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 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 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 Ehrlichia sp. (secondary IMHA), 21.4% of dogs with IMHA had azotemia, and 51.8% had increased urine protein creatinine ratio.Discussion: The FC was confirmed as a more sensitive technique for the diagnosis of IMHA compared to auto-agglutination and Coomb’s tests. The auto-agglutination test was more specific than the Coomb’s test, however the last one was more sensitive. The similar prevalence of IgG and IgM in IMHA did not indicate which class of immunoglobulin would be a better choice for diagnosis by the FC technique. Antibodies are produced against normal red cells (primary or idiopathic IMHA) or to red cells that are antigenical changed by the action of drugs, neoplasia or infectious diseases (leptospirosis, babesiosis, canine ehrlichiosis), known as secondary IMHA. This study alerts for the high prevalence of IMHA in dogs, in most cases characterized by a regenerative anemia associated with intense thrombocytopenia and secondary to Ehrlichia sp. in areas endemic to this infectious disease. In Brazil, the presence of endemic areas for various infectious diseases may contribute to the high prevalence of secondary IMHA. Kidney damage may occurs because tissue hypoxia increases the risk of progressive injury, due to acute hematocrit decrease (below 22%), leading to renal tubular necrosis, whereas the deposition of immune complexes, mainly in the renal parenchyma, may aggravate the renal injury, further complicating the clinical state of the animal. Direct renal injury caused by crystallization of free hemoglobin in the renal tubules may result in renal azotemia. Hypoxia and nephrotoxicity caused by hemoglobinemia reflects increased liver enzymes and azotemia, respectively. Significant proteinuria and the increase in urine protein creatinine ratio revealed evidence of renal injury in dogs with IMHA

Cinnamic Acid Is Partially Involved in Propolis Immunomodulatory Action on Human Monocytes

Propolis is a beehive product used in traditional medicine due to its biological properties. It shows a complex chemical composition including phenolics, such as cinnamic acid (Ci). The mechanisms of action of propolis have been the subject of research recently; however, the involvement of Ci on propolis activity was not investigated on immune cells. Ci effects were evaluated on human monocytes, assessing the expression of Toll-like receptors (TLRs), HLA-DR, and CD80. Cytokine production (TNF-α and IL-10) and the fungicidal activity of monocytes were evaluated as well. Data showed that Ci downregulated TLR-2, HLA-DR, and CD80 and upregulated TLR-4 expression by human monocytes. High concentrations of Ci inhibited both TNF-α and IL-10 production, whereas the same concentrations induced a higher fungicidal activity against Candida albicans. TNF-α and IL-10 production was decreased by blocking TLR-4, while the fungicidal activity of monocytes was not affected by blocking TLRs. These results suggest that Ci modulated antigen receptors, cytokine production, and the fungicidal activity of human monocytes depending on concentration, and TLR-4 may be involved in its mechanism of action. Ci seemed to be partially involved in propolis activities

Use of In Vivo and In Vitro Systems to Select Leishmania amazonensis Expressing Green Fluorescent Protein

Various Leishmania species were engineered with green fluorescent protein (GFP) using episomal vectors that encoded an antibiotic resistance gene, such as aminoglycoside geneticin sulphate (G418). Most reports of GFP-Leishmania have used the flagellated extracellular promastigote, the stage of parasite detected in the midgut of the sandfly vector; fewer studies have been performed with amastigotes, the stage of parasite detected in mammals. In this study, comparisons were made regarding the efficiency for in vitro G418 selection of GFP-Leishmania amazonensis promastigotes and amastigotes and the use of in vivo G418 selection. The GFP-promastigotes retained episomal plasmid for a prolonged period and G418 treatment was necessary and efficient for in vitro selection. In contrast, GFP-amastigotes showed low retention of the episomal plasmid in the absence of G418 selection and low sensitivity to antibiotics in vitro. The use of protocols for G418 selection using infected BALB/c mice also indicated low sensitivity to antibiotics against amastigotes in cutaneous lesions

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 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 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 Ehrlichia sp. (secondary IMHA), 21.4% of dogs with IMHA had azotemia, and 51.8% had increased urine protein creatinine ratio.Discussion: The FC was confirmed as a more sensitive technique for the diagnosis of IMHA compared to auto-agglutination and Coomb’s tests. The auto-agglutination test was more specific than the Coomb’s test, however the last one was more sensitive. The similar prevalence of IgG and IgM in IMHA did not indicate which class of immunoglobulin would be a better choice for diagnosis by the FC technique. Antibodies are produced against normal red cells (primary or idiopathic IMHA) or to red cells that are antigenical changed by the action of drugs, neoplasia or infectious diseases (leptospirosis, babesiosis, canine ehrlichiosis), known as secondary IMHA. This study alerts for the high prevalence of IMHA in dogs, in most cases characterized by a regenerative anemia associated with intense thrombocytopenia and secondary to Ehrlichia sp. in areas endemic to this infectious disease. In Brazil, the presence of endemic areas for various infectious diseases may contribute to the high prevalence of secondary IMHA. Kidney damage may occurs because tissue hypoxia increases the risk of progressive injury, due to acute hematocrit decrease (below 22%), leading to renal tubular necrosis, whereas the deposition of immune complexes, mainly in the renal parenchyma, may aggravate the renal injury, further complicating the clinical state of the animal. Direct renal injury caused by crystallization of free hemoglobin in the renal tubules may result in renal azotemia. Hypoxia and nephrotoxicity caused by hemoglobinemia reflects increased liver enzymes and azotemia, respectively. Significant proteinuria and the increase in urine protein creatinine ratio revealed evidence of renal injury in dogs with IMHA

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 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 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 Ehrlichia sp. (secondary IMHA), 21.4% of dogs with IMHA had azotemia, and 51.8% had increased urine protein creatinine ratio.Discussion: The FC was confirmed as a more sensitive technique for the diagnosis of IMHA compared to auto-agglutination and Coomb’s tests. The auto-agglutination test was more specific than the Coomb’s test, however the last one was more sensitive. The similar prevalence of IgG and IgM in IMHA did not indicate which class of immunoglobulin would be a better choice for diagnosis by the FC technique. Antibodies are produced against normal red cells (primary or idiopathic IMHA) or to red cells that are antigenical changed by the action of drugs, neoplasia or infectious diseases (leptospirosis, babesiosis, canine ehrlichiosis), known as secondary IMHA. This study alerts for the high prevalence of IMHA in dogs, in most cases characterized by a regenerative anemia associated with intense thrombocytopenia and secondary to Ehrlichia sp. in areas endemic to this infectious disease. In Brazil, the presence of endemic areas for various infectious diseases may contribute to the high prevalence of secondary IMHA. Kidney damage may occurs because tissue hypoxia increases the risk of progressive injury, due to acute hematocrit decrease (below 22%), leading to renal tubular necrosis, whereas the deposition of immune complexes, mainly in the renal parenchyma, may aggravate the renal injury, further complicating the clinical state of the animal. Direct renal injury caused by crystallization of free hemoglobin in the renal tubules may result in renal azotemia. Hypoxia and nephrotoxicity caused by hemoglobinemia reflects increased liver enzymes and azotemia, respectively. Significant proteinuria and the increase in urine protein creatinine ratio revealed evidence of renal injury in dogs with IMHA

TRANSLOCAÇÃO MICROBIANA EM MULHERES EM IDADE REPRODUTIVA: O PAPEL DA INFECÇÃO POR HIV E O DA GESTAÇÃO NESTE PROCESSO

Introdução/Objetivo: A translocação microbiana e ativação imune podem ocorrer de forma mais exacerbada em gestantes que vivem com HIV, entretanto, ainda não está claro qual seria o principal fator responsável pela intensificação destes processos - infecção viral ou gestação. Assim, objetivamos avaliar a presença proteína de ligação de ácidos graxos intestinais (iFABP), lipopolissacarídeos (LPS), cluster de diferenciação14 (sCD14) e interleucina 6 (IL-6) nos diferentes períodos gestacionais em mulheres com diferentes condições clínicas (infectadas ou não pelo HIV). Métodos: Foram incluídas 39 mulheres, de 2016 a 2019, frequentadoras do Hospital das Clínicas de Botucatu. Grávidas foram analisadas nos momentos M0 (1° semestre), M1 (pré parto) e M2 (pós parto). Elas compunham o G1 (HIV+, n = 13) e o G2 (HIV-, n = 10). Já as não grávidas representaram o G3 (HIV-, n = 10) e G4 (HIV+, n = 4). Além de dados de prontuários eletrônicos, ensaios imunoenzimáticos e citometria de fluxo foram as técnicas laboratoriais utilizadas. Para as análises longitudinais e transversais foram utilizados Teste de Anova seguido de Tukey e Gamma seguido de Wald. Resultado: Os grupos eram homogêneos quanto à terapia antirretroviral (TARV) utilizada e contagem de linfócitos T CD4+. Como algumas mulheres foram diagnosticadas com HIV no pré-natal, esse grupo apresentou maior frequência de carga viral detectável (p = 0,05) e menor tempo de infecção (p = 0,01) e de TARV (p = 0,01) em relação às não grávidas HIV+. G1 mostrou maiores níveis de iFABP em todos os momentos em relação a G2 (p <,001). O LPS esteve elevado apenas no M2 do G2 (p <,001), mas, entre as não grávidas, este valor era maior no G4 (p = 0,004). Este último grupo também apresentou maior sCD14 (p <,0001), mas na condição de gestação, estes níveis diminuíram ao longo do tempo em G1 (p = 0,04), juntamente com o decréscimo nos níveis de IL-6 (p = 0,003). Conclusão: A infecção pelo HIV foi um fator que demonstrou relação com o intenso dano epitelial intestinal e maior ativação imune, em gestantes ou não. Entretanto, a gestação parece ser uma condição que tenta “controlar” este desequilíbrio, pois alguns destes marcadores foram diminuindo ao longo do período gestacional, e foram de modo geral, menores nas gestantes que não gestantes, especialmente nas HIV+. Por fim, no período pós-parto, o aumento de LPS presente em grávidas HIV- não esteve associado à translocação microbiana, indicando que outros componentes podem estar envolvidos neste dinamismo

The immunomodulatory effect of propolis on receptors expression, cytokine production and fungicidal activity of human monocytes

ObjectivesPropolis is a beehive product and its immunomodulatory action has been documented; however, little is known concerning its mechanisms of action on human cells. Propolis influence on the initial events of the immune response was assessed, evaluating cell markers, cytokine production and the fungicidal activity of human monocytes.MethodsToll-like receptor (TLR)-2, TLR-4, human leukocyte antigen-DR and cluster of differentiation (CD)80 expression by human monocytes was assessed using a FACSCalibur flow cytometer, cytokine production (tumour necrosis factor (TNF)- and interleukin (IL)-10) was determined by ELISA and the candidacidal activity was investigated after monocytes incubation with propolis and challenged with Candida albicans. The role of TLR-2 and TLR-4 on propolis action was assessed as well.Key findingsPropolis upregulated TLR-4 and CD80 expression and affected TNF- and IL-10 production, depending on concentration. Propolis also increased the fungicidal activity of monocytes. Cytokine production was decreased by blocking TLR-4, whereas the fungicidal activity was affected by blocking TLR-2.ConclusionsPropolis exerted an immunomodulatory action on cell receptors, cytokine production and fungicidal activity of human monocytes without affecting cell viability and depending on concentration. TLR-2 and TLR-4 may be involved in its mechanism of action.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Isolation and immunophenotypic characterization of mesenchymal stem cells derived from equine species adipose tissue

The purpose of this work was to isolate and cultivate mesenchymal stem cells (MSC) derived from equine adipose tissue and conduct cellular characterization with the following markers: CD90, CD44 and CD13. Adipose tissue collection was performed at the base of the horses' tails, followed by immediate isolation and cultivation of the MSC and posterior characterization by flow cytometry for the interspecies reaction test using mouse anti-rat CD90 monoclonal antibody (mAb), fluorescein isothiocyanate (FITC), and tests with specific mAb mouse anti-horse CD13 and mouse anti-horse CD44. The technique used for isolation and cell cultivation proved to be safe and viable. The CD90 mAb expressed cross-reaction with MSC derived from equine adipose tissue and CD44 showed greater expression in cells as the number of culture passages increased. Although marker CD13 expresses reaction in other studies involving MSC in different species, it presented no expression in the experiment realized. The results obtained revealed the immunophenotypic characterization of the surface of isolated and cultivated MSC, classifying these cells as a promising type of progenitor cells that can be applied in equine cellular therapy. (C) 2009 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP