Efficient isolation and proliferation of human adipose-derived mesenchymal stromal cells in xeno-free conditions.

Classical methods used for culture of adipose-derived mesenchymal stromal cells (ADSCs) use xenobiotic components, which may present a potential risk for biological contamination and/or elicit immunological reactions. Therefore, the aim of this study was to establish a xeno-free methodology for the isolation and proliferation of human ADSCs (hADSCs). hADSCs were isolated by enzymatic digestion or mechanical dissociation and cultured in the presence of fetal bovine serum or human platelet lysate. Proliferation curves were performed as a function of time from the cell culture and used to calculate the population doubling time. Immunophenotyping and differentiation tests were used to identify and characterize the hADSCs. Human ADSCs isolated and cultured in conventional or xenobiotic-free conditions peaked at different days but achieved similar maximum proliferation. The hADSCs differentiation ability was similar in all groups. The characterization of hADSCs by flow cytometry showed low contamination of the cultures by other cell types. The xenobiotic-free methodology described in this study is a feasible and reproducible alternative for isolation and proliferation of hADSCs. This methodology is in accordance with the recommendations of the National Health Surveillance Agency, which proposes avoidance of xenobiotic products.

Nyctinomops laticaudatus bat-associated Rabies virus causes disease with a shorter clinical period and has lower pathogenic potential than strains isolated from wild canids.

Rabies is a lethal viral disease that can affect a wide range of mammals. Currently, Rabies virus (RABV) in some European and American countries is maintained primarily in wild species. The regulation of viral replication is one of the critical mechanisms involved in RABV pathogenesis. However, the relationship between replication and the pathogenesis of RABV isolated from wild animals remains poorly understood. In the present study, we evaluated the pathogenicity of the street viruses Nyctinomops laticaudatus bat-associated RABV (NYBRV) and Cerdocyon thous canid-associated RABV (CECRV). Infection of mice with NYBRV led to 33% mortality with rapid disease evolution and marked histopathological changes in the CNS. In contrast, infection with CECRV led to 67% mortality and caused mild neuropathological lesions. The proportion of RABV antigen was significantly higher in the cytoplasm of neuronal cells of the cerebral cortex and in the meninges of mice infected with CECRV and NYBRV, respectively. Moreover, the replication rate of NYBRV was significantly higher (p < 0.001) than that of CECRV in neuroblastoma cells. However, CECRV replicated to a significantly higher titer in epithelial cells. Our results indicate that NYBRV infection results in rapid disease progression accompanied by frequent and intense histopathological alterations in the CNS in mice, and in a high replication rate in neuroblastoma cells. Although, CECRV is more pathogenic in mice, it caused milder histopathological changes in the CNS and replicated more efficiently in epithelial cells. Our data point to a correlation between clinical aspects of disease and the replication of RABV in different cell lines.

Street rabies virus strains associated with insectivorous bats are less pathogenic than strains isolated from other reservoirs.

Rabies is a fatal and viral zoonosis that causes acute, progressive encephalitis and remains an important concern in public health. In the last few years, there has been a change in the epidemiological profile of rabies after implementing canine rabies control in the Americas, which has led to a significant increase in both human and pet cases of rabies associated with insectivorous bats. Thus, it is important to understand the pathogenesis caused by Rabies virus (RABV) isolates from insectivorous bats. Viral growth kinetics, cell-to-cell spread and virus uptake in vitro were analyzed for RABV isolates from Eptesicus furiralis and Myotis nigricans. For pathogenesis evaluation, mice were inoculated with RABV isolates from Eptesicus furiralis and Myotis nigricans, and clinical signs were observed for 40 days. We observed that the insectivorous bat strains showed a higher replication rate, faster cell-to-cell spread and delayed virus uptake in N2a cells. Furthermore, after the first sign of a clinical infection, mice infected with Myotis nigricans and Eptesicus furiralis isolates succumbed rapidly (6 ±â€¯9 days) compared with RABV strains associated with other reservoirs. Our results show that the insectivorous bat RABV strains are less pathogenic for mice than strains associated with other reservoirs. In addition, this study also indicates that the differences in the biological characteristics of the RABV strains are important to their pathogenicity. An enhanced understanding of rabies pathogenesis may be important for the development of novel therapies for humans and in the implementation of rabies control strategies.

Infection of neuroblastoma cells by rabies virus is modulated by the virus titer.

Rabies is a lethal viral infection that can affect almost all mammals, including humans. To better understand the replication of Rabies lyssavirus, we investigated if the viral load in brains naturally infected with rabies influences viral internalization and viral growth kinetics in neuroblastoma cells, and if the viral load affects mortality in mice after intradermal infection. We noted that high initial viral loads in brains (group II) were unfavourable for increasing viral titers during serial passages in neuroblastoma cells when compared to low initial viral loads in brains (group I). In addition, group I strains showed higher viral growth and enhanced internalization efficiency in neuroblastoma cells than group II strains. However, we observed that the dominant virus subpopulation in group II promoted efficient viral infection in the central nervous system in the new host, providing a selective advantage to the virus. Our data indicate that rabies infection in animal models depends on not only the virus strain but also the amount of virus. This study may serve as a basis for understanding the biologic proprieties of Rabies lyssavirus strains with respect to the effects on viral replication and the impact on pathogenesis, improving virus yields for use in vaccine development.

Proposição de uma nova metodologia para isolamento e cultivo de células-tronco mesenquimais derivadas do tecido adiposo / A new methodology proposition for isolation and culture of adipose-derived mesenchymal stem cells

Introdução: As células-tronco mesenquimais (CTM) têm despertado interesse de vários grupos de pesquisa em função do grande potencial de aplicabilidade em terapia celular e medicina regenerativa. Nesse contexto, o tecido adiposo vem recebendo grande destaque como importante fonte para obtenção de CTM. Os protocolos utilizados atualmente para o isolamento das células-tronco derivadas do tecido adiposo (ADSC) empregam, de forma geral, o método de digestão enzimática com colagenase extraída de bactéria (Clostridium histolyticun), que pode conter contaminantes, como endotoxinas e outros peptídeos que, eventualmente, poderão resultar em reações adversas nos procedimentos de terapia celular em pacientes humanos. Objetivo: Pretendeu-se no presente estudo adequar e propor uma nova abordagem empregando a metodologia de dissociação mecânica para isolamento de CTM derivadas de tecido adiposo de ratos. Métodos: As células cultivadas foram analisadas quanto ao potencial de adesão, proliferação e tempo de duplicação celular, por meio de uma curva de crescimento. As células isoladas e cultivadas a partir do tecido adiposo foram também analisadas quanto ao potencial de diferenciação in vitro nas linhagens adipogênica, condrogênica e osteogênica. Resultados: Os resultados mostraram que o tempo de duplicação (velocidade de crescimento) da população celular isolada por dissociação mecânica é mais expressivo quando comparado com a técnica de digestão enzimática. As células isoladas do tecido adiposo apresentaram potencial de diferenciação nas linhagens osteogênica, condrogênica e adipogênica. Conclusão: Os resultados obtidos permitem concluir que a metodologia de dissociação mecânica apresenta-se como uma alternativa viável, de baixo custo e, como tal, extremamente promissora no sentido de permitir que a colagenase de origem bacteriana (Clostridium histolyticun) torne-se um componente prescindível para isolamento e cultivo de células provenientes do tecido adiposo.

Background: Mesenchymal stem cells (MSCs) have attracted interest of several research groups due to the large potential applicability in cell therapy and regenerative medicine. In this context, adipose tissue has received high profile as an important source in order to obtain MSC. The protocols currently suggested for the isolation and culture of adipose- -derived stem cells (ADSC) utilize, in general, the enzymatic digestion method with bacterial collagenase (Clostridium histolyticun) which may contain contaminants such as endotoxin and other peptides that eventually may result in adverse reactions in the cell therapy procedures in human patients. Objective: In this context, it was intended in this study to propose a new methodological approach of mechanical dissociation for isolating and culture of adipose-derived mesenchymal stem cells. Methods: The cultured cells were analyzed for potential adhesion, proliferation and cell doubling time, through a growth curve lineages The cells were also analyzed according to potential for differentiation in adipogenic, chondrogenic and osteogenic lineages. Results: The results showed that the doubling time of the cell population isolated by mechanical dissociation is faster when compared to the enzymatic digestion technique. The isolated cells from adipose tissues howed potential for differentiation in cell lineages osteogenic, adipogenic and chondrogenic. Conclusion: The obtained results allow us to conclude that the methodology of mechanical dissociation, presented in this paper, is a viable, low cost and therefore an extremely promising alternative in order to permit that the bacterial collagenase, from Clostridium histolyticun, become a dispensable component for isolation and cultivation of adipose-derived stem cells.

Delayed progression of rabies transmitted by a vampire bat.

Here, we compared the growth kinetics, cell-to-cell spread, and virus internalization kinetics in N2a cells of RABV variants isolated from vampire bats (V-3), domestic dogs (V-2) and marmosets (V-M) as well as the clinical symptoms and mortality caused by these variants. The replication rate of V-3 was significantly higher than those of V-2 and V-M. However, the uptake and spread of these RABV variants into N2a cells were inversely proportional. Nevertheless, V-3 had longer incubation and evolution periods. Our results provide evidence that the clinical manifestations of infection with bat RABV variant occur at a later time when compared to what was observed with canine and marmoset rabies virus variants.

Cell therapy in experimental model of inflammatory bowel disease / Terapia celular em modelo experimental de doença inflamatória intestinal

Inflammatory bowel disease, which mainly involves Crohn's disease and ulcerative rectocolitis, is an inflammatory condition of the mucosa that can afflict any segment of the gastrointestinal tract. Despite the fact that the existing therapies result in improvement in patient's symptomatology and quality of life, there is no curative treatment. Surgical treatment involves complex procedures associated with high morbidity and mortality rates. In this context, cell therapy with stem cells has emerged as a treatment with broad potential applicability. In this study, we intended to verify the efficacy of transplantation of adipose tissue-derived stem cells in rats with intestinal inflammation induced by trinitrobenzenesulfonic acid. The cell population was isolated from the adipose tissue of inguinal region of rats and processed for culture by mechanical dissociation. The animals were evaluated with respect to clinical and biochemical aspects, as well as by macroscopic, microscopic and histological analyses. In the experimental model of bowel inflammation by 2,4,6-trinitrobenzenesulfonic acid, the infusion of adipose tissue significantly reduced the presence of adhesions in the colon and adjacent organs and decreased the activity of myeloperoxidase, a marker of neutrophil infiltration in the injured mucosa. The results suggest that cell therapy with adipose tissue can promote and/or accelerate the regeneration of damaged intestinal mucosa. It is concluded that the presence of adhesions and the determination of myeloperoxidase activity provide indications that adipose tissue can promote and/or accelerate the regeneration of inflammatory bowel mucosa. (AU)

A Doença Inflamatória Intestinal (DII), consistindo principalmente da doença de Crohn e retocolite ulcerativa, é uma condição inflamatória da mucosa que pode acometer qualquer segmento do trato gastrointestinal. Apesar das terapias existentes resultarem na melhora dos sintomas e da qualidade de vida dos pacientes, não há nenhum tratamento curativo. O tratamento cirúrgico envolve procedimentos complexos associados a altas taxas de morbimortalidade. Neste contexto, a terapia celular com células-tronco desponta como opção de tratamento potencialmente promissora. Em função destes aspectos, pretendeu-se, no presente estudo, verificar a eficácia do transplante de células-tronco derivadas do tecido adiposo (ASC) em ratos com inflamação intestinal induzida por ácido trinitrobenzenosulfonico (TNBS). As ASCs foram obtidas por dissociação mecânica do tecido adiposo da região inguinal de ratos e processadas para cultivo. Os animais foram avaliados, considerando-se os aspectos clínicos e bioquímicos, além de análises macroscópica, microscópica e histológica. No modelo de inflamação intestinal induzida por TNBS, a infusão de ASCs reduziu significativamente a presença de aderências entre o cólon e órgãos adjacentes, bem como diminuiu a atividade da mieloperoxidase (MPO), um marcador da infiltração de neutrófilos na mucosa lesada. Os resultados obtidos permitem concluir que a terapia celular com ASCs pode promover e/ou acelerar o processo de regeneração da mucosa intestinal inflamada. (AU)

Animal models in chronic obstructive pulmonary disease-an overview.

ABSTRACT Chronic obstructive pulmonary disease (COPD) is characterized by progressive airway obstruction resultant from an augmented inflammatory response of the respiratory tract to noxious particles and gases. Previous reports present a number of different hypotheses about the etiology and pathophysiology of COPD. The generating mechanisms of the disease are subject of much speculation, and a series of questions and controversies among experts still remain. In this context, several experimental models have been proposed in order to broaden the knowledge on the pathophysiological characteristics of the disease, as well as the search for new therapeutic approaches for acute or chronically injured lung tissue. This review aims to present the main experimental models of COPD, more specifically emphysema, as well as to describe the main characteristics, advantages, disadvantages, possibilities of application, and potential contribution of each of these models for the knowledge on the pathophysiological aspects and to test new treatment options for obstructive lung diseases.