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

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

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

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

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

Bone marrow mesenchymal stem cells stimulated by bFGF up-regulated protein expression in comparison with periodontal fibroblasts in vitro

Objective: The aim of this study was to evaluate, in vitro, the role of bFGF in the proliferation and expression of collagen type I and fibronectin of dog bone marrow mesenchymal stem cells (dBMMSCs) in comparison with the expression of the same proteins in dog periodontal fibroblasts (dPLFs).Design: dBMMSCs from the iliac crest were cultivated in Dulbecco's Modified Eagle's Medium (DMEM). Flow cytometry analysis (FCA) was used to characterize dBMMSC. Cells were stimulated with bFGF (1, 5 and 10 ng/mL) after 24 and 48 h. Real time RT-PCR was performed to verify collagen type I and fibronectin expressions. MTT assay was used to confirm cellular proliferation. Statistical analyses were performed (ANOVA and Kruskal Wallis tests; p < 0.05).Results: FCA showed 55.98% of CD34+ and 32.67% of CD90+ after bone marrow aspiration; 3.33% of CD34+ and 33.0% of CD90+ before P1. After P2, 10.54% of dBMMSCs expressed CD90, whereas after P3, this number decreased to 1.58%. dPLFs presented 4.04% of CD90+ and 1.05% of CD34+ after P3. MU evaluation showed increase in dBMSC proliferation with 5 ng/mL bFGF-stimulus after 24-h. Both collagen land fibronectin expression were very similar between the two cells groups after 24-h stimulation with 1 ng/mL bFGF concentration. Fibronectin and collagen I expressions were higher after 24-h stimulation with 5 ng/mL bFGF.Conclusion: dBMMSCs (1 ng/mL-bFGF stimulus after 24 h) are very similar to dPLFs as regards morphological and immunostaining characteristics, and collagen and/or fibronectin production. The dBMMSCs presented the highest protein expression rates with 5 ng/mL-bFGF stimulus after 24-h. (C) 2013 Elsevier Ltd. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Phenotypic and functional evaluations of peripheral blood monocytes from chronic-form paracoccidioidomycosis patients before and after treatment

Background: Paracoccidioidomycosis (PCM) is systemic mycosis caused by the thermal dimorphic fungus of genus Paracoccidioides, leading to either acute/subacute (AF) or chronic (CF) clinical forms. Numerous CF patients after treatment exhibit sequels, such as pulmonary and adrenal fibrosis. Monocytes are cells that are involved in the inflammatory response during active infection as well as in the fibrogenesis. These cells comprise a heterogeneous population with distinct phenotypic and functional activities. The scope of this study was to identify changes regarding functional and phenotypical aspects in monocytes comparing CF PCM patients on antifungal treatment versus non-treated patients (PMC-p).Methods: Twenty-three CF PCM composed of 11 non-treated patients (NTG) and 12 patients in apparent cure (ACG) were studied. Sixteen healthy individuals were used as control group (CG). Monocyte subsets were determined by immunophenotyping based on CD14 and CD16 expression. Cellular function was measured in vitro with and without stimulation with lipopolysaccharide (LPS) and P. brasiliensis exoantigen (PbAg) for 24 hours. Independent samples were compared using unpaired t tests, dependent samples were analyzed by paired t-test. Groups of more than two independent samples were analyzed using an ANOVA, with Tukey's post-test. Significance was set up at p < 0.05.Results: Our results showed high counts of peripheral blood CD14(+)CD16(+) and CD14(+)CD16(++) monocytes in untreated PCM-p accompanied by intense production of pro-inflammatory cytokines (IL-1 beta and TNF-alpha) and profibrotic growth factors (TGF-beta 1 and bFGF) by monocytes challenged with P. brasiliensis antigens. After the introduction of antifungal therapy, the counts of CD14(+)CD16(+) cells returned to baseline while CD14(+)CD16(++) counts remained high. Interestingly, counts of CD14(+)CD16(++) monocytes remained elevated even 52 +/- 7 months after successful antifungal treatment. Furthermore, the ACG-patients showed preserved pro-inflammatory activity in the presence of specific antigen stimuli and high spontaneous production of TNF-a by monocytes.Conclusions: Infection with Paracoccidioides leads to initiation of a specific proinflammatory response by monocytes of PCM-p during active disease and in the apparent cure. A profibrotic profile by monocytes was observed only at admission. Furthermore, PCM-p with apparent cure showed high spontaneous production of TNF-alpha and high counts of CD14(+)CD16(++) monocytes, probably induced by hypoxia duo to fibrotic sequelae.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP