Priscila Cardoso Cristovam

Transplante de células-tronco epiteliais límbicas alógenas expandidas ex vivo sobre membrana amniótica: relato de caso

Case report of a patient who developed failure of an allogenic living related conjunctival limbal transplantation in the right eye after an episode of epidemic keratoconjunctivitis. After a few months, she underwent transplantation of allogenic limbal epithelial stem cells cultivated ex vivo on amniotic membrane (first case in Brazil). The patient evolved with total corneal epithelialization and improvement of the visual acuity. Three months after the surgery, peripheral superficial neovascularization with worsening of the corneal transparency was observed. The vision remained 0.1 after one year of the transplantation.

Importância do co-cultivo com fibroblastos de camundongo 3T3 para estabelecer cultura de suspensão de células epiteliais do limbo humano

PURPOSEnTo evaluate the importance of the presence of 3T3 fibroblasts for establishing limbal epithelial cultures from cell suspension obtained from corneo-scleral rims (CSR).nnnMETHODSnCorneo-scleral rims from different donors (n=6) had their posterior stroma and endothelium stripped away. Each corneo-scleral rim was divided into three equal segments that were set up in tissue culture in three different conditions: one of the segments was placed with the epithelial side up on the bottom of a 6-well culture plate (Group A). The other two fragments were trypsinized and the obtained cell suspension was cultured with (Group B) or without (Group C) irradiaded 3T3 cells. The cells were cultured in supplemental hormonal epithelial medium (SHEM), the epithelial migration and clone formation in groups A, B and C were evaluated with phase contrast microscopy and rodamine B staining.nnnRESULTSnEpithelial cell growth was observed in 4/6 rims (Group A). All epithelial cell suspensions that were cultured with 3T3 cells (Group B) formed clones. No adhesion or true clone formation (holo- or meroclones) was observed in the cell suspensions that were cultivated without 3T3 (Group C) (p=0.009).nnnCONCLUSIONSnEpithelial cell suspension obtained from corneo-scleral rims in this model needs to be cultivated with 3T3 cells in order to form clones and establish limbal epithelial cell colonies with the potential to be used for ocular surface reconstruction.

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.

Minor Salivary Gland Transplantation for Severe Dry Eyes

Abstract: Dry eye is a multifactorial disease comprising a wide spectrum of ocular surface alterations and symptoms of discomfort. In most patients with aqueous-deficient dry eye, pharmaceutical tear substitutes are used to control symptoms and prevent ocular surface damage. However, in severe dry eye conditions caused by cicatricial disorders, such as Stevens–Johnson syndrome and ocular cicatricial mucous membrane pemphigoid, noninvasive treatments are insufficient, and patients are at risk of developing complications that can lead to blindness. The use of salivary glands as a source of lubrication to treat severe cases of dry eye has been proposed by different authors. The first reports proposed parotid or submandibular gland duct transplantation into the conjunctival fornix. However, complications limited the functional outcomes. Minor salivary gland autotransplantation together with labial mucosa has been used as a complex graft to the conjunctival fornix in severe dry eye with a good outcome. Our group demonstrated significant improvements in best-corrected visual acuity, Schirmer I test score, corneal transparency, and neovascularization after using this technique. A symptoms questionnaire applied to these patients revealed improvements in foreign body sensation, photophobia, and pain. Similar to tears, saliva has a complex final composition comprising electrolytes, immunoglobulins, proteins, enzymes, and mucins. We demonstrated the viability of minor salivary glands transplanted into the fornix of patients with dry eye by performing immunohistochemistry on graft biopsies with antibodies against lactoferrin, lysozyme, MUC1, and MUC16. The findings revealed the presence of functional salivary gland units, indicating local production of proteins, enzymes, and mucins.

Genetic analysis and cAMP measurement: comparison between lean and obese anovulating mice

PURPOSEnTo evaluate genes differentially expressed in ovaries from lean (wild type) and obese (ob/ob) female mice and cyclic AMP production in both groups.nnnMETHODSnThe expression on messenger RNA levels of 84 genes concerning obesity was analyzed through the PCR array, and cyclic AMP was quantified by the enzyme immunoassay method.nnnRESULTSnThe most downregulated genes in the Obesity Group included adenylate cyclase-activating polypeptide type 1, somatostatin, apolipoprotein A4, pancreatic colipase, and interleukin-1 beta. The mean decrease in expression levels of these genes was around 96, 40, 9, 4.2 and 3.6-fold, respectively. On the other hand, the most upregulated genes in the Obesity Group were receptor (calcitonin) activity-modifying protein 3, peroxisome proliferator activated receptor alpha, calcitonin receptor, and corticotropin-releasing hormone receptor 1. The increase means in the expression levels of such genes were 2.3, 2.7, 4.8 and 6.3-fold, respectively. The ovarian cyclic AMP production was significantly higher in ob/ob female mice (2,229 ± 52 fMol) compared to the Control Group (1,814 ± 45 fMol).nnnCONCLUSIONSnObese and anovulatory female mice have reduced reproductive hormone levels and altered ovogenesis. Several genes have their expression levels altered when leptin is absent, especially adenylate cyclase-activating polypeptide type 1.

Variation in the volume of lubricating eyedrops available in the brazilian market

Objetivo: Avaliar a variacao intra e interexaminadores do volume de gotas dispensados de frascos de colirios lubrificantes disponiveis no mercado. Metodos: Foram estudados cinco frascos de colirios lubrificantes e dezenove voluntarios participaram deste estudo. A massa media de gotas de 20µl dos colirios foi obtida utilizando micropipeta e balanca de precisao e como padrao para comparacao com a massa das gotas obtidas pelos voluntarios. Cinco gotas de cada frasco foram pesadas individualmente com o tubo de colirio perpendicular a balanca, usando o primeiro e segundo dedos da mao direita, de forma que a pressao fosse aplicada somente no meio do frasco. Os experimentos foram realizados em uma sala climatizada a temperatura ambiente (21±1°C). Resultados: Todos os frascos de colirios apresentaram variacao estatisticamente significante das massas das gotas obtidas pelos examinadores quando comparadas com a massa media padrao de 0,0182±0,0014g, com excecao da comparacao entre os dados do colirio A com o colirio D, que nao apresentou variacao estatisticamente significante. Conclusao: O presente estudo demonstra a ausencia de uniformidade das gotas dispensadas pelos frascos de colirios disponiveis no mercado e a sua inadequacao a real necessidade, uma vez que as gotas dispensadas sao maiores do que o indicado. Esse fato torna-se um problema quando se trata de periodo de tratamento prolongado, especialmente com colirios dispendiosos como os indicados para a terapeutica do glaucoma. Nesse sentido, a padronizacao das gotas de colirios se faz necessaria.