Culture Filtrate Antigen Research Articles

Characterization of cellular infiltrates and cytokine production during the expression phase of the anticryptococcal delayed-type hypersensitivity response

Cryptococcosis, an increasingly important opportunistic infection caused by the encapsulated yeast-like organism Cryptococcus neoformans, is limited by an anticryptococcal cell-mediated immune (CMI) response. Gaining a thorough understanding of the complex anticryptococcal CMI response is essential for developing means of controlling infections with C. neoformans. The murine cryptococcosis model utilizing footpad swelling to cryptococcal antigen (delayed-type hypersensitivity [DTH]) has proven to be a valuable tool for studying the induction and regulation of the anticryptococcal CMI response, but this technique has limitations with regard to evaluating the role of the final effector cells recruited by an ongoing CMI response. The purpose of this study was to assess the types of cells and cytokines induced into the site of cryptococcal antigen deposition in C. neoformans-infected and -immunized mice compared with those for control mice. We used a gelatin sponge implant model to examine the cells and cytokines present at the site of an anticryptococcal DTH response. Sponges implanted in infected mice and injected with cryptococcal culture filtrate antigen (CneF) 24 h before assessment had significantly increased numbers of infiltrating leukocytes compared with saline-injected sponges in the same animals. Exaggerated influxes of neutrophils and mononuclear cells were the major contributors to the increase in total numbers of cells in the DTH-reactive sponges. The numbers of CD4+ and LFA-1+ cells were found to be significantly increased in the CneF-injected sponges of infected and immunized mice over the numbers in control sponges. The numbers of large granular lymphocytes were also increased in DTH-reactive sponges compared with control sponges. Gamma interferon, interleukin 2 (IL-2), and IL-5 are clearly relevant cytokines in the anticryptococcal CMI response, since they were produced in greater amounts in the CneF-injected sponges from C. neoformans-infected and -immunized mice than in control sponges. IL-4 was not associated with the expression of DTH to cryptococcal antigen. The gelatin sponge model is an excellent tool for studying cells and cytokines involved in specific CMI responses.

Influence of genes from the major histocompatibility complex on the antibody repertoire against culture filtrate antigens in mice infected with live Mycobacterium bovis BCG.

C57BL/10 and C57BL/6 mice (H-2b); B10 congenic mice with f, k, p, q, r, and s H-2 haplotypes; B10 mice with recombinant g2, o2, a, h2, h4, i5, and bq1 H-2 haplotypes; and B6 mice with major histocompatibility complex (MHC) mutant bm1 and bm13 (class I) and bm12 (class II) haplotypes were infected intravenously with 4 x 10(6) CFU of live Mycobacterium bovis BCG and examined by Western immunoblot analysis for serum antibodies against BCG culture filtrate antigens, following a boost injection with live BCG or with BCG culture filtrate. Parental B10 and B6 mice reacted very intensely with three culture filtrate protein bands with estimated molecular masses of 37, 38, and 40 kDa. Response against the 40-kDa protein was stronger following a boost injection with live BCG than following a boost with culture filtrate. Sera from mice with f, p, i5, bm1, and bm13 haplotypes reacted strongly, with both the 37-38- and 40-kDa antigens, and sera from mice with q and bq1 haplotypes showed a somewhat weaker reaction. Sera from mice with r, s, and bm12 haplotypes reacted against the 37-38-kDa antigen but not against the 40-kDa antigen, and sera from mice with the h2 haplotype reacted only with the 40-kDa antigen but not with the 37-38-kDa antigen. Sera from mice with the k, g2, o2, a, and h4 haplotypes showed, at most, a very weak reaction with the 37-38- and 40-kDa antigens. These results demonstrate that MHC genes profoundly affect the antibody repertoire used against culture filtrate antigens in mice infected with live M. bovis BCG. In particular, as shown in mice with the recombinant H-2 haplotype and in class II mutant bm12 mice, the I-A heterodimer controls the recognition of the immunodominant 40-kDa antigen. By using crossed immunoelectrophoresis, this 40-kDa antigen was identified as antigen 88 according to the reference system of Closs et al. for BCG antigens.

Development of a serologic panel for the recognition of nocardial infections in a murine model

A murine model was used to develop a sensitive and specific serologic test for clinical and subclinical infections caused by Nocardia. The following tests were used: (a) enzyme-linked immunosorbent assays (ELISAs) with culture filtrate and cytoplasmic extract antigens from Nocardia asteroides; (b) ELISA with N. asteroides trehalose dimycolate (cord factor); (c) indirect immunofluorescent antibody assay with whole cells of N. asteroides; and (d) Western-blot analysis for the 54 to 55-kD, 35-kD, and 62-kD proteins of N. asteroides. The sera from BALB/c mice, experimentally infected with nonlethal doses of three species of Nocardia, nine species of Mycobacterium, Rhodococcus equi, two species of Actinomadura, and two species of Streptomyces were tested using this panel. The serologic tests did, indeed, identify mice infected with nocardiae and could differentiate them from mice infected with the other actinomycetes, including mycobacteria.

Candida-specific cell-mediated immunity is demonstrable in mice with experimental vaginal candidiasis

Women with recurrent vulvovaginal candidiasis often demonstrate a down-regulation of cell-mediated immunity (CMI) to Candida albicans detected by a lack of cutaneous delayed-type hypersensitivity (DTH) to Candida antigens. However, the role of systemic CMI as a host defense mechanism against recurrent vulvovaginal candidiasis is not well understood, in part because of the lack of a well-defined murine model of vaginal candidiasis. The present study was undertaken to determine: (i) whether soluble Candida culture filtrate antigens (CaCF) could be used to induce and detect Candida-specific CMI in mice and (ii) whether these antigens would be useful in detecting systemic CMI in mice given an experimental Candida vaginal infection. To this end, mice were immunized subcutaneously with CaCF in complete Freund's adjuvant, and within 7 days they developed Candida-specific DTH reactivity detected by footpad swelling (increase in footpad thickness, 0.36 mm) 24 h after footpad challenge with CaCF. Adoptive transfer studies showed that the DTH responsiveness was elicited by CD4+ DTH T cells. In mice given a vaginal inoculum of C. albicans blastoconidia (5 x 10(5)), footpad challenge with CaCF resulted in positive DTH responses (0.24 mm) as early as 1 week, responses similar to immunization in 2 to 3 weeks (0.33 mm), and sustained low levels of DTH reactivity (0.15 mm) through 10 weeks of vaginal infection. Vaginal lavage cultures revealed that peak vaginal Candida burden occurred 1 week post-vaginal inoculation (10(5) CFU) and declined 16-fold by week 10. These results provide evidence that Candida-specific systemic CMI is generated and can be detected longitudinally in mice with Candida vaginitis by a multiantigen preparation of Candida organisms which both initiates and detects Candida-specific CMI.

The major secreted antigen complex (Ag 85) from Mycobacterium bovis bacille Calmette-Guérin is associated with protective T cells in leprosy: a follow-up study of 45 household contacts.

T cell proliferation and interferon (IFN)-gamma secretion were analyzed in 45 leprosy contacts stimulated with antigen 85 (Ag85), the major culture filtrate antigen from Mycobacterium bovis bacille Calmette-Guérin. All 14 Mitsuda reaction-positive contacts reacted to Mycobacterium leprae and Ag85. Three Mitsuda reaction-negative contacts reacted weakly to M. leprae and Ag85. The other 28 Mitsuda reaction-negative contacts did not react to M. leprae, but 9 reacted to Ag85. Thirty-four contacts were retested 16 months later. Eleven contacts initially positive by the Mitsuda test remained lepromin positive and reactive to M. leprae and Ag85. Fourteen contacts initially negative by the Mitsuda test converted, and all reacted in vitro to M. leprae and Ag85. Finally, 9 contacts remained Mitsuda test-negative, and 7 were unreactive to Ag85. In vitro reactivity to Ag85 at baseline in Mitsuda test-negative contacts was associated with subsequent conversion to lepromin reactivity in 7 of 9 subjects. These data suggest that reactive T cells against Ag85 develop very early during M. leprae infection and that Ag85 is a potentially protective T cell immunogen.

Treatment and serological studies of an Italian case of penicilliosis marneffei contracted in Thailand by a drug addict infected with the human immunodeficiency virus

A case of disseminated penicilliosis marneffei, the first to be diagnosed in Italy, is described in a male HIV-positive drug addict. The patient had visited Thailand several times in the two years prior to his hospitalization. The presenting signs were fever, productive cough, facial skin papules and pustules, nodules on both thumbs and oropharyngeal candidiasis. Penicillium marneffei was isolated from a series of blood specimens with the lysis centrifugation procedure. Septate, yeast-like cells were observed in histological sections of the nodules and sputum smears. The patient was treated for 6 weeks with amphotericin B (total dosage 1,400 mg) and flucytosine (150 mg/kg/die) for the first 3 weeks. Prompt clinical improvement and sterilization of all biological specimens were attained. Itraconazole was administered as maintenance therapy (400 mg/die for the first month and 200 mg afterward). During the follow-up period, no relapse was observed. The patient, however, did succumb to a variety of non-mycotic infections and died nine months after start of therapy. At autopsy, P. marneffei was not detected in his tissues. Serological studies were performed with a micro-immunodiffusion procedure using a mycelial culture filtrate antigen of P. marneffei. Sera taken early in the course of the disease gave positive antibody reactions. Whereas sera taken 3-5 months following therapy were negative. All known cases of penicilliosis marneffei in bamboo rats and in humans among the inhabitants and visitors to the endemic areas of P. marneffei in South East Asia and Indonesia are summarized.

Immunoblot analysis of Aspergillus fumigatus antigen with human antibodies and lectin probes.

Components of culture filtrate antigen of Aspergillus (A.) fumigatus were electrophoretically separated, transferred to hydrophobic durapore membrane, and then probed with patients' sera and lectins. Using human sera from patients with aspergilloma and allergic bronchopulmonary aspergillosis (ABPA), more than 13 IgG binding components were identified. IgG binding to high molecular weight components (HMW) of over 30 kDa was observed in all patient sera tested, but IgG binding to low molecular weight components (LMW) of approximately 18 kDa was observed in 7 out of 8 patients' sera which showed a positive precipitin reaction by means of counterimmunoelectrophoresis. Concanavalin A and lens culinaris agglutinin showed strong binding to HMW but no binding to LMW. Further, strong IgE binding to the 18 kDa component was observed in serum from a patient with ABPA during the acute stage, but not during the convalescence stage. It is suggested that the 18 kDa protein from A. fumigatus is the primary antigen in the serological antibody responses observed in patients with aspergilloma or ABPA.

Spleen cell cytokine secretion in Mycobacterium bovis BCG-infected mice.

Three susceptible mouse strains, i.e., BALB/c (H-2d), C57BL/6 (H-2b), and major histocompatibility complex-congenic BALB.B10 (H-2b), were infected intravenously with 4 x 10(6) CFU of live Mycobacterium bovis BCG and analyzed 4 weeks later for in vitro spleen cell cytokine secretion in response to purified protein derivative (PPD), BCG culture filtrate (CF), BCG cellular extract, total BCG, the purified extracellular 30-32-kDa antigen (the fibronectin-binding antigen 85), or the intracellular 65-kDa heat shock protein. C57BL/6 and BALB.B10 mice produced 5- to 10-fold more gamma interferon and interleukin-2 (IL-2) when stimulated with CF, PPD, and antigen 85 than BALB/c mice did. When stimulated with BCG extract and whole BCG, gamma interferon and IL-2 levels were generally lower and comparable in the three strains. IL-4 was detected in spleen cell culture supernatants from infected BALB/c mice but not from C57BL/6 or BALB.B10 mice. IL-5 could not be detected. C57BL/6 and BALB.B10 spleen cells also produced more tumor necrosis factor alpha and IL-6 after stimulation with PPD and CF than BALB/c cells did. Finally, BCG vaccination generated efficient protective immunity in C57BL/6 and BALB.B10 mice but not in BALB/c mice. These data suggest that secreted mycobacterial CF antigens selectively induce a strong TH1 response in BCG-infected C57BL/6 and BALB.B10 mice, whereas in BALB/c mice this response is partly counterbalanced by TH2 cells.

Immunodiffusion test for diagnosing basidiobolomycosis.

An immunodiffusion test was developed for the diagnosis of basidiobolomycosis. When culture filtrate antigen (CFA) from Basidiobolus ranarum was reacted against two human patient and two rabbit antisera, 2 precipitin bands, inner (N) and outer (Y), were revealed for both patient and rabbit antisera. A line of identity was also observed between precipitin bands obtained with patient and rabbit sera. When CFA from B. ranarum (B CFA) was reacted against rabbit sera which contained antibody to Conidiobolus coronatus and Pythium insidiosum, 1 precipitin band corresponding to inner band (N) was observed. This finding showed that B. ranarum, C. coronatus and P. insidiosum shared at least one common antigen. After B CFA was absorbed with Pythium rabbit antiserum, the inner precipitin line that occurred between B CFA and rabbit antisera of Pythium and Conidiobolus disappeared. However, with Basidiobolus rabbit antiserum, the result did not change. The antigens which could be demonstrated by inner (N) and outer (Y) precipitin bands were heat stable at 56 degrees C for 30 min. The titer of the antibodies specific to these antigens decreased as the lesions subsided. When B. ranarum CFA was reacted against sera from 20 apparently normal persons, 20 diabetes mellitus patients, 5 aspergillosis patients, 2 candidosis patients and 3 pythiosis patients, no precipitin band was found. B. ranarum CFA was also treated with each rabbit antiserum specific to Candida albicans, Malassezia furfur and Aspergillus fumigatus. No precipitin bands occurred with any of these antisera. Thus, this test was found to be practical, sensitive and specific, and can be used to monitor patients infected with Basidiobolus ranarum.

Immunodiagnosis of nocardiosis

A specific immunodominant 54-kDa antigen was purified from a cultured filtrate of Nocardia asteroids by immunoaffinity chromatography. The chromatography column was prepared with immunoglobulin G obtained from sera from parients with lepromatous leprosy. Unbound solutes consisted of specific, partially purified N. asteroides antigens, primarily a 54-kDa band, accompanied by two others of 31 and 62 kDa. The Western blot (immunoblot) technique was applied to detecting the immunologic response to nocardiae in the serum of nocardiosis patients. Each of the serum samples from immunosuppressed or immunocompetent patients infected with N. asteroids reacted with the 54-kDa band, and two reacted with the 31- and 62-kDa bands. There was no reaction to either the 54- or the 31-kDa antigen will all serum samples obtained from patients with tuberculosis, except for one, will all serum samples obtained from patients with leprosy, or with all sera obtained from healthy controls. The partially purified 54-kDa antigen, specific for N. asteroides, was used as the immunogen to generate monoclonal antibodies (mAbs) and two mAbs were selected. As determined by Western blot, both mAbs reacted with the 54-kDa band. Using indirect immunofluorescence or enzyme immunoassay with whole N. asteroides microorganisms, the mAbs did not react with N. asteroides cells. No cross-reactivity with mycobacterial antigens, either culture-filtrate antigens or tuberculin, was exhibited with any of the two mAbs. These mAbs are candidates to be used for the development of a sensitive and specific diagnostics test for nocardiosis.

Entomophthoromycosis due to Conidiobolus.

Entomophthoromycosis due to Conidiobolus coronatus is a granulomatous infection characterized by lesions that originate in the inferior turbinate, spread through ostia and foramina to involve the facial and subcutaneous tissues and paranasal sinuses. The majority of the cases have been described from areas of tropical rainforest in West Africa, agricultural and outdoor workers (aged 20-60 years) being the ones most frequently affected. The fungus is common in soil and decaying vegetation. Infection probably occurs by implantation of the spores of the fungus in nasal mucosa. C. incongruus is a rare agent of the disease, so far known only from two cases with lesions involving the pericardium, mediastinum, lungs, liver, oesophagus and jejunum. C. coronatus is known to cause a clinically similar disease in horses, mules, a dolphin and a chimpanzee. A characteristic histological feature is the presence of thin-walled, broad, often septate hyphae or hyphal fragments with a thick eosinophilic sheath, frequently phagocytosed within giant cells. The fungus is known to produce in vitro several enzymes, e.g., elastase, esterase, collagenase and lipase, which have a possible role in pathogenicity. A concentrated brain heart infusion culture filtrate antigen is useful for immunodiagnosis. Several drugs e.g., potassium iodide, cotrimoxazole, amphotericin B, ketoconazole and itraconazole have been tried with varying success. Investigations on the immunology of disease and the role of proteases and lipases in the pathogenesis of infection is an important area of further research.

Immunological characterization of protective antigens prepared by alkaline treatment of whole cells and from the culture filtrate of Erysipelothrix rhusiopathiae

Culture filtrate and alkaline-extracted antigens from whole cells of an attenuated strain of Erysipelothrix rhusiopathiae (strain Koganei: serovar la) were fractionated with ammonium sulfate; both induced protective immunity in mice. Sephadex G-200 gel filtration revealed three protein fractions in the alkaline-extracted antigen and four protein fractions in the culture filtrate antigen. A fraction in the alkaline extract (NaOH P-2) and in the culture filtrate (CF P-2) induced protection in mice against challenge with a different serovar strain (strain Agata: serovar 5). Anti-NaOH P-2 and anti-CF P-2 mouse sera were protective against different serovars. Glycoprotein fraction derived from CF P-2 antigen by affinity chromatography with Con A-Sepharose 4B did not show protective activity. Western blotting between the antisera (anti-NaOH P-2, Anti-CF P-2 and anti-Koganei strain) and the antigens (NaOH P-2, and sonicated antigens of Agata, Fujisawa and Koganei strains) showed strong recognition of the same bands at 62, 42 and 41 kDa.

Antibody Levels to Whole Culture Filtrate Antigens and to Purified P32 during Treatment of Smear-Positive Tuberculosis

Antimycobacterial IgG levels were measured repeatedly during treatment in 12 patients with moderate or severe pulmonary tuberculous disease using a dot immunobinding assay. We used reflectance densitometry equipment to quantify the immunoperoxidase staining and a Mycobacterium bovis BCG culture filtrate and the purified P32 protein as antigens. Antibody response against whole culture filtrate and P32 antigen increased after a three-month period of treatment. After this antibiotherapy was completed, the estimated amount of antibodies directed against the P32 decreased while those against the whole culture filtrate remained at the same level. A serologic test using P32 as the antigen seems to allow a better discrimination between active and healed tuberculosis.

Production of species-specific murine monoclonal antibodies against Cryptococcus neoformans which recognize a noncapsular exoantigen

Three monoclonal antibodies (MAbs), designated 7C5, 7C9, and 5G8, against a cytoplasmic antigen of Cryptococcus neoformans were produced. MAbs 7C5 and 7C9 recognize culture filtrate antigen (exoantigen) of both encapsulated and nonencapsulated isolates of this pathogen, which suggests that they do not recognize capsular polysaccharide material. This is supported by immunofluorescence data which show reactivity of all 3 MAbs to cytoplasm and cell membranes only. MAb 7C9 also recognized C. neoformans var. gattii antigens but no other fungal pathogens tested in an enzyme-linked immunosorbent assay, while 7C5 and 5G8 recognized antigens of the cross-reactive pathogen Trichosporon beigelii but did not recognize either C. neoformans var. gattii isolates or any other fungal antigens. By Western blot (immunoblot), 7C9 detected antigen at 110 to 120, 65 to 70, 45 to 50, and 36 to 38 kDa; in addition to the latter band, the other two MAbs recognized a band at approximately 30 kDa. All three MAbs were of the immunoglobulin G1 subclass. The two MAbs which are capable of reacting with noncapsular culture supernatant antigen have possible uses in serodiagnosis, particularly in AIDS patients infected with C. neoformans, since in this group the present latex agglutination test has some limitations.

Preparation and purification of monoclonal antibodies for Aspergillus fumigatus.

Monoclonal antibodies were raised against Aspergillus fumigatus, a fungus which is an etiological agent of aspergillosis. Male SPF BALB/c mice were immunized subcutaneously with A. fumigatus culture-filtrate or mycelial antigen. Their splenocytes were hybridized with mouse myeloma cells, P3 X 63 Ag 8.653 The hybridoma supernatants were screened by enzyme-linked immunosorbent assay (ELISA), using A. fumigatus culture-filtrate antigen. Of the 123 hybridoma clones, 27 cell lines secreting antibodies positive for A. fumigatus were obtained. Two of these cell lines were further grown to larger volumes. The monoclonal antibodies obtained were immunoglobulin-M (IgM) antibodies, which were then purified by ammonium sulphate precipitation and Mono Q anion exchange chromatography. The purified monoclonal antibodies showed reactivity against the protein constituent of the culture-filtrate antigen.

5 Concanavalin a nonbinding aspergillus fumigatus antigen: A major immunogen in allergic broncho-pulmonary aspergillosis

Humoral immune responses in allergic bronchopulmonary aspergillosis (ABPA) have been well studied. However, reports of cell-mediated immune responses in ABPA are conflicting and not well documented, perhaps because well-characterized antigens are not available. In the present study, we assessed the role of peripheral blood mononuclear cells (PBMCs) from patients with ABPA in their ability to respond to both crude and semipurified Aspergillus fumigatus antigens by in vitro proliferation and immunoglobulin E synthesis. Eight patients with ABPA, eight patients with immediate wheal and flare skin reactivity to A. fumigatus, and ten healthy control subjects with nonreactive skin tests were included in this study. Four A. fumigatus antigens were tested in vitro. Antigens included culture filtrate, mycelial extract, concanavalin A-nonbinding, and concanavalin A-binding antigens. There was a wide range of response to each antigen by each group of subjects. However, PBMCs from patients with ABPA showed greater response to the antigens than did those from the healthy control subjects when evaluated by lymphoproliferation (tritiated thymidine uptake) and immunoglobulin E synthesis (isotype-specific enzyme-linked immunosorbent assay). The concanavalin A-nonbinding antigen fraction had the ability to specifically stimulate proliferation of PBMCs from seven of eight patients with ABPA and from four of eight skin-reactive subjects; none of the healthy control subjects responded. Significantly high levels of immunoglobulin E were detected in unstimulated PBMC cultures from five patients with ABPA when compared with those from healthy control subjects. These results indicate that concanavalin A-nonbinding A. fumigatus antigens may be significant in the cellular immune response of ABPA; such results are similar to those from previous humoral studies of the disease.

Immunodiffusion test for serodiagnosing subcutaneous zygomycosis

Culture filtrate antigens of Basidiobolus ranarum and Conidiobolus coronatus were analyzed by immunodiffusion (ID) with homologous rabbit antisera. B. ranarum and C. coronatus were each found to have five specific antigens. Results of tests with heterologous antisera indicated that all of the species shared at least one antigen. ID tests incorporating the specific precipitin bands as references were developed for detection of basidiobolomycosis and conidiobolomycosis. These tests were performed with sera from humans and horses with proven basidiobolomycosis and conidiobolomycosis as well as with control sera from humans and animals with and without heterologous mycotic and oomycotic infections. Only sera from cases of basidiobolomycosis and conidiobolomycosis produced lines of identity with the reference precipitates of B. ranarum and C. coronatus, respectively. The ID tests were found to be completely sensitive and specific for determining the etiology of zygomycosis caused by these two species. In addition they appeared useful for monitoring resolution of the infections.

Repertoires of antibodies to culture filtrate antigens in different mouse strains infected with Mycobacterium bovis BCG

Two susceptible (Bcgs) mouse strains, BALB/c and C57BL/6, were compared by Western blot (immunoblot) analysis for their immunoglobulin G response to 14-day-old BCG culture filtrate (CF) following intravenous infection with live Mycobacterium bovis BCG. The two strains demonstrated a completely different antibody repertoire. BALB/c antibodies were directed against a wide range of CF antigens between 20 and about 100 kilodaltons (kDa), with a preferential recognition of the 65-kDa heat shock protein and the 32-kDa fibronectin-binding protein. C57BL/6 sera, on the other hand, showed a much more restricted antibody pattern, almost exclusively directed against three antigens with estimated molecular sizes of 37, 38, and 40 kDa. Whereas the 37- and 38-kDa antigens were also recognized by BALB/c mice, the 40-kDa antigen was very intensely stained by C57BL/6 sera only. F1 mice had the restricted antibody pattern of C57BL/6 after one injection of BCG and had a hybrid BALB/c-C57BL/6 phenotype following a boost injection of BCG 2 months after the initial infection. Analysis of seven recombinant inbred strains derived from the BALB/c x C57BL/6 cross and of congenic mice differing in major histocompatibility complex-coding chromosome 17 fragments suggests that a gene in the K-IA region of the H-2 locus is associated with the preferential recognition of certain CF antigens. Inoculation with the same dose of killed BCG failed to elicit an antibody response to these filtrate antigens.

Importance of Serotype-related Antigen in the Induction of Experimental Hypersensitivity Pneumonitis byTrichosporon cutaneumin Rabbits: Correlation between Granulomatous Alveolitis and Cellular and Humoral Immune Responses to Polysaccharide-rich Antigen

We evaluated the immunopathogenic properties of Trichosporon cutaneum, a major etiologic agent of Japanese summer-type hypersensitivity pneumonitis (HP). When the culture filtrate antigen of T. cutaneum was chromatographed on DEAE-cellulose, two peaks of polysaccharide, fractions A and B, were obtained. Fraction B was highly reactive to the specific IgG and IgA antibodies in the serum and bronchoalveolar lavage fluid samples obtained from sensitized rabbits, whereas fraction A was mainly reactive to the IgG antibodies. When the rabbits sensitized by intratracheal injection with the particulate antigen of T. cutaneum were challenged intratracheally by these antigenic fractions, fraction B induced granulomatous alveolitis, but fraction A induced alveolitis rather than granuloma. Lymphocytes of the mediastinal lymph nodes responded prominently to fraction B, but less to fraction A, as assessed by proliferative response. Each of the fractions B obtained from two strains of different serotypes (TIMM 1573, serotype I and TIMM 1318, serotype II) was reactive in enzyme-linked immunosorbent assay to the serum samples from the rabbits sensitized with homologous antigen, but far less to that from the rabbits sensitized with heterologous antigen, that is, the antigenic specificity of fraction B was related to the serotype of T. cutaneum. Fractionation by gel filtration on Sepharose CL-4B revealed that the molecular weight of the antigenic components in fraction B was larger than 1,000,000 and that the components consisted mainly of polysaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a cell population which amplifies the anticryptococcal delayed-type hypersensitivity response

Cell-mediated immunity to Cryptococcus neoformans can be detected by delayed-type hypersensitivity (DTH) to a culture filtrate antigen of C. neoformans. Recently, we have identified a population of cells in spleens of mice immunized with cryptococcal antigen that, when transferred to recipient mice at the time of immunization, amplifies the anticryptococcal DTH response. If the cell donor mice are treated with cyclosporin A during induction of the anticryptococcal DTH response, the amplifier cells are not induced, whereas the cells which transfer DTH (TDH cells) are induced. The purpose of this study was to characterize the amplifier cells with respect to their surface and functional properties and, in so doing, determine whether or not the amplifier cells are analogous to long-lived memory cells. We demonstrated that the amplifier cells were nylon-wool-nonadherent, antigen-specific, CD4 (L3T4+ Lyt-2-) T lymphocytes which appear in the spleens of mice 5 days postimmunization with cryptococcal culture filtrate antigen in complete Freund adjuvant. The amplifier T (Tamp) cells are not considered to be memory cells because they are relatively short-lived, being present 14 but not 18 days after the stimulating immunization. Moreover, the amplified anticryptococcal DTH response does not fulfill the criteria of the typical secondary immune (anamnestic) response in that the amplified response does not appear early relative to the appearance of the primary anticryptococcal DTH response, and it does not persist longer than the primary DTH response. We speculate that Tamp cells are not long-lived memory cells but rather act in a T-helper cell capacity to amplify the anticryptococcal DTH response.