Histological and ultrastructural study of the inflammation evoked by Paracoccidioides brasiliensis antigen in previously immunized mice

Bentonite particles uncoated and coated with soluble antigen of Paracoccidioides brasiliensis (Pb) were intravenously injected into mice with and without previous immunization with Pb antigen. The inflammatory reaction around the bentonite emboli in small lung vessels was quantitated and morphologically studied by light and electron (EM) microscopy, 2 to 8 days after challenge. In control nonimmunized animals, coated and uncoated bentonite particles caused mild and nonspecific inflammation made up by macrophages. By EM, they formed loosely aggregated clusters with cytoplasm containing few organelles and borders without interdigitation. In immunized mice injected with coated bentonite particles, the inflammatory area was significantly greater than that in nonimmunized animals in all periods of study with maximum difference at day 2. The inflammatory process at days 2 and 4 was characterized as mature granulomata, composed of macrophages with great number of organelles in the cytoplasm, large euchromatic nuclei and prominent nucleoli. Altogether these findings indicated a lesion with high metabolic activity, compatible with a granulomatous hypersensitivity reaction. At days 6 and 8, there was a change from mature to epithelioid granulomata, well demonstrated by EM which showed macrophages with characteristically interdigitated cytoplasmic borders. The results strengthen the importance of cellular immunity in the genesis of epithelioid granuloma in paracoccidioidomycosis and reinforce the usefullness of the present model in studies of the inflammatory cellular sequency and events in this mycosis.     

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 pulmonary paracoccidioidomycosis in mice: Morphology and correlation of lesions with humoral and cellular immune response

The present paper describes a murine model for pulmonary paracoccidioidomycosis injecting 6×10⁵ yeast forms ofParacoccidioides brasiliensis (Pb) by the direct intratracheal route. The sequential histopathology of lung and dissemination lesions together with humoral (immunodiffusion test) and cellular immune response (footpad test and macrophage inhibition factor assay — MIF assay) were investigated since the 1^st to the 360^th day after infection. All infected animal showed pulmonary Pbmycosis up to Day 30; onwards the lesions subsided being found only in one mouse at Day 360. Dissemination lesions were observed in paratracheal and cervical lymph nodes in 9 out of 68 infected animals. Histologically early lesions were rich in polymorphonuclear cells and evolved to a macrophage desquamative pneumonitis at Day 15 and to typical epithelioid granulomata from Day 30 up to Day 360. Specific precipitating antibodies were first detected 15 days after infection, peaked from Day 30 to 60 and were not observed at Day 360. Significant cell-mediated immunity to Pb was noted at Day 15 with the peak reaction at Day 60 and 90.The intratracheal route represents a highly effective way of infecting mouse with Pb. This experimental pulmonary Pbmycosis is a granulomatous inflammation which courses with specific humoral and cellular immune response. It may be a good tool for further investigation in the pathogenesis and natural history of the disease.     

"Labrador lung": an unusual mixed dust pneumoconiosis.

Forty-eight workers involved for various periods from 1962 in mining and milling iron-containing rock in the Carol Lake area of western Labrador were found in late 1974 and early 1975 to have pneumoconiosis. Their annual chest roentgenograms demonstrated irregular nodular densities, and several revealed mild hilar lymphadenopathy or pleural thickening. All 48 workers were asymptomatic and had normal pulmonary function; their average age was 38 years. At work they had been exposed to high levels of dust containing iron, silica and anthophyllite, a type of asbestos. Lung biopsy specimens from 13 of them showed widespread focal fibrosis, large amounts of hemosiderin and silica and many ferruginous bodies; one ferruginous body was identified as having a core of anthophyllite. A granulomatous reaction was seen in two biopsy specimens and typical silicotic nodules were found in two others. The relatively brief exposure, especially in ones case (11 months), and the short latent interval before the development of the pneumoconiosis are reasons for concern for the health of iron ore workers.     

Coccidioidomycosis and other endemic mycoses in Mexico

The endemic mycoses traditionally include coccidioidomycosis, histoplasmosis, blastomycosis and paracoccidioidomycosis. Although sporotrichosis and chromomycosis are technically not included among the endemic mycoses, they are frequently diagnosed in Mexico. Most systemic endemic mycoses are a consequence of inhaling the fungi, while subcutaneous mycoses are acquired through the inoculation of vegetable matter or soil containing the organism. Coccidioidomycosis is caused by Coccidioides spp., a dimorphic pathogenic fungus. Approximately 60% of exposures result in asymptomatic infection; in the rest there are protean manifestations that range from a benign syndrome also known as "Valley Fever" to progressive pulmonary or extrapulmonary disease. Histoplasmosis, caused by the dimorphic fungus Histoplasma capsulatum, is endemic to the Americas. Pulmonary histoplasmosis manifestations are protean, ranging from a brief period of malaise to a severe, prolonged illness. The spectrum of illness in disseminated histoplasmosis ranges from a chronic, intermittent course to an acute and rapidly fatal infection. Paracoccidioidomycosis is a chronic, granulomatous systemic disease caused by Paracoccidioides brasiliensis that characteristically produces a primary pulmonary infection, often asymptomatic, and then disseminates to form ulcerative granulomata of the oral, nasal and occasionally the gastrointestinal mucosa. Sporotrichosis, caused by Sporothrix schenckii, has diverse clinical manifestations; the most frequent is the lymphocutaneous form. Generally, infection results from inoculation of the fungus through thorns, splinters, scratches and small traumas. Chromomycosis (Chromoblastomycosis) is a slowly progressive cutaneous and subcutaneous mycosis attributed to various saprophyte Hypomycetes fungi. The primary lesion is also thought to develop as a result of percutaneous traumatic inoculation.     

First report of a lyase for cepacian, the polysaccharide produced by Burkholderia cepacia complex bacteria

Bacteria belonging to the Burkholderia cepacia complex (Bcc) are interesting for their involvement in pulmonary infections in patients affected by cystic fibrosis (CF) or chronic granulomatous disease. Many Bcc strains isolated from CF patients produce high amounts of exopolysaccharides (EPS). Although different strains sometimes biosynthesise different EPS, the majority of Bcc bacteria produce only one type of polysaccharide, which is called cepacian. The polymer has a unique heptasaccharidic repeating unit, containing three side chains, and up to three O-acetyl substituents.. We here report for the first time the isolation and characterisation of a lyase active towards cepacian produced by a Bacillus sp., which was isolated in our laboratory. The enzyme molecular mass, evaluated by size-exclusion chromatography, is 32,700+/-1500Da. The enzyme catalyses a beta-elimination reaction of the disaccharide side chain beta-d-Galp-(1-->2)-alpha-d-Rhap-(1--> from the C-4 of the glucuronic acid residue present in the polymer backbone. Although active on both native and de-acetylated cepacian, the enzyme showed higher activity on the latter polymer.     

Measurement of the immunoperoxidase staining of macrophages within liver granulomata of mice infected with Mycobacterium tuberculosis.

A quantitative image analysis technique developed for the measurement of the extent of macrophage activation and epithelioid cell differentiation was performed on mice infected experimentally with Mycobacterium tuberculosis. The granulomatous inflammatory response within the liver reached a peak at day 23 and declined by day 33. Animals of strain B10.BR (H-2k) showed an increased granuloma fraction as compared to Balb/k (H-2k) mice, thus confirming the influence of non-H2 genes in the control of granuloma formation in mice. Using a monoclonal antibody against CD11b/CD18 (Mac1;CR3), we observed two subpopulations of macrophages within the granulomata. The small, darkly staining cells at the periphery of granulomata appear to be newly recruited macrophages. Larger, paler staining cells toward the center of granulomata represent activated and mature epithelioid macrophages. Using a semiautomated image analyzer (Quantimet 970), we measured the relative numbers of these macrophage subpopulations. There were more activated macrophages (epithelioid cells) associated with the increased granuloma fraction in the B10.BR mice than in the Balb/k. However, similar numbers of newly recruited peripheral macrophages were found in both Balb/k and B10.BR strains. This technique has shown qualitative as well as quantitative differences in the granulomatous inflammatory response in this murine model of tuberculosis in strains of mice with quite different antibody repertoires to mycobacterial antigens.     

Paragonimus kellicotti infection in wild carnivores in southwestern Ontario: II. Histopathologic features.

Pulmonary lesions associated with naturally acquired Paragonimus kellicotti infection were studied in mink (Mustela vison), striped skunks (Mephitis mephitis), red foxes (Vulpes vulpes) and a coyote (Canis latrans). In ,ink a fibrous capsule was formed around mature flukes in dilated bronchioles or bronchi, and there was mild focal interstitial pneumonitis adjacent to fluke eggs in alveoli and migrating parasites. A thick wall, infiltrated heavily with mononuclear cells and eosinophils, surrounded mature P. kellicotti in skunks and an extensive inflammatory reaction was found around fluke eggs. In red foxes the wall was thin, hemorrhagic and contained little collagen; necrosis was associated with migrating parasites. Thick capsules formed by marked fibroplastic changes in the lamina propria of affected bronchi were found in the coyote. In raccoons with pleural adhesions massive fibroplasia with eosinophil infiltration beneath the pulmonary pleura suggested a reaction to a migrating parasite entering the lung from the thoracic cavity. It was thought that immature P. kellicotti may have caused the lesion.     

Circulating cytokines in sarcoidosis: Phenotype-specific alterations for fibrotic and non-fibrotic pulmonary disease

AimsSarcoidosis is a granulomatous disease of unknown etiology marked by tremendous clinical heterogeneity. Many patients enter remission with good long-term outcomes. Yet, chronic disease is not uncommon, and this important phenotype remains understudied. Identified alterations in local and circulating cytokines—specifically targeted for study, and often in the acute phase of disease—have informed our growing understanding of the immunopathogenesis of sarcoidosis. Our aim was to evaluate a broad panel of circulating cytokines in patients with chronic sarcoidosis. Among those with chronic disease, pulmonary fibrosis occurs in only a subset. To gain more insight into the determinants of the fibrotic response, we also determined if the phenotypes of fibrotic and non-fibrotic pulmonary sarcoidosis have distinct cytokine profiles.ResultsIn patients with sarcoidosis compared to controls, IL-5 was decreased, and IL-7 was increased. Both of these comparisons withstood rigorous statistical correction for multiple comparisons. GM-CSF met a nominal level of significance. We also detected an effect of phenotype, where IL-5 was significantly decreased in non-fibrotic compared to fibrotic pulmonary sarcoidosis, and compared to controls. Compared to controls, there was a trend towards a significant increase in IL-7 in fibrotic, but not in non-fibrotic pulmonary sarcoidosis. In contrast, compared to controls, there was a trend towards a significant increase in GM-CSF in non-fibrotic, but not in fibrotic pulmonary sarcoidosis.ConclusionsIn a comprehensive evaluation of circulating cytokines in sarcoidosis, we found IL-5, IL-7, and GM-CSF to be altered. These findings provide a window into the immunopathogenesis of sarcoidosis. IL-7 is a novel sarcoidosis cytokine and, as a master regulator of lymphocytes, is an attractive target for further studies. By observing an effect of phenotype upon cytokine patterns, we also identify specific immune alterations which may contribute to clinical heterogeneity.     

The ability to form biofilm influences Mycobacterium avium invasion and translocation of bronchial epithelial cells

Organisms of the Mycobacterium avium complex (MAC) are widely distributed in the environment, form biofilms in water pipes and potable water tanks, and cause chronic lung infections in patients with chronic obstructive pulmonary disease and cystic fibrosis. Pathological studies in patients with pulmonary MAC infection revealed granulomatous inflammation around bronchi and bronchioles. BEAS-2B human bronchial epithelial cell line was used to study MAC invasion. MAC strain A5 entered polarized BEAS-2B cells with an efficiency of 0.1 +/- 0.03% in 2 h and 11.3 +/- 4.0% in 24 h. In contrast, biofilm-deficient transposon mutants 5G4, 6H9 and 9B5 showed impaired invasion. Bacteria exposed to BEAS-2B cells for 24 h had greater ability to invade BEAS-2B cells compared with bacteria incubated in broth. M. avium had no impact on the monolayer transmembrane resistance. Scanning electron microscopy showed that MAC A5 forms aggregates on the surface of BEAS-2B cell monolayers, and transmission electron microscopy evidenced MAC within vacuoles in BEAS-2B cells. Cells infected with the 5G4 mutant, however, showed significantly fewer bacteria and no aggregates on the cell surface. Mutants had impaired ability to cause infection in mice, as well. The ability to form biofilm appeared to be associated with the invasiveness of MAC A5.     

Pulmonary paracoccidioidomycosis in immunized mice

Paracoccidioidomycosis was induced in immunized (IM) and non-immunized (NI) mice. The histopathology, the number of fungi in the lungs, the cellular (footpad test — FPT and macrophage inhibition factor assay — MIF) and humoral (immunodiffusion test) immune response were investigated serially postinfection. In the IM mice, at days 1 and 3, there was intense and predominant macrophagic-lymphocytic alveolitis with loose granulomatous reaction; at day 30, inflammation was mild. In the NI group, up to day 3, the lesions were focal; later there was formation of extensive epithelioid granuloma. The number of fungi in IM mice were always smaller than those of NI group. Immunization alone induced positive FPT and MIF indices with low titer of antibody. After infection, there was a significant decrease of the FPT indices in the IM group, which we interpreted as desensitization due to trapping of sensitized lymphocytes in the lungs. In conclusion, (1) The lesional pattern of pulmonary paracoccidioidomycosis in IM mice was similar to that of a hypersensitivity pneumonitis. This reaction was probably effective in reducing the extension of the infection and decrease the number of fungi. (2) In this model, pulmonary resistance against P. brasiliensis seems to be related to local and systemic delayed-type hypersensitivity reaction.     

Histopathologic and immunologic effects of the itraconazole treatment in a murine model of chronic pulmonary paracoccidioidomycosis

A comparative study, based on histopathologic findings (inflammation, cellularity, and fibrosis) and immunologic parameters (pro-inflammatory and anti-inflammatory cytokines), was carried out in order to evaluate the effects of itraconazole (ITC) treatment and its starting time in a BALB/c murine model of chronic pulmonary paracoccidioidomycosis (PCM), induced by intranasal inoculation of Paracoccidioides brasiliensis (Pb) conidia. Two different groups of mice were exposed to ITC therapy beginning at the 4th or 8th week after Pb infection, respectively. ITC was administered daily, via gavage, for a period of sixty days. At weeks 0, 4, 8, 12 and 16 the animals were sacrificed and their lungs removed for histology staining with hematoxylin and eosin (H&E), Masson’s trichromic and Gomori–Grocott; pulmonary levels of IL-1β, TNF-α, IFN-γ, IL-13 and TGF-β were also measured by ELISA. The development or absence of the principal pulmonary PCM sequela, lung fibrosis, was directly related to the therapy’s starting time. This and other histopathologic findings were related to the behavior of cytokine levels.     

Reassessment of inflammation of airways in chronic bronchitis.

The term chronic bronchitis has been criticised because it is associated with hypersecretion of mucus rather than bronchial inflammation. This study was designed to establish the presence or absence of clinical chronic bronchitis and measure pulmonary function in 45 patients about to undergo resection of the lung. The condition in the cartilaginous and small airways and the severity of the emphysema were then measured in the resected specimen. The results from 20 patients who had clinical chronic bronchitis were compared with those in 25 patients who did not. The data show that patients with chronic bronchitis had greater inflammation (a) on mucosal surfaces (p less than 0.05) of all bronchi larger than 2 mm luminal diameter and (b) around glands (p less than 0.005) and gland ducts (p less than 0.05) in bronchi larger than 4 mm diameter. A variable degree of inflammation was present in the submucosa of smaller bronchi. The groups had equivalent proportions of mucous glands and Reid''s indices in central airways, and no differences were noted in measurements of pulmonary function, condition of small airways, or emphysema. These data show that the term chronic bronchitis is justified by inflammation of cartilaginous airways and suggest that this abnormality may be the cause of the chronic productive cough.     

Liver in Leprosy: Histological and Biochemical Findings

The histological findings and their correlation with biochemical functions of the liver in 240 leprosy patients are presented. In 21% with tuberculoid leprosy and in 62% with lepromatous leprosy leprous granulomata were found in the liver. A significant prevalence of granulomatous lesions in the liver among patients with tuberculoid and borderline-tuberculoid leprosy of less than one year''s duration suggests that bacillaemia occurs early in all forms of leprosy.There was a direct correlation between bacterial index and the presence of acid-fast bacilli in the liver. Of 50 patients with negative skin smears seven had acid-fast bacilli at liver biopsy. From none of these liver homogenates were acid-fast bacilli grown on culture in Löwenstein-Jensen medium.The alterations in liver functions were more consistently seen when acid-fast bacilli were associated with the presence of leprous granulomatous lesions. The acid-fast bacilli were found to persist even after one to five years of specific antileprosy therapy and after the bacilli in the skin had cleared up. This may explain the relatively frequent recrudescence or relapse of the bacillated types of leprosy when specific antileprosy therapy is stopped soon after bacterial negativity is attained on skin smears.     

Scedosporium apiospermum Complex: Diagnosis and Species Identification

Fungi belonging to the Scedosporium apiospermum species complex have been known since the early 20th century as etiologic agents of human infections occurring in immunocompetent individuals as well as immunocompromised people. Additionally, these fungi are now recognized as common colonizers of the airways in patients with cystic fibrosis (CF). The prevalence and importance of these opportunistic pathogens have likely been underestimated, due to the absence of specific features on histological examination of biopsy specimens and the rapid and extensive growth of the aspergilli, which are frequently associated with Scedosporium species in respiratory secretions from CF patients or patients with chronic obstructive pulmonary disease. In this article, the literature on diagnostic and/or identification methods has been reviewed. Development of molecular tools brings about novel challenges in the field of diagnosis, and further studies are required to transfer these methods to routine clinical use.     

Strain variation in BCG-induced chronic pulmonary inflammation in mice. I. Basic model and possible genetic control by non-H-2 genes.

C57BL/6 mice (haplotype H-2b) responded in a dose-dependent fashion to killed BCG by marked enlargement of the spleen and lung. Neither CBA nor C3H mice (haplotype H-2k) responded to such treatment. Pulmonary inflammation in responder B6 animals was characterized by a marked chronic interstitial and alveolar granulomatous process, and was accompanied by occasional granulomata, hyperemia, and loss of architecture in the spleen. Inflammation in non-responder CBA and C3H animals was minimal in both the lung and spleen. The response does not appear to be controlled by genes within the major histocompatibility complex, but is associated with a C57 background. B10.BR mice (responder background, H-2k) were responder animals and C3H.SW mice (nonresponder background, H-2b) were nonresponders. In addition, all animals tested with a C57 background were responders even though two of these strains were not H-2b (C57BL/Ks, H-2d and C57Br/cd, H-2k). The resolution of the mechanism of genetic control of this response in mice may provide information relevant to possible genetic control of chronic pulmonary inflammation in man.     

Dysfunctional inhibitory muscarinic receptors mediate enhanced histamine release in isolated human bronchi

In human airways mucosal mast cells are under the control of inhibitory muscarinic receptors. The described experiments tested, whether the inhibitory potency of two muscarinic receptor agonists (oxotremorine, acetylcholine) becomes impaired in advanced chronic obstructive pulmonary disease (COPD). Isolated human bronchi obtained from 26 patients with lung cancer were separated into two groups. Group 1 patients suffered from moderate COPD (mean FEV1 56%; range 34-71%; mean pack years of cigarette smoking 50, range 20-96; one non-smoker). Group 2 patients had no or only a mild form of COPD; mean FEV1 was 82% (62-97%) and the number of pack years 22 (6-45; 3 non-smoker). The calcium ionophore A23187 induced a maximal histamine release of 4100+/-870 pmol/g/5 min in group 1 bronchi, in contrast to only 1730+/-240 pmol/g/5 min in group 2 bronchi (p<0.02). Oxotremorine (1 nmol/L) reduced the stimulated histamine release by 81+/-5% in group 2 bronchi, but did not produce a significant effect in group 1 bronchi (11+/-14%). In conclusion, the present experiments show an enhanced histamine release in advanced COPD, which can be explained by a dysfunction of inhibitory muscarinic receptors.     

Anorexigen-induced pulmonary hypertension and the serotonin (5-HT) hypothesis: lessons for the future in pathogenesis

Pulmonary surfactant is a surface active material composed of both lipids and proteins that is produced by alveolar type II pneumocytes. Abnormalities of surfactant in the immature lung or in the acutely inflamed mature lung are well described. However, in a variety of subacute diseases of the mature lung, abnormalities of lung surfactant may also be of importance. These diseases include chronic obstructive pulmonary disease, asthma, cystic fibrosis, interstitial lung disease, pneumonia, and alveolar proteinosis. Understanding of the mechanisms that disturb the lung surfactant system may lead to novel rational therapies for these diseases.