Edna Freymuller Haapalainen

Ultrastructural and cytochemical identification of megasome in Leishmania (Leishmania) chagasi

The present work showed the presence of a megasome in Leishmania (Leishmania) chagasi amastigotes. Transmission electron microscopy analysis of ultrathin serial sections and three-dimensional reconstruction allowed visualization of large structures in amastigote forms of L. (L.) chagasi and a multivesicular tubule–lysosome structure in metacyclic promastigotes. Morphometric data showed that the relative volume occupied by the megasome and the multivesicular tubule (MVT)–lysosome structures was about 5% and 3.2%, respectively, in amastigotes and promastigotes of L. (L.) chagasi. Further characterization of the megasome in L. (L.) chagasi amastigotes was carried out by immunolabeling of cysteine proteinase, whereas the lysosomal content of amastigotes and promastigotes was confirmed by arylsulfatase cytochemistry.

Assessment of the use of cryopreserved x freeze-dried amniotic membrane (AM) for reconstruction of ocular surface in rabbit model

PURPOSE To determine the efficacy of freeze-dried amniotic membrane (AM) for reconstruction of the ocular surface in rabbit eyes. METHODS The sterilized, freeze-dried amniotic membrane (lyophilized or FD-AM) is a preservative method that uses the drying by freezing process to maintain the AM well preserved for a long time even at room temperature. This paper is an experimental animal interventional study. One eye of each of 15 male New Zealand rabbits (1.5 - 3.0 kg) had the central cornea marked with a 6.0 mm trephine. The marked area was deepithelialized with a No.15 blade. The denuded corneal surface was covered as follows: Group 1: cryopreserved AM (n=6); Group 2: freeze-dried AM (n=6); and Group 3: not covered (control group, n=3). The AM in group 1 and 2 and the periphery of the denuded area in group 3 were secured with continuous 10-0 nylon sutures. The clinical evaluation was made by a blinded observer and graded on a four-point scale (1= minimal, 4= marked) for conjunctival and ciliary hyperemia, eyelid edema, corneal neovascularization, corneal opacity and reepithelialization on postoperative (PO) days 1, 7 and 30 . After PO day 30, the rabbits were euthanized and their corneas were sent for histopathological and ultrastructural analysis to evaluate tissue inflammation, reepithelialization, and basement membrane integrity. RESULTS Two eyes in group 2 had a corneal infection and were excluded from the analysis. No statistically significant differences among the three groups were found (p>0.05) regarding the clinical evaluation on 1st, 7th and 30th PO days. On transmission electron microscopy, the basement membrane in lyophilized and control groups was more continuous and homogeneous than in the glycerol group. CONCLUSIONS The freeze-drying method seems to be a good option to preserve human amniotic membrane to be used in ocular surface reconstruction. This preservative method reduces the preservation costs and may enhance the use of AM, facilitating its storage and transport.

Avaliação morfológica de diferentes técnicas de desepitelização da membrana amniótica humana

PURPOSE: To evaluate the morphological features of the amniotic membrane denuded by different techniques. METHODS: Human amniotic membrane was collected at the time of delivery, fixed in increasing concentrations of glycerol (0-50% in DMEM) and preserved at -80oC until the time of use. The study consisted of 4 groups: intact epithelium (control) and denuded by trypsin (2 mg/mL at 1:250), dispase (1.2 U/mL in Mg2+ and Ca2+ free Hanks balanced salt solution) or ethylenediaminetetraacetic acid (EDTA), 0.02%. Specimens were submitted to electron (scanning and transmission) microscopy analysis. RESULTS: Scanning electron microscopy disclosed intact epithelium in the control group and its absence in the amniotic membranes denuded by trypsin and dispase. In those denuded by ethylenediaminetetraacetic acid there were areas with and without epithelium. When assessed by transmission electron microscopy, the epithelium was intact and firmly adhered to the basement membrane by hemidesmossomes in controls and in parts of ethylenediaminetetraacetic acid group. There were only collagen fibers in the dispase- and trypsin-treated groups. CONCLUSIONS: Trypsin and dispase treatment of the amniotic membrane may cause complete denuding of the epithelium and basement membrane whereas ethylenediaminetetraacetic acid may leave some intact epithelium-areas and partially destroy the basement membrane in others.

Análise ultraestrutural e de fatores de crescimento de diferentes métodos de preservação da membrana amniótica utilizada em cirurgia ocular

PURPOSE: To compare the anatomical structure and the presence of growth factors and cytokines of amniotic membrane preserved in glycerol/MEM (1:1) or undiluted dimethyl sulfoxide through electron microscopy. METHODS: Amniotic membrane preserved in glycerol/MEM (1:1) or undiluted dimethyl sulfoxide were processed for transmission and scaning electron microscopy. As control, freshly collected amniotic membrane was fixed and processed for electron microscopy. The cytokines and growth factors assessed were: TGF-b (transforming growth factor b); TGF-b activ (activated transforming growth factor b); EGF (epidermal growth factor); FGF-4 (fibroblast growth factor 4); bFGF (basic fibroblast growth factor); IL-4 (interleukin 4); PGE2 (prostaglandin E2); IL-10 (interleukin 10); KGF (keratinocyte growth factor); HGF (hepatocyte growth factor). RESULTS: Amniotic membrane from the control group showed intact epithelium, with surface microvilli and junctional complexes between the cells and the basal membrane. Glycerol/MEM preserved amniotic membrane had similar aspect to the control, with higher epithelial cells. Those amniotic membranes preserved in dimethyl sulfoxide disclosed less intercellular junction and detachment of the epithelium from the basal membrane. The cytokines and growth factors did not disclose significant differences, except for FGF-4, bFGF, PGE2 and KGF. CONCLUSIONS: Amniotic membrane preserved in glycerol/MEM showed a better tissue structure, with less detachment of the epithelium from the basal membrane, in comparison to undiluted dimethyl sulfoxide. The majority of the growth factors and cytokines were kept with both techniques of preservation.

Toxicity and retinal penetration of infliximab in primates

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.

Myriocin, a Serine Palmitoyltransferase Inhibitor, Blocks Cytokinesis in Leishmania (Viannia) braziliensis Promastigotes

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.

Comparação entre membrana amniótica com e sem epitélio como substrato para cultura de células epiteliais do limbo ex vivo

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.

Tridimensional ultrastructure and glycolipid pattern studies of Trypanosoma dionisii.

Trypanosoma (Schizotrypanum) dionisii is a non-pathogenic bat trypanosome closely related to Trypanosoma cruzi, the etiological agent of Chagas disease. Both kinetoplastids present similar morphological stages and are able to infect mammalian cells in culture. In the present study we examined 3D ultrastructure aspects of the two species by serial sectioning epimastigote and trypomastigote forms, and identified common carbohydrate epitopes expressed in T. dionisii, T. cruzi and Leishmania major. A major difference in 3D morphology was that T. dionisii epimastigote forms present larger multivesicular structures, restricted to the parasite posterior region. These structures could be related to T. cruzi reservosomes and are also rich in cruzipain, the major cysteine-proteinase of T. cruzi. We analyzed the reactivity of two monoclonal antibodies: MEST-1 directed to galactofuranose residues of glycolipids purified from Paracoccidioides brasiliensis, and BST-1 directed to glycolipids purified from T. cruzi epimastigotes. Both antibodies were reactive with T. dionisii epimastigotes by indirect immunofluorescense, but we noted differences in the location and intensity of the epitopes, when compared to T. cruzi. In summary, despite similar features in cellular structure and life cycle of T. dionisii and T. cruzi, we observed a unique morphological characteristic in T. dionisii that deserves to be explored.

Ultrastructural transneuronal degeneration study of axonal elements within the paratrigeminal nucleus in sinoaortic deafferented rats

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.

Escherichia coli enteroagregativa como agente provocador de diarreia persistente: modelo experimental utilizando microscopia óptica de luz

RESUMO Objetivo: Avaliar interacoes de amostras de Escherichia coli enteroagregativa com tecido intestinal humano, a fim de documentar potenciais alteracoes em diferentes regioes do trato digestivo. Metodos: Amostras de Escherichia coli enteroagregativa isoladas das fezes de criancas com diarreia persistente e a amostra prototipo 042, isolada de uma crianca com diarreia em Lima, no Peru (controle positivo), foram analisadas por microscopia optica de luz apos semeadura em cultura de orgao in vitro de fragmentos de mucosa ileal e colonica. Foram analisadas as interacoes entre as diferentes cepas de Escherichia coli enteroagregativa e as mucosas ileal e colonica. Resultados: A analise por microscopia optica de luz indicou associacao destes micro-organismos com o epitelio, provocando alteracoes. As cepas estudadas aderiram a ambas as regioes avaliadas (intestino delgado distal e grosso) e causaram alteracoes, especialmente naquelas areas onde interagiram diretamente com o epitelio. No ileo, algumas regioes mostraram internalizacao secundaria. Conclusoes: Esses agentes podem causar diarreia persistente por meio de alteracoes no intestino delgado, no qual ocorrem as funcoes digestivo-absortivas. As lesoes inflamato rias descritas na mucosa colonica poderiam explicar a colite mostrada em algumas criancas 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.