Distinct effects of intravenous bone marrow-derived mesenchymal stem cell therapy on ischemic and non-ischemic lungs after ischemia-reperfusion injury.

BACKGROUND: The preclinical efficacy of mesenchymal stem cell (MSC) therapy after intravenous infusion has been promising, but clinical studies have yielded only modest results. Although most preclinical studies have focused solely on the ischemic lung, it is crucial to evaluate both lungs after ischemia-reperfusion injury, considering the various mechanisms involved. This study aimed to bridge this gap by assessing the acute effects of bone marrow MSC(BM) infusion before ischemic insult and evaluating both ischemic and non-ischemic lungs after reperfusion. METHODS: Eighteen male Wistar rats (403 ± 23 g) were anesthetized and mechanically ventilated using a protective strategy. After baseline data collection, the animals were randomized to 3 groups (n = 6/group): (1) SHAM; (2) ischemia-reperfusion (IR), and (3) intravenous MSC(BM) infusion followed by IR. Ischemia was induced by complete clamping of the left hilum, followed by 1 h of reperfusion after clamp removal. At the end of the experiment, the right and left lungs (non-ischemic and ischemic, respectively) were collected for immunohistochemistry and molecular biology analysis. RESULTS: MSC(BM)s reduced endothelial cell damage and apoptosis markers and improved markers associated with endothelial cell integrity in both lungs. In addition, gene expression of catalase and nuclear factor erythroid 2-related factor 2 increased after MSC(BM) therapy. In the ischemic lung, MSC(BM) therapy mitigated endothelial cell damage and apoptosis and increased gene expression associated with endothelial cell integrity. Conversely, in the non-ischemic lung, apoptosis gene expression increased in the IR group but not after MSC(BM) therapy. CONCLUSION: This study demonstrates distinct effects of MSC(BM) therapy on ischemic and non-ischemic lungs after ischemia-reperfusion injury. The findings underscore the importance of evaluating both lung types in ischemia-reperfusion studies, offering insights into the therapeutic potential of MSC(BM) therapy in the context of lung injury.

Pharmacological evaluation of a traditional Brazilian medicinal plant, Monteverdia ilicifolia. Part I - Preclinical safety study.

ETHNOPHARMACOLOGICAL RELEVANCE: Monteverdia ilicifolia (Maytenus ilicifolia, Celastraceae), known as "espinheira-santa", has been widely used in Brazil to manage mainly gastrointestinal diseases. This species has been listed in the Brazilian Pharmacopeia and in the National List of Essential Medicines (RENAME). Considering that clinical studies about M. ilicifolia are rare, our group has been performing a broader project designed to evaluate the efficacy of M. ilicifolia capsules in a clinical trial, for this reason, approaches to provide safety to those patients are relevant. AIM OF THE STUDY: We aimed to investigate the potential pharmacokinetic interaction and hepatotoxicity and intestinal toxicity of an aqueous extract of M. ilicifolia and its main phytocompounds, catequin, epicatequin, and quercetin. METHODS AND MATERIALS: Slices of liver and intestine of Wistar rats were incubated with different concentrations of M. ilicifolia extract or isolated compounds (catechin, epicatechin and quercetin). Commercial kits were used to evaluate enzyme activities of CYP2D6 and CYP3A4 isoforms, as well as cell viability (MTT) assay and intracellular enzymes leakage, specifically lactate dehydrogenase (LDH), alkaline phosphatase (AP), aspartate aminotransferase (AST), alanine aminotransferase (ALT) were studied. RESULTS: Incubation with M. ilicifolia extract, catechin, epicatechin and quercetin did not affect significantly any evaluated parameter in intestines. The intracellular enzymes leakages, CYP2D6, LDH and AST, were increased with M. ilicifolia extract and quercetin in liver slices. CONCLUSIONS: Our in vitro findings highlighted, for the first time, the potential hepatotoxicity induced by an aqueous extract of M. ilicifolia, consequently this species and its products should be avoided in liver diseases, supporting that studies of safety must be performed including in the context of traditional medicinal plants.

Cytotoxicity profile of Cronobacter species isolated from food and clinical specimens in Brazil.

AIMS: The objective of this study was to evaluate the cytotoxic activity of Cronobacter strains isolated from foods (n = 50) and clinical samples (n = 6) in Brazil and genotype selected strains (n = 18) using multi-locus sequence typing (MLST) METHODS AND RESULTS: The cytotoxic activity of C. sakazakii (n = 29), C. dublinensis (n = 13), C. malonaticus (n = 6), C. turicensis (n = 6) and C. muytjensii (n = 2) was screened using Vero, RK13, Hep2c, NCTC clone 929 and BHK-21 cell lines. Selected Cronobacter strains were assigned to C. sakazakii ST 21, C. turicensis ST 252, C. sakazakii ST 647, and three newly assigned STs: C. turicensis STs 738-740. The maximum death caused by non-heat-treated filtrates was 20·4, 86·2, 47·0 and 84·0%, in Vero, RK13, Hep2c and NCTC clone 929 cells, respectively. These were caused by C. sakazakii strains C291 and C292 (ST 494) which had been isolated during neonatal Cronobacter meningitis infection, and C110 (ST 395) isolated from flaxseed flour. Thermal treatment (100°C/20 min) significantly reduced the cytotoxicity activity in NCTC clone 929 and Vero cells (P ≤ 2 × 10-6 ), but not in RK13 (P = 0·12) and Hep2c (P = 0·85), indicating the cytotoxin(s) were probably proteinaceous. Electron microscopy revealed that cytotoxic compounds from C. sakazakii induced several cell death characteristics, including loss of cell-cell contact, microvilli reduction and cellular lysis. Autophagic vacuoles and mitochondrial damage were the most common ultrastructural features observed. CONCLUSIONS: It was concluded that Cronobacter strains, especially C. sakazakii, could produce heat-labile cytotoxic compounds in cell filtrates. SIGNIFICANCE AND IMPACT OF THE STUDY: This study providing insights into the pathogenesis of the Cronobacter genus. Cytotoxins were identified in excreted filtrates of C. sakazakii strains isolated from food and clinical specimens. The presence of Cronobacter strains that can produce cytotoxins in foods can be a potential threat to human health and highlight the need for high levels of hygiene.

Successive exposure to Mentha piperita L. essential oil affects the culturability and induces membrane repair in a persister epidemic Salmonella Typhimurium PT4.

This study had as aims to evaluate the effects of successive exposures to Mentha piperita L. essential oil (MPEO) on culturability and physiological functions of Salmonella Typhimurium PT4. S. Typhimurium PT4 cells (108 log CFU/mL) were exposed to the same (1.25 µL/mL) or increasing MPEO concentrations (1.25-80 µL/mL) during 252 h. At each 36-h interval, the viable cell counts, and distinct cell functions were assessed using plate counting and flow cytometry, respectively. As the exposure time to the same MPEO concentration increased, the population of S. Typhimurium PT4 cells with damaged, permeabilized and depolarized membrane, and compromised efflux activity decreased. Otherwise, S. Typhimurium PT4 cells with damaged membrane physiological functions increased over the exposure to increasing concentrations of MPEO. Genomic analyses showed that the strain carries 17 genes associated with stress responses and the persistence of the tested strain among sources associated with poultry spanning more than 16 years and its virulence for humans. Therefore, successive exposure to a sublethal concentration of MPEO induced S. Typhimurium PT4 cells capable of maintaining the membrane integrity and its functions despite their non-culturable state.

Efficacy of oregano and rosemary essential oils to affect morphology and membrane functions of noncultivable sessile cells of Salmonella Enteritidis 86 in biofilms formed on stainless steel.

AIMS: This study evaluated the efficacy of essential oil from Origanum vulgare L. (oregano; OVEO) and Rosmarinus officinalis L. (rosemary; ROEO) to inactivate sessile cells of Salmonella enterica serovar Enteritidis 86 (SE86) in young and mature biofilms formed on stainless steel. METHODS AND RESULTS: Ultrastructural alterations and damage in different physiological functions caused by OVEO and ROEO in noncultivable sessile cells of SE86 were investigated using scanning electron microscopy and flow cytometry. OVEO (2·5 µl ml-1 ) and ROEO (40 µl ml-1 ) were effective to eradicate young and mature biofilms formed by SE86 sessile cells on stainless steel surfaces; however, the efficacy varied with exposure time. OVEO and ROEO caused alterations in morphology of SE86 sessile cells, inducing the occurrence of bubbles or spots on cell surface. OVEO and ROEO compromised membrane polarization, permeability and efflux activity in noncultivable SE86 sessile cells. These findings show that OVEO and ROEO act by a multitarget mechanism on SE86 membrane functions. CONCLUSIONS: ROEO and OVEO showed efficacy to eradicate SE86 sessile cells in preformed biofilms on stainless steel, displaying a time-dependent effect and multitarget action mode on bacterial cell membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides for the first time the effects of OVEO and ROEO on morphology and physiological functions of noncultivable sessile cells of S. Enteritidis biofilms preformed on stainless steel surfaces.

UDCA, NorUDCA, and TUDCA in Liver Diseases: A Review of Their Mechanisms of Action and Clinical Applications.

Bile acids (BAs) are key molecules in generating bile flow, which is an essential function of the liver. In the last decades, there have been great advances in the understanding of BA physiology, and new insights have emerged regarding the role of BAs in determining cell damage and death in several liver diseases. This new knowledge has helped to better delineate the pathophysiology of cholestasis and the adaptive responses of hepatocytes to cholestatic liver injury as well as of the mechanisms of injury of biliary epithelia. In this context, therapeutic approaches for liver diseases using hydrophilic BA (i.e., ursodeoxycholic acid, tauroursodeoxycholic, and, more recently, norursodeoxycholic acid), have been revamped. In the present review, we summarize current experimental and clinical data regarding these BAs and its role in the treatment of certain liver diseases.

Bile Acids in Cholestasis and its Treatment.

Bile acids (BA) are key molecules in generating bile flow, which is an essential function of the liver. In the last decades there have been great advances in the understanding of the role of a number of specific transport proteins present at the sinusoidal and canalicular membrane domains of hepatocytes and cholangiocytes in generating and maintaining bile flow. Also, a clearer understanding on how BA regulate their own synthesis and the expression and/or function of transporters has been reached. This new knowledge has helped to better delineate the pathophysiology of cholestasis and the adaptive responses of hepatocytes to cholestatic liver injury as well as of the mechanisms of injury of biliary epithelia. In this context, therapeutic approaches including new hydrophilic BA such as the conjugation-resistant nor- ursodeoxycholic acid, nuclear receptors (FXR, PPAR-alpha) agonists, FGF19 analogues, inhibitors of the apical sodium-dependent bile acid transporter [ASBT] and modulators of the inflammatory cascade triggered by BAs are being studied as novel treatments of cholestasis. In the present review we summarize recent experimental and clinical data on the role of BAs in cholestasis and its treatment.

VMP1-related autophagy induced by a fructose-rich diet in ß-cells: its prevention by incretins.

The aim of the present study was to demonstrate the role of autophagy and incretins in the fructose-induced alteration of ß-cell mass and function. Normal Wistar rats were fed (3 weeks) with a commercial diet without (C) or with 10% fructose in drinking water (F) alone or plus sitagliptin (CS and FS) or exendin-4 (CE and FE). Serum levels of metabolic/endocrine parameters, ß-cell mass, morphology/ultrastructure and apoptosis, vacuole membrane protein 1 (VMP1) expression and glucose-stimulated insulin secretion (GSIS) were studied. Complementary to this, islets isolated from normal rats were cultured (3 days) without (C) or with F and F + exendin-4 or chloroquine. Expression of autophagy-related proteins [VMP1 and microtubule-associated protein light chain 3 (LC3)], apoptotic/antiapoptotic markers (caspase-3 and Bcl-2), GSIS and insulin mRNA levels were measured. F rats developed impaired glucose tolerance (IGT) and a significant increase in plasma triacylglycerols, thiobarbituric acid-reactive substances, insulin levels, homoeostasis model assessment (HOMA) for insulin resistance (HOMA-IR) and ß-cell function (HOMA-ß) indices. A significant reduction in ß-cell mass was associated with an increased apoptotic rate and morphological/ultrastructural changes indicative of autophagic activity. All these changes were prevented by either sitagliptin or exendin-4. In cultured islets, F significantly enhanced insulin mRNA and GSIS, decreased Bcl-2 mRNA levels and increased caspase-3 expression. Chloroquine reduced these changes, suggesting the participation of autophagy in this process. Indeed, F induced the increase of both VMP1 expression and LC3-II, suggesting that VMP1-related autophagy is activated in injured ß-cells. Exendin-4 prevented islet-cell damage and autophagy development. VMP1-related autophagy is a reactive process against F-induced islet dysfunction, being prevented by exendin-4 treatment. This knowledge could help in the use of autophagy as a potential target for preventing progression from IGT to type 2 diabetes mellitus.

Bile Acids in Cholestasis and its Treatment.

Bile acids (BA) are key molecules in generating bile flow, which is an essential function of the liver. In the last decades there have been great advances in the understanding of the role of a number of specific transport proteins present at the sinusoidal and canalic-ular membrane domains of hepatocytes and cholangiocytes in generating and maintaining bile flow. Also, a clearer understanding on how BA regulate their own synthesis and the expression and/or function of transporters has been reached. This new knowledge has helped to better delineate the pathophysiology of cholestasis and the adaptive responses of hepatocytes to cholestatic liver injury as well as of the mechanisms of injury of biliary epithelia. In this context, therapeutic approaches including the use of new hydrophilic BA such as the conjugation-resistant nor- ursodeoxycholic acid, nuclear receptor (FXR, PPAR-alpha) agonists, FGF19 analogues, inhibitors of the apical sodium-depend bile acid transporter (ASBT) and modulators of the inflammatory cascade triggered by BA are being studied as novel treatments of cholestasis. In the present review we summarize recent experimental and clinical data on the role of BA in cholestasis and its treatment.

Side Effects of Neem Oil on the Midgut Endocrine Cells of the Green Lacewing Ceraeochrysa claveri (Navás) (Neuroptera: Chrysopidae).

We described the ultrastructure of Ceraeochrysa claveri (Navás) midgut endocrine cells in larva, pupa, and adult, and evaluated the side effects of ingested neem oil, a botanical insecticide obtained from the seeds of the neem tree (Azadirachta indica), on these cells. During the larval period, C. claveri were fed (ad libitum) Diatraea saccharalis (F.) eggs treated with neem oil at concentrations of 0.5%, 1%, or 2%. Transmission electron microscopy showed that two subtypes of endocrine cells, namely granular and vesicular, occurred in the midgut epithelium during the three stages of the life cycle. Both cell types did not reach the midgut lumen and were positioned basally in the epithelium. The endocrine cells did not show extensive infoldings of the basal plasma membrane, and there were numerous secretory granules in the basal region of the cytoplasm. In the granular endocrine cells, the granules were completely filled with a dense matrix. In the vesicular endocrine cells, the main secretory products consisted of haloed vesicles. Ultrastructural examination indicated that only the granular endocrine cells exhibited signs of morphologic changes of cell injury present in all life cycle stages after the larvae were chronically exposed to neem oil by ingestion. The major cellular damage consisted of dilatation and vesiculation of the rough endoplasmic reticulum and the development of smooth endoplasmic reticulum and mitochondrial swelling. Our data suggest that cytotoxic effects on midgut endocrine cells can contribute to a generalized disruption of the physiological processes in this organ due to a general alteration of endocrine function.

The influence of cell dimensions on the vulnerability of ventricular myocytes to lethal injury by high-intensity electrical fields / Influência das dimensões celulares sobre a vulnerabilidade de miócitos ventriculares ao efeito letal de campos elétricos de alta intensidade

Application of high intensity electric fields (HIEF) to the myocardium is commonly used for cardiac defibrillation/cardioversion. Although effective at reversing life-threatening arrhythmias, HIEF may cause myocyte damage due to membrane electropermeabilization. In this study, the influence of cell length and width on HIEF-induced lethal injury was analyzed in isolated rat cardiomyocytes in parallel alignment with the field. The field-induced maximum variation of membrane potential (ΔVmax) was estimated with the Klee-Plonsey model. The studied myocyte population was arranged in two group pairs for comparison: the longest vs. the shortest cells, and the widest vs. narrowest cells. Threshold field intensity was significantly lower in the longest vs. shortest myocytes, whereas cell width influence was not significant. The threshold ΔVmax was comparable in all groups. Likewise, a significant leftward shift of the lethality curve (i.e., relationship of the probability of lethality vs. field intensity) of the longest cells was observed, evidencing greater sensitivity to HIEF-induced damage. However, the lethality curve as a function of ΔVmax was similar in all groups, confirming a prediction of the Klee-Plonsey model. The similar results for excitation and injury at threshold and HIEF stimulation, respectively, indicate that: a) the effect of cell length on the sensitivity to the field would be attributable to differences in field-induced membrane polarization that lead to excitation or lethal electroporation; b) the Klee-Plonsey model seems to be reliable for analysis of cell interaction with HIEF; c) it is possible that increased cell length in hypertrophied hearts enhances myocyte fragility upon defibrillation/cardioversion.

Campos elétricos de alta intensidade (HIEF) são aplicados ao miocárdio durante desfibrilação e cardioversão. Embora eficazes na reversão de arritmias potencialmente letais, HIEF podem lesar cardiomiócitos por eletropermeabilização da membrana. Neste estudo, a influência das dimensões celulares sobre o efeito letal de HIEF foi estudada em cardiomiócitos isolados de rato alinhados paralelamente ao campo. A máxima variação do potencial de membrana induzida pelo campo (ΔVmax) foi calculada com o modelo de Klee-Plonsey. As células estudadas foram distribuídas em dois pares de grupos de acordo com seu comprimento e largura. A intensidade limiar do campo não dependeu da largura celular, mas sim do comprimento (menor nas células mais longas, p < 0.001), enquanto ΔVmax no limiar foi comparável entre os grupos. Nas células mais longas, observou-se desvio à esquerda (p < 0.01) da curva que descreve a relação entre probabilidade de letalidade e a intensidade do campo, evidenciando maior sensibilidade à ação deletéria de HIEF. Porém, a curva de letalidade em função de ΔVmax foi semelhante em todos os grupos, o que confirma a predição pelo modelo de Klee-Plonsey. A similaridade de resultados com estimulação limiar e com HIEF indica que: a) o efeito do comprimento celular sobre a sensibilidade ao campo poderia ser atribuído a diferenças no grau de polarização da membrana durante a aplicação do estímulo; b) o modelo de Klee-Plonsey parece ser confiável para a análise da interação espacial da célula com HIEF; c) é possível que o maior comprimento celular em miócitos hipertrofiados os torne mais susceptíveis a lesão durante desfibrilação/cardioversão.

Propuesta de protocolo de biopsia cardíaca. Servicio de Anatomía Patológica y Departamento de Cardiología / Proposal of cardiac biopsy protocol. Service of Pathology and Department of Cardiology / Proposta de protocolo de biópsia cardíaca. Serviços de anatomia patológica e departamento de cardiologia

Introducción: Las biopsias endomiocárdicas se utilizan habitualmente para el diagnóstico de muchas patologías que han sido agrupadas por Billigham y Tazelaar en inflamatorias, metabólicas, endocrinas, neuromusculares, tóxicas, procesos linfoproliferativos y el diagnóstico de rechazo a trasplante cardíaco para evaluar la cardiotoxicidad por drogas (antraciclinas, dexoxirubina, cocaína, alcohol, entre otras); también como análisis de miocardio isquémico y su zona limítrofe. Objetivo: Trataremos de evaluar desde un punto de vista cuantitativo, la amplitud de observaciones que se han hecho de la biopsia cardíaca, en un innumerable listado de patologías, desde la MO a la ME (Microscopía óptica a microscopía electrónica). Se intentará graduar los hallazgos morfológicos de la misma, enlazándolos con la topografía y función, teniendo en cuenta, además, los factores bioquímicos y genéticos, organismos vivos, drogas, agentes físicos y procedimientos diagnósticos. Material y Métodos: Para el presente estudio se realizó una recopilación bibliográfica teniendo en cuenta los siguientes criterios: 1) cambios morfológicos sub-celulares-matriz tisular; 2) score de necrosis; 3) score inflamatorio, 4) score de fibrosis; 5) depósitos intracelulares y de pigmentos. Procesamiento de las muestran fijadas en formol buffer (tiempo 2-12 hs.) con coloración de rutina: H/E, Pas, Masson, Zihel Neelsen prolongada, Perls, Azul de Toluidina, Rojo Congo, Orceina. Panel con técnicas de I.H.Q.(BIO SB®).Conclusión: Creemos importante establecer un método que permita hacer una adecuada correlación clínico patológica aplicable a mejorar la interpretación de la injuria tisular, celular y facilitar de este modo, una correcta elección terapéutica.

Introdução: As biópsias endomiocárdicas são utilizadas habitualmente para o diagnóstico de muitas patologias, que foram agrupadas por Billigham e Tazelaar em inflamatórias, metabólicas, endócrinas, neuromusculares, tóxicas, processos linfoproliferativos e o diagnóstico de rejeição a trasplante cardíaco, para avaliar a cardiotoxicidade por drogas (antraciclinas, doxorrubicina, cocaína, álcool, entre outras). Também como análise de miocárdio isquêmico e sua zona limítrofe. Objetivo: Trataremos de avaliar sob o ponto de vista quantitativo, a amplidão de observações feitas da biópsia cardíaca, em uma inumerável lista de patologias, de MO a ME.Tentaremos graduar os descobrimentos morfológicos da mesma, enlaçando-os com a topografia e função, levando em consideração também os fatores bioquímicos e genéticos, organismos vivos, drogas, agentes físicos, procedimentos e diagnósticos.Material e Métodos: para o presente estudo realizou-se uma recopilação bibliográfica considerando os seguintes critérios, 1) alterações morfológicas subcelulares-matriz tissular; 2) escore de necrose; 3) escore inflamatório, 4) escore de fibrose; 5) depósitos intracelulares e de pigmentos. Processamento das amostras fixadas em formol buffer (tempo 2 -12 h.) com coloração de rotina: H/E, Pas, Masson, Zihel Neelsen prolongada, Perls, Azul de Toluidina, Vermelho Congo, Orceína. Painel com técnicas de I.H.Q. (BIO SB®). Conclusão: Achamos importante estabelecer um método que permita fazer uma adequada correlação clínico patológica aplicável a melhorar a interpretação da injúria tisular celular e, deste modo, facilitar uma correta decisão terapêutica.

Introduction: The endomiocárdica biopsies is habitually used for the diagnosis of many pathologies, grouped by Billigham and Tazelaar in inflammatory, metabolic, endocrine, neuromuscular, toxic, processes linfoproliferative and in diagnosis of rejection to heart transplant or to evaluate the cardiotoxicity for drugs (antracicline, dexoxirubine, cocaine, alcohol among other). Also as analysis of cardiac ischemia and their bordering area. Objective: We will try to evaluate from a quantitative point of view, the width of observations that you/they have been made of the heart biopsy in a countless listing of pathologies, from the MO to ME. We will try to graduate the morphologic discoveries of the same one, connecting them with the topography and function, also keeping in mind the biochemical and genetic factors, alive organisms, you drug, physical agents, procedures diagnoses. (Sherman Bloom, pag 329, I Diagnose of Cardiovasc. Pathology).Material and Methods: for the present study was carried out a bibliographical summary keeping in mind the following approaches, 1) changes morphologic subcellular-main tissue; 2) necrosis score; 3) inflammatory score 4) fibrosis score; 5) deposits intracellular and of pigments. Prosecution of they show them fixed in formol buffer (time 2 -12 hs.) with routine coloration: H/E, Pas, Masson, Zihel lingering Neelsen, Perls, Blue of Toluidine, Red Congo, Orceine. Panel with technical of I.H.Q. (BIO SB®). Conclusion: We believe as very important to establish a methodology eich allow an applicable pathological clinical appropriate correlation to improve the interpretation of the tissue insult and to facilitate in this way a correct therapeutic election.