Biomedical subjects

A Restrepo

Publications and source records attributed to A Restrepo.

At least 73 records · Page 4Linked to original sources

PubMed1992

Killing of Paracoccidioides brasiliensis conidia by pulmonary macrophages and the effect of cytokines.

The ability of conidia, the infectious form of the dimorphic fungus Paracoccidioides brasiliensis, to be killed in vitro by murine pulmonary macrophages was studied. Mice were immunized by intravenous injection of killed conidia, which resulted in cellular immunity demonstrated by delayed type hypersensitivity in vivo and macrophage migration inhibition factor production in vitro. Resident pulmonary macrophages from non-immune mice were able to significantly kill the conidia (28%). Such macrophages treated with supernatants (cytokines) from antigen-stimulated immune mononuclears had a markedly enhanced ability to kill conidia (73%). These results show that activated pulmonary macrophages are potent killers of conidia of P. brasiliensis and that immune mononuclears play a role in activation of macrophages. Activated macrophages may be important for pulmonary defense against the initial stages of infection with this fungus.

Animals↗

PubMed1991

Scanning electron microscopy of the conidia produced by the mycelial form of Paracoccidioides brasiliensis.

The conidia produced by the mycelial form of Paracoccidioides brasiliensis were examined by scanning electron microscopy for the first time. Several different conidial types were characterized. These included intercalary arthroconidia, several types of septate conidia that are formed from other conidia, pedunculate conidia, and terminal hyphal conidia. In addition, the ultrastructure of the supporting pedestal of the pedunculate conidium was found to be separated from the mother conidium by a septum in some instances, and at other times it was not.

Microscopy, Electron, Scanning↗

PubMed1991

Electron microscopic study of conidia produced by the mycelium of Paracoccidioides brasiliensis.

The ultrastructure of asexual spores (conidia) produced by the mycelial form of Paracoccidioides brasiliensis was studied for the first time with transmission electron microscopy, using thin sections of aldehyde-osmium-fixed and epoxy-resin-embedded samples. The various types of conidia observed in the sections correlated well with previous light-microscopic descriptions. These types were intercalary or apical conidia, depending on their location along the originating hyphae. As in previous studies they were characterized as arthroconidia, aleuriospores and sessile or pedunculate pyriform conidia. The sporogenous cells were clearly distinguished from hyphal cells by the thickness and appearance of their cell walls. Copious fibrillar material (glycocalyx) detected at the cell surface was stained with ruthenium red during the fixation process. Typical subcellular organelles (nucleus, nucleolus, mitochondria, ribosomes, etc) were found in most of the sections. It was concluded that the spores produced by the mycelial phase of P. brasiliensis possess all attributes of viable and physiologically competent eukaryotic cells.

Humans↗

PubMed1990

Virulence of Paracoccidiodes brasiliensis: the influence of in vitro passage and storage.

Stability of virulence in P. brasiliensis isolates was studied with respect to the in vitro culture history and methods used for storage. Virulence in yeast-form P. brasiliensis isolates was tested in a chronic pulmonary murine model of paracoccidiodomycosis where progression of disease was quantitated in terms of colony forming units recoverable from lungs. Four isolates of P. brasiliensis, including recently isolated form patients or experimental animals, caused chronic progressive disease. Two isolates with a history of subculturing showed attenuation by causing resolving but chronic disease. An attenuated isolate became avirulent subsequent to 15 more years of subculturing. These findings suggest that virulence of P. brasiliensis can be attenuated or lost subsequent to cycles of subculturing over long periods. Our data suggest that the use of fresh P. brasiliensis isolates may be needed to provide reproducible virulence for experimental systems.

Animals↗

PubMed1990

Treatment of paracoccidioidomycosis with itraconazole.

Forty-seven patients with active paracoccidioidomycosis were treated with itraconazole. The majority of the patients (45) had the chronic adult form of the disease while the remaining exhibited the juvenile form. All of the patients received itraconazole, 100 mg day-1, with the exception of two for whom the clinical condition necessitated a larger dose. The mean duration of therapy was 6 months, with a range of 3-24 months. Clinically, the skin and mucous membrane lesions as well as abdominal pain, auscultatory alterations and dysphonia improved markedly during treatment with a concomitant increase in weight. However, other symptoms (cough, expectoration and dyspnoea) persisted as sequelae in some cases. As expected adrenal insufficiency also persisted. The radiological lesions showed a gradual decrease of both scattered and confluent infiltrates, present in 67% of patients before, and in 13% at the end of treatment. On the other hand, fibrosis became more evident at the termination of therapy, increasing from 62% of patients at the beginning of therapy to 81% at the end. The mycological tests (direct examination and cultures) became negative during the first month of treatment in 42 patients (87%). A decline in specific antibody titres was observed in 72% of patients by the end of treatment. Evaluation of therapy by means of a scoring system indicated complete resolution of the disease in one patient (2%), marked improvement in 42 (89%) and minor improvement in four (8.5%); none of the patients showed a deterioration of their disease during therapy. Of fifteen patients who were followed up for 12 months post-therapy none showed clinical relapse during this period. The results of this study confirm the efficacy of itraconazole in the treatment of paracoccidioidomycosis.

Acute Disease↗

PubMed1989

Histopathology of chromoblastomycosis.

A study aimed at determining the histopathologic appearance of chromoblastomycotic lesions was undertaken. Biopsies from 26 patients with the disease were examined. It was found that 23 cases (88.46%) exhibited the organized mixed mycotic granuloma - OMMA -, a granuloma modified by the presence polymorphonuclear neutrophils - PMN's. An equal proportion of cases exhibited pseudoepitheliomatous hyperplasia, with the epithelium playing an important role in the transepidermic elimination of the fungus. Healing of the lesions took place by fibrosis which was observed in 21 (80.77%) patients; this type of reaction was more common in the deeper areas of the dermis.

Biopsy↗

PubMed1989

Intracellular multiplication of Paracoccidioides brasiliensis in macrophages: killing and restriction of multiplication by activated macrophages.

The effect of coculturing yeast-form Paracoccidioides brasiliensis with murine cells was studied. Coculture of resident peritoneal or pulmonary macrophages with P. brasiliensis for 72 h dramatically enhanced fungal multiplication 19.3 +/- 2.4- and 4.7 +/- 0.8-fold, respectively, compared with cocultures with lymph node cells or complete tissue culture medium alone. Support of P. brasiliensis multiplication by resident peritoneal macrophages was macrophage dose dependent. Lysates of macrophages, supernatants from macrophage cultures, or McVeigh-Morton broth, like complete tissue culture medium, did not support multiplication of P. brasiliensis in 72-h cultures. Time course microscopic studies of cocultures in slide wells showed that macrophages ingested P. brasiliensis cells and that the ingested cells multiplied intracellularly. In sharp contrast to resident macrophages, lymphokine-activated peritoneal and pulmonary macrophages not only prevented multiplication but reduced inoculum CFU by 96 and 100%, respectively, in 72 h. Microscopic studies confirmed killing and digestion of P. brasiliensis ingested by activated macrophages in 48 h. These findings indicate that resident macrophages are permissive for intracellular multiplication of P. brasiliensis and that this could be a factor in pathogenicity. By contrast, activated macrophages are fungicidal for P. brasiliensis.

Animals↗

PubMed1989

Virulence of a variant of Paracoccidioides brasiliensis that exists in the yeast form at room temperature.

Paracoccidioides brasiliensis (ATCC 60885) produces conidia that, when plated in enriched media and incubated at 21-25 degrees C, give rise to a yeast-form variant (YRT) (ATCC 46678). The virulence of this variant for BALB/c mice has been compared with that of the yeast and conidia produced by the parent isolate. Adult female mice were inoculated intravenously with 10(7) viable cells of YRT or yeast, or 5 x 10(5) viable conidia. Mice were weighed weekly, and three mice were sacrificed at 1, 2, 3, 4 and 8 weeks post-inoculation and their organs weighed and prepared for determination of viable counts (colony forming units; c.f.u.). Animals infected with yeast cells or with conidia exhibited little weight loss in comparison with that seen in the YRT-infected mice which began to lose weight 1 week post-challenge. Organ weights increased in the YRT group, while the changes in the remaining two groups were minor. The c.f.u. from these organs also increased in the YRT-infected mice, whereas the infection in the yeast-and conidia-infected mice was self-limiting. A proportion (16.6%) of the YRT-infected animals died during the observation period. Conversely, no deaths occurred in the remaining groups. These results indicate that the YRT variant possesses increased virulence.

Animals↗

PubMed1988

In vivo and in vitro activation of pulmonary macrophages by IFN-gamma for enhanced killing of Paracoccidioides brasiliensis or Blastomyces dermatitidis.

The fungicidal capacity of murine pulmonary macrophages (PuM) activated in vitro with IFN or lymphokines or in vivo with IFN was studied. PuM treated overnight with IFN (1000 U/ml), Con A-stimulated spleen cell culture supernatants, or lymph node cells plus Con A significantly killed yeast cells of the Gar w isolate of Paracoccidioides brasiliensis 45.5 +/- 2.1%, 72.0 +/- 4.2%, and 51.5 +/- 0.7% respectively. Two other isolates of P. brasiliensis (Ru and LA) were also killed (45 and 34%) by PuM activated by lymph node cells plus Con A. Control PuM had lesser but significant capacity for killing of P. brasiliensis isolates, ranging from 15 to 22%. Killing of P. brasiliensis by PuM activated by Con A-stimulated spleen cell culture supernatants could not be significantly inhibited by superoxide dismutase, catalase, or azide. When mice were treated in vivo with 4 X 10(5) IFN U i.p. and PuM isolated 24 h later, the PuM had significantly enhanced ability to kill P. brasiliensis (47.0 +/- 6.3%) compared with PuM from control mice (25.0 +/- 4.2%). PuM thus activated also showed enhanced killing (43%) of a second isolate compared with control PuM (22%). PuM from IFN-treated mice were able to significantly kill Blastomyces dermatitidis (37.5 +/- 0.7%) compared with control PuM (4.5 +/- 6.3%). These results show that PuM can be activated in vitro and in vivo by IFN for enhanced fungicidal activity against two pulmonary fungal pathogens and suggests that immunologic production of IFN could be an important factor in host defenses against these diseases.

Animals↗

PubMed1988

Morphological study of a variant of Paracoccidioides brasiliensis that exists in the yeast form at room temperature.

Incubation of Paracoccidioides brasiliensis conidia at 20-25 degrees C (RT) results in two types of colonies, mycelial (M) and yeast (YRT). A study of the latter colonies was undertaken. Conidia were plated in complex (BHI) and chemically-defined media (CDCM), with and without fetal calf serum (FCS). Incubation was carried out at 21 degrees C for 4 weeks. The mean number of YRT colonies was approximately 18.5% on BHI agar. Selected YRT colonies were transferred to liquid CDCM, incubated 7 days at 21 degrees C and transferred to solid media; YRT appearance was preserved in 95% of the colonies if media were FCS-supplemented; otherwise, most colonies reverted to the M form. When FCS was replaced by bovine albumin or by alpha-globulin, 63% and 68% respectively of the colonies obtained after plating YRT cells, became mycelial. Comparative morphologic studies of both YRT and yeasts grown at 37 degrees C suggested that there were no major differences between these two types of yeast cells when size and budding were taken into consideration. The results indicate that in this particular variant, dimorphism is not exclusively temperature-dependent.

Culture Media↗

PubMed1988

Treatment of chromoblastomycosis with itraconazole.

The results of long-term itraconazole therapy in 10 patients with active chromoblastomycosis due to F. pedrosoi were reported. Therapy consisted of 100 or 200 mg/day of itraconazole, the length of therapy depending on the patient's response (12 to 24 months). This new triazole proved effective in reducing the number, size, and severity of the lesions in nine of the patients. Those patients with minor involvement profited more from therapy and were cured; patients with moderate involvement achieved either minor or major improvement. In most cases, signs and symptoms began to improve after 6 months of therapy. Mycological tests (in which tissue samples were treated with potassium hydroxide and cultured) became negative in six patients, but the fungus was eradicated in only three patients. Itraconazole produced no side effects. In spite of the need for long-term therapy, this new azole derivative effectively controls the disease.

Antifungal Agents↗

PubMed1988

Inhibition by estrogens of conidium-to-yeast conversion in the fungus Paracoccidioides brasiliensis.

Conidia produced by Paracoccidioides brasiliensis are inhibited by mammalian estrogens in their in vitro conversion into yeast-form cells. This was demonstrated with four different isolates. In these experiments, conversion was reduced to 10.7 and 34.4% of the control values by 17-beta-estradiol at 10(-6) and 10(-8) M, respectively. At the same concentrations, the synthetic estrogen diethylstilbestrol was slightly less inhibitory. In contrast, other sex hormones and analogs, i.e., testosterone, 17-alpha-estradiol, tamoxifen, and hydroxytamoxifen, had no effect on conidium-to-yeast conversion. Previous studies have shown that estrogens similarly inhibit mycelium-to-yeast-form transition in P. brasiliensis. Conidia, and not mycelial fragments, are believed to be the natural infectious propagules. These findings with conidia support the hypothesis that estrogens, affecting the initial host-parasite interactions by suppressing conversion to the parasitic form of the organism, are, at least in part, responsible for the greater resistance of females to paracoccidioidomycosis.

Estrogens↗

PubMed1988

Growth and production of iron chelants by Paracoccidioides brasiliensis mycelial and yeast forms.

The mycelial and yeast forms of the fungus Paracoccidioides brasiliensis were cultured in a chemically defined liquid medium with different iron concentrations and the growth measured spectrophotometrically. The iron binding capacity of culture supernatants was measured by a colorimetric assay. Both the mycelial and yeast forms were able to grow in media containing trace amounts of iron (0.02 mgl-1) but when the iron chelant 1.10 phenantroline was added there was a delay in the initiation of growth of the yeast and almost total inhibition of the mycelium. When iron excess was added to media containing phenantroline, this inhibitory effect was reversed, partially for the mycelial, and completely for the yeast form. For both mycelial and yeast forms, the iron binding capacity of the culture supernatants was greater in media with low iron concentrations.

Culture Media↗

PubMed1987

Experimental murine paracoccidiodomycosis induced by the inhalation of conidia.

Adult BALB/c mice of both sexes were infected intranasally with 10(6) viable P. brasiliensis conidia. Animals were sacrificed at intervals up to 6 months and studied by histopathology and organ cultures. At the time of challenge lung sections showed that instilled conidia had reached the alveoli; at 12 h such conidia were transforming into yeast cells, with multiple buds appearing by 18 h. Initially, the cellular infiltrate was composed of polymorphonuclear leukocytes; 6 days later, lymphocytes, plasmocytes and macrophages predominated. Multinucleated giant cells appeared only after 6 weeks. The rate of pulmonary infection as determined by organ culture was high (82 of the 83 mice studied). The experimental infection was progressive as indicated by increasing numbers of viable fungi with time. The results of this study demonstrate that the conidia produced by P. brasiliensis mycelial form are infectious, producing active disease in healthy animals.

Animals↗

PubMed1987

Effect of murine polymorphonuclear leukocytes on the yeast form of Paracoccidioides brasiliensis [corrected].

The fungicidal activity of murine polymorphonuclear neutrophils from the peripheral blood or elicited intraperitoneally with thioglycollate or with antigen in Paracoccidioides brasiliensis-sensitized [corrected] or nonsensitized mice was studied. Although peripheral blood, thioglycollate-elicited, and antigen-elicited neutrophils from normal mice or thioglycollate-elicited neutrophils from P. brasiliensis-sensitized [corrected] mice killed Candida albicans (57% to 84%), they failed to significantly reduce inoculum colony forming units of P. brasiliensis [corrected] (0% to 13%). In contrast, antigen-elicited neutrophils from sensitized mice reduced colony forming units of P. brasiliensis [corrected] by 40%, and exhibited significantly enhanced candidacidal activity compared to thioglycollate-elicited neutrophils from normal or sensitized mice but not peripheral blood neutrophils from normal mice. Fresh serum, but not specific antibody, was required for optimal killing of P. brasiliensis [corrected], presumably representing an essential role for complement. Killing of P. brasiliensis [corrected] by antigen-elicited neutrophils from sensitized mice correlated with their ability to produce an enhanced oxidative burst, as measured by luminol-assisted chemiluminescence, when interacting with killed P. brasiliensis [corrected] cells. These results indicate that in P. brasiliensis-sensitized [corrected] hosts an inflammatory reaction to P. brasiliensis [corrected] results in activation of neutrophils for significant killing of the pathogen.

Animals↗

PubMed1987

Nuclear staining of Paracoccidioides brasiliensis conidia.

More than 80% of the conidia produced by two different isolates of Paracoccidioides brasiliensis, were found to be uninucleate; however, when they were incubated at 37 degrees C and began to transform into yeast cells, they became bi- or multi-nucleated, so that when most of the conidia had already transformed into yeast cells (72-96 h), there were at least four or five nuclei per cell in approximately 80% of the conidia examined.

Cell Nucleus↗

PubMed1986

Water as a substrate for the development of Paracoccidiodes brasiliensis mycelial form.

Two isolates of P. brasiliensis in the mycelial form were studied for their capacity to survive and grow in sterile distilled water (SDW). Inoculum for the experiments consisted of a spectrophotometrically-standardized suspension of washed and homogenized mycelial fragments; these had been obtained from 2-week old cultures grown in a synthetic medium (SM). Series of tubes with SDW and SM were incubated with the above suspension and kept stationary for 6 months at either 4 degrees C or room temperature (RT). Growth was measured by dry weight (DW) and turbidity (OD) determinations; additionally, CFU and ultrastructural appearance by transmission electron microscope (TEM) were assessed for one of the isolates. In general, cultures in SM at RT, grew exponentially after 2 weeks, becoming stationary for 7 weeks and then, declining abruptly. In SDW, fungal development was slow for 5 months when an increase in mass was recorded. When incubated at 4 degrees C, both SDW and SM cultures required longer time to develop but mass also increased. Morphologically, mycelial elements in SDW at RT exhibited increased lipid vacuoles and glycogen deposits but were otherwise normal up to 6 weeks when they presented the inter-hyphae-hyphae phenomenum. In SDW P. brasiliensis appears to utilize debris from its degenerated fungal partners to continue growing.

Colony-Forming Units Assay↗

PubMed1986

Pathogenesis of paracoccidioidomycosis: a histopathological study of the experimental murine infection.

The pathogenesis of primary pulmonary P. brasiliensis infection, the systemic dissemination which followed, and the histopathology of the main organs involved was studied in a murine model of chronic paracoccidioidomycosis. Adult male BALB/C mice, were challenged intranasally with 26 X 10(-6) viable P. brasiliensis yeast cells. We inoculated 86 animals which were sacrificed from 0 h to 20 weeks. As controls, 11 mice were instilled with saline solution, and 48 with 26 X 10(-6) heat-killed. P. brasiliensis yeast cells. None of the animals receiving saline, exhibited pathologic alterations; 11.6% of those inoculated with the heat-killed cells, revealed mild, transitory acino-pulmonary neutrophilic infiltrates. The animals infected with viable cells, developed a systemic process affecting mainly the lungs (46.5%), liver (18.6%), lymph-nodes (18.6%), and spleen (3.5%). In this group of animals, lung lesions were detected regularly at all time periods from 3 h to 20 weeks. A multiple bronchopneumonic process was initially observed at 6 h, reached its maximum intensity around the third day, subsided thereafter but did not disappear and reactivated after the fifth week to become stationary until the end of experiments. Dissemination to other organs occurred early, and apparently by the hematogenous route. Initially the inflammatory cell infiltrate was mainly neutrophilic. With time, these cells were gradually replaced by lymphocytes, histiocytes and plasmocytes. Granuloma configuration of the cell infiltrate was distinctly seen around the fifth week, with multinucleated giant cells appearing at the ninth week. Hiliary lymph-node involvement was rare (7%) and primary lung lesions, as seen in tuberculosis and histoplasmosis, were not observed.

Animals↗