Bilateral Acute Depigmentation of the Iris (BADI) following Covid-19 Infection.

PURPOSE: To present a case of bilateral acute iris depigmentation after covid 19 infection. CASE REPORT: A 55-year-old female presented with binocular pain and blurred vision a month after being diagnosed with severe acute respiratory syndrome - coronavirus-2 (SARS-CoV-2). She presented pigment dispersion in the anterior chamber and pigment depositions on the corneal endothelium. The patient was treated with dexamethasone and during follow-up visits, the pigment dispersion decreased and the symptoms ceased. CONCLUSIONS: Covid-19 infection may be associated with rare ocular disorders such as BADI.

Histological analysis of elastic cartilages treated with alkaline solution / Análise histológica de cartilagens elásticas tratadas com solução alcalina

The elastic cartilage is composed by chondroblasts and chondrocytes, extracellular matrix and surrounded by perichondrium. It has a low regeneration capacity and is a challenge in surgical repair. One of obstacles in engineering a structurally sound and long-lasting tissue is selecting the most appropriate scaffold material. One of the techniques for obtaining biomaterials from animal tissues is the decellularization that decreases antigenicity. In this work, alkaline solution was used in bovine ear elastic cartilages to evaluate the decellularization and the architecture of the extracellular matrix. The cartilages were treated in alkaline solution (pH13) for 72 hours and lyophilized to be compared with untreated cartilages by histological analysis (hematoxylin-eosin, Masson's trichrome and Verhoeff slides). Areas of interest for cell counting and elastic fiber quantification were delineated, and the distribution of collagen and elastic fibers and the presence of non-fibrous proteins were observed. The results demonstrated that the alkaline solution caused 90% decellularization in the middle and 13% in the peripheral region, and maintenance of the histological characteristics of the collagen and elastic fibers and non-fibrous protein removal. It was concluded that the alkaline solution was efficient in the decellularization and removal of non-fibrous proteins from the elastic cartilages of the bovine ear.(AU)

A cartilagem elástica é composta por condroblastos e condrócitos, matriz extracelular e envolta por pericôndrio. Possui uma baixa capacidade de regeneração e é um desafio em reparos cirúrgicos. Um dos obstáculos na engenharia de tecido estruturalmente sólido e de longa duração é a seleção do material de arcabouço mais adequado. Uma das técnicas para obtenção de biomateriais oriundos de tecidos animais é a descelularização, que diminui a antigenicidade. Neste trabalho, foi utilizada solução alcalina em cartilagem elástica auricular bovina para avaliar a descelularização e a arquitetura da matriz extracelular. As cartilagens foram tratadas em solução alcalina (pH13) durante 72 horas e liofilizadas, e comparadas com cartilagens não tratadas por análise histológica (hematoxilina-eosina, tricrômio de Masson e Verhoeff). Foram determinadas as áreas de interesse para contagem celular e quantificação de fibras elásticas, observada a distribuição de colágeno e fibras elásticas e a presença de proteínas não fibrosas. Os resultados demonstraram que a solução alcalina causou 90% de descelularização na região central e 13% na região periférica, manutenção das características histológicas do colágeno e fibras elásticas e remoção das proteínas não fibrosas. Concluiu-se que a solução alcalina foi eficiente na descelularização e retirada de proteínas não fibrosas de cartilagens elásticas da orelha de bovinos.(AU)

Histological evaluation and E-cadherin and ß-catenin expression in kidney of dogs submitted to renal ischemia and reperfusion after chlorpromazine administration / Avaliação histológica e imunomarcação de E-caderina e ß-catenina em rins de cães submetidos a isquemia e referfusão renal após administração de clorpromazina

Renal ischemia can be associated with some urological procedures, such as renovascular surgery or kidney transplantation, that are often followed by acute renal failure. The aim of this study was to verify the E-cadherin and ß-catenin localization in canine kidney in different times of renal ischemia and reperfusion after chlorpromazine application. Twelve dogs were randomly distributed equally into two groups. GroupA with ischemia and reperfusion without chlorpromazine and groupB with ischemia and reperfusion treated by chlorpromazine. GroupB received intravenous chlorpromazine, 15 min before the artery obstruction, which lasted 1 hour. After this period, the clamps in the renal arteries were released and the organ remained in reperfusion for 2 hours. In each group, anti-E-cadherin and anti-ß-catenin antibodies were made in six tissue samples from renal parenchyma. E-cadherin and ß-catenin are differentially expressed in segments from cortex and medulla in dog's kidneys and the use of chlorpromazine did not alter the expression of both proteins. Occlusion of the left renal artery in dogs causes morphological alterations mainly in proximal convoluted tubules, beginning 30min after the start of ischemia and being aggravated after two hours of reperfusion. These results reveal that chlorpromazine did not change kidneys' histological aspect nor E-cadherin and ß-catenin expression.(AU)

A lesão renal isquêmica pode estar associada a procedimentos urológicos, tais como cirurgia renovascular, cirurgia renal extracorpórea ou transplante renal. Essa injúria, muitas vezes, é seguida de insuficiência renal aguda. O objetivo deste trabalho foi observar a localização da E-caderina e da ß-catenina em rim de cães, além de relacionar a expressão dessas proteínas das junções de aderência em diferentes tempos de isquemia e reperfusão com ou sem a aplicação de clorpromazina. Para tanto, foram utilizados 12 cães, distribuídos aleatoriamente em dois grupos de seis indivíduos: grupo A, com isquemia e reperfusão sem tratamento por clorpromazina, e o grupo B, com isquemia e reperfusão tratado por clorpromazina. No procedimento cirúrgico, foi realizada uma incisão paracostal esquerda para identificação e isolamento do rim esquerdo e da artéria renal esquerda. Após o isolamento da artéria, os animais de todos os grupos tiveram o vaso ocluído. Os animais do grupo B receberam clorpromazina via endovenosa, na dose de 5mg/kg, 15min antes da clampagem do vaso, que durou uma hora. Após este período, as artérias renais foram desobstruídas e os órgãos permaneceram em reperfusão por duas horas. Em cada grupo, foram extraídas seis amostras de parênquima renal, com utilização de agulha tru-cut, para marcação com anticorpos anti-E-caderina e anti-ß-catenina por meio de imunoistoquímica. E-caderina e ß-catenina são diferencialmente expressas em segmentos do córtex e da medula em rim de cães e o uso da clorpromazina não alterou a expressão das duas proteínas.(AU)

Metabolic Reprogramming, Autophagy, and Reactive Oxygen Species Are Necessary for Primordial Germ Cell Reprogramming into Pluripotency.

Cellular reprogramming is accompanied by a metabolic shift from oxidative phosphorylation (OXPHOS) toward glycolysis. Previous results from our laboratory showed that hypoxia alone is able to reprogram primordial germ cells (PGCs) into pluripotency and that this action is mediated by hypoxia-inducible factor 1 (HIF1). As HIF1 exerts a myriad of actions by upregulating several hundred genes, to ascertain whether the metabolic switch toward glycolysis is solely responsible for reprogramming, PGCs were cultured in the presence of a pyruvate kinase M2 isoform (PKM2) activator, or glycolysis was promoted by manipulating PPARγ. Conversely, OXPHOS was stimulated by inhibiting PDK1 activity in normoxic or in hypoxic conditions. Inhibition or promotion of autophagy and reactive oxygen species (ROS) production was performed to ascertain their role in cell reprogramming. Our results show that a metabolic shift toward glycolysis, autophagy, and mitochondrial inactivation and an early rise in ROS levels are necessary for PGC reprogramming. All of these processes are governed by HIF1/HIF2 balance and strict intermediate Oct4 levels. Histone acetylation plays a role in reprogramming and is observed under all reprogramming conditions. The pluripotent cells thus generated were unable to self-renew, probably due to insufficient Blimp1 downregulation and a lack of Klf4 and cMyc expression.

Cell metabolism under microenvironmental low oxygen tension levels in stemness, proliferation and pluripotency.

Hypoxia is defined as a reduction in oxygen supply to a tissue below physiological levels. However, physiological hypoxic conditions occur during early embryonic development; and in adult organisms, many cells such as bone marrow stem cells are located within hypoxic niches. Thus, certain processes take place in hypoxia, and recent studies highlight the relevance of hypoxia in stem cell cancer physiology. Cellular response to hypoxia depends on hypoxia-inducible factors (HIFs), which are stabilized under low oxygen conditions. In a hypoxic context, various inducible HIF alpha subunits are able to form dimers with constant beta subunits and bind the hypoxia response elements (HRE) in the genome, acting as transcription factors, inducing a wide variety of gene expression. Typically, the HIF pathway has been shown to enhance vascular endothelial growth factor (VEGF) expression, which would be responsible for angiogenesis and, therefore, re-oxygenation of the hypoxic sites. Embryonic stem cells inhibit a severely hypoxic environment, which dictates their glycolytic metabolism, whereas differentiated cells shift toward the more efficient aerobic respiration for their metabolic demands. Accordingly, low oxygen tension levels have been reported to enhance induced pluripotent stem cell (iPS) generation. HIFs have also been shown to enhance pluripotency-related gene expression, including Oct4 (Octamer-binding transcription factor 4), Nanog and Wnt. Therefore, cell metabolism might play a role in stemness maintenance, proliferation and cell reprogramming. Moreover, in the hypoxic microenvironment of cancer cells, metabolism shifts from oxidative phosphorylation to anaerobic glycolysis, a process known as the Warburg effect, which is involved in cancer progression and malignancy.

Histopathological detection of entry and exit holes in human skin wounds caused by firearms.

The judiciary needs forensic medicine to determine the difference between an entry hole and an exit hole in human skin caused by firearms for civilian use. This important information would be most useful if a practical and accurate method could be done with low-cost and minimal technological resources. Both macroscopic and microscopic analyses were performed on skin lesions caused by firearm projectiles, to establish histological features of 14 entry holes and 14 exit holes. Microscopically, in the abrasion area macroscopically observed, there were signs of burns (sub-epidermal cracks and keratinocyte necrosis) in the entrance holes in all cases. These signs were not found in three exit holes which showed an abrasion collar, nor in other exit holes. Some other microscopic features not found in every case were limited either to entry holes, such as cotton fibres, grease deposits, or tattooing in the dermis, or to exit holes, such as adipose tissue, bone or muscle tissue in the dermis. Coagulative necrosis of keratinocytes and sub-epidermal cracks are characteristic of entry holes. Despite the small sample size, it can be safely inferred that this is an important microscopic finding, among others less consistently found, to define an entry hole in questionable cases.

c-Kit identifies a subpopulation of mesenchymal stem cells in adipose tissue with higher telomerase expression and differentiation potential.

The stromal vascular fraction (SVF) of adipose tissue is an easy to obtain source of adipose tissue-derived stem cells (ADSCs). We and others have achieved significant but suboptimal therapeutic effects with ADSCs in various settings, mainly due to low rates of differentiation into specific cell types and with the downside of undesired side effects as a consequence of the undifferentiated ADSCs. These data prompted us to find new stem cell-specific markers for ADSCs and/or subpopulations with higher differentiation potential to specific lineages. We found a subpopulation of human ADSCs, marked by c-Kit positiveness, resides in a perivascular location, and shows higher proliferative activity and self-renewal capacity, higher telomerase activity and expression, higher in vitro adipogenic efficiency, a higher capacity for the maintenance of cardiac progenitors, and higher pancreatogenic and hepatogenic efficiency independently of CD105 expression. Our data suggests that the isolation of ADSC subpopulations with anti-c-Kit antibodies allows for the selection of a more homogeneous subpopulation with increased cardioprotective properties and increased adipogenic and endodermal differentiation potential, providing a useful tool for specific therapies in regenerative medicine applications.

Double port injector device to reduce endothelial damage in DMEK.

OBJECTIVE: To study endothelial injury from a newly designed asymmetric double port Descemet Membrane Endothelial Keratoplasty (DMEK) injector, both ex-vivo and in clinical practice. DESIGN: Laboratory investigation with an interventional case series study. METHOD: Sixteen rabbit endothelial rolls were tested for injection using a no-touch technique. For each pair of rolls, one endothelial graft underwent injection with a single port Pasteur pipette twice, wheras the other was injected with a novel asymmetric double port injector with a larger diameter entry port than the exit port also twice. Each graft was stained with 4-6-diamidino-2-phenylinidole dihydrochloride and was counted under a fluorescence-inverted microscope before and after injection. The proportion of graft injury was calculated and the differences were analyzed. Subsequently, six patients requiring DMEK underwent surgery using this novel insertion device and endothelial cell loss was calculated 3 months after the surgery. RESULTS: After injection, the mean proportion of endothelial cell survival with the single port pipette was 78.8% (n=8; SD: ±20.9%), whereas the double port injector yielded a survival rate of 96.8% (n=8; SD: ±8.4%). This difference was statistically significant (P=0.008), representing less endothelial injury with the double port device. Early endothelial cell loss after 3 months in the DMEK patients was 26.1% (SD: ±6.1%). CONCLUSION: In our injection model, using a double port injector created significantly less endothelial cell damage than with the single port pipette. Clinically, this device yielded early endothelial cell loss comparable to that of the series performed by experienced DMEK surgeons.

Clinical and histological effects of the temporary occlusion of the rabbit nasolacrimal duct and point using cyanoacrylate adhesives / Efeitos clínicos e histológicos da oclusão temporária do ponto e do duto nasolacrimais de coelhos com adesivos de cianoacrilato

The objective of this study was to evaluate the clinical and histological effects of occluding the nasolacrimal ducts and points of rabbits. For this study, 20 adult New Zealand rabbits, both males and females, weighing 3.2±0.4kg were allocated into two groups for n-butyl-cyanoacrylate occlusion (GB, n=10) or 2-octyl-cyanoacrylate occlusion (GO, n=10). The contralateral eyes served as the controls. The persistence of tears was evaluated daily using the Schirmer I test. Discomfort, eye discharge, epiphora, and conjunctival hyperemia were assessed prior to the procedure (T0) and during the 14 subsequent days (T1-T14). On days seven and 14, five animals from each group were euthanized, and their nasolacrimal ducts were collected, processed and analyzed by histology. In the GB group, the Schirmer test values differed from that at T0 at all of the subsequent time points, whereas there was no difference in the values observed from the GO group. Compared with the corresponding controls, the GO and GB groups differed significantly at almost all of the time points. When comparing the treatment groups, differences were found at T6, T7, T9, T10, T11, T12 and T14, with higher Schirmer values in the GB group. Epiphora was observed in the GB group from T1 to T8 and in the GO group from T1 to T6. Within seven days post-occlusion, histology revealed a moderate foreign body reaction, with marked necrosis and sloughing of the canalicular epithelium, in the GO group, which was absent at day 14. In the GB group, a marked inflammatory reaction and a mild foreign body reaction were found at day seven, and the foreign body reaction was prevalent at day 14. This study demonstrated that both adhesives were effective in obstructing the nasolacrimal ducts and points of rabbits and that their application and handling are easy and free of complications. However, both adhesives promoted inflammatory and foreign body reactions that evolved to repair and regeneration at day 14 of evaluation.

Avaliaram-se os efeitos clínicos e histológicos da oclusão do ponto e do duto nasolacrimais de coelhos. Para isso, utilizaram-se 20 coelhos adultos da raça Nova Zelândia, machos e fêmeas, com peso de 3,2±0,4kg, distribuídos em dois grupos: oclusão com n-butil cianoacrilato (GB, n=10) e oclusão com 2-octil cianoacrilato (GO, n=10). Os olhos contralaterais foram utilizados como controle. A permanência da lágrima foi avaliada diariamente pelo teste de Schirmer I. Foram avaliados o desconforto, secreção ocular, epífora e hiperemia conjuntival, previamente ao procedimento (T0) e durante 14 dias (T1-T14). Aos sete e aos 14 dias, cinco animais de cada grupo foram submetidos à eutanásia, e os dutos nasolacrimais, colhidos, processados e analisados à histologia. Em GB, os valores de Schirmer diferiram de T0 em todos os momentos; em GO não houve diferença. Na comparação com o respectivo controle, GB e GO diferiram significativamente em quase todos os momentos. Ao compararem-se os tratamentos, houve diferença em T6, T7, T9, T10, T11, T12 e T14, sendo os valores de Schirmer superiores em GB. Epífora esteve presente em GB de T1 a T8 e em GO de T1 a T6. À histologia, em GO, aos sete dias, notou-se moderada reação de corpo estranho com marcante necrose e descamação do epitélio canalicular; tais alterações estiveram ausentes aos 14 dias. Em GB, verificou-se, aos sete dias, acentuada reação inflamatória e discreta reação de corpo estranho; aos 14 dias, houve predomínio da reação de corpo estranho. Concluiu-se que ambos os adesivos foram eficazes na obstrução do ponto e do duto nasolacrimais de coelhos, sendo sua aplicação e manuseio fáceis e livres de intercorrências e ambos promoveram reação inflamatória e de corpo estranho que evoluíram para reparação e regeneração aos 14 dias de avaliação.

Intoxicação crônica por cobre em ovinos: conduta para o diagnóstico conclusivo / Chronic copper toxicities in sheep: conduct for diagnostic confirmation

Descreveram-se os sinais clínicos e achados anatomopatológicos da intoxicação crônica por cobre em um ovino da raça Texxel e definiu-se a conduta diagnóstica correta para confirmação da enfermidade. Um ovino foi encaminhado ao setor de patologia com histórico de apatia, hemoglobinúria e morte em dois a três dias. No exame necroscópico, observaram-se icterícia e edema subcutâneo, fígado aumentado de volume e amarelado e rins escuros. No exame histológico, observaram-se necrose zonal aleatória e acentuada no fígado, necrose epitelial tubular, gotas hialinas e cilindros marrom-alaranjados em túbulos coletores dos rins. O histórico alimentar, a sensibilidade de espécie/raça, o quadro clínico, as alterações macroscópicas e microscópicas sugeriram o quadro de intoxicação crônica por cobre. A confirmação diagnóstica somente foi possível após a marcação de pigmentos de cobre pela técnica histoquímica de Ulzmann e pela quantificação de cobre em matéria seca de fígado e rins, cujos valores foram mais altos que o normal.

The present work describes the clinical signs and anatomopathological findings of chronic copper toxicities in a Texxel breed sheep and defines the optimal diagnostic procedure for confirmation of the disorder. A sheep was sent to pathology analysis service with a history of apathy, hemoglobinuria and death within two to three days. Necropsy showed jaundice and subcutaneous edema, enlarged yellow liver and dark kidneys. The histologic examination showed random zonal necrosis, marked necrosis in the liver and tubular epithelial and orange-brown spotted hyaline cylinders in the collecting tubules of the kidneys. The dietary history, sensitivity of species/breed, clinical, macroscopic and microscopic alterations suggested the framework of chronic copper poisoning. Diagnostic confirmation was only possible after staining copper pigments trough the Ulzmann technique and quantification of copper in the dry liver and kidney, which were higher than normal levels.

IGFBP-3 methylation-derived deficiency mediates the resistance to cisplatin through the activation of the IGFIR/Akt pathway in non-small cell lung cancer.

Although many cancers initially respond to cisplatin (CDDP)-based chemotherapy, resistance frequently develops. Insulin-like growth factor-binding protein-3 (IGFBP-3) silencing by promoter methylation is involved in the CDDP-acquired resistance process in non-small cell lung cancer (NSCLC) patients. Our purpose is to design a translational-based profile to predict resistance in NSCLC by studying the role of IGFBP-3 in the phosphatidyl inositol 3-kinase (PI3K) signaling pathway. We have first examined the relationship between IGFBP-3 expression regulated by promoter methylation and activation of the epidermal growth factor receptor (EGFR), insulin-like growth factor-I receptor (IGFIR) and PI3K/AKT pathways in 10 human cancer cell lines and 25 NSCLC patients with known IGFBP-3 methylation status and response to CDDP. Then, to provide a helpful tool that enables clinicians to identify patients with a potential response to CDDP, we have calculated the association between our diagnostic test and the true outcome of analyzed samples in terms of cisplatin IC50; the inhibitory concentration that kills 50% of the cell population. Our results suggest that loss of IGFBP-3 expression by promoter methylation in tumor cells treated with CDDP may activate the PI3K/AKT pathway through the specific derepression of IGFIR signaling, inducing resistance to CDDP. This study also provides a predictive test for clinical practice with an accuracy and precision of 0.84 and 0.9, respectively, (P=0.0062). We present a biomarker test that could provide clinicians with a robust tool with which to decide on the use of CDDP, improving patient clinical outcomes.

Avaliação da toxicidade aguda e da atividade cicatrizante dos extratos etanólicos das folhas e raízes da Memora nodosa (Silva Manso) Miers (Bignoniaceae) / Evaluation of acute and healing activity of ethanol extracts of Memora nodosa (Silva Manso) Miers (Bignoniaceae) leaves and roots

Memora nodosa (Silva Manso) Miers é uma espécie da flora do Cerrado cujas folhas e caules são utilizados popularmente no tratamento de feridas e úlceras externas, enquanto as raízes são empregadas para dores abdominais e no tratamento da sarna. O objetivo desse trabalho foi avaliar a toxicidade aguda dos extratos etanólicos das folhas e raízes nas doses de 2000 e 5000 mg kg-1 em ratos e camundongos e a atividade cicatrizante das soluções aquosas contendo 2% desses extratos em feridas cutâneas em ratos. A contração das bordas das feridas foi avaliada por análises histológicas e morfométricas após 4, 7 e 14 dias de tratamento e por reação imunohistoquímica após 7 dias de tratamento. Os extratos etanólicos das folhas e raízes não apresentaram toxicidade na dose de 2000 mg kg-1 para ratos e camundongos e na dose de 5000 mg kg-1 para ratos. Nos camundongos, a dose de 5000 mg kg-1 dos extratos das folhas e raízes provocou alterações histológicas no fígado. Não foram observadas diferenças significativas na contração das feridas entre os grupos tratados com os extratos das folhas e das raízes e o controle após 4 e 7 dias de tratamento. Após 14 dias de tratamento, 50% dos animais tratados com o extrato das raízes apresentaram reepitelização total das feridas e reconstrução parcial dos anexos. A alantoína, isolada do extrato etanólico da raiz, pode ser considerada como um dos metabólitos secundários responsáveis pela aceleração da reepitelização.

Memora nodosa (Silva Manso) Miers is a Brazilian Cerrado species whose leaves and stems are commonly used to treat external wounds and ulcers and the roots are used for abdominal pain and to treat scabies. The purpose of this study was to evaluate the acute toxicity of ethanol extracts from the leaves and roots of M. nodosa at 2000 and 5000 mg kg-1 doses in rats and mice and to evaluate the healing activity of aqueous solutions containing 2% of these extracts on skin wounds in rats. The contraction of the wounds was evaluated by histological and morphometric analysis after 4, 7 and 14 days of treatment and by immunohistochemistry analysis after 7 days of treatment. The ethanol extracts of leaves and roots presented no toxicity at a 2000 mg kg-1 dose for rats and mice, and at a 5000 mg kg-1 dose for rats. Histological changes in the liver of mice were verified with a 5000 mg kg-1 dose of the leaf and root extracts. Macroscopic and histological differences were not observed in the contraction of wounds between the groups treated with leaf and root extracts and the control group after 4 and 7 days of treatment. After 14 days of treatment, 50% of the animals treated with the root extract presented total re-epithelialization of wounds and partial reconstruction of the annexes. Allantoin, isolated from the root ethanol extract, can be considered one of the secondary metabolites responsible for accelerating re-epithelialization.

Immunosuppressive properties of mesenchymal stem cells: advances and applications.

Mesenchymal stem cells (MSCs) have been isolated from a variety of tissues, such as bone marrow, skeletal muscle, dental pulp, bone, umbilical cord and adipose tissue. MSCs are used in regenerative medicine mainly based on their capacity to differentiate into specific cell types and also as bioreactors of soluble factors that will promote tissue regeneration from the damaged tissue cellular progenitors. In addition to these regenerative properties, MSCs hold an immunoregulatory capacity, and elicit immunosuppressive effects in a number of situations. Not only are they immunoprivileged cells, due to the low expression of class II Major Histocompatibilty Complex (MHC-II) and costimulatory molecules in their cell surface, but they also interfere with different pathways of the immune response by means of direct cell-to-cell interactions and soluble factor secretion. In vitro, MSCs inhibit cell proliferation of T cells, B-cells, natural killer cells (NK) and dendritic cells (DC), producing what is known as division arrest anergy. Moreover, MSCs can stop a variety of immune cell functions: cytokine secretion and cytotoxicity of T and NK cells; B cell maturation and antibody secretion; DC maturation and activation; as well as antigen presentation. It is thought that MSCs need to be activated to exert their immunomodulation skills. In this scenario, an inflammatory environment seems to be necessary to promote their effect and some inflammation-related molecules such as tumor necrosis factor-α and interferon-γ might be implicated. It has been observed that MSCs recruit T-regulatory lymphocytes (Tregs) to both lymphoid organs and graft. There is great controversy concerning the mechanisms and molecules involved in the immunosuppressive effect of MSCs. Prostaglandin E2, transforming growth factor-ß, interleukins- 6 and 10, human leukocyte antigen-G5, matrix metalloproteinases, indoleamine-2,3-dioxygenase and nitric oxide are all candidates under investigation. In vivo studies have shown many discrepancies regarding the immunomodulatory properties of MSCs. These studies have been designed to test the efficacy of MSC therapy in two different immune settings: the prevention or treatment of allograft rejection episodes, and the ability to suppress abnormal immune response in autoimmune and inflammatory diseases. Preclinical studies have been conducted in rodents, rabbits and baboon monkeys among others for bone marrow, skin, heart, and corneal transplantation, graft versus host disease, hepatic and renal failure, lung injury, multiple sclerosis, rheumatoid arthritis, diabetes and lupus diseases. Preliminary results from some of these studies have led to human clinical trials that are currently being carried out. These include treatment of autoimmune diseases such as Crohn's disease, ulcerative colitis, multiple sclerosis and type 1 diabetes mellitus; prevention of allograft rejection and enhancement of the survival of bone marrow and kidney grafts; and treatment of resistant graft versus host disease. We will try to shed light on all these studies, and analyze why the results are so contradictory.

The interstitial lymphatic peritoneal mesothelium axis in portal hypertensive ascites: when in danger, go back to the sea.

Portal hypertension induces a splanchnic and systemic low-grade inflammatory response that could induce the expression of three phenotypes, named ischemia-reperfusion, leukocytic, and angiogenic phenotypes.During the splanchnic expression of these phenotypes, interstitial edema, increased lymph flow, and lymphangiogenesis are produced in the gastrointestinal tract. Associated liver disease increases intestinal bacterial translocation, splanchnic lymph flow, and induces ascites and hepatorenal syndrome. Extrahepatic cholestasis in the rat allows to study the worsening of the portal hypertensive syndrome when associated with chronic liver disease. The splanchnic interstitium, the mesenteric lymphatics, and the peritoneal mesothelium seem to create an inflammatory pathway that could have a key pathophysiological relevance in the production of the portal hypertension syndrome complications. The hypothetical comparison between the ascitic and the amniotic fluids allows for translational investigation. From a phylogenetic point of view, the ancestral mechanisms for amniotic fluid production were essential for animal survival out of the aquatic environment. However, their hypothetical appearance in the cirrhotic patient is considered pathological since ultimately they lead to ascites development. But, the adult human being would take advantage of the potential beneficial effects of this "amniotic-like fluid" to manage the interstitial fluids without adverse effects when chronic liver disease aggravates.

Reacciones adversas psiquiátricas asociadas a nuevos macrólidos. A propósito de tres casos / Psychiatric adverse reactions associated to new macrolides. Three case reports

Los macrólidos representan el 10-15% del mercado mundial de antibióticos orales. Son uno de los grupos de antibióticos más seguros; las reacciones adversas graves son muy raras. Pueden producir reacciones gastrointestinales, hepatotoxicidad y ototoxicidad. Además, las reacciones psiquiátricas se encuentran entre los efectos adversos esporádicamente asociados con su administración, aunque su mecanismo no está bien establecido. Casos descritos en la Food and Drug Administration (FDA) de EE.UU. muestran que la claritromicina y el ciprofloxacino son los antibióticos más frecuentemente asociados con el desarrollo de manía. Este término ha sido denominado antibiomanía. Presentamos tres casos clínicos vistos en una consulta de Atención Primaria en el período de un año con cuadros similares de hiperactividad y agresividad, coincidiendo con la administración de antibióticos de la familia de los macrólidos (AU)

Macrolides represent the 10-15% of the world-wide market of oral antibiotics. They are one of the safer groups of antibiotics, being the severe adverse reactions very rare. They can produce gastrointestinal reactions, hepatotoxicity and ototoxicity. The psychiatric reactions are found sporadically among the adverse effects. Cases reported to the FDA showed that clarithromycin and ciprofloxacin are the most frequent antibiotics associated with the development of mania. The syndrome has been termed antibiomania. We present three clinical cases seen in a Primary Care office in the last year with similar pictures of hyperactivity and aggressiveness coinciding with the administration of antibiotics of the family of the macrolides (AU)

A5 region modulation of the cardiorespiratory responses evoked from parabrachial cell bodies in the anaesthetised rat.

We have examined the importance of the A5 region modulating cardiorespiratory responses evoked from the parabrachial complex (PB) in spontaneously breathing rats. Cardiorespiratory changes were analyzed in response to electrical stimulation and glutamate microinjections into the PB (10-20 nl, 1-2 nmol) before and after ipsilateral microinjection of muscimol (50 nl, 0.25 nmol) or lidocaine (50 nl, 0.5 nmol) within the A5 region. Stimulation of medial parabrachial and Kölliker-Fuse nuclei (mPB-KF) evoked a decrease in respiratory rate (P<0.001) with a rise in blood pressure (P<0.001) and heart rate (P<0.05). After muscimol or lidocaine microinjections within the A5 region, the pressor and heart rate responses to mPB-KF stimulation were reduced (P<0.05, both cases). Muscimol within the A5 region altered the respiratory response to glutamate stimulation of mPB-KF, evoking an increase in respiratory rate (P<0.05). Lidocaine abolished the respiratory response to mPB-KF stimulation. Stimulation of the lateral parabrachial nuclei (lPB) caused an increase in respiratory rate (P<0.001) with a rise in blood pressure (P<0.001) and heart rate (P<0.05). Muscimol or lidocaine microinjections within A5 region decreased heart rate (P<0.05) and pressor responses (P<0.05) evoked from lPB. The increase of respiratory rate persisted unchanged. To confirm functional interactions between A5 and PB, extracellular recordings of putative A5 neurones were obtained during PB stimulation. Eighty-three A5 cells were recorded, 35 were activated from the mPB-KF (42%). The results indicate that neurones of the A5 region participate in the cardiorespiratory response evoked from the different regions of the PB complex. The possible mechanisms involved in these interactions are discussed.

[Clinical characteristics of children with chronic pain in a pediatric pain unit: oncologic pain versus non-oncologic pain]. / Análisis de las características clínicas de los pacientes con dolor crónico tratados por la unidad de dolor infantil: dolor oncológico frente a no oncológico.

BACKGROUND: Despite undoubted scientific advances in the field of chronic pain in children, there is no evidence of clinical application of this knowledge. OBJECTIVE: To describe the experience of a pediatric pain unit (PPU) specifically dedicated to the treatment of chronic pain in children. MATERIAL AND METHODS: We performed an analytic, observational, retrospective, cohort study of the clinical features of the first 42 patients treated for chronic pain in the PPU during a two-year period. The patients were assigned to two groups: an oncologic group and a non-oncologic group. ANOVA was used to analyze quantitative variables and the Chi-square test was used to analyze qualitative variables. RESULTS: No significant differences were found between the two groups in the demographic variables studied (age and sex). Concerning the type of treatment used, no significant differences were found in effectiveness or compliance. However, treatment duration was significantly longer in the non-oncologic group than in the oncologic group (74.2 days vs 37.5 days, p(0.008). The duration of non-oncologic chronic pain before attending the PPU (mean: 557 days) influenced the effectiveness (r 5 0.781; p 5 0.0001) and duration of treatment (r 5 0.61; p 5 0.0051). However, the duration of previous chronic oncologic pain was significantly shorter (mean: 34 days) and showed no influence on treatment effectiveness or duration. CONCLUSIONS: The pediatric population presents chronic pain syndromes that can be appropriately treated in a PPU with conventional, easy to manage analgesics. We recommend the establishment of pediatric pain units similar to those for adults, using a multidisciplinary approach to mitigate children's suffering.

Lack of germline transmission of vector sequences following systemic administration of recombinant AAV-2 vector in males.

A potential consequence of systemic administration of viral vectors is the inadvertent introduction of foreign DNA into recipient germ cells. To evaluate the safety of in vivo recombinant adeno-associated virus (rAAV) mediated gene transfer approaches for hemophilia B, we explored the risk of germline transmission of vector sequences following intramuscular (IM) injection of rAAV in four species of male animals (mouse, rat, rabbit and dog). In vector biodistribution studies in mice and rats, there is a dose-dependent increase in the likelihood that vector sequences can be detected in gonadal DNA using a sensitive PCR technique. However, in dogs DNA extracted from semen is negative for vector sequences. To address this discrepancy, studies were done in rabbits, and both semen and testicular DNAs were analyzed for the presence of vector sequences. These studies showed that no AAV vector sequences were detected in DNA extracted from rabbit semen samples collected at time points ranging from 7 to 90 days following IM injection of 1 x 10(13) vector genomes rAAV (vg) per kg. In contrast, DNA extracted from gonadal tissue was positive for vector sequences, but the positive signals diminished in number and strength with time. By FISH analysis, AAV signals were localized to the testis basement membrane and the interstitial space; no intracellular signal was observed. We observed similar findings following hepatic artery administration of rAAV in rats and dogs, suggesting that our findings are independent of the route of administration of vector. Attempts to transduce isolated murine spermatogonia directly with AAV-lacZ were unsuccessful. In clinical studies human subjects injected IM with an AAV vector at doses up to 2 x 10(12) vg/kg have shown no evidence of vector sequences in semen. Together, these studies suggest that rAAV introduced into skeletal muscle or the hepatic artery does not transduce male germ cells efficiently. We conclude that the risk of inadvertent germline transmission of vector sequences following IM or hepatic artery injection of AAV-2 vectors is extremely low.

Germ cell biology--from generation to generation.

Germ cells hold a unique place in the life cycle of animal species in that they are the cells that will carry the genome on to the next generation. In order to do this they must retain their DNA in a state in which it can be used to recapitulate embryonic development. In the normal life cycle, the germ cells are the only cells that retain this ability to recapitulate development, referred to as developmental totipotency. The molecular mechanisms regulating developmental potency are poorly understood. Recently its has been shown that germ cells can be turned into pluripotent stem cells when cultured in specific polypeptide growth factors that affect their survival and proliferation. The ability to manipulate developmental potency in germ cells with growth factors allows the underlying mechanisms to be dissected. Germ cells are also the only cells that undergo the unique reductive division of meiosis. This too is essential for the ability of germ cells to form the gametes that will carry the genome into the next generation. Arguably meiosis is the most important division in the life of a nascent organism. Defects in meiosis can result in embryonic or fetal loss or, if the animal survives, in the birth of an individual with chromosomal abnormalities. Recent advances in our understanding of meiosis have come from knockout mice and studies on genes identified through studies of human infertility. This review will focus on these two key aspects of germ cell biology, developmental potency and meiosis.