T. De Brito

Cardiovascular involvement in human and experimental leptospirosis: pathologic findings and immunohistochemical detection of leptospiral antigen

Twenty hearts from patients dying of leptospirosis were studied. Interstitial myocarditis was found in 50% of the cases, and a significant statistical correlation was observed between myocarditis and the inflammatory involvement of the conduction tissue. Acute coronary arteritis, affecting the main branches of the coronary arteries, was observed in 70% of the cases, and this finding also correlates significantly with interstitial myocarditis. Aortitis was found in 57.8% of the cases. When serum against L. interrogans serovar icterohaemorrhagiae was used, focal IP antigen deposits were observed in the coronary arteries and in the aorta. Experimental data from 12 guinea-pigs inoculated with L. interrogans serovar icterohaemorrhagiae showed a focal myocarditis involving mainly the subendocardial and pericoronary heart tissue, with IP antigen deposits in the same sites. Leptospirosis might be visualized as a generalized illness resembling other infectious vasculitides. The heart and main vessels are involved during the septicaemic phase of the disease, and bacterial migration, toxin(s), enzymes and/or antigenic products liberated by bacterial lysis might account for the increased endothelial permeability with antigen deposits and inflammation.

Pathology of the human pulmonary paracoccidioidomycosis

Lungs of twelve patients with chronic paracoccidioidomycosis (Pb) were studied in an attempt to understand the pathogenesis of the pulmonary disease. Ribbons of the lung parenchyma including the hilar region and directed towards apical, basal and lateral regions were subdivided into sections from the hilar, intermediate and peripheral segments. The following histopathological reactions directly or indirectly related to P. brasiliensis were described and analysed in relation to the number of slides studied and the pulmonary region involved: (1) pneumonic reaction; (2) early granulomatous formation; (3) mature and healed granulomata; (4) mixed pattern (early and mature granuloma in the same pulmonary area visualized in the slide); (5) pulmonary fibrosis.It was concluded that chronic pulmonary Pb is a recurrent disease affecting equally both lungs. Fibrosis was connected mainly with the progressive evolution of the granulomata towards cicatrization and to a lesser degree probably to a direct induction by the fungi. Based chiefly on the tendency of the fibrosis to run around bronchi and to make up septa interconnecting bronchi and vessels it was hipothesized that these findings were the result of a previous chronic specific lymphangitis by the fungi. Hilar fibrosis would be the result of this lymphangitis and/or of the progression of the specific granulomatous reaction seen in the hilar lymph nodes.Non specific forms of arteritis and areas of destructive emphysema related to granulomatous inflammation and fibrosis were described. Three cases developed pulmonary hypertension.

Antigen distribution and antigen-presenting cells in skin biopsies of human chromoblastomycosis.

Background:  Chromoblastomycosis is a chronic, suppurative, granulomatous mycosis usually confined to skin and subcutaneous tissues. The host defense mechanisms in chromoblastomycosis have not been extensively investigated. The purpose of the present study was to determine the distribution and pathways of the fungal antigen(s) and the possible role of the different immunocompetent cells in antigen processing in skin lesions.

Pathology of the kidney and liver in the experimental leptospirosis of the guinea-pig. A light and electron microscopy study.

A light and electron microscopy study of the experimental leptospirosis of the guinea pig wasdone. The earliest lesion found was located at the cell membrane, with partial or total disappearance of the brush border of the cells of the proximal tubuli as well as partial disappearance and distortion of the microvilli of the hepatic cells. Intercellular spaces were found to be enlarged both in the liver and kidney. Capillaries showed endothelial cell tumefaction and, sometimes, disjunction of the endothelial lining, a finding also in accordance with the basic pathology of the disease. Only at the late phase of the disease, mainly at the agonic period, that pathology of the organelles such mitochondria was found. However, a definite increase of “dense bodies” whose origin was discussed, was found since the early phase of the disease. Also described, a mild but definite focal glomerular lesion, which provides anatomical basis for the proteinuria seen in the disease. The above described basic pathology of the disease is in accordance with the possibility of a toxin as the main mechanism acting for leptospiral pathogenicity. Die erste Veränderung wurde an der Zellmembran beobachtet, zusammen mit teilweiser oder vollständiger Zerstörung des Bürstensaumes der proximalen Nierenkanälchen sowie der Mikrovilli der Leberzellen. Die intercellulären Räume der Leber und Niere waren erweitert. An den Blutcapillaren fanden sich endotheliale Schwellungen und manchmal auch Loslösung des Endothels. Eine pathologische Veränderung der Mitochondrien wurde nur während der Endphase der Krankheit gesehen, doch waren „dense bodies“ schon von Anfang an vorhanden. Außerdem wurde auch eine leichte glomeruläre Läsion gefunden, welche die anatomische Grundlage der Proteinurie darstellen mag. Diese Befunde lassen die Annahme einer toxischen Wirkung als Grundlage der Pathogenese der Leptospirose zu.

Immunohistochemical and in situ hybridization studies of the liver and kidney in human leptospirosis

An in situ hybridization (ISH) assay for the detection of leptospiral DNA in tissues was described and its diagnostic and pathogenetic usefulness in combination with immunohistochemistry (IHC) was evaluated in formalin-fixed, paraffin-embedded liver and kidney samples from human fatal cases of leptospirosis. IHC assays with anti-E-cadherin antibodies assessed the liver-plate disarray frequently observed in leptospirosis. Immunohistochemistry detected leptospiral antigen (LAg) in macrophages, both in human liver and kidney. In guinea pigs, in addition to these findings, staining on cell membranes of hepatocytes and, occasionally, in apical membrane of kidney tubular cells was demonstrated. Positive ISH signal was observed chiefly in the nuclei of human hepatocytes and in the cytoplasm and nuclei of liver cells of experimentally infected guinea pigs. Loss of E-cadherin membrane expression is associated with liver-plate disarray. These findings were discussed in the contention that, in leptospirosis, cell membrane damage might be important for the pathogenesis of the disease. Finally, it was suggested that both IHC and/or ISH might be used for both diagnostic and research purposes.

Liver biopsy in human leptospirosis: A light and electron microscopy study

Sixteen human liver biopsies of patients with leptospirosis were studied both by light and electron microscopy. The sinusoidal pole of the liver cell show distortion and/or partial disappearence of the microvilli. There is hypertrophy and hyperplasia of Kupffers cells. These data are suggestive of one or more circulating toxins. Complete cell necrosis is not prominent in human leptospirosis, this finding being in accordance with the low level of serum transaminase seen in the disease. Bile capillaries show microvilli disappearence or distortion, a non specific finding seen both in the extra and intrahepatic forms of cholestasis. No explanation for jaundice mechanism in leptospirosis was found. Sechzehn Leberbiopsien wurden untersucht. Der sinusoidale Pol der Leberzellen zeigt Strukturänderungen mit/oder teilweisem Verlust der Mikrovilli, die Kupfferschen Sternzellen weisen Hypertrophie und Hyperplasie auf. Diese Befunde sprechen für das Vorkommen von einem oder mehreren Toxinen. Vollständige celluläre Nekrose wurde nicht oft gefunden, was mit den niedrigen Serum-Transaminase-Werten bei dieser Krankheit übereinstimmt. Die Gallencapillaren zeigen Formveränderung bis Zerstörung der Mikrovilli, ein unspezifischer Befund der sowohl bei extraals auch intrahepatischen Formen der Cholestase gesehen wird. Es wurde keine Erklärung für den Ikterus von Leptospirosekranken gefunden.

Leptospiral antigens in the liver of experimentally infected guinea pig and their relation to the morphogenesis of liver damage

In order to investigate the morphogenes of experimental leptospirosis by morphologic and immunohistologic methods, 24 guinea-pigs were inoculated intraperitoneally with L. interrogans serogroup Icterohaemorrhagiae. They were divided in 6 groups, sacrificed from the 1st to the 6th day of infection. Semiquantitative analyses of histopathological liver lesions were performed in 1 micron sections of tissue embedded in glycol-methacrylate. The distribution of leptospiral antigen (L. Ag) and its glycolipoprotein (GLP) was demonstrated by peroxidase-antiperoxidase on paraffin embedded tissue. Significant lesions appeared at the 4th day of infection, progressing to a peak on the 6th day. Inflammation was associated with injury of the portal triad. Liver cells showed either swelling or acidophilic degeneration and necrosis, together with loss of cell cohesion, leading to disarray of liver cell plates. Mitochondria were found progressively enlarged and irregularly distributed. L. Ag expression was parallel to the morphological changes. Portal distribution was significant at the 4th day and on later stages centrilobular localization became predominant. Spiral forms suggestive of intact leptospires were initially found but, chiefly at the 6th day, L. Ag was seen in granules, probably resulting from phagocytosis. GLP staining was similar to granular L. Ag in morphology, and distribution. Cytokeratin condensation was seen in liver cells with acidophilic necrosis and was marked in areas of disorganization of cell plates. Our findings lead us to hypothesize a direct leptospiral cytotoxic effect on endothelial and on liver-cell membranes. At first, leptospires themselves would induce subcellular changes acting mainly on membrane permeability. Afterwards, their granular forms, including GLP, would act as adjuvant factors. These findings demonstrate that the disarray of liver cell plates at the late phase of the disease is genuine.

Kidney biopsies in human leptospirosis: A biochemical and electron microscopy study

Die Niere von Patienten mit Leptospirose wurde mittels Elektronenmikroskopie und biochemisch-enzyniatischer Bestimmungen untersucht. Verminderte enzymatische Aktivität wurde bei einer morphologischen Läsion der Tubuluszellen gefunden. Cytosome und Cytosegresome wurden mit größerer Häufigkeit in einigen veränderten Tubuluszellen nachgewiesen. Ferner werden pathologische Veränderungen von Mitochondrien, Bürstensaum, Capillaren und Glomerula beschrieben. Diese Befunde sind mit einer toxischen Wirkung auf Blutcapillaren und auf Tubuluzellen vereinbar, welche die Mereninsuffizienz der Leptospirose erklären kann. Biochemical enzymatic assays with electron microscopic studies of biopsied human kidney were made in leptospirosis. There was decreased enzymatic activity together with morphological evidence of tubular cell injury. Cytosomes and cytosegresomes were more abundant in some of the altered cells; mitochondrial and brush border injury were present. Damage to capillaries and a mild glomerular alteration were noted. These data are consistent with a circulating toxin which, acting on capillaries and/or directly on the tubular cell, might be responsible for the kidney failure seen in leptospirosis.

Intestinal spirochetosis: first cases reported in Brazil and the use of immunohistochemistry as an aid in histopathological diagnosis.

Colonization of the colon and rectum by intestinal spirochetes is detected for the first time in Brazil in 4 of 282 (1.41%) patients who had undergone sigmoidoscopy and/or colonoscopy with a histopathological diagnosis of chronic non specific-colitis. This frequency is probably underestimated, since surgically obtained specimens were not considered in the present study. Histopathological diagnosis was performed using routine stains like hematoxylin-eosin which showed the typical, of 3-microns thick hematoxyphilic fringe on the brush border of the surface epithelium, and by silver stains like the Warthin-Starry stain. Immunohistochemical procedures using two, polyclonal, primary antibodies, one against Treponema pallidum and the other against Leptospira interrogans serovar copenhageni serogroup Icterohaemorrhagiae cross-reacted with spirochetal antigen/s producing a marked contrast of the fringe over the colonic epithelium, preserving the spiral-shaped morphology of the parasite. In one case with marked diarrhea, immunohistochemistry detected spirochetal antigen/s within a cell in an intestinal crypt, thus demonstrating that the infection can be more widely disseminated than suspected using routine stains. Immunohistochemical procedures, thus, greatly facilitate the histological diagnosis of intestinal spirochetosis and may contribute to a better understanding of the pathogenesis of the disease. Transmission and scanning electron microscopy performed in one case showed that the spirochete closely resembled the species designated as Brachyspira aalborgi.

The structure and reproduction of Paracoccidioides brasiliensis in human tissue

The structure and reproduction of Paracoccidioides brasiliensis (Splendore) Almeida, the causal agent of South American Blastomycosis, were studied under the electron microscope, in material taken from human tissue. The yeast-like cells of the fungus show all major cellular components. The reproduction of the organism is accomplished by two kinds of sporulation. One is represented by regular budding observed in yeasts or yeast-like organisms, with formation of chains of cells. The other is characterized by production of numerous small blastospores around the mother cell and presenting an appearance in optical median section of a fan or of a ships steering wheel.