Detection of antibody responses and delayed dermal hypersensitivity with Blastomyces dermatitidis yeast and mycelial lysate antigens

Yeast cell lysate and mycelial lysate antigens prepared from one strain (T-58) of Blastomyces dermatitidis were evaluated with respect to the detection of antibodies and delayed dermal hypersensitivity. Comparable ELISA sensitivity values were evidenced with the two antigens when assayed against serum specimens from dogs with blastomycosis, sera from non-infected dogs residing in endemic and nonendemic areas for blastomycosis and sera from rabbits that were hyperimmunized with B. dermatitidis antigens. Specificity determinations with anti -Histoplasma capsulatum rabbit sera indicated that both reagents exhibited only minimal cross-reactivity; the mycelial antigen was slightly more specific than the yeast phase reagent. Similar sensitivity and specificity results were experienced when the two antigens were used to detect delayed dermal hypersensitivity in guinea pigs previously sensitized with B. dermatitidis or H. capsulatum.     

Secondary Intracerebral Blastomycosis with Giant Yeast Forms

Secondary central nervous system (CNS) blastomycosis is an unusual manifestation of blastomycosis. We report a case of recurrent intracerebral blastomycosis that presented histopathologically with giant yeast-like cells and multinucleation that mimicked Coccidioides immitis. The yeast forms of Blastomyces dermatitidis usually range in size from 8 to 20 μm in diameter. Large or giant yeast forms (20–40 μm) are rare. The four cases previously reported in the literature involving giant yeast cell forms of B. dermatitidis are reviewed here. Intracerebral blastomycosis should be suspected in patients with signs and symptoms of CNS lesions and histories of primary blastomycosis, or treatment with corticosteroids, or comprised immune systems. The diagnosis should be confirmed by culture which presents typical biphasic microbiologic features.     

Dialysable leukocyte extracts modify the course of experimental paracoccidioidomycosis in the Syrian hamster

The effect of dialysable leukocyte extracts (DLE) obtained from hamsters immunized withParacoccidioides brasiliensis (immune DLE) and from non-immunized hamsters (non-immune DLE) was studied in hamsters inoculated withP. brasiliensis by the intratesticular route. Treatment with immune or non-immune DLE was started during the third week of infection and was repeated at 7, 11, 15 and 19 weeks. A group of untreated infected animals was used as control. Animals were submitted to the delayed hypersensitivity skin test toP. brasiliensis antigen (PbAg) in vivo and assayed in vitro by the macrophage migration inhibition test in the presence of Phytohemagglutinin (PHA) and PbAg and by immunodiffusion for specific antibody. The animals were sacrificed at 4, 8, 12, 16 and 20 weeks. The morphology and extension of the lesions were studied at the inoculation site, and in lymph nodes, lungs, liver, spleen and kidneys. In contrast to the controls, animals treated with both DLEs maintained a positive cell-mediated immune response throughout the experiment and developed less extensive infection with a significantly lower number of fungi in the lesions. The results suggest that immune and non-immune DLE preparations modified the evolution of experimental paracoccidioidomycosis with equal efficiency. This similarity may be explained by the immunoregulatory activities of both extracts.     

Experimental paracoccidioidomycosis in the syrian hamster: Morphology,ultrastructure and correlation of lesions with presence of specific antigens and serum levels of antibodies

Male hamsters (134) received intratesticular injection of a live cerebriform culture of Paracoccidioides brasiliensis and were sacrificed from 6 hours up to 123 days onwards. Tissues from testis, lymph nodes, lungs, liver, spleen, kidneys and intestines were examined microscopically; presence of specific antigens was saught in lesions of testis, regional lymph nodes and liver by indirect immunofluorescence (IF); inoculation site lesions were studied electron microscopically and circulating specific antibodies measured by complement fixation and IF tests.Up to 24 hours inoculation site lesions showed fungi surrounded by PMNs; 48 hours latter macrophages accumulated forming loose nodules; epithelioid granulomata appeared after 5 days. Fungi, scarce in early lesions, increased in numbers up to the time when epithelioid granulomata dominated the picture; in young granulomata fungi were abundant and small; older granulomata contained rare, vacuolated fungi. Ultrastructurally the space between fungi and host-cells was larger around reproducing forms decreasing in size as the parasites grew larger and being a virtual slit around old degenerated fungi. Immunofluorescence studies revealed that fungal walls were brightly fluoerescent; in early lesions macrophages surrounding fungi or free in the intersticium contained fluorescent antigenic material in the cytoplasm; similar macrophages were observed in draining lymph nodes as early as 18 hours after inoculation, and latter, in macrophage nodules and Kupffer cells in the liver; epithelioid and giant cells appear to block diffusion of antigens, since in epithelioid granulomata fluorescence was limited to fungal walls.Disseminated paracoccidioidomycosis occurred in 100% of animals after day 5 of infection. Besides specific lesions (containing fungi), antigens were identified by immunofluorescence in non specific lesions in the liver (diffuse or nodular Kupffer cell hyperplasia) and in the lymph nodes (histiocytic hyperplasia). Serum antibodies appeared in low titers, up to day 20, increasing onwards. From day 70 on, titers decreased and lesions changed from confluent epithelioid to loose granulomata infiltrated by PMNs; fungi that before were large and quiescent now were small and in active reproduction. Secondary amyloidosis was present in 85% of the animals.In the hamster, Paracoccidioidomycosis develops as a chronic progressive disease and the lesions are related both to fungi and its antigens.     

Experimental paracoccidioidomycosis in the Syrian hamster

Male hamsters (105) received intratesticular injection of suspension of a live yeast phase culture ofParacoccidioides brasiliensis and were sacrificed weekly during 20 weeks. Humoral immunity was studied by the agar-gel immunodiffusion (ID) and indirect immunofluorescence (IF) tests. Cell-mediated immunity was determined by the macrophage migration inhibition test in the presence of phytohemagglutinin (PHA) andParacoccidioides brasiliensis soluble antigen (PbAg). The morphology of the lesions was studied in the inoculation site, lymph nodes, lung, liver, spleen and kidneys.Disseminated paracoccidioidomycosis was observed in 100% of the animals after the first week. The lesions were initially made up of fungi surrounded by polymorphonuclear neutrophils and macrophages. Up to the 10th week the majority of the lesions appeared as compact confluent ephitelioid granulomas containing rare large fungi, some showing signs of degeneration. At this time, the specific antibody titers and the cellular immune response to PHA and PbAg were highest.From the 11th week on the granulomas became less compact, edematous with the epithelioid cells loosely arranged. This change was accompanied by an increase in the number of fungi showing reproductive activity and was associated with renal amyloidosis and progressive decline of cellular immune response both to PHA and PbAg. Contrariwise the titers of circulating antibodies were maintained.In the present model, disseminated paracoccidioidomycosis of the hamster was associated with depression of cellular immunity, change in the pattern of the granuloma, intense fungi proliferation and amyloidosis.     

Application of an enzyme‐labeled antigen method for visualizing plasma cells producing antibodies against Strep A,a carbohydrate antigen of Streptococcus pyogenes,in recurrent tonsillitis

Streptococcus pyogenes is the main causative pathogen of recurrent tonsillitis. Histologically, lesions of recurrent tonsillitis contain numerous plasma cells. Strep A is an antigenic carbohydrate molecule on the cell wall of S. pyogenes. As expected, plasma cells in subjects with recurrent tonsillitis secrete antibodies against Strep A. The enzyme‐labeled antigen method is a novel histochemical technique that visualizes specific antibody‐producing cells in tissue sections by employing a biotin‐labeled antigen as a probe. The purpose of the present study was to visualize plasma cells producing antibodies reactive with Strep A in recurrent tonsillitis. Firstly, the lymph nodes of rats immunized with boiled S. pyogenes were paraformaldehyde‐fixed and specific plasma cells localized in frozen sections with biotinylated Strep A. Secondly, an enzyme‐labeled antigen method was used on human tonsil surgically removed from 12 patients with recurrent tonsillitis. S. pyogenes genomes were PCR‐detected in all 12 specimens. The emm genotypes belonged to emm12 in nine specimens and emm1 in three. Plasma cells producing anti‐Strep A antibodies were demonstrated in prefixed frozen sections of rat lymph nodes, 8/12 human specimens from patients with recurrent tonsillitis but not in two control tonsils. In human tonsils, Strep A‐reactive plasma cells were observed within the reticular squamous mucosa and just below the mucosa, and the specific antibodies belonged to either IgA or IgG classes. Our technique is effective in visualizing immunocytes producing specific antibodies against the bacterial carbohydrate antigen, and is thus a novel histochemical tool for analyzing immune reactions in infectious disorders.     

Impact of anti-sandfly saliva antibodies on biological aspects of Phlebotomus papatasi (Diptera: Psychodidae), vector of cutaneous leishmaniasis

Sandflies are the main vectors of Leishmania parasites in tropical and subtropical areas. The immunization of vertebrate hosts with vector components through repeated bites may offer an alternative method for sandfly control. Aliquots of female Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae) were weekly blood fed on 12 individual hamsters throughout 18 successive weeks. Significant biological and biochemical changes resulting from antibodies developed by immunized host sera against repeated biting were observed in sandfly females. Blood feeding and fertility rates of females significantly gradually declined to the end of the study period. No appreciable difference was observed in mortality rates among flies repeatedly fed on individual hamsters throughout weeks 9 and 18, compared to flies fed on naïve hamsters. Total salivary gland proteins of female sandflies were compared to proteins in sera of sensitized hamsters. SDS-page revealed bands common to both flies and hosts, indicating the development of anti-saliva antibodies in hamster sera. The importance of anti-sandfly saliva antibodies as a potential tool for vector control leading to the interruption of leishmaniasis is discussed.     

Live Blastomyces dermatitidis yeast-induced responses of immune and nonimmune human mononuclear cells

Peripheral blood mononuclear cells from humans with treated blastomycosis or from normal persons were cultured with live Blastomyces dermatitidis yeast. There was no inhibition of growth of the fungus in this suspension culture technique but morphologic and functional differences of the human cells were great between the two groups. Lymphocyte stimulation by live Blastomyces yeast was found in the patient group but not in the normal donors. These events add to the observations that cellular immunity is expressed in blastomycosis.     

Characterization and Pathogenicity of Hemolysis Mutants of Mycoplasma pneumoniae

Hemolysis mutants were produced by treating Mycoplasma pneumoniae FH-P24 strain with N-methyl-N-nitro-nitrosoguanidine and were classified into three different groups. The first group of mutants, strains P24-L1, L2, and L11, showed wide and clear hemolytic zones. Their attachment ability to erythrocytes of various animals and to hamster lung cells were the same as those of the parent strain. The second group, strain P24-S1, showed non-hemolysis and non-hemadsorption, but retained the attachment ability to lung cells, although not to erythrocytes. The third group, strain P24-S11, was non-hemolytic, had completely lost the attaching ability, and did not proliferate in vivo. Strains in the first group produced significant microscopic pneumonic lesions in hamsters while strain P24-S1 produced milder lung lesions. Strain P24-S11 did not cause any lung lesions, and organisms were not recovered from the lungs of hamsters. The attachment of M. pneumoniae to respiratory epithelium as a cause of infection and the existence of a relationship between the hemolytic abilities of the organisms and histopathogenicity in the hamster lung tissue were further supported by the present data. It was also shown that the use of hemolysis mutants is useful for the elucidation of pathogenesis in mycoplasmal infections.     

Disruption of masking by hypothalamic lesions in Syrian hamsters

Negative masking of locomotor activity by light in nocturnal rodents is mediated by a non-image-forming irradiance-detection system in the retina. Structures receiving input from this system potentially contribute to the masking response. The suprachiasmatic nucleus (SCN) regulates locomotor activity and receives dense innervation from the irradiance-detection system via the retinohypothalamic tract, but its role in masking is unclear. We studied masking in adult Syrian hamsters (Mesocricetus auratus) with electrolytic lesions directed at the SCN. Hamsters were exposed to a 3.5:3.5 ultradian light/dark cycle and their wheel-running activity was monitored. Intact hamsters showed robust masking, expressing less than 20% of their activity in the light even though light and dark occurred equally during their active times. In contrast, hamsters with lesions showed, on average, as much activity in the light as in the dark. Tracing of retinal projections using cholera toxin subunit showed that the lesions damaged retinal projections to the SCN and to the adjacent subparaventricular zone. Retinal innervation outside the hypothalamus was not obviously affected by the lesions. Our results indicate that retinohypothalamic projections, and the targets of these projections, to the SCN and/or adjacent hypothalamic areas play an important role in masking.     

Specific Immunodiffusion Test for Blastomycosis

A specific immunodiffusion test for blastomycosis has been developed. The test permitted the detection of approximately 80% of 113 proven cases of blastomycosis. Two diagnostically important precipitins designated A and B were frequently recognized in patients with blastomycosis. Routine use of reference sera containing the A and B precipitins in immunodiffusion tests would permit the specific diagnosis of blastomycosis without the need for parallel tests with coccidioidin and histoplasmin.     

Experimental infection withBasidiobolus haptosporus

The authors tried to reproduce experimentally theBasidiobolus haptosporus infection. Culture forms of the fungus were inoculated in 26 adult hamsters, in two newborn hamsters and in two marmosets. Oral, intratesticular, intrahepatic, intraperitonial and subcutaneous inoculations were made. Bethametasone was given prior to inoculation in a group of animals.The lesions produced were only of the foreign body type and there was no development of the fungus in the animal tissues. The AA concluded that an experimental model for theB. haptosporus infection has not yet been found.     

Isoelectric focusing and ELISA evaluation of a <Emphasis Type="Italic">Blastomyces dermatitidis</Emphasis> human isolate

Blastomyces dermatitidis is a dimorphic fungal organism and the causative agent of blastomycosis. This organism is endemic east of the Mississippi river as is the fungal organism Histoplasma capsulatum. This study was performed to determine if sensitive and specific antigens from the B. dermatitidis yeast phase lysate (human isolate 592) could be separated using isoelectric focusing (IEF) to eliminate antigens that are cross-reactive with H. capsulatum. Indirect enzyme linked immunosorbent assays were performed to test for reactivity and cross-reactivity and indicate that certain fractions (4–6) were highly reactive. Fraction 16 exhibited a high degree of cross-reactivity with H. capsulatum. This study indicates that IEF may be a useful method for the separation of B. dermatitidis proteins.     

Immunochemical detection of DNA damage induction and repair at different cellular stages of spermatogenesis of the hamster after in vitro or in vivo exposure to ionizing radiation

An immunochemical method has been used to detect quantitatively DNA damage caused by ionizing radiation in germ cells. With this method, DNA strand breaks as well as lesions converted into breaks in alkaline medium are measured as a function of controlled partial unwinding of the DNA, a time-dependent process starting at each breakage site, followed by the determination of the relative amount of single-stranded regions by use of a single-strand specific monoclonal antibody. With this method the induction and repair of DNA damage in different cellular stages of spermatogenesis (spermatocytes, round and elongated spermatids) of the hamster were investigated. Germ cells were irradiated in vitro with ⁶⁰Co-γ-rays, at doses between 0 and 5 Gy. A linear dose-response relationship was observed. Spermatocytes and round spermatids had normal, fast repair of the lesions when compared with the repair of these sites in cultured V79 or CHO cells and human lymphocytes. The elongated spermatids, however, showed hardly any repair. Similar results were obtained after the in vivo γ-irradiation of hamsters with doses of 0, 4, and 8 Gy and subsequent isolation of germ cells. The damage was still detectable in the elongated spermatids at 24 h after exposure. The results of the experiments show substantial differences in repair capacity between different stages of germ cell development. Because DNA is the major target for mutation induction, this assay may be useful for assessment of the genetic risk of exposure of male germ cells to ionizing radiation, in relation to the stage of development.     

Isolation of an antigen fromBlastomyces dermatitidis that is specific for the diagnosis of blastomycosis

The A antigen ofBlastomyces dermatitidis has been isolated and purified by DEAE column chromatography. In the complement-fixation test, the antigen reacted with 10 of 16 sera from patients with proven cases of blastomycosis and was negative with known positive sera from 7 cases of histoplasmosis, 5 cases of coccidioidomycosis, 5 cases of candidiasis, and 5 cases of cryptococcosis. In the enzyme-immunoassay test, 25 of 27 sera from cases of blastomycosis were positive, but all heterologous and normal sera tested were negative. The antigen gave a positive skin test with guinea pigs sensitized with killed yeast-phase cells ofB. dermatitidis and negative skin tests with guinea pigs sensitized with killed yeast-phase cells ofHistoplasma capsulatum.     

Enfermedad de Jorge Lobo

Sumario Se presenta el segundo caso colombiano de Enfermedad de Lobo o Blastimicosis queloidiana, en un agricultor de 46 años de edad, procedente del departamento del Chocó. Presentaba lesión nodular de tipo queloidiano, exulcerada localizada en el dorso del pie derecho, de un año de evolución, sin compromiso del estado general, sin adenopatía satélite y sin compromiso pulmonar. Se hace la descripcíon de las lesiones histopatológicas producidas por el hongo y la descripción de la morfología del parásito en fresco y teñido por diversos métodos. Cultivos e inoculaciones a cobayos y ratones fueron negativos.Se hace énfasis en las diferencias clínicas, histológicas, micológicas y experimentales que existen entre la Paracoccidioidomicosis o Blastomicosis suramericana y la Enfermedad de Lobo y se concluye que la Enfermedad de Lobo o Blastomicosis queloidiana es una enfermedad autónoma y completamente diferente a la Blastomicosis suramericana y producida por un hongo diferente alP. brasiliensis.

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Summary The second known case of keloid Blastomycosis or Lobo's disease in Colombia is reported. The patient, a 46 year old male farmer from the State of Chocó (northwest region of Colombia), had been suffering for a year from a keloid-like ex-ulcerated, lobulated lesion located on the dorsal aspect of the right foot. Neither regional adenopathy nor lesions were found.The histopathological changes were studied by means of serial sections taken from the surgical specimen. The epidermis was atrophic, and had neither pseudo-epitheliomatous hyperplasia nor intra-epidermal abscesses. The dermis showed a marked keloid like fibrosis with abundant collagenous bands, there were many foreign body type granulomas without necrosis, with histiocytes and abundant giant cells which contained many parasites. In the H-E stain, the fungus cells were poorly stained, nevertheless they were readily recognized due to the refracting capsule and to the central slightly basophilic protoplasmic material. There were also many free parasites intermingled with the collagenous tissue. With the P.A.S. technique the fungi were intensely stained, showing a pink capsule and a dark red protoplasmic material. With the Grocott technique the fungus capsule, but not the protoplasmic mass, was very sharply stained; we consider this method the best to study the external morphology of the fungus.The fungus cells were very uniform in size (8–10µ), neither dwarf nor giant forms were seen; they were generally rounded but occasionaly pear-shaped (Fig. 6), and semilunar cells (Figs. 6, 7) were present. Chains of buds were commonly observed and multiple budding forms were also abundant (Figs. 3, 4, 8). The daughter cells were attached to the mother one by an easily visible bridge (Fig. 8).The differences from South American blastomycosis are discussed and a brief review of the literature concerning keloid blastomycosis is made.The conclusion is that keloid blastomycosis is an autonomous disease and not a clinical form of South American blastomycosis and the etiologic agent is different fromP. brasiliensis.

     

Preparation and characterization of monoclonal antibodies against a form of hamster liver cytochrome P-450 highly specific to aflatoxin B1

Monoclonal antibodies were prepared against a form of cytochrome P-450 (designated as cytochrome P-450-I) purified from 3-methylcholanthrene-treated hamster livers which is highly specific to aflatoxin B1. The cytochrome P-450-I was detected in ELISA and Western blots in liver microsomes from 3-methylcholanthrene-treated hamsters and also from non-treated and phenobarbital-treated hamsters in smaller amounts. However, none of the liver microsomes from 3-methylcholanthrene-treated rat, rabbit, guinea pig and Suncus murinus contained the cytochrome P-450-I. These results suggest that cytochrome P-450-I is specific to hamster and is induced mainly by 3-methylcholanthrene.     

Treatment of Blastomycosis

Blastomycosis, disease caused by the thermally dimorphic fungus Blastomyces dermatitidis, occurs predominantly in the Midwest, south central, and northeastern United States. Spores from the environment are inhaled into the lungs where they may cause subclinical infection, acute or chronic pneumonia, or disseminated disease. The Infectious Diseases Society of America has recently published updated guidelines on the management of blastomycosis. Antifungal therapies that have proven effective for blastomycosis include itraconazole and amphotericin B. The lipid formulations of amphotericin B are preferred owing to reduced nephrotoxicity, especially when prolonged intravenous therapy is necessary. The more recently approved triazole voriconazole may play a greater role in treating blastomycosis, especially central nervous system disease, as more clinical data become available.     

Development of Leishmania (Viannia) panamensis lesions and relationship of numbers of amastigotes to lesion area on antimony-treated and untreated hamsters

Young adult (60-70-g) male golden hamsters (Mesocricetus auratus) each were injected intradermally at the dorsal base of the tail with 15 x 10(6) promastigotes of Leishmania (Viannia) panamensis (MHOM/PA/83/WR539), and progression and regression of subsequent lesions were evaluated for up to 17 wk postinfection (PI) as to area, weight, and number of amastigotes within lesions in untreated hamsters and in hamsters treated with meglumine antimoniate (Glucantime). In untreated hamsters total area of lesion, weight, and numbers of amastigotes generally increased rapidly and concomitantly up to 3-4 wk PI. Amastigote numbers tended to decrease from 4 to 11 wk PI and subsequently the numbers of amastigotes within the lesions decreased rapidly, whereas relatively little change occurred in the area and weight of the lesions. Meglumine antimoniate treatment of cutaneous hamster lesions resulted in marked concomitant decrease in size of the lesions and numbers of amastigotes within the lesions examined 1 wk after treatment. Measurement of the area of cutaneous leishmanial lesions thus would appear to be a valid method of evaluating the efficacy of promising compounds against L. panamensis in hamsters when measurements are taken 3-5 wk after experimental infection and reflects the number of amastigotes present in the lesion.