Isolamento e purificação de uma neurotoxina crotoxina do veneno de Crotalus durissus cascavella : caracterização bioquimica e biologica / \ c Daniela Gois Beghini

Orientador: Sergio MarangoniDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A serpente Crotalus durissus cascavel/a é encontrada no cerrado do nordeste do Brasil (BARRA VI ERA, 1990) e apesar de pouco estudada sua picada constitui um importante problema de saúde pública (MARTINS et aI., 1998). A crotoxina é um complexo heterodimérico com atividade de fosfolipase A2 sendo o principal componente neurotóxico do veneno da Cascavel Sul Americana Crotalus durissus terrificus. A crotoxina atua bloqueando a transmissão de impulsos nervosos na junção neuromuscular causando uma paralisia flácida nas vítimas. O complexo crotoxina consiste em uma fosfolipase A2 (PLA2), componente básico, e um componente ácido, não enzimático conhecido como crotapotina. Acrotoxina do veneno de C. d. terrificus tem sido extensivamente estudada, enquanto que a crotoxina de outros venenos de Crotalus durissus sp tem sido pouco investigada.O objetivo deste trabalho foi a caracterização bioquímica e biológica do veneno e da crotoxina de Crotaus durissus cascavel/a, permitindo também um melhor entendimento do complexo crotoxina (PLA2 e crotapotina). PLA2 e crotapotina, foram purificadas através de dois passos cromatográficos: cromatografia de exclusão molecular em Superdex G-75 e cromatografia em HPLC de fase reversa. A análise cromatográfica em HPLC de fase reversa revelou quatro isoformas de crotapotina (F2, F3, F4 e F5) e apenas uma PLA2 (F6) na crotoxina do veneno de C. d. cascavel/a. A crotoxina de C. d. terrificus, ao contrário, contém três isoformas de PLA2 (Cdt F15, F16 e F17) e somente duas isoformas de crotapotina (Cdt F5 e F7). O grau de pureza das frações foi confirmado pela eletroforese em PAGE-SDS-Tricina onde mostrou que PLA2 e crotapotina migram como uma única banda com massa molecular estimada de 15 kDa e 9 kDa, respectivamente. A composição de aminoácidos da PLA2 mostrou uma grande quantidade de resíduos de meia-cisteína e um alto conteúdo de resíduos básicos, enquanto que as crotapotinas F3 e IF4 mostraram maior quantidade de resíduos carregados negativamente. Para uma melhor caracterização bioquímica avaliou-se a cinética da enzima PLA2 de C. d. cascavel/a. A PLA2 mostrou um comportamento alostérico, com atividade máxima em pH 8,3 e 35-40°C. A PLA2 de C. d. cascavel/a requer Ca2+ para exibir sua atividade, mas foi inibida por Zn2+ e CU2+. Crotapotina e heparina inibiram a atividade catalítica da enzima atuando como inibidores alostéricos. Com o objetivo de estudar a atividade biológica do veneno e da crotoxina de C. d. cascavel/a foi verificado a neurotoxicidade em preparação nervo frênico-diafragma de camundongo e preparação biventer cervicis de pintainho. O veneno de C. d. cascavel/a aqui reportado, bem como seu componente crotoxina, apresentou potente atividade neurotóxica, principalmente em preparação biventer cervicis de pintainho. O estudo miográfico, portanto, revelou que a preparação biventer cervicis de pintainho é mais sensível ao efeito paralisante do veneno e crotoxina quando comparada à preparação nervo frênico-diafragma de camundongo. A principal conclusão deste trabalho é que o veneno de C. d. cascavel/a contém pelo menos quatro isoformas de crotoxina formadas por diferentes combinações das crotapotinas com uma única isoforma de PLA2. Essa situação difere da crotoxina do veneno de C. d. terrificus, onde a heterogeneidade resulta da presença de três ou quatro isoformas de PLA2. Desde que a toxicidade do veneno depende principalmente de seu conteúdo de crotoxina, a presença das isoformas pode contribuir para variações na potência da neurotoxicidadeAbstract: Crotalus durissus cascavel/a is a snake usually found in scrubland of Brazilian Northeast (BARRA VI ERA, 1990) and in spite of little studied its bite constitutes an important public health problem (MARTINS et aI., 1998). The crotoxin is a heterodimeric complex with phospholipase A2 activity and is the main neurotoxic component in venom of the South American rattlesnake Crotalus durissus terrifícus. Crotoxin acts blocking the transmission of nervous pulses in the neuromuscular junction causing a flaccid paralysis in the victims. The crotoxin complex consists of a phospholipase A2 (PLA2), basic component, and an acid component, nonenzymatic known as crotapotin. The crotoxin of the C. d. terrifícus venom has been studied extensively, while crotoxin of others Crotalus durissus sp venoms have been poorly investigated. The objective of this work was the biochemical and biological characterization of the venom and of the crotoxin of Crotaus durissus cascavel/a, also allowing a better understanding of the crotoxin complex (PLA2 and crotapotin). PLA2 and crotapotin, were purified through two chromatography's steps: molecular exclusion chromatography in Superdex G-75 and in HPLC of reverse phase. The chromatographyc analysis in HPLC of reverse phase revealed four crotapotin isoforms (F2, F3, F4 and F5) and just a PLA2 (F6) in the crotoxin of the C. d. cascavel/a venom. Crotoxin from C. d. terrifícus contained three PLA2 isoforms (Cdt F15, F16 and F17), but only two crotapotin isoforms (Cdt F5 e F7). The degree of purity of the fractions was confirmed by the electrophoresis in Page-SDS-Tricina that showed that PLA2 and crotapotin migrated as single band with estimated molecular masses of 15 kDa and 9 kDa, respectively. The composition of amino acids of PLA2 showed a great amount of residues of half-cysteines and a high content of basic residues, while the crotapotins F3 and F4 showed larger amount of residues carried negatively. For a better biochemical characterization, the kinetics of the enzyme PLA2 of C. d cascavella was evaluated. The PLA2 showed allosteric behavior, with maximal activity at pH 8,3 and 35-40°C. The C. d. cascavella PLA2 required Ca2+ for activity, but was inhibited by Cu2+ and Zn2+. Crotapotin and Heparin inhibited the catalytic activity of the enzyme by acting as allosteric inhibitors. Aiming to study the biological activity of the venom and of the crotoxin of C. d. cascavella the neurotoxicity was verified in the mouse phrenic nerve diaphragm preparation and chick biventer cervicis preparation. The C. d. cascavella venom here reported, as well as its crotoxin component, presented potent neurotoxic activity, mainly in chick biventer cervicis preparation. The myographic study also revealed remarkable differences in the sensibility of the mouse phrenic nerve diaphragm preparation and chick biventer cervicis preparation. The preparation mouse nerve-muscle was shown less sensitive to the action neuromuscular blocking of both crotoxin and total venom, when submitted to indirect stimulus. The main conclusion of this work it is that C. d. cascavella venom contains at least four crotoxin isoforms formed by different combinations of crotapotins with the single PLA2 isoform. This situation differs from that of crotoxin in C. d. terrificus venom, where the heterogeneity results from the presence of three or four PLA2 isoforms. Since the toxicity of the venom depends mainly on its content of crotoxin, the presence of isoforms may contribute to variations in neurotoxicityMestradoBioquimicaMestre em Biologia Funcional e Molecula

Mesenchymal Stem Cells in the Treatment of COVID-19, a Promising Future

Mesenchymal stem cells (MSCs) are multipotent adult stem cells present in virtually all tissues; they have a potent self-renewal capacity and can differentiate into multiple cell types. They also affect the ambient tissue by the paracrine secretion of numerous factors in vivo, including the induction of other stem cells’ differentiation. In vitro, the culture media supernatant is named secretome and contains soluble molecules and extracellular vesicles that retain potent biological function in tissue regeneration. MSCs are considered safe for human treatment; their use does not involve ethical issues, as embryonic stem cells do not require genetic manipulation as induced pluripotent stem cells, and after intravenous injection, they are mainly found in the lugs. Therefore, these cells are currently being tested in various preclinical and clinical trials for several diseases, including COVID-19. Several affected COVID-19 patients develop induced acute respiratory distress syndrome (ARDS) associated with an uncontrolled inflammatory response. This condition causes extensive damage to the lungs and may leave serious post-COVID-19 sequelae. As the disease may cause systemic alterations, such as thromboembolism and compromised renal and cardiac function, the intravenous injection of MSCs may be a therapeutic alternative against multiple pathological manifestations. In this work, we reviewed the literature about MSCs biology, focusing on their function in pulmonary regeneration and their use in COVID-19 treatment

Neutralização de atividades farmacologicas e enzimaticas de venenos crotalicos perante antivenenos

Orientadores: Sergio Marangoni, Stephen HysloTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Crotoxina, a principal neurotoxina do veneno das cascavéis Crotalus durissus terrificus e Crotalus durissus cascavella, é um complexo protéico que contém uma fosfolipase A2 (PLA2) básica e uma proteína ácida, crotapotina. O veneno e a crotoxina purificada do veneno de C. d. cascavella foram estudados quanto aos efeitos neurotóxico e miotóxico na preparação biventer cervicis de pintainho. O veneno e a crotoxina mostraram bloqueio neuromuscular nessa preparação em concentrações tão baixas quanto 0,2 mg/ml e 1 mg/ml. Quanto ao efeito miotóxico, a mionecrose foi mais marcante com altas concentrações de veneno e crotoxina (1, 5 e 25 mg/ml). Assim, o veneno de C. d. cascavella e sua crotoxina possuem um efeito neurotóxico preponderante e bastante potente nessa preparação, e uma ação miotóxica que foi observada somente em altas concentrações. Para melhor entender o mecanismo de neutralização da crotoxina por anticorpos foram produzidos antissoros específicos contra a crotoxina e PLA2 do veneno de C. d. cascavella. O título de anticorpos e a especificidade dos antisoros produzidos foram avaliados por ELISA e immunoblotting, respectivamente. A neutralização da atividade neurotóxica foi avaliada por intermédio de técnica miográfica nas preparações biventer cervicis de pintainho e nervo frênico diafragma de camundongo. A capacidade neutralizante dos antissoros na preparação de camundongo foi comparável à do soro comercial anticrotálico produzido contra o veneno de C. d. terrificus. O antissoro anti-crotoxina foi um pouco menos potente do que o antissoro anti-PLA2 no processo de neutralização e isso confirma o papel central da PLA2 no mecanismo de neurotoxicidade da crotoxina. Neste trabalho, nós também avaliamos a habilidade do antissoro produzido em coelho contra a crotapotina do veneno C. d. cascavella em neutralizar a neurotoxicidade e miotoxicidade deste veneno e crotoxina, e inibir a atividade enzimática do complexo crotoxina e da PLA2. Este antissoro neutralizou parcialmente o bloqueio neuromuscular causado pelo veneno e crotoxina na preparação frênico nervo-diafragma de camundongo, sem prevenir o dano morfológico resultante ou inibir a atividade fosfolipásica. Em contraste, antissoros contra a PLA2 e crotoxina neutralizaram o bloqueio neuromuscular e a atividade enzimática efetivamente. A neutralização parcial do bloqueio neuromuscular pelo anti-crotapotina sugere que este antissoro pode prevenir a interação da crotoxina com seu receptor causando dissociação do complexo crotoxina por se ligar a crotapotina. Neste estudo, nós também examinamos a habilidade dos antissoros contra a crotoxina e PLA2 em neutralizar a neurotoxicidade dos venenos de Crotalus durissus terrificus e Bothrops jararacussu e suas principais toxinas na preparação frênico nervo-diafragma de camundongo. Immunoblotting mostrou que o anti-crotoxina de C. d. cascavella reconheceu a crotoxina de C. d. terrificus e BthTX-I de B. jararacussu, enquanto que o antissoro contra a PLA2 reconheceu a PLA2 de C. d. terrificus e a BthTX-I de B. jararacussu. ELISA confirmou esta reatividade cruzada. Estes antissoros neutralizaram eficazmente o bloqueio neuromuscular causado pelo veneno e crotoxina de C. d. terrificus na preparação de camundongo em proporções semelhantes às usadas para neutralizar o veneno e a crotoxina de C. d. cascavella. Anti-crotoxina e anti-PLA2 também neutralizaram eficientemente o bloqueio produzido pelo veneno e principal toxina de Bothrops jararacussu, porém doses mais altas dos antissoros foram necessárias para essa neutralização. Então, os resultados mostram reatividade cruzada entre esses venenos e suas toxinas principais e também mostra que o antissoro produzido contra PLA2 eficazmente neutraliza a neurotoxicidade dos venenos de C. d. terrificus e B. jararacussu e suas toxinas PLA2. Esses resultados, portanto, confirmam o importante papel da PLA2 no mecanismo de neurotoxicidade dos venenosAbstract: Crotoxin, the main neurotoxin from venom of the rattlesnakes Crotalus durissus terrificus and Crotalus durissus cascavella, is a protein complex that contains a phospholipase A2 (PLA2) basic and an acid protein, crotapotin. The venom and the purified crotoxin from C. d. cascavella venom were studied with relationship to the neurotoxic and myotoxic effects in the chick biventer cervicis preparation. The venom and crotoxin showed neuromuscular blockade in this preparation at doses as low as 0,2 mg/ml and 1 mg/ml. With relationship to the myotoxic effect, the myonecrosis was stronger with higher doses of venom and crotoxin (1, 5 and 25 mg/ml). These results showed that the C. d. cascavella venom and its crotoxin possess a preponderant and quite potent neurotoxic effect in this preparation, and a myotoxic action that was observed only at higher doses. To clarify the crotoxin neutralization mechanism by antibodies, specific anti-sera was produced against the crotoxin and PLA2 from C. d. cascavella venom. The title of antibodies and specificity of the anti-sera raised in rabbits were tested by ELISA and immunoblotting, respectively. The neutralization of the neurotoxic activity was evaluated through myoghraphic technique in the chick biventer cervicis and mouse phrenic nerve diaphragm preparations. The neutralizing capacity of the anti-sera against crotoxin and PLA2 in the mouse preparation was comparable to the commercial crotalic antiserum produced against the C. d. terrificus venom. The anti-crotoxin anti-sera was a little less potent than the anti-PLA2 and this confirms the central role of PLA2 in the mechanism of neurotoxicity of the crotoxin. In this work, we also examined the ability of rabbit anti-serum raised against crotapotin purified from Crotalus durissus cascavella venom to neutralize the neurotoxicity and myotoxicity of this venom and crotoxin, and to inhibit the enzymatic activity of the crotoxin complex and PLA2 alone. This anti-serum to crotapotin partially neutralized the neuromuscular blockade caused by venom and crotoxin in electrically stimulated mouse phrenic nerve-diaphragm preparations, without preventing the resulting morphological damage or inhibiting the PLA2 activity. In contrast, rabbit anti-sera to PLA2 and crotoxin effectively neutralized the neuromuscular and PLA2 activities. The partial neutralization of the neurotoxicity of crotoxin by the anti-serum to crotapotin suggested that this anti-serum may prevent the interaction of intact crotoxin with its recceptor by causing dissociation of the crotoxin complex through binding to crotapotin. In this study, we also examined the ability of anti-sera raised against crotoxin and PLA2 to neutralize the neurotoxicity of Crotalus durissus terrificus and Bothrops jararacussu venoms and their major toxins, crotoxin and bothropstoxin-I (BthTX-I), respectively, in mouse isolated phrenic nerve-diaphragm preparations. Immunoblotting showed that anti-serum to crotoxin from C. d. cascavella recognized crotoxin from C. d. terrificus and BthTX-I from B. jararacussu, while anti-serum to PLA2 from C. d. cascavella recognized PLA2 from C. d. terrificus and BthTX-I from B. jararacussu. ELISA corroborated this cross-reactivity. These anti-sera efficiently neutralized the neuromuscular blockade caused by venom and crotoxin from C. d. terrificus in mouse preparation in similar proportions used to neutralize the venom and crotoxin from C. d. cascavella. Anti-crotoxin and anti-PLA2 anti-sera also efficiently neutralized this blockade produced by venom and main toxin of Bothrops jararacussu, bothropstoxin-I (BthTX-I), however higher doses of anti-sera was necessary for this neutralization. Therefore, the results show cross-reactivity between these venoms and its main toxins and also shown that anti-serum produced against PLA2 efficiently neutralized the neurotoxicity of C. d. terrificus and B. jararacussu venoms and their PLA2 toxins. These results confirms the important role of PLA2 in the neurotoxicity mechanism of the venomsDoutoradoBioquimicaDoutor em Biologia Funcional e Molecula

Induced Pluripotent Stem Cells in Drug Discovery and Neurodegenerative Disease Modelling

Induced pluripotent stem cells (iPSCs) are derived from reprogrammed adult somatic cells. These adult cells are manipulated in vitro to express genes and factors essential for acquiring and maintaining embryonic stem cell (ESC) properties. This technology is widely applied in many fields, and much attention has been given to developing iPSC-based disease models to validate drug discovery platforms and study the pathophysiological molecular processes underlying disease onset. Especially in neurological diseases, there is a great need for iPSC-based technological research, as these cells can be obtained from each patient and carry the individual’s bulk of genetic mutations and unique properties. Moreover, iPSCs can differentiate into multiple cell types. These are essential characteristics, since the study of neurological diseases is affected by the limited access to injury sites, the need for in vitro models composed of various cell types, the complexity of reproducing the brain’s anatomy, the challenges of postmortem cell culture, and ethical issues. Neurodegenerative diseases strongly impact global health due to their high incidence, symptom severity, and lack of effective therapies. Recently, analyses using disease specific, iPSC-based models confirmed the efficacy of these models for testing multiple drugs. This review summarizes the advances in iPSC technology used in disease modelling and drug testing, with a primary focus on neurodegenerative diseases, including Parkinson’s and Alzheimer’s diseases

Induced Pluripotent Stem Cells: Hope in the Treatment of Diseases, including Muscular Dystrophies

Induced pluripotent stem (iPS) cells are laboratory-produced cells that combine the biological advantages of somatic adult and stem cells for cell-based therapy. The reprogramming of cells, such as fibroblasts, to an embryonic stem cell-like state is done by the ectopic expression of transcription factors responsible for generating embryonic stem cell properties. These primary factors are octamer-binding transcription factor 4 (Oct3/4), sex-determining region Y-box 2 (Sox2), Krüppel-like factor 4 (Klf4), and the proto-oncogene protein homolog of avian myelocytomatosis (c-Myc). The somatic cells can be easily obtained from the patient who will be subjected to cellular therapy and be reprogrammed to acquire the necessary high plasticity of embryonic stem cells. These cells have no ethical limitations involved, as in the case of embryonic stem cells, and display minimal immunological rejection risks after transplant. Currently, several clinical trials are in progress, most of them in phase I or II. Still, some inherent risks, such as chromosomal instability, insertional tumors, and teratoma formation, must be overcome to reach full clinical translation. However, with the clinical trials and extensive basic research studying the biology of these cells, a promising future for human cell-based therapies using iPS cells seems to be increasingly clear and close

A Promising Future for Stem-Cell-Based Therapies in Muscular Dystrophies-In Vitro and In Vivo Treatments to Boost Cellular Engraftment

Submitted by Sandra Infurna ([email protected]) on 2020-03-30T20:05:26Z No. of bitstreams: 1 LuizAALves_AndreaHPons_etal_IOC_2019.pdf: 2544451 bytes, checksum: 9f5dedfde163060c5ea2851ce117e481 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2020-03-30T20:13:43Z (GMT) No. of bitstreams: 1 LuizAALves_AndreaHPons_etal_IOC_2019.pdf: 2544451 bytes, checksum: 9f5dedfde163060c5ea2851ce117e481 (MD5)Made available in DSpace on 2020-03-30T20:13:43Z (GMT). No. of bitstreams: 1 LuizAALves_AndreaHPons_etal_IOC_2019.pdf: 2544451 bytes, checksum: 9f5dedfde163060c5ea2851ce117e481 (MD5) Previous issue date: 2019Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Inovações em Terapias, Ensino e Bioprodutos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Inovações em Terapias, Ensino e Bioprodutos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Comunicação Celular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Comunicação Celular. Rio de Janeiro, RJ, Brasil.Binghamton University. School of Pharmacy and Pharmaceutical Sciences. Department of Pharmaceutical Sciences. New York, NY, USA.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Inovações em Terapias, Ensino e Bioprodutos. Rio de Janeiro, RJ, Brasil.Muscular dystrophies (MD) are a group of genetic diseases that lead to skeletal muscle wasting and may affect many organs (multisystem). Unfortunately, no curative therapies are available at present for MD patients, and current treatments mainly address the symptoms. Thus, stem-cell-based therapies may present hope for improvement of life quality and expectancy. Different stem cell types lead to skeletal muscle regeneration and they have potential to be used for cellular therapies, although with several limitations. In this review, we propose a combination of genetic, biochemical, and cell culture treatments to correct pathogenic genetic alterations and to increase proliferation, dispersion, fusion, and differentiation into new or hybrid myotubes. These boosted stem cells can also be injected into pretreate recipient muscles to improve engraftment. We believe that this combination of treatments targeting the limitations of stem-cell-based therapies may result in safer and more efficient therapies for MD patients. Matricryptins have also discussed

Toxoplasma gondii Impairs Myogenesis in vitro, With Changes in Myogenic Regulatory Factors, Altered Host Cell Proliferation and Secretory Profile

Submitted by Sandra Infurna ([email protected]) on 2020-03-26T20:31:17Z No. of bitstreams: 1 HeleneSBarbosa_PCVieira_etal-IOC_2019.pdf: 2526535 bytes, checksum: 4801e366dc3bc1fcea874e83869d4711 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2020-03-26T20:43:59Z (GMT) No. of bitstreams: 1 HeleneSBarbosa_PCVieira_etal-IOC_2019.pdf: 2526535 bytes, checksum: 4801e366dc3bc1fcea874e83869d4711 (MD5)Made available in DSpace on 2020-03-26T20:43:59Z (GMT). No. of bitstreams: 1 HeleneSBarbosa_PCVieira_etal-IOC_2019.pdf: 2526535 bytes, checksum: 4801e366dc3bc1fcea874e83869d4711 (MD5) Previous issue date: 2019Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Estrutural. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genômica Funcional e Bioinformática. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Inovação em Terapias, Ensino e Bioprodutos. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Ciências Biomédicas. Laboratório de Embriologia de Vertebrados. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Ciências Biomédicas. Laboratório de Embriologia de Vertebrados. Rio de Janeiro, RJ, Brasil.Sorbonne Université. INSERM, Institut de Myologie, Myology Research Center. UMRS974, Paris, France.Sorbonne Université. INSERM, Institut de Myologie, Myology Research Center. UMRS974, Paris, France.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Estrutural. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Estrutural. Rio de Janeiro, RJ, Brasil.Toxoplasma gondii is the causative agent of toxoplasmosis, a parasitic disease with a wide global prevalence. The parasite forms cysts in skeletal muscle cells and neurons, although no evident association with inflammatory infiltrates has been typically found. We studied the impact of T. gondii infection on the myogenic program of mouse skeletal muscle cells (SkMC). The C2C12 murine myoblast cell line was infected with T. gondii tachyzoites (ME49 strain) for 24 h followed by myogenic differentiation induction. T. gondii infection caused a general decrease in myotube differentiation, fusion and maturation, along with decreased expression of myosin heavy chain. The expression of Myogenic Regulatory Factors Myf5, MyoD, Mrf4 and myogenin was modulated by the infection. Infected cultures presented increased proliferation rates, as assessed by Ki67 immunostaining, whereas neither host cell lysis nor apoptosis were significantly augmented in infected dishes. Cytokine Bead Array indicated that IL-6 and MCP-1 were highly increased in the medium from infected cultures, whereas TGF-β1 was consistently decreased. Inhibition of the IL-6 receptor or supplementation with recombinant TGF-β failed to reverse the deleterious effects caused by the infection. However, conditioned medium from infected cultures inhibited myogenesis in C2C12 cells. Activation of the Wnt/β-catenin pathway was impaired in T. gondii-infected cultures. Our data indicate that T. gondii leads SkMCs to a pro-inflammatory phenotype, leaving cells unresponsive to β-catenin activation, and inhibition of the myogenic differentiation program. Such deregulation may suggest muscle atrophy and molecular mechanisms similar to those involved in myositis observed in human patients