Analysis of the toxicological and pharmacokinetic profile of Kaempferol-3-O-β-D-(6"-E-p-coumaryl) glucopyranoside - Tiliroside: in silico, in vitro and ex vivo assay / Análise do perfil toxicológico e farmacocinético de Kaempferol-3-O-β D-(6"-E-p-cumaril) glucopiranosídeo - Tilirosídeo: ensaio in silico, in vitro e ex vivo

Tiliroside is a glycosidic flavonoid present in many plants species including Helicteres velutina K. Schum (Malvaceae sensu lato), commonly known in Brazil as "pitó". This molecule has been shown to have many biological activities, however no study has been carried out to investigate the toxicity of this substance. The present work aimed to evaluate the possible cellular toxicity in silico, in vitro and ex-vivo of the kaempferol-3-O-β-D-(6”-E-p-coumaroyl) glucopyranoside (tiliroside), through chemical structure analysis, toxicity assessment and predictive bioactive properties, using human samples for in vitro and ex-vivo tests. The in silico analysis suggests that tiliroside exhibited great absorption index when penetrating biological membranes. In addition, it also displayed considerable potential for cellular protection against free radicals, and anticarcinogenic, antioxidant, antineoplastic, anti-inflammatory, anti-hemorrhagic and antithrombotic activities. The assessment of the hemolytic and genotoxic effects of tiliroside showed low hemolysis rates in red blood cells and absence of cellular toxicity in the oral mucosa cells. The data obtained indicate that this molecule could be a promising therapeutic approach as a possible new drug with biotechnological potential.

O tilirosídeo é um flavonóide glicosídico presente em muitas espécies de plantas, incluindo Helicteres velutina K. Schum (Malvaceae sensu lato), conhecida no Brasil como “pitó”. Esta molécula mostrou ter muitas atividades biológicas, porém nenhum estudo foi realizado para investigar a toxicidade dessa substância. O presente trabalho teve como objetivo avaliar a possível toxicidade celular in silico, in vitro e ex-vivo do kaempferol-3-O-β-D- (6 ”-Ep-coumaroil) glucopiranosídeo (tilirosídeo), por meio de análises de estrutura química, toxicidade avaliação e propriedades bioativas preditivas, utilizando amostras humanas para testes in vitro e ex-vivo. A análise in silico sugere que o tilirosídeo exibe bom índice de absorção para penetrar nas membranas biológicas. Além disso, apresentou considerável potencial de proteção celular contra os radicais livres e com atividades anticarcinogênica, antioxidante, antineoplásica, antiinflamatória, anti-hemorrágica e antitrombótica. A avaliação dos efeitos hemolíticos e genotóxicos do tilirosídeo mostrou baixas taxas de hemólise nas hemácias e ausência de toxicidade em células da mucosa oral. Os dados obtidos indicam que esta molécula pode possuir uma abordagem terapêutica promissora como uma possível nova droga com potencial biotecnológico.

Analysis of the toxicological and pharmacokinetic profile of Kaempferol-3-O--D-(6-E-p-coumaryl) glucopyranoside - Tiliroside: in silico, in vitro and ex vivo assay

Abstract Tiliroside is a glycosidic flavonoid present in many plants species including Helicteres velutina K. Schum (Malvaceae sensu lato), commonly known in Brazil as pitó. This molecule has been shown to have many biological activities, however no study has been carried out to investigate the toxicity of this substance. The present work aimed to evaluate the possible cellular toxicity in silico, in vitro and ex-vivo of the kaempferol-3-O--D-(6-E-p-coumaroyl) glucopyranoside (tiliroside), through chemical structure analysis, toxicity assessment and predictive bioactive properties, using human samples for in vitro and ex-vivo tests. The in silico analysis suggests that tiliroside exhibited great absorption index when penetrating biological membranes. In addition, it also displayed considerable potential for cellular protection against free radicals, and anticarcinogenic, antioxidant, antineoplastic, anti-inflammatory, anti-hemorrhagic and antithrombotic activities. The assessment of the hemolytic and genotoxic effects of tiliroside showed low hemolysis rates in red blood cells and absence of cellular toxicity in the oral mucosa cells. The data obtained indicate that this molecule could be a promising therapeutic approach as a possible new drug with biotechnological potential.

Resumo O tilirosídeo é um flavonóide glicosídico presente em muitas espécies de plantas, incluindo Helicteres velutina K. Schum (Malvaceae sensu lato), conhecida no Brasil como pitó. Esta molécula mostrou ter muitas atividades biológicas, porém nenhum estudo foi realizado para investigar a toxicidade dessa substância. O presente trabalho teve como objetivo avaliar a possível toxicidade celular in silico, in vitro e ex-vivo do kaempferol-3-O--D- (6 -Ep-coumaroil) glucopiranosídeo (tilirosídeo), por meio de análises de estrutura química, toxicidade avaliação e propriedades bioativas preditivas, utilizando amostras humanas para testes in vitro e ex-vivo. A análise in silico sugere que o tilirosídeo exibe bom índice de absorção para penetrar nas membranas biológicas. Além disso, apresentou considerável potencial de proteção celular contra os radicais livres e com atividades anticarcinogênica, antioxidante, antineoplásica, antiinflamatória, anti-hemorrágica e antitrombótica. A avaliação dos efeitos hemolíticos e genotóxicos do tilirosídeo mostrou baixas taxas de hemólise nas hemácias e ausência de toxicidade em células da mucosa oral. Os dados obtidos indicam que esta molécula pode possuir uma abordagem terapêutica promissora como uma possível nova droga com potencial biotecnológico.

Analysis of the toxicological and pharmacokinetic profile of Kaempferol-3-O-ß-D-(6"-E-p-coumaryl) glucopyranoside - Tiliroside: in silico, in vitro and ex vivo assay / Análise do perfil toxicológico e farmacocinético de Kaempferol-3-O-ß-D- (6 "-E-p-cumaril) glucopiranosídeo - Tilirosídeo: ensaio in silico, in vitro e ex vivo

Abstract Tiliroside is a glycosidic flavonoid present in many plants species including Helicteres velutina K. Schum (Malvaceae sensu lato), commonly known in Brazil as "pitó". This molecule has been shown to have many biological activities, however no study has been carried out to investigate the toxicity of this substance. The present work aimed to evaluate the possible cellular toxicity in silico, in vitro and ex-vivo of the kaempferol-3-O-β-D-(6"-E-p-coumaroyl) glucopyranoside (tiliroside), through chemical structure analysis, toxicity assessment and predictive bioactive properties, using human samples for in vitro and ex-vivo tests. The in silico analysis suggests that tiliroside exhibited great absorption index when penetrating biological membranes. In addition, it also displayed considerable potential for cellular protection against free radicals, and anticarcinogenic, antioxidant, antineoplastic, anti-inflammatory, anti-hemorrhagic and antithrombotic activities. The assessment of the hemolytic and genotoxic effects of tiliroside showed low hemolysis rates in red blood cells and absence of cellular toxicity in the oral mucosa cells. The data obtained indicate that this molecule could be a promising therapeutic approach as a possible new drug with biotechnological potential.

Resumo O tilirosídeo é um flavonóide glicosídico presente em muitas espécies de plantas, incluindo Helicteres velutina K. Schum (Malvaceae sensu lato), conhecida no Brasil como "pitó". Esta molécula mostrou ter muitas atividades biológicas, porém nenhum estudo foi realizado para investigar a toxicidade dessa substância. O presente trabalho teve como objetivo avaliar a possível toxicidade celular in silico, in vitro e ex-vivo do kaempferol-3-O-β-D- (6 "-Ep-coumaroil) glucopiranosídeo (tilirosídeo), por meio de análises de estrutura química, toxicidade avaliação e propriedades bioativas preditivas, utilizando amostras humanas para testes in vitro e ex-vivo. A análise in silico sugere que o tilirosídeo exibe bom índice de absorção para penetrar nas membranas biológicas. Além disso, apresentou considerável potencial de proteção celular contra os radicais livres e com atividades anticarcinogênica, antioxidante, antineoplásica, antiinflamatória, anti-hemorrágica e antitrombótica. A avaliação dos efeitos hemolíticos e genotóxicos do tilirosídeo mostrou baixas taxas de hemólise nas hemácias e ausência de toxicidade em células da mucosa oral. Os dados obtidos indicam que esta molécula pode possuir uma abordagem terapêutica promissora como uma possível nova droga com potencial biotecnológico.

Thromboembolism in a bitch - diagnosis by infrared thermography

Background: Thermography is a noninvasive, non-contact, painless, and non-ionizing imaging technique that records cutaneous thermal patterns generated by infrared emission of the surface. The surface heat is closely related to dermal microcirculation. Thromboembolism is responsible for important changes in the thermal pattern of the body surface due to physical obstruction of blood flow, being the main complication in immune-mediated hemolytic anemia. The aim of this paper is to report a dog with thrombus in his left forelimb secondary to idiopathic hemolytic anemia, whose diagnostic screening was performed through infrared thermography. Case: A 9-year-old mixed breed bitch was referred to a veterinary hospital with a history of emesis, diarrhea and dark-colored urine for 2 days. The complete blood count showed hypochromic macrocytic anemia (hematocrit [HTC] 28%, reference: 37 to 55%) with the presence of nucleated erythrocyte (14/100 leukocytes, reference: 0 to 5/100 leukocytes), polychromasia and spherocytes. Leukocytosis (28,300 mm³, reference: 6,000 to 17,000 mm³) by neutrophilia with left deviation and toxic granulations was also present, in addition to hemoglobinuria at urine exam. Therefore, treatment for immune-mediated hemolytic anemia (IMHA) was instituted. After 2 days, the animal returned with acute functional impairment of the left forelimb. Physical examination revealed that the limb was cold, without pulse, proprioception, reflexes, and deep pain. New blood analyses revealed decreased hematocrit (HTC 17%, reference: 37 to 55%), and increased total leukocyte number (57,000 mm3, reference: 6,000 to 17,000 mm³). Infrared thermography revealed an important temperature difference between the limbs, with the affected limb temperature considerably lower (31.3ºC) when compared to the contralateral limb (35.0ºC). Thermography showed the site of the thrombus in the medial portion of the limb (cephalic vein), where the catheter had been placed for fluid therapy. Due to the severity of the condition, the bitch was submitted to amputation surgery, which occurred without complications. The patient had a good response to treatment, with decreased signs of hemolysis and hypercoagulability. The medications were slowly withdrawn, and the clinical discharge occurred after 4 weeks. Discussion: In humans, thermography has been widely used in the assessment of thrombotic diseases, contributing to diagnosis, localization, and prognosis. In veterinary medicine, however, the use of this tool in the diagnosis of thromboembolism is still rare. The difference of 3.7°C between the affected and contralateral limb was objectively verified using thermography. A minimum difference of 2.4°C between limbs has high sensitivity and specificity for diagnosing thromboembolism and occurs due to the reduction in local blood flow. In the present case this tool was essential for the anatomical location of the thrombus, which was in the middle third of the forearm, and allowed an adequate surgical planning. It is known that the main complication of IMHA is thromboembolism. Its predisposing factors include venous stasis, endothelial damage, and hypercoagulability, being exacerbated by cage confinement, decubitus and presence of a peripheral venous catheter. The reported case corroborates at least one of these factors since it had a peripheral venous catheter in the left forelimb, which later showed absence of pulse, spinal reflexes, pain and proprioception. The thermography showed to be an objective, rapid and non-invasive tool to diagnose and precisely locate the thrombus, which allowed for adequate treatment and surgical planning for the case. To the best of our knowledge, this is the first report about use of thermography to diagnose thromboembolism secondary to immune-mediated hemolytic anemia in a dog.

Analysis of the toxicological and pharmacokinetic profile of Kaempferol-3-O-β-D-(6"-E-p-coumaryl) glucopyranoside - Tiliroside: in silico, in vitro and ex vivo assay / Análise do perfil toxicológico e farmacocinético de Kaempferol-3-O-β D-(6"-E-p-cumaril) glucopiranosídeo - Tilirosídeo: ensaio in silico, in vitro e ex vivo

Tiliroside is a glycosidic flavonoid present in many plants species including Helicteres velutina K. Schum (Malvaceae sensu lato), commonly known in Brazil as "pitó". This molecule has been shown to have many biological activities, however no study has been carried out to investigate the toxicity of this substance. The present work aimed to evaluate the possible cellular toxicity in silico, in vitro and ex-vivo of the kaempferol-3-O-β-D-(6”-E-p-coumaroyl) glucopyranoside (tiliroside), through chemical structure analysis, toxicity assessment and predictive bioactive properties, using human samples for in vitro and ex-vivo tests. The in silico analysis suggests that tiliroside exhibited great absorption index when penetrating biological membranes. In addition, it also displayed considerable potential for cellular protection against free radicals, and anticarcinogenic, antioxidant, antineoplastic, anti-inflammatory, anti-hemorrhagic and antithrombotic activities. The assessment of the hemolytic and genotoxic effects of tiliroside showed low hemolysis rates in red blood cells and absence of cellular toxicity in the oral mucosa cells. The data obtained indicate that this molecule could be a promising therapeutic approach as a possible new drug with biotechnological potential.(AU)

O tilirosídeo é um flavonóide glicosídico presente em muitas espécies de plantas, incluindo Helicteres velutina K. Schum (Malvaceae sensu lato), conhecida no Brasil como “pitó”. Esta molécula mostrou ter muitas atividades biológicas, porém nenhum estudo foi realizado para investigar a toxicidade dessa substância. O presente trabalho teve como objetivo avaliar a possível toxicidade celular in silico, in vitro e ex-vivo do kaempferol-3-O-β-D- (6 ”-Ep-coumaroil) glucopiranosídeo (tilirosídeo), por meio de análises de estrutura química, toxicidade avaliação e propriedades bioativas preditivas, utilizando amostras humanas para testes in vitro e ex-vivo. A análise in silico sugere que o tilirosídeo exibe bom índice de absorção para penetrar nas membranas biológicas. Além disso, apresentou considerável potencial de proteção celular contra os radicais livres e com atividades anticarcinogênica, antioxidante, antineoplásica, antiinflamatória, anti-hemorrágica e antitrombótica. A avaliação dos efeitos hemolíticos e genotóxicos do tilirosídeo mostrou baixas taxas de hemólise nas hemácias e ausência de toxicidade em células da mucosa oral. Os dados obtidos indicam que esta molécula pode possuir uma abordagem terapêutica promissora como uma possível nova droga com potencial biotecnológico.(AU)

Acute erythroid leukemia in a cat infected with feline leukemia virus

Background: Erythroid leukemia is a myeloproliferative hematopoietic disorder considered acute when there is a predominance of blasts in the bone marrow. It is frequently reported in cats infected with feline leukemia virus, but it is unclear whether this virus is involved in the oncogenesis. The clinical signs in cats are anorexia, apathy, weight loss, with evolution from 2 weeks to 2 months, pale mucous membranes, hemorrhages, ascites, salivation, and dyspnea due to pleural effusion. This affection responds little to chemotherapy with an unfavorable prognosis. The aim of this study is to report a case of a feline leukemia virus infected cat with the onset of severe hemolytic anemia. Case: A 8-year-old male mixed breed cat was attended with a history of anorexia, oligodipsia, apathy, progressive weight loss, and yellowish color of urine for 7 days. Laboratorial exams showed anemia (with metarubricytes, acanthocytes and ghost cells), leukocytosis and FeLV reagent test. The cat underwent treatment with methylprednisolone acetate and supportive care. One day later, the animal returned with icteric mucous membranes, and emesis. A blood count was performed that found worsening anemia, increased leukocytosis, and lymphocytosis. Abdominal ultrasound showed cholangiohepatitis and lymphadenomegaly in mesenteric lymph nodes. Treatment was started with ondansetron, metronidazole, and amoxicilin with potassium clavulanate. The cat returned after 3 days and laboratorial exams revealed worsening of blood parameters, so blood transfusion was performed. After 2 days, the patient started with dyspnea and hypothermia, that evolved to cardiorespiratory arrest. The body was sent to necropsy and histopathology, where blast cells and rubricytes were found in blood vessels of various organs. The bone marrow was markedly cellular with complete disappearance of adipose tissue. Most of the cells were blasts with abundant and eosinophilic cytoplasm, central nucleus with finely dotted chromatin and a large nucleolus. There were rubricytes, which made possible to confirm acute erythroid leukemia as a morphological diagnosis. Discussion: The clinical signs observed in acute erythroid leukemia are lethargy, inappetence, fever, splenomegaly, mild lymphadenomegaly, associated with leukocytosis, severe anemia, and thrombocytopenia. The reported animal presented signs similar to those described in the literature except that there was no change in platelet counts. The diagnosis of leukemia was reached after histopathology, and it is made when is observed more than 30% of myeloblasts and monoblasts together or when the blast cells count including rubriblasts is greater than 30%. Although chemotherapy, the prognosis is usually poor. It is essential to perform the myelogram for the diagnosis of myeloid leukemias in vivo. In this report, we only achieve final diagnosis after the cat's death, due to the aggressive behavior of the disease. Clinicians must be aware of the likely development of acute erythroid leukemia whenever a feline leukemia virus infected cat presents hemolytic anemia to get an early diagnosis, since this is an extremely aggressive disease, to propose prompt chemotherapy and give the patient a longer survival period.

Crise hemolítica em um cão com hepatite crônica associada ao cobre / Hemolytic crisis in a dog with copper-associated chronic hepatitis

Background: Copper is an essential micronutrient for the body to function properly. However, although it is a vital element,an excess of copper in the body is extremely toxic. Copper toxicity has been reported mainly in sheep. In dogs, clinicopathological signs of toxicity are characterized by chronic liver failure. This means that the hemolytic crisis so commonin sheep is a condition rarely associated with toxicity in dogs, so there are very few descriptions of this condition in theveterinary literature. The purpose of this report is to describe a case of hemolytic crisis in a dog with copper-associatedchronic hepatitis.Case: A medium-sized 6-year-old bitch was brought to the Veterinary Hospital of the Federal University of Santa Maria,with clinical presentation of apathy, anorexia and red urine. A physical examination revealed mildly jaundiced mucosaand dark brown urine. A urinalysis indicated the presence of protein, bilirubin and occult blood. The blood count revealedhypochromic macrocytic anemia, leukocytosis due to left shift neutrophilia and thrombocytopenia. Serum biochemistryshowed elevated levels of alanine aminotransferase and alkaline phosphatase. The animal was given a blood transfusiondue to the severity of her anemia, but her clinical condition worsened and she died, whereupon her body was sent for necropsy. This necropsy revealed conspicuous signs of jaundice, splenomegaly and altered liver and kidney color. The liverwas brownish, with its natural surface firm and slightly irregular. The kidneys were diffusely blackened. The urine wasdark brown. Fragments of different organs were collected, fixed in 10% buffered formalin solution, routinely processedfor histopathology and stained with hematoxylin and eosin. A histological dissection of the liver showed the hepatic lobesdissected by fibrosis, forming islands of hepatocytes and numerous lymphocytes and...

Crise hemolítica em um cão com hepatite crônica associada ao cobre / Hemolytic crisis in a dog with copper-associated chronic hepatitis

Background: Copper is an essential micronutrient for the body to function properly. However, although it is a vital element,an excess of copper in the body is extremely toxic. Copper toxicity has been reported mainly in sheep. In dogs, clinicopathological signs of toxicity are characterized by chronic liver failure. This means that the hemolytic crisis so commonin sheep is a condition rarely associated with toxicity in dogs, so there are very few descriptions of this condition in theveterinary literature. The purpose of this report is to describe a case of hemolytic crisis in a dog with copper-associatedchronic hepatitis.Case: A medium-sized 6-year-old bitch was brought to the Veterinary Hospital of the Federal University of Santa Maria,with clinical presentation of apathy, anorexia and red urine. A physical examination revealed mildly jaundiced mucosaand dark brown urine. A urinalysis indicated the presence of protein, bilirubin and occult blood. The blood count revealedhypochromic macrocytic anemia, leukocytosis due to left shift neutrophilia and thrombocytopenia. Serum biochemistryshowed elevated levels of alanine aminotransferase and alkaline phosphatase. The animal was given a blood transfusiondue to the severity of her anemia, but her clinical condition worsened and she died, whereupon her body was sent for necropsy. This necropsy revealed conspicuous signs of jaundice, splenomegaly and altered liver and kidney color. The liverwas brownish, with its natural surface firm and slightly irregular. The kidneys were diffusely blackened. The urine wasdark brown. Fragments of different organs were collected, fixed in 10% buffered formalin solution, routinely processedfor histopathology and stained with hematoxylin and eosin. A histological dissection of the liver showed the hepatic lobesdissected by fibrosis, forming islands of hepatocytes and numerous lymphocytes and...(AU)

Anemia hemolítica em bovinos de corte em sistema de criação extensiva em Mato Grosso e Rondônia / Hemolytic anemia in beef cattle in extensive system farming in Mato Grosso and Rondônia

Casos de anemia hemolítica em bovinos de corte em sistema de criação extensiva em Mato Grosso e Rondônia são acompanhados desde 2008. Os animais acometidos apresentam fraqueza, mucosas pálidas ou ictéricas, urina enegrecida, depressão e anorexia. Esse quadro muitas vezes evolui para morte, principalmente quando os animais são movimentados. Durante esse período aproximadamente 429 bovinos morreram em diferentes surtos eem alguns casos a mortalidade chegou a 50%. Foi realizada investigação para as principais causas infecciosas como leptospirose, babesiose, tripanossomíase, anaplasmose e hemoglobinúria bacilar, bem como para as possíveis causas toxicas relatadas como etiologia de anemia hemolítica em bovinos por meio de exames hematológicos, bioquímica sanguínea, moleculares e histopatológicos. Não se observou nas propriedades acometidas, nenhuma das plantas já descritas no Brasil como causadora de anemia hemolítica em animais de interesse pecuário no momento das investigações dos surtos. Apesar da etiologia desses casos ainda não ser conhecida, as evidências epidemiológicas, clínicas e patológicas da doença sugerem que a mesma seja causada por uma planta tóxica de ação hemolítica, presente nas propriedades acometidas, mas que ainda não foi confirmada experimentalmente.(AU)

Cases of hemolytic anemia in beef cattle in na extensive breeding system in Mato Grosso and Rondônia have been monitored since 2008. The affected animals presented weakness, pale or icteric mucosas, blackened urine, prostration and anorexia. The outcome of these cases often is death, principally if the animals are forced to walk. Approximately 429 cattle died in these outbreaks andin some of these, mortality rates ranged from 0.26 to 50%. Research was carried out on the main infectious causes such as leptospirosis, babesiosis, trypanosomiasis, anaplasmosis and bacillary hemoglobinuria, as well as the possible toxic causes reported as etiology of hemolytic anemia in cattle through hematology, blood biochemistry, molecular and histopathological examinations. Any plant ready described in Brazil as cause of hemolytic anemia in animals from livestock interest was observed in thosefarms. Although the etiology of these cases is not yet known, clinical and pathological evidences of the disease suggested that it is caused by a toxic plant of hemolytic action, present in the affected properties, but not yet confirmed experimentally.(AU)

Anemia hemolítica em bovinos de corte em sistema de criação extensiva em Mato Grosso e Rondônia / Hemolytic anemia in beef cattle in extensive system farming in Mato Grosso and Rondônia

Casos de anemia hemolítica em bovinos de corte em sistema de criação extensiva em Mato Grosso e Rondônia são acompanhados desde 2008. Os animais acometidos apresentam fraqueza, mucosas pálidas ou ictéricas, urina enegrecida, depressão e anorexia. Esse quadro muitas vezes evolui para morte, principalmente quando os animais são movimentados. Durante esse período aproximadamente 429 bovinos morreram em diferentes surtos eem alguns casos a mortalidade chegou a 50%. Foi realizada investigação para as principais causas infecciosas como leptospirose, babesiose, tripanossomíase, anaplasmose e hemoglobinúria bacilar, bem como para as possíveis causas toxicas relatadas como etiologia de anemia hemolítica em bovinos por meio de exames hematológicos, bioquímica sanguínea, moleculares e histopatológicos. Não se observou nas propriedades acometidas, nenhuma das plantas já descritas no Brasil como causadora de anemia hemolítica em animais de interesse pecuário no momento das investigações dos surtos. Apesar da etiologia desses casos ainda não ser conhecida, as evidências epidemiológicas, clínicas e patológicas da doença sugerem que a mesma seja causada por uma planta tóxica de ação hemolítica, presente nas propriedades acometidas, mas que ainda não foi confirmada experimentalmente.(AU)

Cases of hemolytic anemia in beef cattle in na extensive breeding system in Mato Grosso and Rondônia have been monitored since 2008. The affected animals presented weakness, pale or icteric mucosas, blackened urine, prostration and anorexia. The outcome of these cases often is death, principally if the animals are forced to walk. Approximately 429 cattle died in these outbreaks andin some of these, mortality rates ranged from 0.26 to 50%. Research was carried out on the main infectious causes such as leptospirosis, babesiosis, trypanosomiasis, anaplasmosis and bacillary hemoglobinuria, as well as the possible toxic causes reported as etiology of hemolytic anemia in cattle through hematology, blood biochemistry, molecular and histopathological examinations. Any plant ready described in Brazil as cause of hemolytic anemia in animals from livestock interest was observed in thosefarms. Although the etiology of these cases is not yet known, clinical and pathological evidences of the disease suggested that it is caused by a toxic plant of hemolytic action, present in the affected properties, but not yet confirmed experimentally.(AU)

Hematological and Renal Function Evaluation in Dogs with IMHA

Background: The IMHA is a common cause of anemia in dogs and characterized by direct destruction or phagocytosis of erythrocytes opsonized by IgG, IgM and/or complement. The diagnosis is based on the identification of erythrocytes destruction in the presence of anti-erythrocyte antibodies, producing spherocytes, auto-agglutination, Coombs test or flow cytometry test positive, in addition to anemia and clinical signs of hemolysis. The renal biochemical profile and urinalysis may reveal important changes due to the severity of the kidney demage. The aim of this study were to evaluate the incidence of hematological and renal abnormalities, and the prevalence of immunoglobulins classes involved in IMHA.Materials, Methods & Results: In a total of 87 anemic dogs were selected and tested by Coombs test, flow cytometry (FC), and auto-agglutination, along with CBC, reticulocyte count, renal profile (ureia and creatinine), hemoparasite search in peripheral blood smears, and Ehrlichia sp. and leptospirosis tests. The results were analyzed by t test or Mann-Whitney with 5% of significance. Therefore, 61 dogs (70.11%) were positive for IMHA by FC, 31 (35.63%) by Coombs test, and 24 (27.58%) by auto-agglutination. There was not a predominance of IgG or IgM involvement. The hematological and clinical changes in dogs with IMHA included macrocytic, hypochromic regenerative anemia, and r

Alterações clínicas e laboratoriais em um cão com anemia hemolitica imunomediada: relato de caso

A anemia hemolítica imunomediada (AHIM) é um distúrbio imunológico mais comum em cães que em gatos. Ela se caracteriza pela presença de anemia por destruição direta ou fagocitose de hemácias opsonizadas. Pode ser aguda ou crônica, regenerativa ou arregenerativa. Objetivou-se descrever as alterações clínicas e laboratoriais em um cão com AHIM. O animal apresentou anemia regenerativa, leucocitose com neutrofilia e monocitose. Alterações séricas de ALT, fosfatase alcalina, creatinina e uréia estavam presentes. Apatia, dispneia e icterícia foram visualizadas evoluindo para óbito. Nesse relato, a anemia hemolítica imunomediada induziu alterações clínicas, hematológicas e bioquímicas significativas que comprometeram a vida do cão. (AU)

Immune-mediated hemolytic anemia (AHIM) is a more common immune disorder in dogs than in cats. It is characterized by the presence of anemia by direct destruction or phagocytosis of opsonized red blood cells. It can be acute or chronic, regenerative or arregenerative. The aim of this study was to describe clinical and laboratory changes in a dog with AHIM. The animal presented regenerative anemia, leukocytosis with neutrophilia and monocytosis. Serum alterations of ALT, alkaline phosphatase, creatinine and urea were present. Apathy, dyspnoea and jaundice were visualized evolving to death. In this report, immune-mediated hemolytic anemia induced significant clinical, hematological and biochemical changes that compromised the dog's life. (AU)

Alterações clínicas e laboratoriais em um cão com anemia hemolitica imunomediada: relato de caso

A anemia hemolítica imunomediada (AHIM) é um distúrbio imunológico mais comum em cães que em gatos. Ela se caracteriza pela presença de anemia por destruição direta ou fagocitose de hemácias opsonizadas. Pode ser aguda ou crônica, regenerativa ou arregenerativa. Objetivou-se descrever as alterações clínicas e laboratoriais em um cão com AHIM. O animal apresentou anemia regenerativa, leucocitose com neutrofilia e monocitose. Alterações séricas de ALT, fosfatase alcalina, creatinina e uréia estavam presentes. Apatia, dispneia e icterícia foram visualizadas evoluindo para óbito. Nesse relato, a anemia hemolítica imunomediada induziu alterações clínicas, hematológicas e bioquímicas significativas que comprometeram a vida do cão.

Immune-mediated hemolytic anemia (AHIM) is a more common immune disorder in dogs than in cats. It is characterized by the presence of anemia by direct destruction or phagocytosis of opsonized red blood cells. It can be acute or chronic, regenerative or arregenerative. The aim of this study was to describe clinical and laboratory changes in a dog with AHIM. The animal presented regenerative anemia, leukocytosis with neutrophilia and monocytosis. Serum alterations of ALT, alkaline phosphatase, creatinine and urea were present. Apathy, dyspnoea and jaundice were visualized evolving to death. In this report, immune-mediated hemolytic anemia induced significant clinical, hematological and biochemical changes that compromised the dog's life.

Hematological and renal function evaluation in dogs with IMHA

Background: The IMHA is a common cause of anemia in dogs and characterized by direct destruction or phagocytosis of erythrocytes opsonized by IgG, IgM and/or complement. The diagnosis is based on the identification of erythrocytes destruction in the presence of anti-erythrocyte antibodies, producing spherocytes, auto-agglutination, Coomb’s test or flow cytometry test positive, in addition to anemia and clinical signs of hemolysis. The renal biochemical profile and urinalysis may reveal important changes due to the severity of the kidney demage. The aim of this study were to evaluate the incidence of hematological and renal abnormalities, and the prevalence of immunoglobulin’s classes involved in IMHA. Materials, Methods & Results: In a total of 87 anemic dogs were selected and tested by Coomb’s test, flow cytometry (FC), and auto-agglutination, along with CBC, reticulocyte count, renal profile (ureia and creatinine), hemoparasite search in peripheral blood smears, and Ehrlichiasp. and leptospirosis tests. The results were analyzed by t test or Mann-Whitney with 5% of significance. Therefore, 61 dogs (70.11%) were positive for IMHA by FC, 31 (35.63%) by Coomb’s test, and 24 (27.58%) by auto-agglutination. There was not a predominance of IgG or IgM involvement. The hematological and clinical changes in dogs with IMHA included macrocytic, hypochromic regenerative anemia, and reticulocytosis, as well as icterus, fever, auto-agglutination, hyperglobulinemia and bilirrubinuria. Spherocytosis was found in 9.8% of dogs with IMHA, and 29.5% of dogs had leukocytosis, 39.6% neutrophilia, and 72.1% thrombocytopenia. Mostly of cases of IHMA (74.6%) were attributed to infectious diseases and associated with Ehrlichiasp. (secondary IMHA), 21.4% of dogs with IMHA had azotemia, and 51.8% had increased urine protein creatinine ratio.[...]

Hematological and renal function evaluation in dogs with IMHA

Background: The IMHA is a common cause of anemia in dogs and characterized by direct destruction or phagocytosis of erythrocytes opsonized by IgG, IgM and/or complement. The diagnosis is based on the identification of erythrocytes destruction in the presence of anti-erythrocyte antibodies, producing spherocytes, auto-agglutination, Coombs test or flow cytometry test positive, in addition to anemia and clinical signs of hemolysis. The renal biochemical profile and urinalysis may reveal important changes due to the severity of the kidney demage. The aim of this study were to evaluate the incidence of hematological and renal abnormalities, and the prevalence of immunoglobulins classes involved in IMHA. Materials, Methods & Results: In a total of 87 anemic dogs were selected and tested by Coombs test, flow cytometry (FC), and auto-agglutination, along with CBC, reticulocyte count, renal profile (ureia and creatinine), hemoparasite search in peripheral blood smears, and Ehrlichiasp. and leptospirosis tests. The results were analyzed by t test or Mann-Whitney with 5% of significance. Therefore, 61 dogs (70.11%) were positive for IMHA by FC, 31 (35.63%) by Coombs test, and 24 (27.58%) by auto-agglutination. There was not a predominance of IgG or IgM involvement. The hematological and clinical changes in dogs with IMHA included macrocytic, hypochromic regenerative anemia, and reticulocytosis, as well as icterus, fever, auto-agglutination, hyperglobulinemia and bilirrubinuria. Spherocytosis was found in 9.8% of dogs with IMHA, and 29.5% of dogs had leukocytosis, 39.6% neutrophilia, and 72.1% thrombocytopenia. Mostly of cases of IHMA (74.6%) were attributed to infectious diseases and associated with Ehrlichiasp. (secondary IMHA), 21.4% of dogs with IMHA had azotemia, and 51.8% had increased urine protein creatinine ratio.[...](AU)

Avaliação das alterações hemostáticas e do risco tromboembólico em cães com AHIM / Evaluation of hemostatic abnormalities and thromboembolic risk in dogs with IMHA

A anemia hemolítica imuno-mediada (AHIM) é a causa mais comum dentre as anemias hemolíticas e a doença imuno-mediada de maior prevalência em cães, incluindo causas primárias e secundárias. As AHIM tem sido associadas a estados de hipercoagulabilidade, sendo o tromboembolismo a complicação mais comum. Este estudo teve como objetivo correlacionar as possíveis alterações hemostáticas e o risco tromboembólico nas AHIM e nas anemias por outras etiologias. Para tanto, foram selecionados 76 cães anêmicos (hematócrito ≤ 20%) somados ao menos um sinal clínico comumente associado à AHIM ou possuir pré-disposição racial. Foram realizados os seguintes testes para os animais selecionados: teste de citometria de fluxo e avaliação do perfil hemostático (contagem de plaquetas, TP, TTPA, TT, AT, PDF e Dímeros D); além de hemograma com contagem de reticulócitos, pesquisa de hematozoários em sangue periférico, PCR para Ehrlichia sp. e sorologia para leptospirose. 59 cães foram positivos para AHIM. O estado tromboembólico foi caracterizado pela presença de alteração em três ou mais testes do perfil hemostático. 74,6% casos de AHIM foram atribuídos às doenças infecciosas, sendo em sua maioria associados à Ehrlichia sp. (88,6%). 72,1% apresentaram trombocitopenia e 57,6% apresentaram anemia regenerativa com valores significativamente maiores de metarrubrícitos e contagem de reticulócitos. Não houve diferença estatística entre os grupos de cães anêmicos (positivos e negativos para AHIM). Os cães anêmicos apresentaram valores médios maiores de TTPA e menores de AT e contagem de plaquetas quando comparados aos cães saudáveis (p < 0,05). 25 cães positivos e sete negativos apresentaram estado tromboembólico. A especificidade de PDF foi menor (30,2%) quando comparada outros estudos. A escolha da classe de Ig não interfere no diagnóstico de AHIM. [...](AU)

Immune-mediated hemolytic anemia (IMHA) is the most common cause of hemolytic anemia and the most prevalent immune-mediated disease in dogs, which is classified in primary or secondary. IMHA has been associated with hypercoagulability state and thromboembolism has been referred as the most common complication. The purpose of this study was to correlate the possibility of hemostatic abnormalities and the thromboembolic risk in IMHA and in anemias of other etiologies. 76 dogs were selected, all exhibiting hematocrit lower than 20% and at least one clinical sign commonly associated with IMHA or breed predisposition. These dogs were tested for IMHA by flow cytometry. Hemostatic tests (platelet count, PT, APTT, TT, AT, PDF and D-dimer) as well as CBC, reticulocyte count, blood parasite search in peripheral blood smear, Ehrlichia sp. and leptospirosis tests were performed in all dogs. 59 dogs were positive for IMHA. The thromboembolic risk was characterized by the presence of three or more changes in the tests of the hemostatic profile. 74.6% cases of IMHA were attributed to infectious diseases and was mostly associated with Ehrlichia sp. (88.6%). 72.1% of dogs had thrombocytopenia and 57.6% had regenerative anemia with significantly higher values of metarubricyte and reticulocyte count. There were no significant changes between two anemic groups (positive and negative for IMHA). Anemic dogs had higher APTT mean value and lower AT and platelet counts mean values than the control group (p<0.05). 25 dogs with IMHA and seven dogs without IHMA had thromboembolic state. The specificity of PDF was lower (30.2%) than previous studies. The choice of the Ig class does not affect the diagnosis of IMHA. There were no correlation between the presence of antibodies bound to the RBC surface and the hemostatic tests, but there were a week correlation (p<0,05) between Ht(%) and APTT (r=-0.2621), AT (r=0.4297) and platelets count (r=0.5349) values. [...](AU)

Detecção dos genes codificantes da toxina CDT, e pesquisa de fatores que influenciam na produção de hemolisinas em amostras de Campylobacter jejuni de origem avícola / Detection of the genes encoding the toxin CDT, and research factors which influence the production of hemolysin in Campylobacter jejuni from poultry products

Membros termofílicos do gênero Campylobacter são reconhecidos como importantes enteropatógenos para o ser humano e animais. A grande diversidade ecológica destes micro-organismos em diferentes habitats tais como água, animais e alimentos predispõem ao aparecimento de novos fatores de virulência. Este trabalho teve por objetivo detectar os genes codificantes da Toxina Distensiva Citoletal (CDT) por meio da técnica de PCR, pesquisar a atividade de hemolisinas e a influência de soluções quelantes e de íons nesta atividade. Foram utilizadas 45 amostras de Campylobacter jejuni de origem avícola para pesquisa de atividade hemolítica, cultivadas em Caldo Triptona de Soja (TSB). Após o crescimento bacteriano, as amostras foram semeadas em Ágar tríptico de soja (TSA) contendo 5% de sangue de ovino. Para verificar a influência de agentes quelantes e solução de íons na atividade hemolítica, as amostras de C. jejuni foram cultivadas em TSB contendo separadamente os quelantes EDTA, ácido acético, soluções de íons CaCl2, MgCl2 e FeCl3, em atmosfera de microaerofilia. Quanto à atividade de hemolisina de C. jejuni em placas de TSA - sangue ovino foi possível observar que houve hemólise em 40% das amostras analisadas apenas com caldo TSB. Somente o ácido acético apresentou ação quelante sobre a atividade de hemolisinas em amostras de C. jejuni semeadas em placas de TSA - sangue ovino. Para detecção dos genes cdtA, cdtB e cdtC através da técnica da Reação em Cadeia da Polimerase (PCR) foram utilizadas 119 amostras de C. jejuni de origem avícola. Foi possível observar que 37,8% possuíam o perfil de genes cdtABC. Os resultados demonstraram em amostras avícolas a presença de cepas de C. jejuni com potencial virulento, devido à presença dos genes da toxina CDT e potencial hemolítico, que apresentou ação reduzida in vitro com ácido acético.(AU)

Thermophilic members of the Campylobacter genus are recognized as important enteropathogenics for humans and animals. The great variety of ecological habitats, such as water, food and milk, may promote new virulence factors. To detect the encoding genes distending cytolethal toxin (CDT) by PCR and study the hemolytic activity with influence of chelation solutions and ions, 45 Campylobacter jejuni samples from poultry production origin were used to perform the hemolytic research. To check the influence of chelation agents and solution of ions in the hemolytic activity, samples of C. jejuni strains were grown in tryptone soy broth TSB containing chelation agents separately EDTA, acetic acid, CaCl2, MgCl2 and FeCl3 ions solutions in microaerophilic atmosphere and then streaked on 5% sheep blood tryptic soy agar (TSA). To perform the detection of cdtA, cdtB and cdtC genes the technique of Polymerase Chain Reaction (PCR) was used in 119 samples of C. jejuni from poultry production origin. We found 40% of samples showing hemolysis after growing with TSB. Only the acetic acid showed reduction in hemolysis. The prevalent gene profile was cdtABC in 37.8 % of the samples. It was observed that the results showed the presence of C. jejuni strains with virulent potential, due to presence of the CDT toxin genes and the hemolytic activity, which showed in vitro reduced when acetic acid was added.(AU)

Icterícia em gatos: estudo retrospectivo e prospectivo.

Foram necropsiados 44 felinos domésticos (Felis catus) com icterícia, no período de junho de 2014 a dezembro de 2019. Os gatos eram provenientes da região metropolitana de Belo Horizonte, Minas Gerais (n= 31) e de Teresina, Piaui (n= 13). Desses 44 felinos, 34,1% eram fêmeas, 56,8% eram machos e em 9,1% não havia informações sobre o sexo do animal. As idades variavam de seis meses a 13 anos. Destes, cinco eram adultos, e em 11 gatos a idade não foi informada. A maioria dos gatos examinados eram sem raça definida (35/44). As raças especificadas foram Angorá (2/44), Oriental Short Hair (1/44) e Persa (1/44), e em cinco gatos, a raça não foi informada. Todos os animais tinham icterícia, com intensidade que variavam de leve a acentuada. Dos 44 animais, 39 foram diagnosticados com anemia leve a acentuada. A classificação do tipo de icterícia (pré-hepática, hepática e pós-hepática) foi baseada nos achados macroscópicos e microscópicos. Dos 44 animais, 10 foram classificados com icterícia pré-hepática, 33 com icterícia hepática e sete com icterícia pós-hepática. Contudo, em alguns gatos foram encontrados dois tipos de icterícia, dos quais cinco apresentaram icterícia pré-hepática e icterícia hepática e um caso de icterícia hepática e icterícia pós-hepática. De acordo com os achados macroscópicos e microscópicos, a causa da icterícia pré-hepática foi a anemia hemolítica idiopática. Para a icterícia hepática, a causa mais frequente foi a lipidose hepática (26/44), em seguida peri-hepatite e hepatite compatível com o vírus da peritonite felina, linfoma, degeneração glicogênica, colangiocarcinoma e um diagnóstico sugestivo de leucemia mieloide. Nos animais com icterícia pós-hepática as causas foram colangite devido a infecção por Platynosomum spp., colangioma do ducto hepático comum e colangite crônica.

Forty-four domestic cats (Felis catus) with icterus were necropsied from June 2014 to December 2019. The cats were from the metropolitan region of Belo Horizonte Minas Gerais (n = 31) and Teresina, Piaui (n = 13). Of these 44 cats, 34.1% were females, 56.8% were males and for 9.1% there was no information about the gender. Ages ranged from six months to 13 years. Of these, five cats were adults and, in 11 cats, the age was not informed. Most of the cats examined were mixed breed (35/44). Other breeds were Angora (2/44), Oriental Short Hair (1/44) and Persian (1/44); in five cats the breed was not informed. All animals had icterus, with intensity ranging from mild to severe. Of the 44 animals, 39 had mild to severe anemia. The classification of the type of icterus (pre-hepatic, hepatic and post-hepatic) was based on macroscopic and microscopic findings. Of the 44 animals, 10 were classified as pre-hepatic icterus, 33 with hepatic icterus and seven with post-hepatic icterus. Nevertheless, in some cats, two types of icterus were found, of which five cases were classified with pre-hepatic and hepatic icterus, and one case with hepatic and post-hepatic icterus. According to gross and microscopic findings, the cause of pre-hepatic icterus was idiopathic hemolytic anemia. For hepatic icterus, the most frequent causes were liver lipidosis (26/44), followed by peri-hepatitis and hepatitis compatible with feline peritonitis virus infection, lymphoma, glycogenic degeneration, cholangiocarcinoma and myeloid leukemia. In animals with post hepatic icterus, the causes were colangitis due Platynosomum spp. infection, cholangioma of the common hepatic duct and chronic cholangitis.

Biological characterization of compounds from Rhinella schneideri poison that act on the complement system

Background The skin secretions of toads of the family Bufonidae contain biogenic amines, alkaloids, steroids (bufotoxins), bufodienolides (bufogenin), peptides and proteins. The poison of Rhinella schneideri, formerly classified as Bufo paracnemis, presents components that act on different biological systems, including the complement system. The aim of this study was to isolate and examine the activity ofRhinella schneideri poison (RsP) components on the complement system.Methods The components active on the complement system were purified in three chromatographic steps, using a combination of cation-exchange, anion-exchange and gel filtration chromatography. The resulting fractions were analyzed by SDS-PAGE and screened for their activity in the hemolytic assay of the classical/lectin complement pathways. Fractions active on the complement system were also assessed for their ability to generate C3 fragments evaluated by two dimensional immunoelectrophoresis assay, C3a and C5a by neutrophil chemotaxis assay and SC5b-9 complex by ELISA assay.Results The fractionation protocol was able to isolate the component S5 from theRsP, as demonstrated by SDS-PAGE and the RP-FPLC profile. S5 is a protein of about 6000 Da, while S2 presents components of higher molecular mass (40,000 to 50,000 Da). Fractions S2 and S5 attenuated the hemolytic activity of the classical/lectin pathways after preincubation with normal human serum. Both components stimulated complement-dependent neutrophil chemotaxis and the production of C3 fragments, as shown by two-dimensional immunoelectrophoresis. S2 showed a higher capacity to generate the SC5b- 9 complex than the other fractions. This action was observed after the exposure of normal human serum to the fractions.Conclusions This is the first study to examine the activity of RsP components on the complement system. Fractions S2 and S5 reduced the complement hemolytic activity, stimulated complement-dependent neutrophil chemotaxis and stimulated the production of C3 fragments, indicating that they were able to activate the complement cascade. Furthermore, fraction S2 was also able to generate the SC5b-9 complex. These components may be useful tools for studying dysfunction of the complement cascade.(AU)

Biological characterization of compounds from Rhinella schneideri poison that act on the complement system

Background The skin secretions of toads of the family Bufonidae contain biogenic amines, alkaloids, steroids (bufotoxins), bufodienolides (bufogenin), peptides and proteins. The poison of Rhinella schneideri, formerly classified as Bufo paracnemis, presents components that act on different biological systems, including the complement system. The aim of this study was to isolate and examine the activity ofRhinella schneideri poison (RsP) components on the complement system.Methods The components active on the complement system were purified in three chromatographic steps, using a combination of cation-exchange, anion-exchange and gel filtration chromatography. The resulting fractions were analyzed by SDS-PAGE and screened for their activity in the hemolytic assay of the classical/lectin complement pathways. Fractions active on the complement system were also assessed for their ability to generate C3 fragments evaluated by two dimensional immunoelectrophoresis assay, C3a and C5a by neutrophil chemotaxis assay and SC5b-9 complex by ELISA assay.Results The fractionation protocol was able to isolate the component S5 from theRsP, as demonstrated by SDS-PAGE and the RP-FPLC profile. S5 is a protein of about 6000 Da, while S2 presents components of higher molecular mass (40,000 to 50,000 Da). Fractions S2 and S5 attenuated the hemolytic activity of the classical/lectin pathways after preincubation with normal human serum. Both components stimulated complement-dependent neutrophil chemotaxis and the production of C3 fragments, as shown by two-dimensional immunoelectrophoresis. S2 showed a higher capacity to generate the SC5b- 9 complex than the other fractions. This action was observed after the exposure of normal human serum to the fractions.Conclusions This is the first study to examine the activity of RsP components on the complement system. Fractions S2 and S5 reduced the complement hemolytic activity, stimulated complement-dependent neutrophil chemotaxis and stimulated the production of C3 fragments, indicating that they were able to activate the complement cascade. Furthermore, fraction S2 was also able to generate the SC5b-9 complex. These components may be useful tools for studying dysfunction of the complement cascade.(AU)