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.     

Effect of levamisole on experimental paracoccidioidomycosis in the Syrian hamster: Immunologic and histopathologic correlation

The effect of levamisole (LMS) was studied in hamsters inoculated with live yeast phase culture of Paracoccidioides brasiliensis by intratesticular route. One group started LMS therapy at an early stage of infection (LMS₃ group), when the animals were immunocompetent, and another group was treated in a later stage, when the immune response was already depressed (LMS₁₂ group). As control, one group was not treated.The alterations induced by levamisole were studied by immunologic and histopathologic parameters. Compared to controls, the LMS₃ group presented normal levels of cellular immune response and inflammatory reaction characterized by compact epithelioid granuloma during a longer period of time. In addition, this group showed a lower incidence of amyloidosis and lower fungi proliferation in the lesions. In the LMS₁₂ group a transient enhancement was noteworthy of cellular immune response with maintenance of the compact pattern of the epithelioid granuloma as in the LMS₃ group; however, the number of fungi and incidence of amyloidosis were similar to controls. The differences between both treated groups may be accounted for by some factors such as host immune competence, timing and total dose of LMS administered. Levamisole may be of value as additional therapy in paracoccidioidomycosis.     

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.     

Ketoconazole in the treatment of experimental murine paracoccidioidomycosis

In a murine model of chronic disseminated paracoccidioidomycosis (strain 18; intravenous route), Ketoconazole (200 mg/kg in 0.2% agar) was given daily by gavage in three different schedules. Continuous treatment from an early stage of infection (day 3) up to week 20 was the most effective protocol, leading to remission of histopathological lesions and of both humoral and cellular anti-P. brasiliensis immune response, and clearance of the fungus in lungs; only 1 treated animal at week 20 showed pulmonary granulomas, although less extensive than control mice. Continuous treatment from early stage up to week 8, followed by a 16 week-period of drug discontinuity, caused remission of lesions in all but 3 treated mice which showed active pulmonary paracoccidioidomycosis similar to controls (14.2% of unresponsiveness to treatment). The continuous Ketoconazole protocol since a late stage of infection (week 4) up to week 20 produced a slower remission of lesions and immune response when compared with the first drug schedule. In this model of paracoccidioidomycosis, Ketoconazole showed no detectable side-effects and was a very effective drug especially in a prolonged administration protocol from an early stage of infection.     

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 26×10^–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 26×10^–6 heat-killed. P. brasiliensis yeast cells. None of the animals receiving saline, exhibited pathologic alterations; 11.6% of those inoculated with the heatkilled cells, revealed mild, transitory acinopulmonary neutrophilic infiltrates. The animals infected with viable cells, developed a systemic process affecting mainly the lungs (46.5%), liver (18.6%), lymphnodes (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 bronchoneumonic 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 lymphnode involvement was rare (7%) and primary lung lesions, as seen in tuberculosis and histoplasmosis, were not observed.     

Pathology of experimental Babesia microti infection in the Syrian hamster

Pathologic changes produced after 4 weeks of infection by Babesia microti in Syrian hamsters are described and compared to babesiosis of humans. Following intraperitoneal inoculation, both intravascular and extravascular hemolysis developed. Up to 70% of red blood cells were parasitized. The principal morphologic abnormalities were an increase in extramedullary hematopoiesis and hyperplasia of the mononuclear phagocytic cells of the red pulp manifested grossly as splenomegaly, marked renal tubular hemosiderosis and hypertrophy of Kupffer cells. The disease was not fatal to any hamsters during the 4 week study. The clinical signs and lesions were less severe than fatal babesiosis of asplenic humans and similar to severe, but nonfatal disease in spleen intact humans. The hamster may serve as an animal model for the studying the pathophysiology of human babesiosis and for studying potential chemotherapeutic agents.     

The Syrian hamster as a model for the dilated cardiomyopathy of Chagas' disease: a quantitative echocardiographical and histopathological analysis

Chronic Chagas' disease cardiomyopathy (CCC) is caused by the protozoan Trypanosoma cruzi, and it affects 30% of the 16-18 million people infected in Latin America. A good rodent model that develops a dilated cardiomyopathy closely resembling human CCC after T. cruzi infection is still needed. We compared the cardiomyopathy developed by T. cruzi-infected Syrian hamsters with human Chagas' disease cardiomyopathy using quantitative methods. Female hamsters were infected with 3.5 x 10(4) (G1, n = 10) or 10(5) (G2, n = 10) T. cruzi Y strain blood trypomastigotes. Control animals (C, n = 10) were injected with saline solution. Cardiac function was assessed by echocardiography at 4, 8 and 12 months post-infection. Heart sections were submitted to histopathological/morphometric analysis 12 months post-infection. At this time, ventricular dysfunction and diffuse or multi-focal myocarditis were observed in 91% and 100% of G1 and G2 infected groups, respectively. Median interstitial collagen volumes in groups C, G1 and G2 were 1.2%, 1.9% and 3.9%, respectively, and were significantly higher in group G2 than in group C. Among infected animals, myocarditis showed a positive correlation with interstitial fibrosis. Deaths in the chronic phase (8-12 months post-infection) were more frequent among G2 than G1, and were associated with macroscopic ventricular dilation, severe myocarditis and increased fibrosis values, along with an earlier onset of ventricular dysfunction. The T. cruzi chronically infected Syrian hamster develops a cardiomyopathy which resembles human Chagas' disease cardiomyopathy, and might be an adequate tool to investigate pathogenic mechanisms of this disease and to search for novel therapeutic strategies.     

Additive effect of rPb27 immunization and chemotherapy in experimental paracoccidioidomycosis

Paracoccidioidomycosis, PCM, the major systemic mycosis in Latin America, is caused by the termally dimorphic fungus Paracoccidioides brasiliensis and requires extended periods of chemotherapy with a significant frequency of relapsing disease. The search for new alternatives of treatment is necessary. rPb27 is an antigenic protein from P. brasiliensis that already showed a significant protective activity as a vaccine for PCM in experimental models. The cDNA of rPb27 was subcloned into a pET-DEST 42 plasmid, expressed in E. coli with a his-tag and purified by affinity chromatography. Immunization with this recombinant protein and chemotherapy were used together in an attempt to improve treatment of PCM. For this, BALB/c mice were challenged with pathogenic P. brasiliensis strain and after immunized with rPb27, in the presence of Corynebacterium parvum and Al(OH)(3), some groups were also treated with fluconazole. After 40 days of treatment, the combined drug/rPb27 administration controlled PCM in the liver and spleen, with long lasting protection, and largely preserved tissues structures of these organs. Additionally, in the lungs after 40 days of treatment there was a significant reduction in the fungal load and size of lesions. At the same time, the levels of TNF-α were higher than infected-only mice. Moreover, significant levels of anti-rPb27 specific IgG1, IgG2a and IgG2b isotypes were detected in the sera of mice immunized with rPb27 fluconazole treated or not. These results showed an additive protective effect of rPb27 immunization and chemotherapy, suggesting that an rPb27-based vaccine can be used to enhance PCM antifungal treatment.     

Immunohistochemical renin study of DES-induced renal tumor in the Syrian hamster

Diethylstilbestrol (DES) treatment of a male Syrian hamster resulted in the development of a renal tumor and its widely scattered serosal metastases. Cells in both the primary tumor and metastatic nodules contained secretory granules. The tumors were transplanted serially into DES-supported and non-DES-supported host hamsters until DES-independent tumors developed. Rabbit antiserum to mouse salivary renin and rabbit antiserum to rat kidney resin were reacted with sections of the primary tumor, metastatic nodules, and all transport tumors. The sections were stained by the PAP and Vector-ABC-AP procedures. Renin-positive material was observed in all tumors. Plasma renin activity (PRA) was determined for the host hamsters carrying the renal tumor transplants and compared to the PRA values that had been determined for normal non-DES-treated male and female hamsters. It was found that the average PRA values of host hamsters carrying the tumor transplants were significantly higher than the normal PRA values.     

Electron microscopic study on the innervation of Peyer's patches of the Syrian hamster

Summary Electron microscopic investigations have been carried out on the innervation of Peyer's patches. Bundles of axons partly devoid of Schwann satelite cells have been observed. The intercellular space between free axons which contain synaptic vesicles and lymphatic respectively reticulum cells is 200 Å.Apart from the possibility of neuronal transmission at these contact points the hypothesis is established that a temporary contact between lymphocytes and the surface of nerve cells of the own body is necessary for maintainance of immune tolerance of some specific neuronal antigenic structures. Peyer's patches may grant a suitable environment for this purpose.For stimulating discussions we are much obliged to Prof. Dr. med. K. Lennert and to Prof. Dr. med. Dr. med. dent W. Müller-Ruchholtz.     

Experimental paracoccidioidomycosis of hamster inoculated in the cheek pouch

We compared the granuloma morphology and immune response of hamsters inoculated withParacoccidioides brasiliensis (Pb) into the cheek pouch, which lacks lymphatic drainage, and into the footpad, which is rich in lymphatics. Our objective was to better understand the modulation ofPb granuloma in an immunocompetent animal inoculated in an immunologically privileged site. The humoral immune response (ELISA) and cell mediated immunity (footpad test) became positive on days 7 and 14, respectively in animals inoculated into footpad and on days 35 and 60 in animals inoculated into the pouch. Typical epithelioid granulomas were observed at both sites on day 14. The number of fungi gradually decreased from the beginning of the experiment in footpad lesions, but only after day 35 in pouch granulomas, when cell mediated immunity was detectable. The results indicate that typical epithelioid paracoccidioidomycotic granulomas may develop in the absence of a detectable immune response; however, they are incapable of controlling fungal reproduction. Lack of lymphatic drainage delays the appearance of a detectable immune response, but with time fungi escape from the pouch, elicit an immune response and reach other organs. Our results further indicate the importance of the lymphatics in the pathogenesis of paracoccidioidomycosis.Abbreviations HCP hamster cheek pouch - Pb Paracoccidioides brasiliensis - Pbmycosis Paracoccidioidomycosis     

Protective effect of L-carnitine on experimental lead toxicity in rats: a clinical,histopathological and immunohistochemical study

Abstract

Female Wistar-albino rats were given lead acetate (PbAc) for 60 days to investigate the protective effects of L-carnitine (CA) clinically and histopathologically on PbAc-induced tissue damage. Blood samples were obtained from the jugular vein for hemoglobin (HB), hematocrit (HCT), red blood cells (RBC), white blood cells (WBC), platelets (PLT), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and creatinine. PbAc treatment caused a significant decrease in HB, HCT and RBC, a significant increase in WBC, AST, ALT and creatinine compared to controls. Although administration of CA did not reverse HB and HCT values, it reversed both the decrease in RBC and the increase in WBC, AST, ALT and creatinine. After the experimental period, all rats were weighed, then decapitated for pathological examination. Control rat liver, kidney and brain showed normal histological architecture. Lead-induced nephropathic kidneys; degenerative changes, inflammation and portal edema of the liver; and brain neuropil vacuolation, neuronal vacuolation, satellitosis and neuronophagia were observed in experimental groups. All changes were reduced in the PbAc group treated with CA (PbAc + CA). PbAc caused copper/zinc superoxide dismutase (Cu/Zn-SOD) expression in both the hepatocytes and tubular epithelium of the kidney. PbAc + CA exposure caused moderate Cu/Zn-SOD immunoreactivity. While in the brain sections of the PbAc group the degenerative neurons were stained intensely with anti-ubiquitin antibody, PbAc + CA rats showed moderate staining in neurons with anti-ubiquitin antibody. These results show that CA as a food additive reduced the severity of tissue damage caused by PbAc.     

A comparative study on neoplastic transformation of human and Syrian hamster cells

Three approaches have been taken to study simultaneously Syrian hamster cells and human cells in order to develop an extrapolation from the more established hamster system to human cells. On the Characterization of normal cells in comparison to tumor cells, human tumor cells and hamster tumor cells showed similarity in displaying anchorage independence, growth in suspension as micro tumor spheroids, and xenotumorigenicity in contrast to their respective normal cells; in addition, these tumor cells exhibited shorter population doubling time, higher saturation density, higher cloning efficiency, and higher fibrinolytic activity relative to their respective normal cell types. Other differences including ploidy change, contact inhibition on growth, serum requirement, and morphological transformation were also noted between human and hamster cells. On the application of microcarrier culture for a xenotumorigenicity test, the microcarrier technique seemed to have enhanced the sensitivity by reducing the number of inoculated tumor cells required for tumor formation. On the in vitro transformation of normal human and hamster cells, the highest efficiency of morphological transformation of hamster cells has been observed in the group treated with N-methyl-N'-nitro-N-nitrosoguanidine followed by griseofulvin which was employed to enhance the transformation by disturbing the chromosome apparatus. However, no evidence of transformation was observed in the treated human cells thus far.     

Genetic studies of the Syrian hamster

A new mutant allele of the Syrian hamster is described. It is inherited as an autosomal recessive and is probably an homologue of the gene for brown pigment, a mutational step which is known to occur in a number of rodent species. In animals homozygous for the mutant allele, all the normal black eumelanin is changed to brown. The new coat colour engendered in this manner is described in detail. The brown allele has been tested for linkage against the genes cream and ruby-eye but the results were negative.