antigens by Western blot (immunoblot). The CFA preparation contained components ranging in molecular mass from 18 to 102 kDa. The immunoglobulin G (IgG) reactivity profiles were similar for patients with both forms of the disease, and the 43-kDa component was recognized by 100% of the sera. IgM antibodies from the AF- and the CF-PCM sera recognized 21 and 20 components, respectively, the AF-PCM sera reacting preferentially with components with molecular masses above 50 kDa. None of the AF-PCM sera (IgM) reacted with the 43-kDa component, and only 10%Yl of the CF-PCM sera recognized this molecule. The IgA response was more significant in the CF-PCM group than in the AF-PCM group, and the 43- and 74-kDa components were the most reactive ones (about 40%o each). Our results showed that the cell-re antigen preparation is very appropriate for the immunoblotting analysis of PCM sera, and they also showed that the detection of IgG anti-gp43 is the best marker for the diagnosis and the following up of patients with the acute or the chronic form of the disease. The systemic mycosis paracoccidioidomycosis (PCM) is caused by the dimorphic fungus Paracoccidioides brasilien- sis, and it presents a wide spectrum of clinical manifesta- tions. Over the years, it has been suggested that infection with P. brasiliensis occurs by inhalation of the airborne mycelial structures (not yet defined) of the saprobic phase of the fungus. Once in the lungs, the propagules convert themselves into the yeast phase, initiating the infection, and then they eventually disseminate themselves to the other parts of the body (9). Two main clinical forms of the disease are recognized by the International Committee of PCM (10), namely, the acute or subacute form (AF) and the unifocal or multifocal chronic form (CF). The pathogen is endemic to many regions of Latin America, mainly in Argentina, Brazil, Colombia, and Venezuela. The diagnosis of PCM can be made by direct observation of the characteristic multiple-budding cells in clinical mate- rials, by biopsy, or by culture. However, when these proce- dures are not available, the detection of antibodies to P. brasiliensis antigens is a valuable aid for diagnosing PCM, mainly by the immunodiffusion test (6). In general, the antigenic preparations used in serological assays for P. brasiliensis antibodies have been undefined, and it is unknown if there is any relationship between the various antigens and the clinical forms of PCM. We have recently described a simple method for obtaining antigen from the surface of the fungus, which was designated "cell- free antigen" (CFA) (5). In this study, the Western blot (immunoblot) assay was used to analyze the main components of P. brasiliensis CFA recognized by the individual immunoglobulin G (IgG), IgM, and IgA in sera from patients with the acute and the chronic forms of PCM, because of the fact that the CFA preparation is composed by the cell surface-associated antigens which might represent the first molecules to have contact with the host defense cells. MATERIALS AND METHODS Fungal strain. P. brasiliensis B-339 (kindly provided by A. Restrepo, Corporacion para Investigaciones Biologicas, Medellin, Colombia) was maintained by frequent subcultur- ing on Sabouraud glucose agar (Difco Laboratories). The fungus was converted into its yeast form by being grown on kDa - 66 - 43 - 29 FIG. 1. Components of the CFA of P. brasiliensis after SDS- PAGE and silver staining. Molecular mass standards are indicated at the right. * Corresponding author. 671 Vol. 31, No. 3 on June 25, 2020 by guest http://jcm.asm.org/ Downloaded from

Transcript of Immunological Response to Cell-Free of Paracoccidioides ...IMMUNOLOGICAL RESPONSE TOP. BRASILIENSIS...

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JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1993, p. 671-6760095-1137/93/030671-06$02.00/0Copyright © 1993, American Society for Microbiology

Immunological Response to Cell-Free Antigens ofParacoccidioides brasiliensis: Relationship with

Clinical Forms of ParacoccidioidomycosisM. H. S. L. BLOlTA' AND Z. P. CAMARGO2*

Departamento de Patologia Clinica, Faculdade de Ciencias Medicas, UNICAMP,' and

Disciplina de Biologia Celular, Escola Paulista de Medicina,04023-062 Rua Botucatu 862,2 Sdo Paulo, Brazil

Received 24 August 1992/Accepted 15 December 1992

Sera from patients with the acute (AF) and chronic (CF) forms of paracoccidioidomycosis (PCM) were testedagainst Paracoccidioides brasiiensis cell-free antigens by Western blot (immunoblot). The CFA preparationcontained components ranging in molecular mass from 18 to 102 kDa. The immunoglobulin G (IgG) reactivityprofiles were similar for patients with both forms of the disease, and the 43-kDa component was recognized by100% of the sera. IgM antibodies from the AF- and the CF-PCM sera recognized 21 and 20 components,respectively, the AF-PCM sera reacting preferentially with components with molecular masses above 50 kDa.None of the AF-PCM sera (IgM) reacted with the 43-kDa component, and only 10%Yl of the CF-PCM sera

recognized this molecule. The IgA response was more significant in the CF-PCM group than in the AF-PCMgroup, and the 43- and 74-kDa components were the most reactive ones (about 40%o each). Our results showedthat the cell-re antigen preparation is very appropriate for the immunoblotting analysis of PCM sera, andthey also showed that the detection of IgG anti-gp43 is the best marker for the diagnosis and the following up

of patients with the acute or the chronic form of the disease.

The systemic mycosis paracoccidioidomycosis (PCM) iscaused by the dimorphic fungus Paracoccidioides brasilien-sis, and it presents a wide spectrum of clinical manifesta-tions. Over the years, it has been suggested that infectionwith P. brasiliensis occurs by inhalation of the airbornemycelial structures (not yet defined) of the saprobic phase ofthe fungus. Once in the lungs, the propagules convertthemselves into the yeast phase, initiating the infection, andthen they eventually disseminate themselves to the otherparts of the body (9). Two main clinical forms of the diseaseare recognized by the International Committee of PCM (10),namely, the acute or subacute form (AF) and the unifocal ormultifocal chronic form (CF). The pathogen is endemic tomany regions of Latin America, mainly in Argentina, Brazil,Colombia, and Venezuela.The diagnosis of PCM can be made by direct observation

of the characteristic multiple-budding cells in clinical mate-rials, by biopsy, or by culture. However, when these proce-dures are not available, the detection of antibodies to P.brasiliensis antigens is a valuable aid for diagnosing PCM,mainly by the immunodiffusion test (6).

In general, the antigenic preparations used in serologicalassays for P. brasiliensis antibodies have been undefined,and it is unknown if there is any relationship between thevarious antigens and the clinical forms of PCM. We haverecently described a simple method for obtaining antigenfrom the surface of the fungus, which was designated "cell-free antigen" (CFA) (5).

In this study, the Western blot (immunoblot) assay wasused to analyze the main components of P. brasiliensis CFArecognized by the individual immunoglobulin G (IgG), IgM,and IgA in sera from patients with the acute and the chronicforms of PCM, because of the fact that the CFA preparation

is composed by the cell surface-associated antigens whichmight represent the first molecules to have contact with thehost defense cells.

MATERIALS AND METHODS

Fungal strain. P. brasiliensis B-339 (kindly provided by A.Restrepo, Corporacion para Investigaciones Biologicas,Medellin, Colombia) was maintained by frequent subcultur-ing on Sabouraud glucose agar (Difco Laboratories). Thefungus was converted into its yeast form by being grown on

kDa

- 66

- 43

- 29

FIG. 1. Components of the CFA of P. brasiliensis after SDS-PAGE and silver staining. Molecular mass standards are indicated atthe right.* Corresponding author.

671

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J. CLIN. MICROBIOL.672 BLOTTA AND CAMARGO

IgG-CFIkDa

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FIG. 2. IgG, IgM, and IgA immunoblots of the CFA of P. brasiliensis B-339, revealed with 30 PCM-AF and 30 PCM-CF serum samples.

such medium at 35°C and was maintained in such form bybeing subcultured every third day.

Preparation of antigen. P. brasiliensis B-339 was grown onSabouraud glucose agar at 35°C for 3 days. The fungalgrowth from three randomly selected tubes (about 300 mg,wet weight) was collected by gently scraping the surface.The cell mass was suspended in 1 ml of 0.15 M phosphate-buffered saline (PBS), pH 7.4, mixed for 30 s in a Vortexmixer (Tecnal, Sao Paulo, Brazil), and immediately centri-fuged at 10,000 x g in an Eppendorf tabletop centrifuge(Sorvall MC 12V; Dupont, Newtown, Conn.) for 60 s. Theresulting supernatant fluid contained the antigen (CFA). Theprotein concentration in CFA was determined by the methodof Bradford (4).

Sera. Serum samples were obtained from 60 PCM patientsfrom the Hospital das Clinicas da UNICAMP, Campinas,Brazil. The patients were all Caucasians, grouped according

to the clinical form of the disease as follows: 30 patients (30serum samples) were 6 to 32 years of age with the acute form(PCM-AF), and 30 patients (30 serum samples) were 35 to 69years of age with the chronic form (PCM-CF). Most of themwere rural workers from the region of Campinas. These serawere taken before antimycotic therapy. Ten patients in eachgroup were serologically monitored for 2 years during thecourse of therapy (adding up to 50 serum samples).SDS-PAGE. CFA obtained as described above was mixed

with the reducing sample buffer containing 62.5 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 10% glyc-erol, 10% 2-mercaptoethanol, and 0.05% bromophenol blue.The polypeptides were separated by SDS-polyacrylamidegel electrophoresis (SDS-PAGE) (11) on 10% acrylamidegels. Protein standards with the following molecular masseswere used: bovine serum albumin, 67,000 Da; ovalbumin,43,000 Da; and concanavalin A, 29,000 Da (Pharmacia,

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IMMUNOLOGICAL RESPONSE TO P. BRASILIENSIS CFA 673

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FIG. 3. Histograms of the frequencies of binding of IgG, IgM, and IgA from 30 PCM-AF and 30 PCM-CF serum samples to thecomponents of the CFA of P. brasiliensis B-339.

Uppsala, Sweden). Proteins were visualized by silver nitratestaining (1).

Immunoblotting. After electrophoresis, the CFA proteinswere transferred overnight to nitrocellulose paper (NCP) at100 mA in a transblotting chamber (Bio-Rad, Melville, N.Y.)(15, 16). The transfer buffer consisted of 25 mM Tris, 192mM glycine, and 20% (vol/vol) methanol, pH 8.3. Prior tothe immunological staining, the free sites on the NCP were

blocked with 2% bovine serum albumin (BSA; Sigma) inPBS and 0.2% Nonidet P-40 (Sigma), pH 7.4, for 4 h. TheNCP was sliced vertically, and the strips were incubatedindividually for 60 min at room temperature with sera frompatients with PCM diluted at 1:100 in PBS containing 0.02%Tween 20 and 0.25% gelatin (PBS-T-G). Then, each stripwas washed four times for 15 min in PBS-T-G and afterwardsincubated individually with peroxidase anti-human IgG, IgA,or IgM (Sigma) for 60 min. Afterwards, each strip was

washed four times for 15 min in PBS-T-G and incubated witha freshly prepared mixture of 5 mg of DAB (3,3-diaminoben-zidine-4HCl; Sigma) in 50 ml of 0.1 M Tris buffer, pH 7.5,plus 5 pl of 30% H202. After color development, the stripswere rinsed in 10% acetic acid.

Scanning densitometry. After immunoblotting, each NCPstrip incubated with the sera obtained from patients moni-

tored during antimycotic therapy was analyzed by scanningdensitometry (CS 9000; Shimadzu Corporation Spectropho-tometric Instrument Plant, Kyoto, Japan) at 550 nm for

quantitative evaluation of the band intensity.

RESULTS

The components of the CFA of P. brasiliensis B-339 wereanalyzed by SDS-PAGE. At least 42 components (18 to 102kDa) were observed after silver staining (Fig. 1).

Fig. 2 shows IgG, IgM, and IgA blots of CFA componentsexposed to 30 individual AF-PCM serum samples and to 30individual CF-PCM serum samples (all obtained beforetherapy). Several components of the following molecularmasses were detected in IgG-positive patient serum samples:18, 20, 22, 24, 28, 29, 33, 34, 39, 43, 50, 52, 54, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 86, 88, 90, 96, and 102 kDa.The number of components varied with the PCM serum. TheAF-PCM sera (IgG) recognized 30 components, whereas theCF-PCM sera recognized 23 components. Patients with bothforms of the disease had similar IgG reactivity profiles. Themost frequent components that reacted with the IgG-AFserum group were 24 and 28 kDa (20%), 20, 22, and 78 kDa(25%), 76 kDa (28%), 80 kDa (30%), 70 kDa (45%), and 43kDa (100%), whereas for the IgG-CF serum group, thecomponents that reacted most frequently were 28 kDa(20%), 22 and 88 kDa (25%), 70 kDa (35%), and 43 kDa(100%).IgM blots of the CFA exposed to 30 individual AF-PCM

serum samples and to 30 individual CF-PCM serum samplesrecognized 21 and 19 components, respectively. Note thatthe components with molecular masses of 22, 24, 28, 29, 34,35, 36, 39, 43, 47, 50, 52, 54, 58, 60, 62, 64, 66, 70, 72, 74, 76,80, 82, 86, 88, and 90 kDa were recognized, but the numberof antigens varied with the PCM serum. Sera from patients

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VOL. 31, 1993

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J. CLIN. MICROBIOL.674 BLOTIA AND CAMARGO

A

Ig43

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FIG. 4. Immunoblots (C) and corresponding densitometries of

the IgG responses of AF-PCM sera (patient 8) before (A) and after

(B) 1 year of antimycotic therapy.

with AF-PCM reacted preferentially with components with

molecular masses above 50 kDa. The most frequent reactive

components for IgM-AF serum group were 50, 58, and 66

kDa (20%); 64, 82, and 88 kDa (40%); 90 kDa (55%); 80 kDa

(60%); and 70 kDa (70%), whereas those for the IgM-CFgroup were 70 and 80 kDa (20%) and 88 and 90 kDa (30%).IgA blots of CFA exposed to the same serum groups

recognized 10 and 26 components, respectively. Various

antigens with molecular masses of 20, 22, 24, 26, 28, 30, 32,

36, 38, 43, 46, 48, 50, 54, 56, 58, 60, 62, 64, 66, 70, 72, 74, 76,

80, and 82 kDa were detected. The number of antigens also

varied with the individual PCM serum used. IgA antibodieswere absent in 73% of the sera from patients with the

AiF-PCM, whereas the CF-PCM sera were able to recognizea more extensive set of antigens. The most frequent reactivecomponents for the IgA-AF serum group were 24, 43, 70,and 76 kDa (10%), whereas those for the IgA-CF group were

46, 60, and 80 kDa (25%), 24 kDa (30%), and 43 and 74 kDa(40%).

Figure 3 presents the individual frequencies of recognitionof CFA components by IgG, IgM, and IgA of AF-PCM and

.4# gewA: B :.'....FIG. 5. Immunoblots (C) and corresponding densitometries of

the IgG responses of CF-PCM sera (patient 9) before (A) and after(B) 1 year of antimycotic therapy.

CF-PCM sera. The 43-kDa component was recognized byIgG in 100% of both groups of sera, followed by the 70-kDacomponent, which was recognized by 50% of the IgG sera.On the other hand, none of the IgM AF-PCM sera reactedwith the 43-kDa component.

Profiles of immunoblots (IgG antibodies) obtained withsera from patients 8 (AF-PCM) and 9 (CF-PCM) undergoingtreatment are shown in Fig. 4 and 5, respectively. Thecorresponding densitometries of IgG blots showed a de-crease in antibodies against the gp43 molecule. Beforetherapy, patient 8 (AF) registered 18,408 mm2 (IgG anti-gp43), whereas 1 year later, patient 8's corresponding read-ing decreased to 11,851 mm2; enzyme-linked immunosorbentassay (ELISA) anti-gp43 titers were 1:102,000 and 1:12,800,respectively (data not shown), in agreement with the densi-tometric analysis. Patient 9 (CF) had antibody densitome-tries of 8,428 mm2 before treatment and 5,108 mm2 after 1year of therapy and ELISA titers of 1:51,600 and 1:6,400,respectively (data not shown). Similar results were obtainedwith the other patients (data not shown), indicating that theresponse to gp43 is a good indication of clinical improve-ment.

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IMMUNOLOGICAL RESPONSE TO P. BRASILIENSIS CFA 675

DISCUSSION

Identification of antigens eliciting an antibody response to

an infectious agent can lead to understanding of the immuneresponse to a pathogenic organism at the level of individualmolecules or epitopes. Such knowledge is the basis ofcurrent strategies for the development of simplified subunitvaccines to give protection against diseases (2). The P.brasiliensis CFA is composed of various molecules looselybound to the yeast cell surface which are easily detached bybeing suspended in an aqueous solution. These moleculesare probably more protected against endogenous proteasesthan those excreted into liquid culture medium, in whichthere are proteases released from dead cells during cellgrowth. These presumably better-preserved CFA moleculesmay mimic the antigens released in vivo from the cells in thesite of the infection or released into the circulation, wherethey become available for interaction with the cells of theimmune system.Various investigators have used the immunoblot approach

to study the humoral response to PCM. However, theantigenic preparations used for this purpose were exoanti-gens from aged filtrate cultures (7, 8, 12). The CFA, a young

growth preparation, is constituted of 42 components rangingfrom 18 to 102 kDa, the 43-kDa component being the majorglycoprotein excreted by the fungus (5).Our results indicate that despite the clinical form of the

disease, the IgG antibodies were able to recognize quitesimilar components of the CFA. In both forms, the 43- andthe 70-kDa major components were recognized by 100 and50% of the sera studied, respectively. Molecules 70, 74, 76,78, and 80 kDa in size were more reactive in the acute formthan in the chronic form.Vaz et al. (17) showed with a murine experimental PCM

that a diffuse 43- to 47-kDa band was recognized by all sera

tested, and the IgG against it was present at a high frequencyduring the whole period of infection. In a previous study,Camargo et al. (7) found 100% reactivity of IgG anti-gp43 inPCM sera. Casotto et al. (8) showed that the IgG sera from92% of the PCM patients recognized the 43-kDa componentfrom P. brasiliensis B-339.We found that IgM response to the CFA antigens was

more intense against components with molecular masses

greater than 50 kDa, mainly 54, 58, 60, 70, 80, 82, 86, 88, and90 kDa, in the acute form of PCM than in the chronic form.No response against the 43-kDa component in the AF-PCMsera was verified, and fewer than 10% of the CF-PCM sera

recognized the 43-kDa component. On the other hand,Mendes-Giannini et al. (12) found 100% reactivity with IgManti-gp43 in both clinical forms of the disease. Vaz et al. (17)in their murine experimental PCM study found great reac-

tivity of IgM against the diffuse 43- to 47-kDa band duringthe period of infection (16 weeks). The differences betweenour results and those obtained by others may be due to

differences in the glycosylation process of the gp43 mole-cule, which seems to be dependent on the strain and on thesubstrate on which the fungi were cultured. In this way,carbohydrate epitopes might be inaccessible to IgM antibod-ies in this kind of P. brasiliensis CFA preparation.The IgA response was significant in the CF-PCM group,

and was directed mainly against the components with mo-lecular masses of 20, 24, 26, 43, 46, 50, 58, 60, 70, 76, and 80kDa, whereas a poor IgA response was obtained in theAF-PCM group. The 43- and 74-kDa components were the

most reactive components in chronic PCM (about 40%reactivity each).

Our study showed that the CFA preparation is very usefulin immunoblot analysis of humoral responses in PCM.Regarding the Ig class, the IgG response has apparently beenthe best one to be studied once it recognizes quite similarantigenic components in both clinical forms of the disease.Regarding the antigenic component to be selected for anal-ysis of serum reactivity, the 43-kDa glycoprotein seems torepresent the most important antigenic component of thePCM system.

Antigenic variability in the murine PCM model and even inthe human PCM, with respect to the specific moleculesinvolved in the P. brasiliensis system, was observed (8, 17).Casotto et al. (8) showed that this variability may be ob-served not only among different isolates ofP. brasiliensis butalso among antigens with the same molecular mass. In theirstudy, the 44-kDa molecule (probably similar to our gp43) ofthree different strains of P. brasiliensis was recognized bythe same serum only in two strains (B339 and 688) and not instrain 1789.88. Vaz et al. (17), working with resistant andsusceptible mice, found significant differences in the IgMresponses to many components. The susceptible mice rec-ognized fewer components than the resistant mice. Thesefindings may explain the different results obtained by variousresearchers.For serological studies, we suggest the use of the P.

brasiliensis B-339 isolate, which has been investigated byvarious groups during the last 20 years (3, 6, 13) and whichcontains specific antigen recognized by 96% (14) to 100% (7,12) of the PCM sera. Casotto et al. (8) verified that freshlyisolated virulent strain 1789.88 did not appear to be useful fordiagnostic purposes, whereas isolates B339 and 688, whichhad been subcultured for many years, appear to be better.On the basis of the present findings, as well as previous

reports, and because the gp43 component is recognized byall or almost all of the PCM sera (IgG), we propose thatmeasurement or titration of IgG anti-gp43 be used for thefollowing up of patients under antimycotic therapy. Thismay be done by the reading of IgG blots by densitometry orby titration of sera by ELISA.

ACKNOWLEDGMENTS

This work was supported by Conselho Nacional de Desenvolvi-mento Tecnico e Cientifico (CNPq) and Fundacao de Amparo aPesquisa do Estado de Sao Paulo (FAPESP).

REFERENCES1. Ansorge, W. 1983. Fast visualization of protein bands by im-

pregnation in potassium permanganate and silver nitrate. Elec-trophoresis 82:235-242.

2. Arnon, R. 1984. Synthetic vaccines, p. 93. In I. M. Roitt (ed.),Immune intervention. Academic Press, London.

3. Blumer, S. O., M. Jalbert, and L. Kaufman. 1984. Rapid andreliable method for production of a specific Paracoccidioidesbrasiliensis immunodiffusion test antigen. J. Clin. Microbiol.19:404-407.

4. Bradford, M. 1976. A rapid and sensitive method for thequantification of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal. Biochem. 72:248-254.

5. Camargo, Z. P., C. P. Taborda, E. C. Rodrigues, and L. R.Travassos. 1991. The use of cell-free antigens of Paracoccidio-ides brasiliensis in serological tests. J. Med. Vet. Mycol.29:31-38.

6. Camargo, Z. P., C. Unterkircher, S. P. Campoy, and L. R.Travassos. 1988. Production of Paracoccidioides brasiliensisexoantigens for immunodiffusion tests. J. Clin. Microbiol. 26:2147-2151.

7. Camargo, Z. P., C. Unterkircher, and L. R. Travassos. 1989.Identification of antigenic polypeptides of Paracoccidioides

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Page 6: Immunological Response to Cell-Free of Paracoccidioides ...IMMUNOLOGICAL RESPONSE TOP. BRASILIENSIS CFA 673 loo0lgG-CF nn Hnn 1I So 3 lilU llinllUlnlHJnldlA[l-1 111i1dnnililinninl-I

676 BLOT1TA AND CAMARGO

brasiliensis by immunoblotting. J. Med. Vet. Mycol. 27:407-412.

8. Casotto, M., S. Paris, and Z. P. Camargo. 1991. Antigens ofdiagnostic value in three isolates of Paracoccidioides brasilien-sis. J. Med. Vet. Mycol. 29:243-253.

9. Franco, M. 1986. Host-parasite relationships in paracoccidio-idomycosis. J. Med. Vet. Mycol. 25:5-18.

10. Franco, M., M. R. Montenegro, R. P. Mendes, S. A. Marques,N. C. Dillon, and N. G. M. Mota. 1987. Paracoccidioidomycosis:a recently proposed classification of its clinical forms. Rev. Soc.Bras. Med. Trop. 20:129-132.

11. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

12. Mendes-Giannini, M. J. S., J. P. Bueno, M. A. Shikanai-Yasuda,A. M. S. Stolf, A. Masuda, V. A. Neto, and A. W. Ferreira. 1990.Antibody response to the 43 kDa glycoprotein of Paracoccidio-ides brasiliensis as a marker for the evaluation of patients undertreatment. Am. J. Trop. Med. Hyg. 43:200-206.

J. CLIN. MICROBIOL.

13. Puccia, R., S. Schenkman, P. A. J. Gorin, and L. R. Travassos.1986. Exocellular components of Paracoccidioides brasiliensis:identification of a specific antigen. Infect. Immun. 53:199-206.

14. Stambuk, B. U., R. Puccia, M. L. C. de Almeida, L. R.Travassos, and S. Schenkman. 1988. Secretion of the 43 kDaglycoprotein antigen by Paracoccidioides brasiliensis. J. Med.Vet. Mycol. 26:367-373.

15. Towbin, H., and J. Gordon. 1984. Immunoblotting and dotimmunoblotting-current status and outlook. J. Immunol.Methods 72:313-340.

16. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulosesheets: procedure and some applications. Proc. Natl. Acad. Sci.USA 76:4350-4354.

17. Vaz, C. A. C., D. W. R. Mackenzie, V. M. Hearn, Z. P.Camargo, L. M. Singer-Vermes, E. Burger, and V. L. G. Calich.1992. Specific recognition pattern of IgM and IgG antibodiesproduced in the course of experimental paracoccidioidomyco-sis. Clin. Exp. Immunol. 88:119-123.

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