Culture Filtrate Antigen Research Articles

Rabbit as an animal model of paranasal sinus mycoses.

Paranasal sinus mycoses are endemic in rural populations of northern India. To study host-parasite interactions, we developed an animal model of paranasal sinus mycoses. After failure in small animals such as mice and rats, we used New Zealand white rabbits weighing 2.5-3 kg. Inoculum sizes consisted of 0.75-1.0 x 10(8), 0.75-1.0 x 10(7), 0.75-1.0 x 10(6) conidia of a clinical isolate of Aspergillus flavus. The inoculum was injected at a spot 0.5 cm in front of the alveolar process of the maxilla and 0.5 cm below the maxillary process of frontal bone and vertically to a depth of 0.5 cm across the bone directly into the nasal sinus. Paranasal sinus mycoses proven by culture and histopathology developed in 67% of animals injected with 0.75-1.0 x 10(8) conidia and 17% of animals with 0.75 x 10(7) conidia. No lesions were found in the group injected with 0.75-1.0 x 10(6) conidia. Precipitating antibody against culture filtrate antigen was found in rabbits with paranasal sinus mycoses. Therefore, rabbits can be used as an animal model to study paranasal sinus mycoses.

Evaluation of Complex and Defined Antigens of <i>Mycobacterium tuberculosi</i><i>s</i> in an IgG-Specific ELISA for the Diagnosis of Tuberculosis

In recent years, several defined antigens of Mycobacterium tuberculosis have become available either by purifying the natural antigens using biochemical procedures, or by producing large quantities of recombinant antigens, using recombinant DNA technology. In this study, we evaluated these defined antigens of M. tuberculosis, along with the complex antigens, for humoral immune responses in an IgG-specific enzyme-linked immunosorbent assay (ELISA). We hoped to develop a tuberculosis-specific ELISA of diagnostic potential. Our results show that IgG antibodies to the complex antigens (i.e. culture filtrate, cell wall and sonicate) and to some of the defined antigens (i.e. 38-kD antigen and lipoarabinomannan) of M. tuberculosis were significantly elevated in the sera of tuberculous patients, as compared to healthy contacts of tuberculous patients and patients suffering from nonmycobacterial infections. Among the IgG subclasses, IgG2 antibody reactive with culture filtrate, cell wall, and lipoarabinomannan antigens was elevated in the sera of most tuberculous patients. Since the complex antigens are difficult to standardize, and batch-to-batch variations may occur with respect to the amount of active components in different batches, the use of defined antigens of M. tuberculosis in ELISA may be a better alternative in the early and specific diagnosis of tuberculosis.

Identification of a second Mycobacterium tuberculosis gene cluster encoding proteins of an ABC phosphate transporter

Following the identification of a M. tuberculosis phosphate transporter belonging to the superfamily of ABC transporters, we report on the cloning and sequencing of two additional genes, called pstS-3 and pstC-2, encoding proteins homologous to PstS and PstC of Escherichia coli, respectively. Together with the previously isolated M. tuberculosis gene similar to the E. coli pstA, these are included in a cluster encoding a second putative phosphate transport system. We demonstrate that pstS-3 encodes the previously described Ag 88, a 40 kDa M. bovis BCG culture filtrate antigen (immunodominant in H-2 b haplotype type mice). Finally, a signature motif identifying integral transmembrane proteins of prokaryotic phosphate binding-dependent permeases is proposed.

Molecular cloning and expression of a recombinant Aspergillus fumigatus protein Asp f II with significant immunoglobulin E reactivity in allergic bronchopulmonary aspergillosis

The cDNA of Aspergillus fumigatus encoding an allergen was cloned and expressed in Escherichia coli. The 987 bp long cDNA clone expressed a recombinant protein Asp f II of 34 kd. This protein exhibited binding to immunoglobulin E (IgE) in the serum samples from patients with allergic bronchopulmonary aspergillosis (ABPA). The patients with ABPA and central bronchiectasis demonstrated high levels of serum IgE antibodies, whereas patients with ABPA without central bronchiectasis, patients with asthma and Aspergillus skin test reactivity but no evidence of ABPA, and patients with aspergilloma showed only low levels of IgE antibody to Asp f II. In two-dimensional electrophoresis, a native antigen electroeluted from an A. fumigatus culture filtrate antigen preparation showed an isoelectric point and molecular weight similar to that of Asp f II. In a competitive enzyme-linked immunosorbent assay (ELISA), the IgE antibody reactivity of Asp f II with patient serum samples could be significantly inhibited by culture filtrate antigens of A. fumigatus. These results indicate that Asp f II has immunologic reactivities comparable to those of native A. fumigatus antigens. The recombinant Asp f II can be expressed in E. coli in large quantities and should prove useful as a standardized allergen for sensitive and specific immunodiagnosis of ABPA, especially in patients with central bronchiectasis.

Isolation and partial characterization of a Paracoccidioides brasiliensis 58 kDa extracellular glycoprotein which is recognized by human immune sera

A novel 58 kDa antigenic determinant of the fungus Paracoccidioides brasiliensis was identified by enzymelinked immunosorbent assay using a panel of species-specific murine monoclonal antibodies (MAbs). Western immunoblot analysis, deglycosylation studies and isoelectric focusing indicated that this 58 kDa antigen is a glycoprotein, with a pI of approximately 5·2. The molecule was purified from P. brasiliensis culture filtrate and yeast cytoplasmic antigens by membrane ultrafiltration, liquid isoelectric focusing and gel filtration; N-terminal amino acid sequence data revealed no substantial homology with known proteins. The presence of the antigen in the cytoplasm of both yeast and mycelial forms of the fungus was demonstrated when these MAbs were used as markers in immunofluorescence, immunoperoxidase and immunoalkaline phosphatase techniques to label P. brasiliensis in cryostat sections. These MAbs also recognized the cytoplasm of P. brasiliensis yeast forms in paraffin-embedded pathological specimens from human cases. A preparation of the 58 kDa component from yeast cytoplasmic antigen was reacted by Western immunoblotting with 26 different serum samples from paracoccidioidomycosis patients, and 81% of them recognized it.

Development of specific fluorescent-antibody test for tissue form of Penicillium marneffei

The diagnosis of penicilliosis marneffei can be difficult because the clinical manifestations mimic those of tuberculosis, histoplasmosis, and other mycotic infections. Furthermore, the tissue form of Penicillium marneffei can be confused with those of Histoplasma capsulatum and Cryptococcus neoformans. To facilitate the rapid detection and identification of P. marneffei in clinical materials, we sought to develop a specific indirect fluorescent-antibody (IFA) reagent for this dimorphic pathogen. Preliminary IFA studies with yeast-like cells (fission arthroconidia) of P. marneffei indicated that these cellular elements stained with antiglobulins against culture filtrate antigens and whole yeast-like cellular antigens. Both types of antiglobulins reacted with the yeast-like cells of P. marneffei and with H. capsulatum, but not with their respective mycelial forms. The antiglobulins also failed to react with the yeast and hyphal forms of a variety of other heterologous fungi. Specific antiglobulins useful in an IFA test for identifying P. marneffei yeast-like cells in culture or in clinical materials were produced by adsorptions with yeast-form cells of H. capsulatum. The yeast-like culture filtrate antigens of P. marneffei are preferred for use in the production of the specific antiglobulins because they stained P. marneffei yeast-like elements more intensely than antiglobulins produced against intact yeast-like cells.

Effects of the two varieties of Cryptococcus neoformans cells and culture filtrate antigens on neutrophil locomotion.

Cryptococcus neoformans var. gattii (serotype B and C) isolates have a relative predilection for immunocompetent hosts, and C. neoformans var. neoformans (serotype A and D) isolates have a relative predilection for immunocompromised hosts, suggesting that normal host resistance to the former may be relatively inefficient compared with that to the latter variety. In order to assess the possibility that normal cellular host defense is inadequate in protecting against C. neoformans var. gattii, we compared the two varieties of C. neoformans cells and their culture filtrate antigens (CneF) with respect to effects on neutrophil (polymorphonuclear leukocyte [PMN]) locomotion. In a 48-well modified Boyden chamber, the cells and CneF of C. neoformans var. neoformans (serotype A and D) isolates stimulated chemotaxis and chemokinesis of human PMN and activated a complement component(s) in pooled human serum to become a chemoattractant(s) for human PMN. In contrast, the cells and CneF of C. neoformans var. gattii (serotype B and C) isolates did not stimulate chemotaxis or chemokinesis in human PMN but rather inhibited chemokinesis and chemotactic responses of PMN to pooled human serum and formylmethionyl leucyl phenylalanine. Neither of the CneF from the C. neoformans var. gattii isolates was cytotoxic to PMN. Furthermore, with the mouse model, we found that CneF from C. neoformans var. neoformans caused migration of PMN into gelatin sponges implanted in naive and immunized mice, whereas CneF from C. neoformans var. gattii inhibited PMN migration into sponges. Our results, combined with findings of others showing reduced PMN infiltration in lungs of mice infected with C. neoformans var. gattii compared with PMN infiltration in lungs of mice infected with C. neoformans var. neoformans, indicate that the relative inadequacy of normal host resistance mechanisms to prevent infection with C. neoformans var. gattii results, in part, from inhibition of PMN migration to the site of the organism.

Effects of immunization with Cryptococcus neoformans cells or cryptococcal culture filtrate antigen on direct anticryptococcal activities of murine T lymphocytes

Immunizing CBA/J mice with intact Cryptococcus neoformans cells or with a cryptococcal culture filtrate antigen (CneF) induces an anticryptococcal delayed-type hypersensitivity response. Recently, it has been shown that two phenotypically different T-cell populations are responsible for delayed-type hypersensitivity reactivity in mice immunized with intact cryptococcal cells, whereas only one of those populations is present in mice immunized with soluble cryptococcal antigens in complete Freund's adjuvant (CFA). The purpose of this study was to determine if differences occur with regard to direct anticryptococcal activity between T-lymphocyte-enriched populations from mice immunized with intact viable or dead cryptococcal cells and similar cell populations from mice immunized with the soluble cryptococcal culture filtrate antigen, CneF, emulsified in CFA. The percentage of lymphocytes which form conjugates with C. neoformans and the percentage of cryptococcal growth inhibition in vitro are greater with T-lymphocyte-enriched populations from mice sublethally infected with C. neoformans or from mice immunized with intact heat-killed cryptococcal cells in the presence or absence of CFA than with lymphocyte populations from mice immunized with CneF-CFA. Enhanced anticryptococcal activity of T lymphocytes could be induced by immunizing mice with heat-killed C. neoformans cells of serotype A, B, C, or D as well as by immunizing with a similar preparation of an acapsular C. neoformans mutant but not by immunizing with CFA emulsified with CneF prepared from any one of the C. neoformans isolates. These data indicate that the soluble cryptococcal culture filtrate antigens do not induce the same array of functional T lymphocytes as whole cryptococcal cells.

Intravascular cryptococcal culture filtrate (CneF) and its major component, glucuronoxylomannan, are potent inhibitors of leukocyte accumulation

Disseminated cryptococcosis is characterized by high titers of cryptococcal polysaccharides in serum and minimal cellular infiltrates in infected tissues of patients. The main objective of this study was to determine whether the circulating cryptococcal polysaccharides could contribute to the lack of cellular infiltration into infected tissues. To assess this possibility, a gelatin sponge implantation model was used. We found that intravenous (i.v.) injection of mice with cryptococcal culture filtrate antigen (CneF) inhibited migration of leukocytes (neutrophils, lymphocytes, and monocytes) into the intrasponge sites of acute inflammation induced by CneF, tumor necrosis factor alpha, or formylmethionyl leucyl phenylalanine. In addition, i.v. administration of CneF inhibited leukocyte migration into the intrasponge sites of a cell-mediated immune reaction irrespective of whether the delayed-type hypersensitivity response was to CneF or the mycobacterial antigen purified protein derivative. Glucuronoxylomannan, a major constituent of CneF and a major cryptococcal antigen detected in the sera of patients with disseminated cryptococcosis, when given i.v. to mice, inhibited leukocyte migration into the sponges. Our results suggest that the minimal cellular infiltrates observed in infected tissues of cryptococcosis patients may be due, in part, to the circulating cryptococcal polysaccharide functioning as we have demonstrated in the mouse model. Furthermore, the high titers of cryptococcal antigen in the sera of patients may diminish leukocyte migration in response to stimuli other than Cryptococcus neoformans, a point that may be relevant in AIDS patients with cryptococcosis.

Mice immunized by primary vaginal Candida albicans infection develop acquired vaginal mucosal immunity.

It has been postulated that systemic cell-mediated immunity (CMI) is an important host defense mechanism against Candida infections of the vagina. However, in an estrogen-dependent murine model of experimental vaginal candidiasis, we recently showed that systemic Candida-specific Th1-type CMI induced by immunization with Candida culture filtrate antigen had no effect on vaginal Candida population levels during the course of a vaginal infection. In the present study, mice given a second vaginal inoculation in the presence of peripheral Candida-specific Th1-type CMI induced by prior vaginal infection had anamnestic-type increased delayed-type hypersensitivity (DTH) responses, concomitant with significantly fewer Candida organisms in the vagina than in primary-infected mice. In addition, organisms in secondary-infected mice were fragmented and superficial penetration into the epithelium was reduced. The systemic presence of Candida-specific T suppressor (Ts) cells that significantly suppressed the infection-derived anamnestic DTH reactivity did not abrogate the protective effect in the vagina. Additional experiments showed that vaginally immunized mice were not protected from gastrointestinal or systemic candidiasis and, in contrast to mice with a second vaginal infection, did not demonstrate anamnestic DTH reactivity. These results suggest that a moderate level of local protection against a Candida vaginal infection can be achieved by vaginal immunization but that the protective role of acquired peripheral Candida-specific Th1-type reactivity at the vaginal mucosa appears to be limited.

Serolodiagnosis of Some Types of Aspergillosis using Culture Filtrate Antigensfrom Aspergillus fumigatus.

Culture filtrate antigens derived from a strain of Aspergillus fumigatus were used in diagnosing aspergillosis, such as aspergilloma, allergic bronchopulmonary aspergillosis and tissue invasive aspergillosis, by the Western immunoblot method detecting the serum specific antibodies, immunoglobulin M (IgM) and immunoglobulin G (IgG). These antibodies in the sera from all patients with those types of aspergillosis were thus detected significantly more clearly than in the healthy, normal controls.Double diffusion tests using the same antigens were also performed to compare the efficacy of these two methods. In several cases showing negative in the double diffusion test, these antibodies were found to react with proteins possessing the molecular weight of 20, 35, 50, 65 and 73kDa, while in other cases showing positive in the double diffusion test, the antibodies could not be distinguished from each other. We therefore used the Western blotting method to distinguish them.From the data obtained it was concluded that serologic diagnosis by the detection of IgM and IgG using the immunoblot method plays an important role in strengthening the clinical diagnosis.

Isolation and Characterization of a 66-kDa Antigen from Culture Filtrate of Aspergillus fumigatus NCPF2109.

The non-dialyzable fraction of culture filtrate antigen of Aspergillus fumigatus was fractionated by anion exchange chromatography and gel filtration, connecting each column of Mono Q and Superose 6 sequentially. Fractions which had a high protein and reacted strongly with an immunoglobulin-M monoclonal antibody (MAb) 8-3C F5 G9, as determined by enzyme-linked immunosorbent assay (ELISA), were pooled to give four antigenic fractions (F1-F4). Gas-liquid chromatography analysis of the 4 antigenic fractions showed the presence of mannose and galactose. An immunoelectron microscopic experiment with MAb revealed the distribution of antigen on the surface of the hyphae. The fractions had no proteolytic activity. Fraction 4, relatively small in its molecular size but with the highest recovery of the reactivity with MAb among F1 to F4, was selected for further characterization. Antigenic reactivity of F4 with MAb was heat-stable but fully retained after sodium metaperiodate treatment. Pronase E almost destroyed the antigenic reactivity of F4 with MAb. MAb affinity chromatography of F4 gave the specific antigen, F4-AF. In both sodium dodecyl sulfate-polyacrylamide gel electrophoresis by Coomassie blue staining and Western blotting with MAb, F4-AF showed a single band with molecular mass of 66 kDa.

Experimental immunity toMicrosporum canisand cross reactions with other dermatophytes of veterinary importance

An inactivated, broad-spectrum dermatophyte vaccine was used to produce an active immunity in guinea-pigs against Microsporum canis. None of the vaccinates developed infection from a contact exposure challenge that produced clinical infections in 70% of the unvaccinated controls. Infection with M. canis induced antibody titres (ELISA) and delayed cutaneous hypersensitivity (DCH) reactions to itself as well as cross-reacting titres to Trichophyton equinum and T. mentagrophytes and DCH reactions to T. mentagrophytes; however vaccinated animals developed significantly higher antibody titres and DCH responses to all of these antigens than did non-vaccinated animals which had been infected or exposed. Rabbits hyperimmunized with culture filtrate antigens to single dermatophyte agents (M. canis, M. gypseum, T. equinum, and T. mentagrophytes) developed positive inter-species and inter-generic DCH cross-reactions to a battery of six skin test antigens (M. canis, M. gypseum, M. equinum, T. equinum, T. mentagrophytes var. mentagrophytes and T. verrucosum). Guinea-pigs vaccinated with a T. equinum vaccine had increased resistance to M. canis infection than did non-vaccinated controls. These findings support clinical observations which suggest establishment of a broad-based immunity in animals following infection with a single dermatophyte.

Studies on Fusarium solani. Cross-reactivity among Fusarium species.

Allergenic cross-reactivity among three Fusarium species (F. solani, F. equiseti, and F. moniliforme) was determined by crossed immunoelectrophoresis (CIE), ELISA inhibition, and immunoprint-inhibition analysis. Inhibiting immunoprint was used to assess the cross-reactivity of individual allergenic proteins in culture filtrate (CF), separated on SDS-PAGE. Dose-response inhibition of band 14 kDa in immunoprints of F. solani CF by antigens of F. solani CF, F. equiseti CF, and F. moniliforme CF demonstrated significant cross-reactivity of allergenic proteins on analysis of densitometric scans. CIE and ELISA inhibition using hyperimmune sera raised in rabbits against F. solani CF antigen also demonstrated shared antigenicity among Fusarium species. Collectively, this study revealed the presence of shared as well as unique antigenic and allergenic determinants in F. solani CF, F. equiseti CF, and F. moniliforme CF antigens.

Polysaccharide antigens of the capsule of Cryptococcus neoformans.

The major significance of the capsular polysaccharide of C. neoformans is its role in potentiating opportunistic infections by the yeast. It has the ability to exert a broad spectrum of influences on the immune response, from activation of phagocytic cells and complement components of the alternative pathway, to the induction of specific antibody, T-suppressor cells, DTH responses, and cytokines (51). These biological properties along with the serotype specificities are all determined by the physical properties and chemical structures of the polysaccharide antigens that compose the capsule. There is evidence not only for an association of lethal infections with serotype A in patients with advanced AIDS (34, 56), but also for a role for the capsule in directly influencing the infection of CD4+ cells by HIV (57). Together, these phenomena raise intriguing questions about the possible connection between the chemistry of these capsular antigens and cryptococcal infections in AIDS patients. One speculation is that AIDS creates the optimal physiological conditions for the establishment and spread of cryptococcosis. It has been observed that during the progression of AIDS there is a shift towards a T-2 response (14). This could lead to conditions that would inhibit the cellular immune responses that block dissemination of cryptococcal infections. Thus, an important consideration in the application of vaccine or immune modulation therapies in the treatment of cryptococcosis in AIDS victims would be the design of vaccines that could boost the T-1 immune response. It has been shown that the form and dose of an antigenic challenge can influence the induction of a T-1 or T-2 immune response (61). Recently, Murphy has reported that gamma interferon and interleukin 2 are up-regulated in the spleens of mice that produce anticryptococcal TDH and TAMP cells in response to immunogenic doses of cryptococcal culture filtrate antigen given with Freund's complete adjuvant (49). Perhaps purified cryptococcal antigens (e.g., MP) conjugated to an appropriate carrier or adjuvant could be used in therapeutic strategies to limit cryptococcosis in immunocompromised individuals. Future investigations of virulence and pathogenicity in the context of defined polysaccharide antigens from encapsulated strains of C. neoformans will contribute to a better understanding of the regulation of cryptococcal infection and immunity at the cellular and molecular levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of preinduced Candida-specific systemic cell-mediated immunity on experimental vaginal candidiasis

It has been postulated that systemic cell-mediated immunity (CMI) is an important host defense factor against recurrent vaginal infections caused by Candida albicans. Using an estrogen-dependent murine model of vaginal candidiasis, we have previously shown that mice inoculated vaginally with C. albicans acquire a persistent vaginal infection and develop Candida-specific Th1-type systemic CMI. In the present study, experimental vaginitis was monitored in the presence of preinduced systemic Candida-specific CMI. Mice immunized systemically with C. albicans culture filtrate antigens (CaCF) in complete Freund's adjuvant (CFA) had Th1-type reactivity similar to that of vaginally infected mice. CaCF given to mice intravenously induced Candida-specific suppressor T (Ts) cells. Mice preimmunized with CaCF-CFA and given a vaginal inoculum of C. albicans had positive delayed-type hypersensitivity (DTH) reactivity from the time of vaginal inoculation through 4 weeks. Conversely, mice infected in the presence of Ts cells had significantly reduced DTH responses throughout the 4-week period in comparison with naive infected mice. However, the presence of Th1-type Candida-specific DTH cells or Ts cells, either induced in mice prior to vaginal inoculation or adoptively transferred at the time of inoculation, had no effect on the vaginal Candida burden through 4 weeks of infection. A similar lack of effects was obtained in animals with lower Candida population levels resulting from a reduction in or absence of exogenous estrogen. These results suggest that systemic Th1-type CMI demonstrable with CaCF is unrelated to protective events at the level of the vaginal mucosa.

沈降抗体検出用アスペルギルス粗抗原に関する若干の検討 補遺

We reported earlier on culture filtrate antigens from A. fumigatus that had been incubated at 25°C for 1-10 weeks (Jpn J Med Mycol 34: 439-444, 1993). Additionally, we prepared five culture filtrate antigens from A. fumigatus ATCC26430 grown by shaking at 25°C for 11-15 weeks in Sabouraud liquid medium. We also prepared a polysaccharide antigen from the same strain by extracting mycelia and conidia. The growth of the fungi became weaker in cultures after 12 weeks, and by stopped at 14 weeks. Sixteen antigens were then employed in agar gel double diffusion (DD) tests against the sera of 18 aspergilloma patients. The results of DD showed three types (A, B, C) of precipitin pattern, with 9 patients belonging to type A, 6 to type B and 3 to type C. In all cases, precipitin lines were formed with the antigens obtained after 6-15 weeks of culture. To avoid contamination, inferior cultures and accumulation of waste, the incubation time should be as short as possible. Thus, as noted in our previous letter, we found that the antigen obtained from 6-week culture filtrates at 25°C is the most useful for demonstrating precipitin reactions against the sera of aspergilloma patients.

<i>Fusarium solani</i>: Immunochemical Characterization of Allergens

Allergenic components of the fungus Fusarium solani were isolated using (NH4)2SO4 precipitation and ion-exchange column chromatography. The allergenicity of fractions was studied by enzyme-linked immunosorbent assay and radioallergosorbent test inhibition techniques. Proteins of culture filtrate (CF), mycelium (MY), and spore (SP) extracts of F. solani were characterized by isoelectrofocusing, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and IgE-specific immunoblotting. CF antigen of F. solani contained more allergenic proteins than MY and SP, visible on immunoblot analysis using allergenic serum pool. A 65-kD protein component of CF was found to be a major allergen, as it was strongly visible on immunoblots of all 15 patient sera tested. Crossed immunoelectrophoresis and enzyme-linked immunosorbent assay inhibition using rabbit antibodies raised against F. solani CF demonstrated shared antigenicity between CF, MY, and SP extracts. It was observed that F. solani is a significant allergen, and most of the allergens of MY and SP extracts were found in CF extract. Therefore, CF alone can be used in the preparation of a standard extract. However, few unique allergenic proteins were observed in MY as well as in SP extracts of F. solani. Hence, the use of combined CF, MY, and SP extracts of F. solani is recommended for diagnosis and immunotherapy.

Mobility of human neutrophils in response to Cryptococcus neoformans cells, culture filtrate antigen, and individual components of the antigen

The mobility of human neutrophils (PMN) in response to encapsulated or nonencapsulated Cryptococcus neoformans cells or cryptococcal culture filtrate (CneF) and its components was studied by using a 48-well modified Boyden chamber. Encapsulated C. neoformans (isolate 184A) cells and CneF-184A stimulated directed migration of human PMN in the absence of serum (direct chemotactic activity) and activated a heat-labile component(s) in fresh human serum to become a chemoattractant(s) for human PMN (indirect chemotactic activity). At a 1:8 dilution (0.25 mg of carbohydrate per ml), CneF-184A displayed chemokinetic activity when assessed with a checkerboard assay. Nonencapsulated C. neoformans isolate 602 cells did not have direct chemotactic activity but did have indirect chemotactic activity. The capsule of C. neoformans is composed predominantly of glucuronoxylomannan (GXM). Purified GXM displayed both direct and indirect chemotactic activity. CneF-184A contains, in addition to GXM, a concanavalin A-binding mannoprotein (MP), whereas CneF-602 contains no GXM but does contain MP. CneF-184A showed direct chemotactic activity and CneF-602 did not. Both CneF-184A and CneF-602 displayed indirect chemotactic activity for human PMN. In addition, purified MP from CneF-184A, like CneF-602, showed only indirect chemotactic activity. These results indicate that GXM contributes to the direct chemotactic activity of PMN observed with the whole encapsulated yeast cells and the unfractionated CneF derived from the encapsulated cells. Both MP and GXM from encapsulated C. neoformans cells mediate indirect chemotactic activity on human PMN.

Cytokine profiles associated with induction of the anticryptococcal cell-mediated immune response

Previous studies with a murine model have shown that immunization with cryptococcal culture filtrate antigen (CneF) emulsified in complete Freund adjuvant (CFA) induces two populations of anticryptococcal reactive CD4+ T cells. One population (TDH cells) transfers anticryptococcal delayed-type hypersensitivity (DTH), and the other population (Tamp cells) amplifies the anticryptococcal DTH response of given to recipient mice at the time of immunization of the recipient. Treatment of mice with cyclosporin A (CsA) ablates the induction of Tamp cells but not TDH cells. The present study focused on assessing the cytokines produced by spleen cells taken from CsA-treated and control (solvent-treated) mice at days 1, 2, 4, and 6 after immunization. Supernatants from the spleen cells cultured in vitro for 24 or 48 h in medium alone or with CneF, concanavalin A, or phorbol 12-myristate 13-acetate plus calcium ionophore were assessed for the presence of interleukin-2 (IL-2), gamma interferon (IFN-gamma), IL-4, IL-5, and tumor necrosis factor. Spleen cells from CneF-CFA-treated mice produced IL-2 and IFN-gamma, but not IL-4 or IL-5, constitutively and in response to CneF, indicating that CneF-CFA induces a Th1 response. Tumor necrosis factor was not produced. Anticryptococcal TDH cells developed in spleens in which there were low levels of IFN-gamma and IL-2 (CsA-treated, immunized mice), whereas anticryptococcal Tamp cells along with TDH cells matured in spleens in which production of IFN-gamma and IL-2 was high (solvent-treated, immunized mice). The data also suggest that IL-2 and IFN-gamma produced by Tamp cells early after adoptive transfer are influential in the development of the amplified anticryptococcal DTH response that has been observed in Tamp cell-recipient mice.