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We studied the effect of myriocin, an inhibitor of serine palmitoyltransferase, on cultured Leishmania (Viannia) braziliensis promastigotes. Myriocin significantly reduced synthesis of inositol phosphorylceramide, the major sphingolipid expressed in promastigotes as characterized by thin layer chromatography and electrospray ionization mass spectrometry. Log-phase promastigotes treated with 1 µM myriocin showed a 52% reduction in growth rate and morphological alterations such as more rounded shape and shorter flagellum. Promastigotes treated with myriocin also displayed a variety of aberrant cell phenotypes. The percentage of cells with one nucleus and one kinetoplast (1N1K), following treatment with 1 or 5 µM myriocin, decreased from 89% (control value) to 27% or 3%, respectively. The percentage of cells with two nuclei (2N2K) varied from 7% (control value) to 19% and 6% for 1 or 5 µM myriocin-treated parasites, respectively. High percentage of myriocin-treated parasites exhibited large atypical cells presenting three or more nucleus (32% and 89% for 1 or 5 µM myriocin, respectively). Transmission electron microscopy following treatment with 1 µM myriocin showed the presence of 4N parasites possibly as a result of an incomplete cytokinesis. Addition of 3-ketodihidrosphingosine to myriocin-treated promastigotes rescue parasite growth and morphology. Addition of ethanolamine did not rescue the myriocin effect on parasite. Our findings indicate that sphingolipids are essential for the completion of cytokinesis, and may play a major role in cell proliferation in L. (V.) braziliensis, thus, differing from data described for Leishmania major sphingolipid-free mutant, where addition of ethanolamine rescue wild-type parasite characteristics.

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OBJECTIVES: To describe HIV children's small intestinal ultrastructural findings. METHODS: Descriptive, observational study of small intestine biopsies performed between August 1994 and May 1995 at São Paulo, SP, Brazil. This material pertained to 11 HIV infected children and was stored in a laboratory in paraffin blocks. Scanning and transmission electronic microscopy were used to view those intestine samples and ultrastructural findings were described by analyzing digitalized photos of this material. Ethical Committee approval was obtained. RESULTS: In most samples scanning microscopy showed various degrees of shortening and decreasing number of microvilli and also completes effacements in some areas. Derangement of the enterocytes was seen frequently and sometimes cells well defined borders limits seemed to be loosened. In some areas a mucous-fibrin like membrane with variable thickness and extension appeared to partially or totally coat the epithelial surface. Fat drops were present in the intestinal lumen in various samples and a bacterium morphologically resembling bacilli was seen in two occasions. Scanning microscopy confirmed transmission microscopy microvilli findings and also showed little "tufts" of those structures. In addition, it showed an increased number of vacuoles and multivesicular bodies inside various enterocytes, an increased presence of intraepithelial lymphocytes, mitochondrial vacuolization and basement membrane enlargement in the majority of samples analyzed. However, some samples exhibited normal aspect. CONCLUSIONS: Our study showed the common occurrence of various important intestinal ultrastructural alterations with variable degrees among HIV infected children, some of them in our knowledge not described before.

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Objectives To describe HIV children's small intestinal ultrastructural findings. Methods Descriptive, observational study of small intestine biopsies performed between August 1994 and May 1995 at São Paulo, SP, Brazil. This material pertained to 11 HIV infected children and was stored in a laboratory in paraffin blocks. Scanning and transmission electronic microscopy were used to view those intestine samples and ultrastructural findings were described by analyzing digitalized photos of this material. Ethical Committee approval was obtained. Results In most samples scanning microscopy showed various degrees of shortening and decreasing number of microvilli and also completes effacements in some areas. Derangement of the enterocytes was seen frequently and sometimes cells well defined borders limits seemed to be loosened. In some areas a mucous-fibrin like membrane with variable thickness and extension appeared to partially or totally coat the epithelial surface. Fat drops were present in the intestinal lumen in various samples and a bacterium morphologically resembling bacilli was seen in two occasions. Scanning microscopy confirmed transmission microscopy microvilli findings and also showed little “tufts” of those structures. In addition, it showed an increased number of vacuoles and multivesicular bodies inside various enterocytes, an increased presence of intraepithelial lymphocytes, mitochondrial vacuolization and basement membrane enlargement in the majority of samples analyzed. However, some samples exhibited normal aspect. Conclusions Our study showed the common occurrence of various important intestinal ultrastructural alterations with variable degrees among HIV infected children, some of them in our knowledge not described before. .

Objetivos Descrever achados ultra-estruturais do intestino delgado de crianças infectadas pelo HIV. Métodos Estudo descritivo, observacional de biopsias do intestino delgado, realizada entre agosto de 1994 e maio de 1995 em São Paulo - Brasil. Este material pertencia a 11 crianças infectadas pelo HIV e foi armazenado em um laboratório em blocos de parafina. As amostras de intestino delgado foram analisadas por microscopia eletrônica de transmissão e de varredura e achados os achados ultra-estruturais foram descritos por meio da análise de fotos digitalizadas desse material. Foi obtida aprovação pelo Comitê de Ética. Resultados Na maioria das amostras a microscopia de varredura mostrou vários graus de encurtamento e diminuição do número das microvilosidades e até o completo apagamento dessas estruturas em algumas áreas. O desarranjo dos enterócitos foi visto com freqüência e, por vezes, os limites celulares estavam imprecisos. Em algumas áreas uma membrana fibrino-mucosa com espessura e extensão variáveis aparentava revestir parcial ou totalmente a superfície epitelial. Gotas de gordura no lúmen intestinal estavam presentes em várias amostras e bactérias morfologicamente semelhantes a bacilos foram observadas em duas amostras. A microscopia eletrônica de varredura confirmou as observações constatadas nas microvilosidades através da microscopia de transmissão e também mostrou pequenos “tufos” dessas estruturas. Além disso, mostrou aumento do número de vacúolos e de formações multivesiculares dentro de vários enterócitos, aumento da presença de linfócitos intraepiteliais, vacuolização mitocondrial e alargamento da membrana basal na maioria das amostras analisadas. ...

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OBJECTIVE: Morphological study that searched to authenticate the presence of sinoaortic baroreceptor inputs within the dorsolateral medullary nucleus under electron microscopy analysis. METHODS: After a 5-day survival period, 9 baroreceptor-denervated rats deeply anaesthetized with equithesin were transcardially perfused and their brains were histologically processed. RESULTS: The neuronal cytoarchitecture of the paratrigeminal nucleus comprehends afferent projections from other nuclei that have a distributive character regarding visceral and nociceptive functions in the cardiovascular reflex integration response. CONCLUSION: The medial portion of the nucleus receives afferent projections of the rostral ventrolateral medulla, as shown by retrograde neurotracing studies. The present results show that the medial extent of the paratrigeminal nucleus contains degenerated axoplasmic cellular components in sinoaortic deafferented rats. The number of degenerated axonal fibers was also larger in this area of the nucleus.

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Objective: Morphological study that searched to authenticate the presence of sinoaortic baroreceptor inputs within the dorsolateral medullary nucleus under electron microscopy analysis. Methods: After a 5-day survival period, 9 baroreceptor-denervated rats deeply anaesthetized with equithesin were transcardially perfused and their brains were histologically processed. Results: The neuronal cytoarchitecture of the paratrigeminal nucleus comprehends afferent projections from other nuclei that have a distributive character regarding visceral and nociceptive functions in the cardiovascular reflex integration response. Conclusion: The medial portion of the nucleus receives afferent projections of the rostral ventrolateral medulla, as shown by retrograde neurotracing studies. The present results show that the medial extent of the paratrigeminal nucleus contains degenerated axoplasmic cellular components in sinoaortic deafferented rats. The number of degenerated axonal fibers was also larger in this area of the nucleus.

Objetivo: Estudo morfológico que buscou verificar, por meio de microscopia eletrônica, a presença de aferências de receptores sino-aórticos em núcleo localizado na região dorso-lateral bulbar. Métodos: Após 5 dias de sobrevida, 9 ratos com desnervação sinoaórtica anestesiados com equitesina foram submetidos à perfusão transcardíaca, e o encéfalo de cada um deles foi processado histologicamente. Resultados: A citoarquitetura neuronal do núcleo paratrigeminal compreende projeções aferentes de outros núcleos que apresentam uma característica distributiva em relação às funções viscerais e nociceptivas na integração do reflexo cardiovascular. Conclusão: A porção medial do núcleo recebe projeções aferentes da região rostro-ventrolateral do troncoencefálico, confirmadas por meio de estudos com rastreadores neuronais. Os resultados indicam que a região medial do núcleo paratrigeminal contém o maior número de fibras axonais degeneradas.

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PURPOSE: To evaluate the retinal penetration and toxicity of two doses of intravitreal infliximab in primates. METHODS: Ten marmosets (Callithrix jacchus) were given intravitreal injection of 100 µg or 400 µg of infliximab, and balanced salt solution served as control. At baseline and after 24 hours (5 animals) and 7 days (the other 5), the eyes were examined by electroretinography. They were then killed (at 24 hours and 7 days) and assessed by light microscopy and transmission electron microscopy for toxicity and immunohistochemistry, using a biotinylated anti-human immunoglobulin G, to evaluate retinal penetration. RESULTS: There was no difference over 50% of the electroretinography b-wave between baseline and the time points studied in all animals. Light and electron microscopy, and electroretinography analysis, showed no signs of toxicity in any of the animals. Strong presence of infliximab was observed in all retinal layers 7 days after intravitreal injection at both doses (100 and 400 µg). CONCLUSION: Infliximab at doses of 100 and 400 µg seemed to cause no damage to the retina 24 hours and 7 days after its intravitreal injection, and deeply penetrated all its layers, in primates. These results encourage future perspectives for the treatment of chronic inflammatory diseases of the retina in humans.

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PURPOSE: To evaluate the efficacy and ultrastructural aspects of human limbal epithelial cells cultured on amniotic membrane (AM) with and without epithelium. METHODS: Limbal epithelial cell cultures were established from cadaveric cor neo-scleral rim explants derived from 6 different donors. The explants from each donor were placed under 3 different groups: on human preserved AM with epithelium (Group 1), AM deepithelialized with trypsin (Group 2) and control (Group 3). The epithelial cell migration was evaluated under phase contrast microscopy. After 15 days, the amniotic membrane with cells cultures were removed and submitted to scanning and transmission electron microscopy to check for epithelial migration and adhesion. RESULTS: All epithelial cell cultures from the controls grew over the botton of the culture plate wells until reaching confluence. Epithelial cultures grew over all but one denuded amniotic membrane. In the group amniotic membrane with epithelium, epithelial cell growing was observed only in 1 well. CONCLUSIONS: Using this model, denuded amniotic membrane appeared to be the best substrate for epithelial cell migration and adhesion comparing to amniotic membrane with epithelium. Removal of amniotic membrane epithelial seems to be an important step for establishing limbal epithelial cell culture on amniotic membrane.

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Trypanosoma cruzi is the protozoan that causes Chagas disease. It divides in the insect vector gut or in the cytosol of an infected mammalian cell. T. cruzi has one mitochondrion, one Golgi complex, one flagellum, and one cytostome. Here, we provide three-dimensional (3D) models of this protozoan based on images obtained from serial sections on electron microscopy at different stages of the cell cycle. Ultrathin serial sections were obtained from Epon™ embedded parasites, photographed in a transmission electron microscope, and 3D models were generated using Reconstruct and Blender 3D modeling softwares. The localization and distribution of organelles was evaluated and attributed to specific morphological patterns and deduced by distribution of specific markers by immunofluorescence analysis. The new features found in the 3D reconstructions are (1) the electron-dense chromatin is interconnected leaving an internal space for a centrally located nucleolus; (2) The kinetoplast is accommodated within a separated branch of the tubular and single mitochondrion; (3) The disk shaped kinetoplast, which is the mitochondrial DNA, duplicates from the interior in G2 phase; (4) The mitochondrion faces the external membrane and shrinks to accommodate an enlarged number of cytosolic vesicles from G1 to G2; (5) The cytostome progress from the parasite surface toward the posterior end contouring the kinetoplast and nucleus and retracts during cell cycle. These new observations might help understanding how organelles are formed and distributed in early divergent eukaryotic cells and provides a useful method to understand the organelle distribution in small eukaryotic cells.

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OBJETIVO: Avaliar a eficácia e aspecto estrutural de células límbicas epiteliais humanas cultivadas sobre membrana amniótica (MA) com e sem epitélio. MÉTODOS: As culturas límbicas foram obtidas a partir de rima corneoescleral remanescentes de transplantes de córnea de 6 diferentes doadores. Cada explante foi cultivado em três diferentes grupos: MA desepitelizada por tripsina (Grupo 1), MA com epitélio íntegro (Grupo 2) e controle (Grupo 3). A migração epitelial foi avaliada por microscopia de contraste de fase. Após 15 dias, as células cultivadas sobre MA foram submetidas à microscopia eletrônica para avaliar migração e adesão epitelial. RESULTADOS: Todas as células do grupo controle cresceram até atingir confluência. Somente uma das culturas em membrana amniótica desepitelizada não apresentou crescimento epitelial. O crescimento de células epiteliais sobre membrana amniótica epitelizada foi observada em apenas uma cultura. CONCLUSÃO: Baseando-se nestes achados, o uso de membrana amniótica desepitelizada aparenta ser o melhor substrato para migração e adesão epitelial comparando com membrana amniótica epitelizada. Remover o epitélio da membrana amniótica demonstra ser um importante passo para estabelecer culturas de células sobre membrana amniótica.

PURPOSE: To evaluate the efficacy and ultrastructural aspects of human limbal epithelial cells cultured on amniotic membrane (AM) with and without epithelium. METHODS: Limbal epithelial cell cultures were established from cadaveric cor neo-scleral rim explants derived from 6 different donors. The explants from each donor were placed under 3 different groups: on human preserved AM with epithelium (Group 1), AM deepithelialized with trypsin (Group 2) and control (Group 3). The epithelial cell migration was evaluated under phase contrast microscopy. After 15 days, the amniotic membrane with cells cultures were removed and submitted to scanning and transmission electron microscopy to check for epithelial migration and adhesion. RESULTS: All epithelial cell cultures from the controls grew over the botton of the culture plate wells until reaching confluence. Epithelial cultures grew over all but one denuded amniotic membrane. In the group amniotic membrane with epithelium, epithelial cell growing was observed only in 1 well. CONCLUSIONS: Using this model, denuded amniotic membrane appeared to be the best substrate for epithelial cell migration and adhesion comparing to amniotic membrane with epithelium. Removal of amniotic membrane epithelial seems to be an important step for establishing limbal epithelial cell culture on amniotic membrane.

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OBJETIVO: Avaliar interações de amostras de Escherichia coli enteroagregativa com tecido intestinal humano, a fim de documentar potenciais alterações em diferentes regiões do trato digestivo. MÉTODOS: Amostras de Escherichia coli enteroagregativa isoladas das fezes de crianças com diarreia persistente e a amostra protótipo 042, isolada de uma criança com diarreia em Lima, no Peru (controle positivo), foram analisadas por microscopia óptica de luz após semeadura em cultura de orgão in vitro de fragmentos de mucosa ileal e colônica. Foram analisadas as interações entre as diferentes cepas de Escherichia coli enteroagregativa e as mucosas ileal e colônica. RESULTADOS: A análise por microscopia óptica de luz indicou associação destes micro-organismos com o epitélio, provocando alterações. As cepas estudadas aderiram a ambas as regiões avaliadas (intestino delgado distal e grosso) e causaram alterações, especialmente naquelas áreas onde interagiram diretamente com o epitélio. No íleo, algumas regiões mostraram internalização secundária. CONCLUSÕES: Esses agentes podem causar diarreia persistente por meio de alterações no intestino delgado, no qual ocorrem as funções digestivo-absortivas. As lesões inflamatórias descritas na mucosa colônica poderiam explicar a colite mostrada em algumas crianças infectadas por Escherichia coli enteroagregativa.

OBJECTIVE: To examine the interactions of Enteroaggregative Escherichia coli strains with small and large intestinal mucosa, in order to detect potential alterations in both regions of the digestive tract. METHODS: Enteroaggregative Escherichia coli strains, isolated from stools of infants with persistent diarrhea and the prototype strain 042 (O44:H18), isolated from a child with diarrhea in Lima, Peru (positive control), were analised by light microscopy after in vitro organ culture assay of ileal and colonic mucosa. The interactions between the different enteroaggregative Escherichia coli strains and the ileal and colonic mucosa were analysed. RESULTS: Light microscopy analysis suggested an association of enteroaggregative Escherichia coli strains with the epithelium, inducing alterations. These bacteria adhered to both small and large bowel mucosa. The enteroaggregative Escherichia coli strains induced alterations in those areas where they were directly interacting with the epithelium. In the ileum, some areas showed a secondary internalization. CONCLUSIONS: The enteroaggregative Escherichia coli strains could cause persistent diarrhea inducing alterations in the small intestinal structures, where the digestive-absorptive functions take place. Inflammatory lesions observed in colons could justify the colitis described in some children infected by enteroaggregative Escherichia coli.

OBJETIVO: Evaluar interacciones de muestras de Escherichia coli enteroagregativa (EAEC) con tejido intestinal humano, a fin de documentar potenciales alteraciones en distintas regiones del tracto digestivo (intestino delgado distal e intestino grueso) y definir, con eso, su rol en la persistencia del proceso diarreico. MÉTODOS: Muestras de EAEC aislada de las heces de niños con diarrea persistente y la muestra prototipo 042, aislada de un niño con diarrea en Lima, Perú (control positivo) fueron analizadas por microscopía óptica de luz (ML) después de siembra en cultura de órgano in vitro de fragmentos de mucosa ileal y del colon. Fueron analizadas las interacciones entra las distintas cepas de EAEC y las mucosas ileal y del colon. RESULTADOS: El análisis por ML indicó asociación de estos microorganismos con el epitelio, provocando alteraciones. Las cepas estudiadas adhirieron a ambas regiones evaluadas: intestino delgado distal y grueso y causaron alteraciones, especialmente en aquellas áreas donde interactuaron directamente con el epitelio. En el íleo, algunas regiones mostraron internalización secundaria. CONCLUSIÓN: Estos agentes pueden causar diarrea persistente mediante alteraciones en el intestino delgado, donde ocurren las funciones digestivo-absortibas. Las lesiones inflamatorias descritas en la mucosa del colon podrían explicar la colitis descrita en algunos niños infectados por EAEC.

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