Oral hairy leukoplakia diagnosis by Epstein-Barr virus in situ hybridization in liquid-based cytology.

OBJECTIVE: To establish a definitive diagnosis of oral hairy leukoplakia (OHL) by in situ hybridization for Epstein-Barr virus (EBV) detection with liquid-based cytology (LBC), using the ThinPrep® Pap Test, and to compare its efficacy with the traditional method of performing biopsy. METHODS: Thirty-three individuals divided into three groups were included in this study. Group 1 consisted of 15 human immunodeficiency virus (HIV)-positive patients with a clinical and histopathological diagnosis of OHL on the lateral border of the tongue. Group 2 consisted of 10 HIV-positive individuals with neither OHL nor other oral lesions. Group 3 consisted of 10 immunocompetent HIV-negative individuals with neither OHL nor other oral lesions. For each patient from the three groups, exfoliative LBC was performed on the lateral border of the tongue using ThinPrep. For the patients from group 1, a 6-mm-diameter punch biopsy was obtained from the same anatomic site as the brush collection to confirm the diagnosis of OHL by histopathology with in situ hybridization. Slides were prepared for morphological cellular analysis using Papanicolaou (Pap) staining, and for EBV detection using in situ hybridization. RESULTS: Thirteen of the 15 patients from group 1 were confirmed on punch biopsy as OHL, providing the gold standard for the study. The sensitivity of LBC followed by a Pap-stained smear was 62% and the specificity was 90%. The sensitivity of LBC followed by in situ hybridization was 100% and the specificity was 100%. CONCLUSIONS: Exfoliative LBC associated with EBV in situ hybridization is a simple, effective and non-invasive diagnostic tool for OHL.

Análise histológica da glândula paratireóide em aves de corte (Gallus gallus), em várias idades / Histological analysis of parathyroid glands in poultry (Gallus gallus) of various ages / Análisis histológica de la glándula paratiroides en aves de corte (gallus gallus), en varias edades

As paratireóides são glândulas derivadas da terceira e da quarta bolsas faringeanas, paratireóides externas e internas, respectivamente, podendo as paratireóides internas serem inexistentes nas aves ou estarem dentro, sobre ou próximas à glândula tireóide. O desequilíbrio destas glândulas é de extrema importância para a fisiologia das aves com a produção do paratormônio afetando economicamente a avicultura industrial, e assim, fazendo crescer os investimentos em pesquisas científicas nesta área. Por meio deste estudo, foi caracterizada a evolução morfológica da glândula paratireóide nas diferentes etapas de vida das aves de corte (Gallus gallus). Foram estudadas 60 aves durante 6 semanas. A cada semana foram submetidas à eutanásia 10 aves (5 machos e 5 fêmeas) e delas retirado em bloco, estruturas de ambos os lados da região cervical, próximo ao esôfago e à traquéia. Das estruturas coletadas, foram confeccionadas lâminas histológicas a fim de encontrar e analisar microscopicamente as glândulas paratireóides, uma vez que não são encontradas macroscopicamente. A análise histológica demonstrou que o parênquima da glândula paratireóide já possui um padrão bem definido a partir do 1º dia de vida da ave com a presença de células principais escuras (ativas) e de células principais claras (inativas), embora possua um menor número de células secretoras que aumenta com a idade. Estes achados demonstram que a glândula já é capaz de produzir o paratormônio deste o início da vida da ave.

The parathyroid are glands derived from the third and fourty pharyngeal bursa, external and internal parathyroid, respectively. The internal parathyroid might be non-existent in fowls or be inside, over or near the thyroid gland. The unbalance of this glands is of utmost importance for the fowls physiology with the production of the parathormone attacking the industrial aviculture economically, and therefore, expanding the investiments in scientifi c researches in this fi eld. By this means, the morphological evolution of the parathyroid gland was characterized in the various stages of life in battery fowls (Gallus gallus). During 6 weeks, 60 fowls were examined. Every week 10 folws (5 male and 5 female) were put into euthanasia and structures of both sides of the cervical region near the esophagus and the trachea were removed in blocks. From the collected structures, histological laminas were prepared in order to fi nd and anlyze the parathyroid glands microscopically. The histological analysis showed that the parenchyma of the parathyroid gland has already got a sharp-cut pattern from the fowl's 1st day of living with the presence of main dark cells (active) and main light cells (inactive), although it owns a minor number of secretory cells which increases with the age. These fi ndings show that the gland is already able to produce the parathormone since the beginning of the fowl's life.

Las glándulas paratiroides son derivadas de la tercera y cuarta bolsas faríngeas, correspondiendo a paratiroides externas e internas, respectivamente. En las aves, las paratiroides internas pueden ser inexistentes o estar dentro, sobre o junto a la glándula tiroides. El desequilibrio de esta glándula es de extrema importancia para la fi siología aviar con la producción de la paratohormona afectando económicamente la avicultura industrial y así aumentando la inversión en investigación científi ca en esta área. Por medio de esta, fue caracterizada la evolución morfológica da la paratiroides en las diferentes etapas de vida de aves de corte. Fueron estudiadas 60 aves durante seis semanas. Semanalmente fueron realizadas eutanasias de 10 aves (5 machos y 5 hembras), siendo retirado un bloque de estructuras en ambos lados de la región cervical junto al esófago y tráquea. De las estructuras retiradas, fueron realizadas láminas histológicas con el objetivo de encontrar y analizarmicroscópicamente las glándulas paratiroides, ya que no son encontradas microscópicamente. La evaluación histológica demostró que aunque el parénquima de la glándula paratiroides posea menor número de células secretoras que aumentan con la edad. Esta posee padrón defi nido a partir del primer día de vida del ave, presentando células principales oscuras (activas) e células principales claras (inactivas). Estos hallazgos demuestran que la glándula es capaz de producir paratohormona desde el inicio de vida del ave.

Value of morphological criteria in diagnosing cervical HPV lesions confirmed by in situ hybridization and hybrid capture assay.

The present study evaluated the value of morphological criteria (binucleation, multinucleation, koilocytosis, spindle koilocytes, abnormal mitosis and dyskeratosis) in the diagnosis of cervical human papillomavirus (HPV) lesions confirmed by in situ hybridization (ISH) and hybrid capture (HC) assay. Colposcopic punch biopsies from a series of 138 women with abnormal Pap smears were examined on light microscopy and in situ hybridization (DAKO widespectrum cocktail probe) for HPV-induced morphological changes and HPV DNA, respectively. Cervical swabs were analyzed for HPV DNA of the oncogenic types using Hybrid Capture. CIN 2 and CIN 3 were found in 44 biopsies, CIN 1 in 62, and no evidence of HPV in 32 cases. HPV was detected by ISH in 51/138 (37%) cases and by HC in 66/138 (48%) lesions. With both tests, HPV DNA detection increased parallel with lesion severity, up to 70% and 59% in CIN 2/3 by HC and ISH, respectively OR 4.6 (1.7-12.1) and 10.1 (3.0-33.8). Among the histological criteria, multinucleation, binucleation and abnormal mitoses were significantly associated with HPV DNA detection. Multinucleation proved to be the strongest predictor of HPV DNA-positivity. Binucleation, abnormal mitosis, koilocytosis and spindle koilocytes were also reliable criteria of HPV lesions. Minor nuclear atypia, and "mild koilocytosis" were of no value in making this diagnosis.

Serious adverse events associated with yellow fever 17DD vaccine in Brazil: a report of two cases.

BACKGROUND: The yellow fever vaccine is regarded as one of the safest attenuated virus vaccines, with few side-effects or adverse events. We report the occurrence of two fatal cases of haemorrhagic fever associated with yellow fever 17DD substrain vaccine in Brazil. METHODS: We obtained epidemiological, serological, virological, pathological, immunocytochemical, and molecular biological data on the two cases to determine the cause of the illnesses. FINDINGS: The first case, in a 5-year-old white girl, was characterised by sudden onset of fever accompanied by headache, malaise, and vomiting 3 days after receiving yellow fever and measles-mumps-rubella vaccines. Afterwards she decompensated with icterus and haemorrhagic signs and died after a 5-day illness. The second patient-a 22-year-old black woman-developed a sore throat and fever accompanied by headache, myalgia, nausea, and vomiting 4 days after yellow fever vaccination. She then developed icterus, renal failure, and haemorrhagic diathesis, and died after 6 days of illness. Yellow fever virus was recovered in suckling mice and C6/36 cells from blood in both cases, as well as from fragments of liver, spleen, skin, and heart from the first case and from these and other viscera fragments in case 2. RNA of yellow fever virus was identical to that previously described for 17D genomic sequences. IgM ELISA tests for yellow fever virus were negative in case 1 and positive in case 2; similar tests for dengue, hantaviruses, arenaviruses, Leptospira, and hepatitis viruses A-D were negative. Tissue injuries from both patients were typical of wild-type yellow fever. INTERPRETATION: These serious and hitherto unknown complications of yellow fever vaccination are extremely rare, but the safety of yellow fever 17DD vaccine needs to be reviewed. Host factors, probably idiosyncratic reactions, might have had a substantial contributed to the unexpected outcome.

Oral focal epithelial hyperplasia in a howler monkey (Alouatta fusca).

Oral focal epithelial hyperplasia is a rare and seldom reported disease in animals and humans induced by a papillomavirus. The present report is the first description of this disease in a Neotropical primate, a howler monkey (Alouatta fusca). The diagnosis was based on gross and microscopic findings. The generic papillomavirus antigen was identified by immunohistochemistry and was found not to be related to any human papillomavirus DNA tested by in situ hybridization. This virus is probably a specific papillomavirus of the howler monkey (HMPV).

In situ hybridization in paracoccidioidomycosis.

In situ hybridization (ISH) was performed using oral biopsies from patients with paracoccidioidomycosis and guinea pig testes inoculated with a culture of Paracoccidioides brasiliensis isolated from soil, employing both a 14 base-pair specific oligoprobe (ACT CCC CCG TGG TC) and its complementary sequence. When combining ISH with the Gridley stain which detects fungal cell walls, about 2-3% of the fungal cells present in the tissues were labelled. When the complementary probe was used, labelling was higher, reaching the 3% level.

Lung lesions in human leptospirosis: microscopic, immunohistochemical, and ultrastructural features related to thrombocytopenia.

Lung fragments from 12 patients were collected immediately after death and studied by light and electron microscopy and by immunohistochemistry to describe the main morphologic and ultrastructural aspects of the lung and platelets in leptospirosis (Weil's syndrome), to search for the possibility of disseminated intravascular coagulation (DIC), and to assess the relationship between endothelial lesions and local platelet aggregation and the leptospiral antigen distribution, as well as its relationship with the intensity of the lesions. The immunohistochemical results for fibrin aggregates were positive in the lumen and/or on the vascular endothelium in nine cases and on the alveolar surface in seven cases, leading to the diagnosis of the adult respiratory distress syndrome in these seven cases. Test results for leptospiral antigen by immunohistochemistry were positive in eight cases with no direct relationship between antigen deposits in the pulmonary vascular endothelium and intensity of the lesions. The ultrastructural findings were uniform and constant. Capillary lesions were characterized by swelling of endothelial cells, an increase in pinocytotic vesicles, and giant dense bodies in the cytoplasm of these cells. No necrosis, rupture, nor exposed subendothelial collagen was observed outside the hemorrhagic areas, and the intercellular junctions were preserved. The lung involvement in severe human leptospirosis presents as hemorrhagic pneumopathy with septal capillary lesions that are the usual cause of death. The thrombocytopenia that was verified in 11 of 12 patients in our study seems to bear no relationship to DIC and seems to be determined by activation, adhesion, and aggregation of platelets to the stimulated vascular endothelium, with an amorphous electron-dense substance between the endothelial cells and the adherent platelets in places where the subendothelial collagen was not exposed.

Trypanosoma cruzi strains and autonomic nervous system pathology in experimental Chagas disease.

Lesions involving the sympathetic (para-vertebral ganglia) and para-sympathetic ganglia of intestines (Auerbach plexus) and heart (right atrial ganglia) were comparatively analyzed in mice infected with either of three different strain types of Trypanosoma cruzi, during acute and chronic infection, in an attempt to understand the influence of parasite strain in causing autonomic nervous system pathology. Ganglionar involvement with neuronal destruction appeared related to inflammation, which most of the times extended from neighboring adipose and cardiac, smooth and striated muscular tissues. Intraganglionic parasitism was exceptional. Inflammation involving peripheral nervous tissue exhibited a focal character and its variability in the several groups examined appeared unpredictable. Although lesions were generally more severe with the Y strain, comparative qualitative study did not allow the conclusion, under the present experimental conditions, that one strain was more pathogenic to the autonomic nervous system than others. No special tropism of the parasites from any strain toward autonomic ganglia was disclosed.

Frequency of Candida sp infection in tuberculous patients with acquired immunodeficiency syndrome: morphological and immunocytochemical study in sputum.

Sputum samples from AIDS patients with and without pulmonary tuberculosis were analyzed morphologically and immunocytochemically to determine the frequency of occurrence of Candida sp. Mycobacterial infection was detected by bacterioscopy and/or culture and cytological evaluation was performed using Papanicolaou and Toluidine Blue staining. Immunoreaction for Candida sp was performed using polyclonal antibody in selected cases with fungal structures in smears stained by the Papanicolaou or Toluidine Blue method. An increased frequency of Candida sp (2.5 times) was observed in the tuberculous group compared to the group of AIDS patients without tuberculosis. The Toluidine Blue stain showed good results for the detection of Candida sp in sputum. Due to the increased risk of this opportunistic infection among more severely immunocompromised patients. Toluidine Blue staining of sputum samples submitted to analysis seems to be a reliable screening method.

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.

Detection of leptospiral antigen (L. interrogans serovar copenhageni serogroup Icterohaemorrhagiae) by immunoelectron microscopy in the liver and kidney of experimentally infected guinea-pigs.

Guinea-pigs were experimentally infected with L. interrogans serovar copenhageni serogroup Icterohaemorrhagiae and their liver and kidney were studied by immunoelectron microscopy using the post embedding indirect immunogold labelling technique. Primary antibody was a purified rabbit anti-serum produced against the same leptospiral strain used in the inoculum. Gold-labelled leptospiral antigen (LAg) was found close to cell membranes of hepatocytes, kidney tubular cells and endothelial cells of the interstitial capillaries of the kidney. Afterwards it was internalized by hepatic and tubular cells, and eventually found in lysosomes. Phagolysosomes of Kupffer cells were also found to contain remnants of degraded leptospires and gold-labelled LAg. Gold-labelled intact leptospires were detected at the enlarged intercellular spaces between hepatocytes at the areas of hepatic cell plate disarray, showing the potential for leptospiral migration during the septicaemic phase of the disease potentially contributing to the pathogenesis of the lesions. The affinity of leptospiral antigenic material for cell membranes suggests an initial interaction with cell surface proteins followed by its internalization and cell damage. The nature of antigenic material detected, however, remains undefined; it may be a toxin, an enzyme or any other factor/s involved in leptospiral virulence.

Human fatal yellow fever. Immunohistochemical detection of viral antigens in the liver, kidney and heart.

An immunohistochemical method to detect yellow fever antigen was developed using immune sera from rabbits and hamsters and hyperimmune ascitic fluid from mice. A search for the antigen was carried out in liver, kidney and heart in three fatal cases of yellow fever. In the liver it was present in the cytoplasm of hepatocytes, Councilman bodies and Kupffer cells. Yellow fever antigen was also detected in renal tubular epithelium and in groups of myocardial fibers. These findings suggest that viral replication occurs at sites other than the liver. Since yellow fever shares many features with other haemorrhagic fevers the use of immunohistochemistry can impart a significant improvement in the accuracy of its histopathological diagnosis.

Use of monoclonal antibodies against human T cells for clinical diagnosis by immunohistochemical procedures.

Monoclonal antibodies (Mabs) were produced against human T cell membrane antigens. Sixteen Mabs were studied and six were selected for immunohistochemical assays on paraffin-embedded tonsil sections. Two Mabs (2D7 and 1E2) specifically recognized T-lymphocyte areas in sections of pathological tissues originating from lymphoproliferative diseases, and reacted with proteins of approximately 80 kDa. Most of the Mabs produced thus far are only suitable for immunohistochemical assays on frozen section. Only a few Mabs recognize lymphoid markers on paraffin-embedded sections, a procedure which permits a more extensive and practical application of Mabs in clinical diagnosis. These antibodies should be valuable in diagnosing T cell-related diseases and their large scale production should reduce laboratory costs because all reagents currently available are imported.

Use of monoclonal antibodies against human T cells for clinical diagnosis by immunohistochemical procedures

Monoclonal antibodies (Mabs) were produced against human T cell membrane antigens. Sixteen Mabs were studied and six were selected for immunohistochemical assays on parffin-embedded tonsil section. Two Mabs (2D7 and 1E2) specifically recognized T-lymphocyte areas in sections of pathological tissues originating from lymphoproliferative diseases, and reacted with proteins of approximately 80 KDa. Most of the Mabs produced thus far are only suitable for immunohistochemical assays on frozen section. Only a few Mabs recognize lymphoid markers on paraffin-embedded sections, a procedure which permits a more extensive and practical application of Mabs in clinical diagnosis. These antibodies should be baluable in diagnosing. T. cell-related diseases and their large scale production should reduce laboratory costs because all reahgents currently avaliable are imported

[High resolution light microscopy: adaptation of the method and its use in the study of experimental leptospirosis in guinea pigs]. / Microscopia óptica de alta resolução: adaptação do método e sua aplicação ao estudo da leptospirose experimental em cobaias.

Morphological lesions in parenchimal and mesenchimal structures of liver and kidney were studied in guinea-pigs experimentally infected with Leptospira interrogans serogroup icterohaemorrhagiae in comparison with a group of non-infected guinea-pigs. All specimens were submitted to conventional light microscopy as well as to high resolution light microscopy, in one micrometer sections of tissue embedded in glycolmethacrylate. High resolution light microscopy, applied for the first time in leptospirosis, was proved very useful, since it enabled us to visualize cellular structures in the same slide used for panoramic view. Cell cohesion, brush borders, pynocytotic vesicles and organellae distributions were parameters especially suitable for analysis at this low-cost, highly precise procedure in microscopy.

Glicolipoproteína de Leptospira interrogans sorogrupo icterohaemorrhagiae: distribuiçäo em fígado e rim de cobaias experimentalmente infectadas / Glycoproteins from Leptospira interrogans serogroup icterohaemorrhagiae: distribution in the liver and kidney of experimentally infected guinea pigs

Acredtita-se que as lesöes teciduais na leptospirose possam decorrer da açäo direta das leptospiras, de toxinas sintetizadas ou liberadas durante sua lise. O presente estudo visou a extraçäo química da glicolipoproteína (GLP) da aleptospira, a produçäo de anti-soro anti-GLP e a avaliaçäo de sua distribuiçäo em cortes de fígado e rim de cobaias inoculadas e sacrificadas em estudo sequencial diário até o 6§ dia de infecçäo, correspondente ao pico da doença. Procurou-se também correlacionar a expressäo tecidual da GLP com o grau de lesöes locais, em busca de novos subsídios para a compreensäo da patogenia da leptospiros. A GLP foi detectada em fígado e rim de 2 dentre 6 cobaias no 5§ dia e em todas as 6 no 6§ dia de infecçäo, sob a forma de grânulos no citoplasma de macrófagos, livres no interstício ou acolados à membrana de células endoteliais e parenquimatosas, especialmente nas regiöes mais lesadas. A cronologia do aparecimento da GLP e sua distribuiçäo sugerem tratar-se de produto de lise de leptospiras fagocitadas por macrófagos e que esta substância, conquanto näo comprovada como iniciadora das lesöes, asocia-se a seu agravamento nas etapas mais avançadas da leptospirose

[Glycoproteins from Leptospira interrogans serogroup icterohaemorrhagiae: distribution in the liver and kidney of experimentally infected guinea pigs]. / Glicolipoproteína de Leptospira interrogans sorogrupo icterohaemorrhagiae: distribuição em fígado e rim de cobaias experimentalmente infectadas.

Tissue damage in leptospirosis has been ascribed to direct effect of the microorganisms and/or their virulence, including products synthetized by leptospires or released during their lysis. This study aimed at chemical extraction of the glycolipoprotein (GLP) from virulent leptospires, production of a rabbit anti-GLP and analysis of its distribution in liver and kidney of inoculated guinea-pigs, sacrificed sequentially from the 1st to 6th day of infection, covering the whole, spectrum of acute leptospirosis. The comparison of GLP expression to local injuries aimed at new pathogenetic data. GLP was detected in liver and kidney in 2 out of 6 guinea-pigs on the 5th day and in all 6 animals on the 6th day of infection. Granular forms were seen in the cytoplasm of macrophages, free in interstitium or adhered to endothelial and parenchymal cell membranes, especially in the most damaged sites. These findings lead us to the hypothesis of GLP as a toxic factor resulting from leptospiral lysis by macrophages. Although it was not proved as a promoter of initial lesions, it seems to be related to the enhancement of tissue damage late in the course of the disease.

Nesidioblastosis associated with congenital malformations of the heart. Morphological and immunohistochemical study of 5 necropsy cases.

Pancreatic tissue from 5 children with congenital heart disease (CHD) with morphological alterations of the endocrine components at autopsy were compared in a double-blind semiquantitative study, which included light microscopy and immunocytochemistry, to pancreatic tissue of 5 children of similar age but without CHD. Hyperplasia and hypertrophy of islets of Langerhans, scattered endocrine cells within the exocrinic acini, cluster of endocrine cells budding from ductular epithelium or within interlobular connective tissue, and hyperplasia of ductular epithelium were found to be present in the patients with congenital cardiac defects. The observed findings resembled those present in a surgically resected pancreas from a child with clinically overt nesidioblastosis. Although this study suggests a higher incidence of endocrine pancreas disturbances in certain types of CHD, the clinical significance of these morphological alterations will require further studies.