UTILIZAÇÃO DE CÉLULAS MONONUCLEARES DE MEDULA ÓSSEA PARA O TRATAMENTO DE SEQUELAS NEUROLÓGICAS DE CINOMOSE CANINA CURITIBA

ABSTRACT

A cinomose é uma doença infecciosa causada por um Morbillivirus pertencente a família Paramyxoviridae. Até 30% dos cães infectados com o vírus da cinomose apresentam complicações neurológicas e aproximadamente 10% morrem por leucoencefalite aguda. Os cães que recuperam podem apresentar sequelas permanentes. Até o momento, não existe nenhum tratamento específico para animais com sinais neurológicos da cinomose, assim, são necessárias novas intervenções terapêuticas. O transplante de células-tronco tem emergido como um novo e promissor modelo de terapia. Células mononucleares derivadas da medula óssea (CMNMO) podem ser isoladas e aplicadas como estratégia terapêutica em estudos clínicos e pré-clínicos. Porém, existem alguns desafios associados a essa abordagem terapêutica. Um deles é a falta de estudos sobre a caracterização das CMNMO dos cães. Heparina e citrato-fosfato-dextrose-adenina-1 (CPDA-1) são comumente utilizados para coletar medula óssea. Porém, o efeito destes anticoagulantes em termos de isolamento dessas células também não é conhecido. Outro ponto que precisa ser mais estudado é a via de administração para ação eficiente das células transplantadas. A via intravenosa (IV) tem sido utilizada porque é o método mais fácil e menos invasivo de transplante de células. A marc

ABSTRACT

Canine distemper is an infectious disease caused by a Morbillivirus belonging to Paramyxoviridae family. Up to 30% of dogs infected with canine distemper virus showed neurological complications and approximately 10% die from acute leukoencephalitis. In addition, dogs that recover may have permanent sequels. There is no specific therapy for animals showing distemper neurological signs, and new therapeutic interventions are necessary, and cell transplantation has emerged as a promising new therapy model. Bone marrow mononuclear cells (BMMNC) can be easily isolated and broadly applied as a therapeutic strategy in a large number of preclinical and clinical studies. However, there are some challenges associated with this therapeutic approach. One of them is the lack of studies on the characterization of canine BMMNC. Heparin and citrate-phosphate-dextrose-adenine-1 (CPDA-1) are commonly used to harvest bone marrow. However, the comparison of effect of anticoagulants at harvest on terms of stem cell yield has not been studied. Another point that needs to be further studied is the route of administration for efficient cell delivery. Intravenous (IV) delivery has been used because it is easier and less invasive method of cell transplantation. The labelling and tracking of cells allow to evaluate if IV transplanted cells are attracted to the site of injury. The PKH26 is a fluorescent labeling molecule, noncytotoxic and is stable for long time period. Because of these characteristics, this dye seems to be ideal for cells tracking. The characterization of subsets of BMMNC and a better understanding of the functions of these cells may contribute to the knowledge of their potential and thus increase the possibilities to their use for clinical trials in veterinary medicine. The BMMMC administered intravenously have a lung passage 30 times larger than the mesenchymal stem cells (MSC), facilitating the migration of these cells to the injured tissues. However, the migration of these cells to the central nervous system (CNS), after intravenous administration, in animals with canine distemper sequels has not been studied. The PKH is a fluorescent labelling molecule, which is incorporated in the lipid bilayer of the cytoplasmic membrane, which has been used for the identification of cell migration. After incorporated into the cells in vitro, the PKH is not transferred to the medium or to unmarked cells, and has no toxic or immunogenic effect. BMMNC can be labeled with PKH and used for transplant intravenously, so this dye appears to be ideal for monitoring the migration of these cells. The aims of this study were the comparison of the yield of bone marrow-derived mononuclear cells harvested from dogs with two different anticoagulants, evaluate cell migration after allogeneic BMMNC transplantation in 10 animals with neurological complications of canine distemper, characterize phenotypically canine BMMNC and, evaluate the safety and efficacy of allogeneic BMMNC transplantation for treatment of neurological sequels of canine distemper in others 23 dogs. For comparison of the anticoagulants, the bone marrow from five dogs was harvest in heparin or CPDA-1, isolated in a density gradient, and stained for CD9 and CD44 for characterization by flow cytometry. The means were compared using Students paired t-test. Samples harvested with CPDA-1 yielded an average of 5.16 x 106 (±1.76 x 106) to 20.20 x 106 (±1.55 x 106) mononuclear cells/mL, whereas the yield of samples harvested with heparin varied between 4.56 x 106 (±0.69 x 106) and 24.30 x 106 (±2.12 x 106) mononuclear cells/mL. By flow cytometry, the mean percentage of double-stained cells varied from 1.96% (±0.64%) to 5.01 % (±0.73%) for CPDA-1 and from 2.23% (±0.70%) to 7.27 % (±0.97%) for heparin. No significant statistical differences were observed on yield or CD9 and CD44 expression. Bone marrow was harvested from eight donors and BMMNC were isolated, labeled with PKH26 dye and IV transplanted into the patients, to assess the labelling and tracking of cells. The cell migration was assessed in the cerebrospinal fluid (CSF) of patients at different periods of time and the percentage of labeled cells with PKH 26 dye was evaluated quantitatively by flow cytometry and qualitatively by fluorescence microscope. In the quantitative analysis by flow cytometer, it was observed a wide variation in the percentage of labeled cells in different CSF collection times. Also, there was an increase on the percentage of PKH26-labeled cells in the CSF, with highest percentage between 4 and 5 ½ hours after infusion with subsequent decrease. In the qualitative analysis, it was showed the presence of labeled BMMNC recruited by central nervous system on CSF. In order to evaluate the safety and efficacy of allogeneic BMMNC transplantation for treatment of neurological sequels of canine distemper, it was used a single blind randomized controlled trial in 46 dogs divided into treatment group and control group with weekly follow-up for 35 days. Bone marrow was harvested from 23 healthy donors and BMMNC were isolated by density gradient centrifugation. BMMNC from four donors were labeled with anti-CD8a, CD9, CD14, CD29, CD34, CD44, CD45, and CD90 for phenotypic characterization. Dogs from the treatment group received 1 x 108 BMMNC intravenously. Regarding the safety of cell transplantation, no serious adverse events related to the procedure were recorded during the 35 days of the study. Functional recovery was evaluated according to the Olby scoring system with some modifications. For the treatment group, the median and interquartile range of the functional score, with 0, 7, 14, 21, 28 and 35 days after injection were 7 (4-13), 9 (5-14), 12 (6-15), 14 (6-15), 14 (6-16), and 14 (6-16) respectively, whereas for the placebo group they were 6 (4-12), 7 (4- 13), 7 (4-12), 7 (4-12), 6 (3-12), and 6 (3-12). The differences observed were statistically significant (p < 0.05), showing the effectiveness of BMMNC transplantation in dogs with distemper leukoencephalitis. In conclusion, this study demonstrates that BMMNC can be efficiently labeled with PKH26 dye and these cells can be monitored after IV transplantation in animals with neurological complications of canine distemper.