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.     

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.     

Increased collagen metabolism in granulomata induced in rats deficient in endogenous prostaglandin precursors

Collagen metabolism was measured (in terms of various hydroxyproline (HP), DNA and protein ratios) in granulomata obtained after s.c. implantation of carrageenan-impregnated and untreated polyether sponges into normal and essential fatty acid deficient (EFAD) rats for 8 and 15 days. Collagen synthesis (HP/protein) in day 8 and 15 untreated granulomata was the same for both normal and EFAD rats, though collagen breakdown (total HP) appeared to be greater in EFAD granulomata on day 15. With carrageenan-impregnated sponges, collagen synthesis in EFAD granulomata was much greater than in normal granulomata on both day 8 and day 15. Ratios of protein and/or HP to DNA (probably indicative of cellular infiltration) were increased in EFAD rats with both sponge types, though this increase was less pronounced with carrageenan-impregnated sponges. It is suggested that endogenous prostaglandin (PG) production (marledly reduced during EFA deficiency) may exert a negative feedback effect on collagen metabolism during proliferative inflammation.     

Increased collagen metabolism in granulomata induced in rats deficient in endogenous prostaglandin precursors

Collagen metabolism was measured (in terms of various hydroxyproline (HP), DNA and protein ratios) in granulomata obtained after s.c. implantation of carrageenan-impregnated and untreated polyether sponges into normal and essential fatty acid deficient (EFAD) rats for 8 and 15 days. Collagen synthesis (HP/protein) in day 8 and 15 untreated granulomata was the same for both normal and EFAD rats, though collagen breakdown (total HP) appeared to be greater in EFAD granulomata on day 15. With carrageenan-impregnated sponges, collagen synthesis in EFAD granulomata was much greater than in normal granulomata on both day 8 and day 15. Ratios of protein and/or HP to DNA (probably indicative of cellular infiltration) were increased in EFAD rats with both sponge types, though this increase was less pronounced with carrageenan-impregnated sponges. Is is suggested that endogenous prostaglandin (PG) production (markedly reduced during EFA deficiency) may exert a negative feedback effect on collagen metabolism during proliferative inflammation.     

Inoculation experimental animals withParacoccidioides brasiliensis strains: An attempt to reestablish the dimorphic process and variation in pathogenicity as a function of time of preservation under mineral oil

In an attempt to reestablish the dimorphic process in strains ofParacoccidioides brasiliensis in the transition phase (Y ⇄ M) and to reisolate them, five strains in the transitional phase due to the long time of preservation under mineral oil and two strains in the yeast-like phase were inoculated into male albino rats. The animals were then studied for the presence of paracoccidioidomycotic granulomata. Of the seven strains inoculated, five caused granulomatous nodules in several organs of the animals and only two of these five strains, which had been preserved for the shortest period of time (9 years) were reisolated in culture. Two strains were unable to provoke infection, with no lesions detected in any organ. It is assumed that the long period of time during which the strains were left under oil favored the alteration of celt wall contents, leading to differences in pathogenicity.     

Cutaneous Sarcoidosis in Blood Donation Venepuncture Sites

Six patients were studied in whom sarcoidosis first showed itself by the development of granulomata at the site of previous venepunctures. The diagnosis was histologically confirmed in all cases. The treatment of the lesions with triamcinolone did not seem to hasten resolution.     

Defensive role of granuloma againstSporothrix schenckii infection

The defensive role of granuloma againstSporothrix schenckii infection was studied histopathologically using nude(nu/ nu) and their heterozygous(nu/+) littermates.Three strains ofS. schenckii (Sp.-1, Sp-17 and Sp-56) were used in this experiment. Each mouse was inoculated into a tail vein with 10⁶ yeast cells of the Sp-1, Sp-17 or Sp-56. The mice were sacrificed at adequate intervals until the 30th day and histopathological sections were prepared from various organs.The numbers of lesions and yeast cells were counted using the liver sections. Furthermore, an experiment of lymph node cell transfer and immunological examinations were carried out.As results the susceptibility of mice to three strains were conspicuously different from each other. The Sp-1 showed the strongest pathogenicity and the Sp-56, the weakest. The susceptibility of the nu/ nu mice inoculated with the Sp-1 was much higher than that of the nu/+ mice and the difference was due to the killing functions of granuloma. Even though about two days' delay was observed in the granuloma formation in the nu/ nu mice in comparison with that in the nu/+ mice, these granulomata could not be distinguished from those of the nu/+ mice. However, functionally there was a definite difference between the granulomata formed in the nu/+ and nu/nu mice. Mononuclear cells forming the granulomata in the nu/ nu mice did not have the ability to kill the yeast cells they had engulfed. Cooperation with T-lymphocytes was necessary for the killing of the yeast cells. A significant response of MIF developed in the immunocompetent mice 11 days after inoculation of the Sp-56, and that day nearly coincided with the day when yeast cells of the Sp-1 began to be destroyed in the granulomata. It was also confirmed by the experiment of lymph node cell transfer that T-cell functions were indispensable for the killing of the yeast cells by mononuclear cells.From these results the authors hypothesize that the mononuclear cells activated with T-lymphocytes could play a leading role as the defense mechanism of mice againstS. schenckii infection.     

Defensive role of granuloma againstSporothrix schenckii infection

The defensive role of granuloma againstSporothrix schenckii infection was studied histopathologically using nude(nu/nu) and their heterozygous(nu/+) littermates. Three strains ofS. schenckii (Sp.-1, Sp-17 and Sp-56) were used in this experiment. Each mouse was inoculated into a tail vein with 10⁶ yeast cells of the Sp-1, Sp-17 or Sp-56. The mice were sacrificed at adequate intervals until the 30th day and histopathological sections were prepared from various organs. The numbers of lesions and yeast cells were counted using the liver sections. Furthermore, an experiment of lymph node cell transfer and immunological examinations were carried out. As results the susceptibility of mice to three strains were conspicuously different from each other. The Sp-1 showed the strongest pathogenicity and the Sp-56, the weakest. The susceptibility of the nu/nu mice inoculated with the Sp-1 was much higher than that of the nu/+ mice and the difference was due to the killing functions of granuloma. Even though about two days’ delay was observed in the granuloma formation in the nu/nu mice in comparison with that in the nu/+ mice, these granulomata could not be distinguished from those of the nu/+ mice. However, functionally there was a definite difference between the granulomata formed in the nu/+ and nu/nu mice. Mononuclear cells forming the granulomata in the nu/nu mice did not have the ability to kill the yeast cells they had engulfed. Cooperation with T-lymphocytes was necessary for the killing of the yeast cells. A significant response of MIF developed in the immunocompetent mice 11 days after inoculation of the Sp-56, and that day nearly coincided with the day when yeast cells of the Sp-1 began to be destroyed in the granulomata. It was also confirmed by the experiment of lymph node cell transfer that T-cell functions were indispensable for the killing of the yeast cells by mononuclear cells. From these results the authors hypothesize that the mononuclear cells activated with T-lymphocytes could play a leading role as the defense mechanism of mice againstS. schenckii infection.     

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.     

Schistosoma mansoni: stimulation of artificial granuloma formation in vivo by carbohydrate determinants

A subset of Schistosoma mansoni egg glycoproteins that share a common carbohydrate epitope recognized by monoclonal antibody 128C3 was shown to induced formation of hepatic granulomata when conjugated to Sepharose beads and injected into the portal circulation of naive mice. Concanavalin-binding egg glycoproteins exhibited more granuloma-inducing activity than did total egg extract, although deglycosylated egg proteins also induced granulomata; thus, both amino acid and carbohydrate epitopes appeared to be involved. Glycoproteins derived from adult male worms also were active, indicating that immunological processes responsible for granuloma formation may not be absolutely stage specific.     

A survey of Encephalitozoon cuniculi in laboratory animal colonies in the United Kingdom

Of 38 animal colonies serologically examined for Encephalitozoon cuniculi, 1 mouse, 2 rat and 4 guineapig colonies were positive. A further survey showed that the prevalence within mouse, rat, guineapig and rabbit colonies varied between 25 and 95%. Guineapigs housed with infected rabbits are at a greater risk of being infected than those housed separately. Nephritis was a common feature, but cerebral granulomata were not seen.     

Studies on naturally-occurring ovine fascioliasis in the Sudan

Haematological, biochemical and pathological changes were investigated in 214 sheep naturally infected with Fasciola gigantica in an endemic area in the Sudan together with 82 uninfected controls. Infected animals showed a clear decrease in erythrocyte counts, haemoglobin concentration and packed cell volume, a normochromic, normocytic anaemia, leucocytosis and eosinophilia. Serum concentrations of the enzymes glutamate dehydrogenase, sorbitol dehydrogenase and glutamate oxaloacetic acid transaminase were also elevated in the infected group, indicating hepatic damage. This was confirmed by histopathological changes, which comprised degenerative and necrotic changes in hepatocytes associated with haemorrhage, fibrosis, increased lobulation of the liver, mononuclear cell infiltration with haemosiderin deposition in fluke tracks and portal areas and the formation of granulomata around fluke eggs and fluke remnants. In the infected group there was slight hyperglobulinaemia and a marked hypoalbuminaemia, with a decrease in A/G ratio. A slight rise in the level of serum bilirubin was also observed.     

Human Leprosy in Normal Mice

It has now been shown that normal mice can be used as models for studying the early stages in the development of leprosy. Inoculation into the foot pads of mice of as few as 10⁴ leprosy bacilli leads to infections which spread to distant sites via the blood stream and after two or more years give rise to granulomata and neural damage at the sites of inoculation. Where the tissue response had fully developed it reproduced exactly the histological features of human leprosy in the borderline range.     

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.     

Clinical utility of fine needle aspiration biopsy in the diagnosis of Wegener's granulomatosis. A report of two cases.

In two patients, pulmonary lesions of Wegener's granulomatosis (WG) were sampled by fine needle aspiration biopsy: one with the clinical diagnosis of primary pulmonary malignancy and the other with a clinical suspicion of WG. In the latter case the smears showed distinctive eosinophilic, collagen necrosis (pathergic necrosis), poorly formed granulomata composed of loose aggregates of elongated, often palisading epithelioid histiocytes, and multinucleate histiocytes. A cell block preparation in this case contained minute tissue fragments illustrating the distinctive, pathergic-type necrosis. In the former case, many of these features were present, but additionally there were several groups, atypical bronchial epithelial cells that, in light of the clinical impression, initially led to an incorrect diagnosis of bronchoalveolar carcinoma. Subsequent review of this case led to the diagnosis of WG. Antineutrophil cytoplasmic antibody (ANCA) serology was later obtained, confirming the diagnosis of WG in both cases. In our experience, the cytomorphologic findings of granular collagen necrosis, granulomata and multinucleate cells, although not specific, should alert the cytopathologist to consider the diagnosis of WG, especially when special stains for microorganisms are negative. A recommendation for ANCA serology testing early in the disease process, particularly in the limited forms of the disease, may lead to early recognition of WG, resulting in prompt institution of immunosuppressive therapy, greatly improving the patient's prognosis.     

MORPHOLOGICAL AND CHEMICAL STUDIES OF COLLAGEN FORMATION : I. The Fine Structure of Guinea Pig Granulomata

This paper describes electron microscopic studies of developing connective tissue in granulomata induced by the subcutaneous injection of carrageenin into guinea pigs. Seven days after injection the granulomata contained many fibroblasts and exhibited rapid production of collagen. The fibroblasts were characterised by an extensively developed endoplasmic reticulum and showed numbers of fine, unstriated filaments in the outer regions of the cytoplasm. The filaments, about 50 A in diameter, tended to lie parallel to and closely adjacent to the cell boundary. The cytoplasmic membrane was frequently ill defined or disrupted, particularly bordering regions in which filaments occurred. In longitudinal sections of extended cell processes, filaments were abundant and, in some instances, the cytoplasmic membrane was barely detectable. In the extracellular space striated collagen fibrils were usually accompanied by filaments, 50 to 100 A in diameter, and these often exhibited the characteristic periodicity of collagen, particularly after intense electron bombardment. Much cellular debris was present in the extracellular space. These observations have led to the suggestion that connective tissue precursors are released from fibroblasts by the disintegration or dissolution of the cytoplasmic membrane and the shedding of cytoplasmic material, as in the apocrine gland cells. In some instances this release may take the form of the elongation from the cell of extended processes; disintegration of the cytoplasmic membrane surrounding these processes then leaves the contents in the extracellular phase.     

Microfilariae in fine needle aspirates from epididymal lesions

Microfilariae of Wuchereria bancrofti were observed in fine needle aspiration smears from three epididymal nodules, and degenerating microfilariae suggestive of Brugia malayi were found in the smears from a fourth case. The smears in all four cases showed a polymorphonuclear inflammatory cell component as well as epithelioid cell granulomata. While blood eosinophilia was present in all four cases, eosinophilia was present in the aspiration smears in only one case. Microfilariae could be demonstrated in the peripheral blood in only one case.     

Pathology of larval Eustrongylides in the rabbit

Three fishermen from Maryland who swallowed live bait-minnows developed severe abdominal pain within 24 hr; 2 required abdominal surgery. Larvae of the nematode Eustrongylides sp. were found in the peritoneal cavity of both (Guerin et al., 1982). In the current study, the lesions produced by Eustrongylides larvae were investigated in New Zealand white rabbits. None of these exhibited any signs of clinical illness; however, postmortem examination within 24 hr of inoculation revealed that larvae had migrated through the walls of the esophagus and stomach and viable larvae were recovered from the pleural and peritoneal cavities as well as from gastric contents. Necropsies performed at different intervals of time postinoculation showed that the migrating larvae had produced multi-focal peritonitis and multiple granulomata in the liver.     

Protection Induced by Simultaneous Subcutaneous and Endobronchial Vaccination with BCG/BCG and BCG/Adenovirus Expressing Antigen 85A against Mycobacterium bovis in Cattle

The incidence of bovine tuberculosis (bTB) in the GB has been increasing since the 1980s. Immunisation, alongside current control measures, has been proposed as a sustainable measure to control bTB. Immunisation with Mycobacterium bovis bacillus Calmette-Guerin (BCG) has been shown to protect against bTB. Furthermore, much experimental data indicates that pulmonary local immunity is important for protection against respiratory infections including Mycobacterium tuberculosis and that pulmonary immunisation is highly effective. Here, we evaluated protection against M. bovis, the main causative agent of bTB, conferred by BCG delivered subcutaneously, endobronchially or by the new strategy of simultaneous immunisation by both routes. We also tested simultaneous subcutaneous immunisation with BCG and endobronchial delivery of a recombinant type 5 adenovirus expressing mycobacterial antigen 85A. There was significantly reduced visible pathology in animals receiving the simultaneous BCG/BCG or BCG/Ad85 treatment compared to naïve controls. Furthermore, there were significantly fewer advanced microscopic granulomata in animals receiving BCG/Ad85A compared to naive controls. Thus, combining local and systemic immunisation limits the development of pathology, which in turn could decrease bTB transmission.     

Perinatal exposure to estradiol masculinizes aspects of sexually dimorphic behavior and morphology in gray short-tailed opossums (Monodelphis domestica)

The effects on adult sexually dimorphic behavior of perinatal exposure to estrogen were examined by treating male and female gray opossums with estradiol (EST), an estrogen receptor antagonist (tamoxifen:TX) or oil control (OIL) during the first week of life, a time period corresponding in this marsupial to late gestation in rodent species. Following gonadectomy and replacement therapy with testosterone in adulthood, males showed more scent-marking behavior than females and EST animals showed more scent marking than TX or OIL animals. Also, phalluses were longer and body weight was higher in males than in females and in EST-treated animals than in TX-treated animals; OIL animals were intermediate in these morphological measures. EST animals of both sexes showed less female-typical screeching threat behavior than OIL or TX animals. Because these hormone manipulations were conducted on the "fetus" directly in this marsupial (rather than via the maternal circulation as in previously studied eutherian species), these findings provide unique confirming evidence for masculinization of aspects of behavior and morphology by early exposure to estradiol in mammals.