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1.
Aquat Toxicol ; 178: 106-17, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27474942

RESUMO

Accumulation and toxicity of cyanobacterial toxins, particularly microcystin-LR (MCLR) have been extensively studied in fish and aquatic invertebrates. However, MCLR excretion mechanisms, which could reduce this toxin's effects, have received little attention. The Patagonian silverside, Odontesthes hatcheri, is an omnivorous-planktivorous edible fish, which has been shown to digest cyanobacterial cells absorbing MCLR and eliminating the toxin within 48h without suffering significant toxic effects. We studied the effects of MCLR on glycoconjugate composition and the possible role of multidrug resistance associated proteins (Abcc) in MCLR export from the cells in O. hatcheri intestine. We treated O. hatcheri with 5µg MCLRg(-1) body mass administered with the food. Twenty four hours later, the intestines of treated and control fish were processed for lectin-histochemistry using concanavalin A (ConA), Triticum vulgaris agglutinin (WGA), and Dolichos biflorus agglutinin (DBA). MCLR affected the distribution of glycoconjugates by augmenting the proportion of ConA-positive at the expense of WGA-positive cells. We studied MCLR effects on the transport of the Abcc-like substrates 2,4-dinitrophenyl-S-glutathione (DNP-SG) and calcein in ex vivo intestine preparations (everted and no-everted sacs and strips). In treated preparations, CDNB together with MCLR (113µg MCLRg(-1) intestine, equivalent to 1.14µmolL(-1) when applied in the bath) or the Abcc inhibitor, MK571 was applied for one hour, during which DNP-SG was measured in the bath every 10min in order to calculate mass-specific DNP-SG transport rate. MCLR significantly inhibited DNP-SG transport (p<0.05), especially in middle intestine (47 and 24%, for luminal and serosal transport, respectively). In middle intestine strips, MCLR and MK571inhibited DNP-SG transport in a concentration dependent fashion (IC50 3.3 and 0.6µmolL(-1), respectively). In middle intestine strips incubated with calcein-AM (0.25µmolL(-1)), calcein efflux was inhibited by MCLR (2.3µmolL(-1)) and MK571 (3µmolL(-1)) by 38 and 27%, respectively (p<0.05). Finally, middle intestine segments were incubated with different concentrations of MCLR applied alone or together with 3µM MK571. After one hour, protein phosphatase 1 (PP1) activity, the main target of MCLR, was measured. 2.5µM MCLR did not produce any significant effect, while the same amount plus MK571 inhibited PP1 activity (p<0.05). This effect was similar to that of 5µM MCLR. Our results suggest that in O. hatcheri enterocytes MCLR is conjugated with GSH via GST and then exported to the intestinal lumen through Abcc-like transporters. This mechanism would protect the cell from MCLR toxicity, limiting toxin transport into the blood, which is probably mediated by basolateral Abccs. From an ecotoxicological point of view, elimination of MCLR through this mechanism would reduce the amount of toxin available for trophic transference.


Assuntos
Transporte Biológico/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Microcistinas/toxicidade , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Smegmamorpha/metabolismo , Poluentes Químicos da Água/toxicidade , Animais , Concanavalina A/metabolismo , Fluoresceínas/metabolismo , Glutationa/metabolismo , Glicosilação/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Toxinas Marinhas , Microscopia de Fluorescência , Lectinas de Plantas/metabolismo , Propionatos/toxicidade , Quinolinas/toxicidade
2.
Tissue Cell ; 48(4): 383-8, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27233914

RESUMO

Paneth cells are secretory epithelial cells of the innate immune system of the intestine of several mammals, including alpacas. Little is known about the latter; thus, in the present study we described the morphology and histochemical characteristics of Paneth cells in healthy fetuses, and young and adult alpacas. For this purpose, samples of duodenum, jejunum and ileum were taken from 6 fetuses at different days of pregnancy (between days 221-330), 66 offsprings (between 0 and 45-days-old) and 5 adult alpacas (>2-years-old). Samples were fixed in 10% buffered formalin and processed for histological and morphometrical analysis using HE and Masson Trichomics technique. Immunohistochemistry was used to identify Paneth cells using anti-lysozyme antibody. In addition, the lectinhistochemichal binding-pattern of Paneth cells granules was evaluated. Lyzozyme was immunohistochemically detected in the granules of Paneth cells from day 283 of pregnancy in all the small intestinal sections of the studied fetuses. In newborn alpacas Paneth cells were initially found in the duodenum, but the following days (days 18-21 after birth) they were also found in the ileum. Their size gradually increased after birth, but then no significant differences were found. In adult alpacas the number was lower than offsprings. We suggest that Paneth cells early differentiate in the small intestine of alpacas, and the increase in their number during the first two weeks of life strongly support their possible involvement in the intestinal defensive functions against the enteric diseases that occur during the lactancy stage.


Assuntos
Camelídeos Americanos , Feto/ultraestrutura , Celulas de Paneth/ultraestrutura , Animais , Grânulos Citoplasmáticos/ultraestrutura , Duodeno/ultraestrutura , Feminino , Íleo/ultraestrutura , Jejuno/ultraestrutura , Celulas de Paneth/metabolismo , Gravidez
3.
Rev. biol. trop ; 64(1): 327-340, ene.-mar. 2016. tab, ilus
Artigo em Inglês | LILACS | ID: biblio-843281

RESUMO

AbstractThe Neotropical catfish Corydoras paleatus is a facultative air-breather and the caudal half of the intestine is involved in gas exchange. In South America, air-breathing fishes are found in tropical or sub-tropical freshwaters where the probability of hypoxia is high. The aim of this study was to characterize by traditional histochemical and lectinhistochemical methods the pattern of carbohydrate in the intestinal mucosa. Intestine samples were taken from 25 healthy adult specimens collected in Buenos Aires (Argentina). Samples were fixed by immersion in 10 % buffered formalin and routinely processed and embedded in paraffin wax. Subsequently, these sections were incubated in the biotinylated lectins battery. Labeled Streptavidin-Biotin (LSAB) system was used for detection, diaminobenzidine as chromogen and haematoxylin as a contrast. To locate and distinguish glycoconjugates (GCs) of the globet cells, we used the following histochemical methods: PAS; PAS*S; KOH/ PA*S; PA/Bh/KOH/PAS; KOH/PA*/Bh/PAS; Alcian Blue and Toluidine Blue at different pHs. Microscopically, the general structure of vertebrate intestine was observed and showed all the cell types characteristic of the intestinal epithelium. The cranial sector of catfish intestine is a site of digestion and absorption and its structure is similar to other fish groups. In contrast, enterocytes of the caudal portion are low cuboidal cells; and between these, globet cells and capillaries are observed, these latter may reach the mucosal lumen. Underlying the epithelium, observed a well-developed lamina propria-submucosa made of connective tissue; this layer was highly vascularized and did not exhibit glands. According to histochemistry, the diverse GCs elaborated and secreted in the intestine are associated with specific functions in relation to their physiological significance, with special reference to their role in lubrication, buffering effect and prevention of proteolytic damage to the epithelium together with other biological processes, such as osmoregulation and ion exchange. The lectinhistochemical analysis of the intestinal mucosa reveals the presence of terminal residues of glucose, mannose and galactose. In conclusion, this study has shown that GCs synthesized in the intestine of C. paleatus exhibit a high level of histochemical complexity and that the lectin binding pattern of the intestinal mucosa is characteristic of each species and the variations are related with the multiple functions performed by the mucus in the digestive tract. The information generated here may be a relevant biological tool for comparing and analyzing the possible glycosidic changes in the intestinal mucus under different conditions, such as changes in diet or different pathological stages. Rev. Biol. Trop. 64 (1): 327-340. Epub 2016 March 01.


ResumenEl pez neotropical Corydoras paleatus, de respiración aérea de tipo facultativa, utiliza el sector caudal del intestino para el intercambio gaseoso. En América del Sur, los peces con respiración aérea se encuentran en las aguas dulceacuícolas tropicales y subtropicales, donde la probabilidad de hipoxia es alta. El objetivo de este trabajo fue caracterizar mediante técnicas histoquímicas tradicionales y de lectinhistoquímica el patrón de carbohidratos de la mucosa intestinal. Para ello se utilizaron muestras de intestino de 25 ejemplares sanos adultos recolectados en la provincia de Buenos Aires (Argentina). Las muestras fueron fijadas en formol amortiguado al 10 % y se procesaron para su inclusión en parafina. Posteriormente, los cortes fueron incubados con una batería de lectinas biotiniladas. Se utilizó el sistema de marcado con estreptavidina-biotina (LSAB) para su detección, diaminobencidina como cromógeno y hematoxilina como colorante de contraste. Para localizar y diferenciar los glicoconjugados (GCs) de las células caliciformes, se utilizaron las siguientes técnicas histoquímicas: PAS, PAS*S, PAPS, KOH/PA*S, PA/Bh/KOH/PAS, KOH/PA*/Bh/PAS, Azul Alcian y Azul de Toluidina a diferentes pHs. Microscópicamente, se observa la estructura general del intestino de los vertebrados y el epitelio intestinal presenta todos los tipos celulares característicos de esta región. El sector craneal del intestino de este teleósteo, es el sitio de digestión y absorción, y posee una estructura similar a la de otros grupos de peces. En cambio, los enterocitos de la porción caudal, son células cúbicas bajas, entre ellos se observan células caliciformes y capilares sanguíneas que llegan hasta el lumen de la mucosa. Por fuera del epitelio, se observa una lámina propia-submucosa muy desarrollada compuesta por tejido conectivo, altamente vascularizada que no presenta glándulas. De acuerdo con las técnicas histoquímicas, los diversos GCs elaborados y secretados por la mucosa intestinal se encuentran asociados con funciones específicas de importancia fisiológica, como su rol en la lubricación, su efecto amortiguador y la prevención de daños proteolíticos del epitelio junto con otros procesos biológicos, tales como la osmorregulación y el intercambio iónico. El análisis lectinhistoquímico de la mucosa intestinal revela la presencia de residuos terminales de glucosa, manosa y galactosa. En conclusión, en este estudio se demuestra que los GCs sintetizados en el intestino de C. paleatus muestran un alto nivel de complejidad histoquímica y que el patrón de unión de lectina de la mucosa intestinal es característico para cada especie y las variaciones se hallan relacionadas con las múltiples funciones realizadas por el mucus en el tracto digestivo. La información brindada en este trabajo es una herramienta de relevancia biológica para comparar y analizar los posibles cambios glicosídicos del mucus intestinal bajo diferentes condiciones como los cambios en la dieta o diferentes estados patológicos.


Assuntos
Animais , Masculino , Feminino , Peixes-Gato/classificação , Glicoconjugados/análise , Intestinos/química , Histocitoquímica , Mucosa Intestinal/citologia , Mucosa Intestinal/química , Intestinos/citologia
4.
Microsc Res Tech ; 77(11): 910-7, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25091902

RESUMO

The extra-cellular matrix of fertilized eggs in the bufonid toads Bufo bufo and Bufotes balearicus was studied to clear the relationships between structural and molecular diversity. Histochemical (PAS, AB pH 2.5 and pH 1.0, Beta-elimination PAS) and lectin-histochemical (Con A, WGA, Succinyl-WGA, PNA, RCA-1, DBA, SBA, AAA, UEA-I, LTA) techniques were used and the observations were made under light and electron microscopy. Both species present a fertilization envelope (FE) and two jelly layers (J1 and J2). The fibers of J2 are shared among the eggs of a clutch in a jelly ribbon. The FE of both species presents neutral glycoproteins, mostly N-linked. In B. bufo there are also residuals of mannose and/or glucose and N-acetylglucosamine. In the FE fibers run parallel to egg's surface or are in bundles or looser hanks with no clear orientation. The J1 layer of both species presents sialosulfoglycoproteins, mostly O-linked, with lactosaminylated, galactosaminylated, glycosaminylated, and fucosylated residuals. A lower amount of galactosaminylated residuals is observed in B. balearicus in respect to B. bufo, whereas the opposite is seen in the amount of fucosylated residuals. The J2 layer is similar in composition to J1 but in B. balearicus there are no glucosaminylated residuals. J layers present fibers and granules that reduce towards J2 . Several microorganisms, in particular blue algae, are observed in the J2 layer of both species. In respect to other species, B. bufo and B. balearicus have a lower number of jelly layers, but a comparable number of glycan types.


Assuntos
Bufo bufo/anatomia & histologia , Bufonidae/anatomia & histologia , Matriz Extracelular/ultraestrutura , Zigoto/ultraestrutura , Animais , Bufo bufo/metabolismo , Bufonidae/metabolismo , Lectinas/metabolismo , Microscopia , Microscopia Eletrônica de Transmissão , Zigoto/metabolismo
5.
Rev. MVZ Córdoba ; 18(3): 3753-3758, set.-dic. 2013. ilus, tab
Artigo em Inglês | LILACS | ID: lil-700559

RESUMO

Objetive. This study was conducted to evaluate, by means of lectinhistochemistry (LHC), the expression of carbohydrates in granulomas induced by the bacillus Calmette-Guerin (BCG) in muscle tissue of Piaractus mesopotamicus after 33 days. Material and methods. Histological sections with 3 μm thick were incubated with the following lectins :WGA (Wheat germ agglutinin), DBA (Dolichos biflorus agglutinin) and HPA (Helix pomatia agglutinin), and the results were evaluated by light microscopy. Results. Acid fast bacilli were stained by Ziehl Neelsen (ZN) and strong labeled by WGA in the cytoplasm of macrophages. Labeling with DBA was intense in fibroblasts and weak in macrophages. On the other hand, HPA binding was stronger in macrophages, especially in those that were in close contact with epithelioid cells, without evidence of binding to fibroblasts. The epithelioid cells were not labeled by the used lectins, but they were identified by Hematoxilin-Eosin (HE). The lectins labeled specific type saccharides in glycoproteins, as N-acetylglucosamine present in bacilli and macrophages, as well as N-acetyl-galactosamine in macrophages. The control group showed no inflammation or lectin binding. Conclusions. This technique may be useful in identifying receptors for WGA, DBA and the HPA lectins in epithelioid granuloma induced by BCG in P. mesopotamicus.


El presente estudio fue realizado para evaluar por medio de lectinhistoquímica (LHC), la expresión de carbohidratos en granulomas inducidos por el bacilo de Calmette-Guérin (BCG) en músculo de Piaractus mesopotamicus después de 33 días. Materiales y métodos. Cortes histológicos de 3 µm de grosor fueron incubados con las siguientes lectinas: WGA (Wheat germ aglutinin), DBA (Dolichos biflorus agglutin) y HPA (Helix pomatia agglutinin), y los resultados evaluados por medio de microscopia de luz. Resultados. Bacilos ácido resistentes fueron identificados por la tinción de Ziehl Neelsen(ZN). Se observó un marcaje intenso con WGA en el citoplasma de macrófagos. El marcaje con DBA fue intenso en fibroblastos y débil en macrófagos. Con la lectina HPA el marcaje fue intenso en macrófagos, principalmente en los que estaban en estrecho contacto con las células epitelióides, externamente se observó marcaje débil en fibroblastos. Las células epitelióides no fueron marcadas por las lectinas, pero fueron identificadas con la tinción de Hematoxilina-Eosina (HE). Las lectinas tuvieron un tipo de marcaje específico en algunos monosacáridos, como N-acetilglucosamina presente en los bacilos y en macrófagos, y N-acetilgalactosamina en macrófagos. En el grupo control no fue observada inflamación así como tampoco marcaje con las lectinas. Conclusiones. Esta técnica resultó eficiente en la identificación de receptores para las lectinas WGA, DBA y HPA en el granuloma epitelióide inducido por BCG en P. mesopotamicus.


Assuntos
Humanos , Animais , Granuloma , Mycobacterium , Polissacarídeos
6.
Pesqui. vet. bras ; 32(3): 227-230, Mar. 2012. ilus, tab
Artigo em Inglês | LILACS | ID: lil-624114

RESUMO

In Brazil, the consumption of Sida carpinifolia by livestock has been associated with neurological diseases linked to lysosomal storage disorders. This paper describes the pathological findings in two caprine fetuses from dams that were experimentally poisoned with S. carpinifolia. The goats were orally dosed with 10 and 13g/kg of a paste of green chopped S. carpinifolia for 30 days and were observed for an additional 15 days period after the last dosage with the plant; thereafter they were euthanized and necropsied. The dams showed only slight clinical signs. The study also includes the findings in one bovine fetus from a naturally S. carpinifolia poisoned cow which showed mild incoordination, generalized tremors, staggering, and frequent falls. The cow was euthanized and necropsied. While there were no significant histopathological changes in the goats, in the cow vacuolation of Purkinje neurons of the cerebellum, pancreatic acinar cells, and thyroid follicular cells were observed. The main microscopic changes observed in the caprine and bovine fetuses were vacuolation in the epithelium of renal tubules, thyroid follicular cells, and Purkinje neurons of the cerebellum. Transmission electron microscopy of sections from CNS of the cow and its fetus revealed vacuoles containing fine granular material surrounded by membrane. Lectin-histochemistry of CNS sections from goat fetuses marked lightly to sWGA lectins, WGA, and Con-A.


No Brasil, o consumo de Sida carpinifolia por animais de produção tem sido associada a doenças neurológicas relacionadas com doença de depósito lisossômico. Este trabalho descreve os achados patológicos observados em dois fetos caprinos de mães que foram experimentalmente intoxicadas por S. carpinifolia. As cabras foram intoxicadas experimentalmente com S. carpinifolia nas doses de 10 e 13g/kg durante 30 dias e foram acompanhadas durante 15 dias após o consumo da planta. Após este período foram eutanasiadas e necropsiadas. O estudo também inclui os achados patológicos encontrados em um feto de uma fêmea bovina intoxicada naturalmente pela planta, que mostrou leve incoordenação, tremores generalizados, andar desequilibrado e quedas frequentes. A vaca foi eutanasiada e necropsiada. Embora não houvesse alterações histológicas significativas nas cabras, vacuolização dos neurônios de Purkinje do cerebelo, das células acinares do pâncreas e nas células foliculares da tireoide foram observadas na vaca. As principais alterações histológicas observadas nos fetos caprinos e no feto bovino foram vacuolização no epitélio dos túbulos renais, nas células foliculares da tireoide e nos neurônios de Purkinje do cerebelo. Na microscopia eletrônica de transmissão do sistema nervoso central da vaca e de seu feto revelaram-se vacúolos contendo material finamente granulado e delimitado por membrana. Na técnica de lectina-histoquímica dos fetos caprinos houve marcação leve no SNC para as lectinas sWGA, WGA e para Con-A.


Assuntos
Animais , Bovinos/embriologia , Feto/fisiopatologia , Malvaceae/intoxicação , Ovinos/embriologia , Swainsonina/intoxicação , Autopsia/veterinária , Células Epiteliais/patologia , Estruturas Celulares/patologia , Microscopia Eletrônica de Transmissão/veterinária
7.
Pesqui. vet. bras ; 29(3): 266-274, mar. 2009. ilus, graf, tab
Artigo em Inglês | LILACS | ID: lil-513302

RESUMO

Solanum glaucophyllum (Sg) [= S. malacoxylon] is a calcinogenic plant inducing "Enzootic Calcinosis" in cattle. The 1,25-dihydroxyvitamin D3, its main toxic principle, regulates bone and calcium metabolism and also exerts immunomodulatory effects. Thymocyte precursors from bone marrow-derived progenitor cells differentiate into mature T-cells. Differentiation of most T lymphocytes is characterized not only by the variable expression of CD4/CD8 receptor molecules and increased surface density of the T cell antigen receptor, but also by changes in the glycosylation pattern of cell surface glycolipids or glycoproteins. Thymocytes exert a feedback influence on thymic non-lymphoid cells. Sg-induced modifications on cattle thymus T-lymphocytes and on non-lymphoid cells were analysed. Heifers were divided into 5 groups (control, intoxicated with Sg during 15, 30 or 60 days, and probably recovered group). Histochemical, immunohistochemical, lectinhistochemical and morphometric techniques were used to characterize different cell populations of the experimental heifers. Sg-poisoned heifers showed a progressive cortical atrophy that was characterized using the peanut agglutinin (PNA) lectin that recognizes immature thymocytes. These animals also increased the amount of non-lymphoid cells per unit area detected with the Picrosirius technique, WGA and DBA lectins, and pancytokeratin and S-100 antibodies. The thymus atrophy found in intoxicated animals resembled that of the physiological aging process. A reversal effect on these changes was observed after suppression of the intoxication. These findings suggest that Sg-intoxication induces either directly, through the 1,25-dihydroxyvitamin D3 itself, or indirectly through the hypercalcemia, the observed alteration of the thymus.


Solanum glaucophyllum (Sg) [= S. malacoxylon] é uma planta calcinogênica que induz "Calcinose Enzoótica" em bovinos. O 1,25-dihidroxivitamina D3, seu principal agente tóxico, regula o metabolismo ósseo, o metabolismo de cálcio e também mostra efeitos na imunomodulação. Precursores de timócitos derivados da medula óssea se diferenciam em linfócitos T maduros. A diferenciação da maioria dos linfócitos T é caracterizada pela expressão variável de moléculas de receptores CD4/CD8 e densidade aumentada dos receptores antigênicos de superfície de células T. Alem disso, há mudanças no padrão de glicosilação de glicolipídeos na superfície celular ou de glicoproteínas. Timócitos mostram uma influência de retro alimentação em células tímicas não-linfóides. Foram analisadas modificações induzidas pelo Sg em linfócitos T e células tímicas não-linfóides de bovinos. Novilhas foram divididas em 5 grupos (controle, intoxicadas com Sg durante 15, 30 ou 60 dias, e grupo provavelmente recuperado). As diferentes populações celulares das novilhas experimentais foram caracterizadas com técnicas histoquímicas, imuno-histoquímicas, lectina-histoquímicas e morfométricas. As novilhas intoxicadas com Sg mostraram uma atrofia cortical progressiva que foi caracterizada usando a lectina aglutinina de amendoim (PNA) que reconhece timócitos imaturos. Estes animais também aumentaram as células não-linfóides tímicas por unidade de área, detectadas com a técnica de Picrosirius, lectinas WGA e DBA e anticorpos antipancitoqueratina e anti-S-100. A atrofia de timo observada nos animais intoxicados foi semelhante àquela do processo de envelhecimento fisiológico. Após supressão da intoxicação, foi observado um efeito de reversão nestas mudanças. Estes resultados sugerem que a intoxicação por Sg induza a alteração observada no timo diretamente, pela ação de 1,25-dihidroxivitamina D3, ou indiretamente, pela ação da hipercalcemia.


Assuntos
Animais , Calcinose , Bovinos , Imuno-Histoquímica , Plantas Tóxicas/efeitos adversos , Solanum glaucophyllum/toxicidade , Timo/patologia
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