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1.
Braz. j. med. biol. res ; 53(7): e9207, 2020. tab, graf
Article in English | ColecionaSUS, LILACS, ColecionaSUS | ID: biblio-1132533

ABSTRACT

The objective of this study was to investigate the relationship between PI3K/mTOR/RhoA signaling regulated cytoskeletal rearrangements and phagocytic capacity of macrophages. RAW264.7 macrophages were divided into four groups; blank control, negative control, PI3K-RNAi, and mTOR-RNAi. The cytoskeletal changes in the macrophages were observed. Furthermore, the phagocytic capacity of macrophages against Escherichia coli is reported as mean fluorescence intensity (MFI) and percent phagocytosis. Transfection yielded 82.1 and 81.5% gene-silencing efficiencies against PI3K and mTOR, respectively. The PI3K-RNAi group had lower mRNA and protein expression levels of PI3K, mTOR, and RhoA than the blank and negative control groups (Р<0.01). The mTOR-RNAi group had lower mRNA and protein levels of mTOR and RhoA than the blank and the negative control groups (Р<0.01). Macrophages in the PI3K-RNAi group exhibited stiff and inflexible morphology with short, disorganized filopodia and reduced number of stress fibers. Macrophages in the mTOR-RNAi group displayed pronounced cellular deformations with long, dense filopodia and an increased number of stress fibers. The PI3K-RNAi group exhibited lower MFI and percent phagocytosis than blank and negative control groups, whereas the mTOR-RNAi group displayed higher MFI and percent phagocytosis than the blank and negative controls (Р<0.01). Before and after transfection, the mRNA and protein levels of PI3K were both positively correlated with mTOR and RhoA (Р<0.05), but the mRNA and protein levels of mTOR were negatively correlated with those of RhoA (Р<0.05). Changes in the phagocytic capacity of macrophages were associated with cytoskeletal rearrangements and were regulated by the PI3K/mTOR/RhoA signaling pathway.


Subject(s)
Humans , Animals , Rats , Phagocytosis/physiology , Cytoskeleton/metabolism , Phosphatidylinositol 3-Kinases/metabolism , rhoA GTP-Binding Protein/metabolism , TOR Serine-Threonine Kinases/metabolism , Macrophages/metabolism , Transfection , Signal Transduction , Blotting, Western , Gene Silencing , RNA Interference , Real-Time Polymerase Chain Reaction , RAW 264.7 Cells , Genetic Vectors
2.
Mem. Inst. Oswaldo Cruz ; 114: e190158, 2019. graf
Article in English | LILACS | ID: biblio-1040629

ABSTRACT

As phagocytosis is the first line of defense against malaria, we developed a phagocytosis assay with Plasmodium vivax (P. vivax) merozoites that can be applied to evaluate vaccine candidates. Briefly, after leukocyte removal with loosely packed cellulose powder in a syringe, P. vivax trophozoites matured to the merozoite-rich schizont stages in the presence of the E64 protease inhibitor. The Percoll gradient-enriched schizonts were chemically disrupted to release merozoites that were submitted to merozoite opsonin-dependent phagocytosis in two phagocytic lines with human and mouse antibodies against the N- and C-terminus of P. vivax Merozoite Surface Protein-1 (Nterm-PvMSP1 and MSP119). The resulting assay is simple and efficient for use as a routine phagocytic assay for the evaluation of merozoite stage vaccine candidates.


Subject(s)
Animals , Female , Mice , Phagocytosis/physiology , Plasmodium vivax/immunology , Antibodies, Protozoan/immunology , Protozoan Proteins/immunology , Merozoites/immunology , Plasmodium vivax/physiology , Merozoites/cytology , Flow Cytometry , Mice, Inbred BALB C
3.
Rev. Soc. Bras. Med. Trop ; 52: e20190089B, 2019. tab, graf
Article in English | LILACS | ID: biblio-1041519

ABSTRACT

Abstract INTRODUCTION The relationships between phagocytosis, and mucoid phenotype, plasmid profile and virulence, and resistance genetic characteristics of Klebsiella pneumoniae clinical isolates were evaluated. METHODS Thirty isolates were used to determine the mucoid aspect. Four were selected for analysis of phagocytosis by alveolar macrophages. RESULTS Thirty percent of the samples presented the mucoid phenotype. The phagocytosis rate ranged from 21.5% to 43.43%. Phagocytosis was not correlated with the plasmid profile, but was apparently correlated with mucoid phenotype and antibiotic susceptibility. CONCLUSIONS: Several virulence factors act in parallel in K. pneumoniae to impair host defense.


Subject(s)
Humans , Phagocytosis/genetics , Virulence/genetics , Drug Resistance, Microbial/genetics , Virulence Factors/genetics , Klebsiella pneumoniae/genetics , Anti-Bacterial Agents/pharmacology , Phagocytosis/physiology , Phenotype , Plasmids , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/pathogenicity
4.
J. appl. oral sci ; 25(3): 265-273, May-June 2017. tab, graf
Article in English | LILACS, BBO | ID: biblio-893620

ABSTRACT

Abstract Elderly denture wearers are commonly affected by Candida-associated denture stomatitis (DS), an inflammatory process of the oral mucosa strongly associated with Candida spp and other microorganisms, as well as local and systemic factors. The impaired immune response against pathogens is among the inherent host factors that have been also associated with the pathogenesis of DS. Mononuclear phagocytes respond to the pathogens through phagocytosis followed by the production of several substances inside the phagosomes, among them are the reactive nitrogen species (RNS). A failure in these mechanisms may contribute to the DS development. Objective The aim of this study was to investigate the influence of aging on the internalization and the production of nitric oxide (NO) by peritoneal adherent cells (PAC), in response to Candida albicans (C. albicans). Material and methods PAC obtained from young and aged mice were challenged with dead or viable C. albicans by using predetermined proportions (cells:yeast) for 30 and 120 minutes. Phagocytosis was analyzed by acridine orange dye, and NO production by the Griess reaction. Results C. albicans phagocytosis by PAC from aged mice was similar to that of young mice, although the cells from older mice cells present more internalized fungi compared with matched control. In addition, a tendency towards impaired NO production by peritoneal mononuclear phagocytes from aged mice was observed. Conclusions PAC from aged mice may capture and store many fungi, which in turn may mean that these cells are effectively unable to eliminate fungi, probably due to impaired NO production. Therefore, considering the important role of C. albicans overgrowth in the pathogenesis of DS and the aspects observed in this study, aging may favor the onset and severity of local candidosis such as DS and its systemic forms.


Subject(s)
Animals , Male , Phagocytosis/physiology , Stomatitis, Denture/metabolism , Stomatitis, Denture/microbiology , Candida albicans/pathogenicity , Aging/physiology , Cell Adhesion/physiology , Nitric Oxide/biosynthesis , Peritoneum/cytology , Reference Values , Time Factors , Candida albicans/isolation & purification , Candidiasis/microbiology , Age Factors , Mice, Inbred C57BL , Nitric Oxide/analysis
5.
Bauru; s.n; 2016. 82 p. tab, ilus, graf.
Thesis in English | LILACS, BBO | ID: biblio-881929

ABSTRACT

Enterococcus faecalis (E. faecalis) é um microrganismo presente em lesões endodônticas persistentes, mostrando maior resistência do que outras bactérias ao Hidróxido de Cálcio, um medicamento alcalino que consegue eliminar diversos microrganismos durante o tratamento endodôntico. Assim, os objetivos desse estudo foram: (a) avaliar a resposta de E. faecalis isolados de canal radicular, após estresse alcalino, quanto sobrevivência, crescimento, alteração do pH, resistência/susceptibilidade antimicrobiana e formação de biofilme sobre discos de dentina; (b) avaliar a capacidade fagocítica e produção de óxido nítrico (NO) por macrófagos humanos, frente a bactérias E. faecalis de canais radiculares, submetidas a estresse alcalino; (c) avaliar a expressão de TLR2 e CD14 na superfície dos macrófagos desafiados com as diferentes cepas bacterianas. As cepas utilizadas foram: ATCC4083 (CANAL 1) e uma cepa clínica, obtida por nós, a partir de uma lesão endodôntica primária (CANAL 2), ambas isoladas de canais radiculares; e ATCC29212 isolada de urina (URINA), utilizada como controle. O estresse alcalino foi obtido através da inoculação das bactérias em meio BHIalcalino por 4, 24, 48 e 72 horas. As bactérias alcalino-resistentes foram semeadas em ágar, com ou sem troca do meio, e quantificadas por CFU/mL. A susceptibilidade antimicrobiana das diferentes cepas, estressadas ou não (controle), foi determinada pelo Etest; e o biovolume do biofilme foi quantificado microscopicamente. Para avaliar a capacidade fagocítica, macrófagos obtidos a partir de monócitos do sangue periférico foram desafiados com as diferentes cepas, estressadas ou não em meio BHI-alcalino, por 30 minutos, na proporção 5:1 (bactéria/macrófago), e corados com Laranja de Acridina. Foi contado o total de macrófagos com bactérias internalizadas, considerando o número de bactérias internalizadas por célula (<5 e =5). A concentração de NO foi medida em sobrenadantes, através da reação de Griess, e a expressão de TLR2 e CD14 pelos macrófagos foi analisada por citometria de fluxo. Os resultados revelaram que Enterococcus oriundos de canal radicular foram menos resistentes ao estresse alcalino e mais susceptíveis aos antibióticos testados, do que as bactérias oriundas de urina. A falta de nutrientes foi um fator determinante para o crescimento bacteriano de todas as cepas. O biovolume dos biofilmes foi semelhante para todas as cepas estudadas, e não foi alterado após exposição ao BHI-alcalino. Na presença de bactérias submetidas ao estresse alcalino, houve um menor número de macrófagos com bactérias internalizadas, em comparação ao controle. No entanto, a produção de NO e a expressão de TLR2 e CD14 não foram alteradas. Independentemente da cepa utilizada e da presença de estresse alcalino, a maioria dos macrófagos apresentavam-se com =5 bactérias internalizadas por célula. Na ausência de estresse, as cepas de urina resultaram em maior produção de NO que aquelas oriundas do canal radicular; entretanto, a produção deste gás foi semelhante entre as cepas após estresse alcalino. A partir desses resultados, podemos concluir que bactérias E. faecalis de urina diferem daquelas oriundas do canal radicular, principalmente quanto a susceptibilidade/resistência microbiana; assim sugerimos que estudos envolvendo o campo da Endodontia devam ser realizados com cepas oriundas de canal radicular, preferencialmente que de urina. Concluiu-se ainda que um ambiente alcalino associado a falta de nutrientes pode reduzir o crescimento de E. faecalis. Adicionalmente, o estresse alcalino pode levar a alterações na estrutura da parede de E. faecalis, o que dificulta o seu reconhecimento, reduzindo sua fagocitose, mas não a sua capacidade de ativar a produção de NO, pelos macrófagos. Assim, uma medicação intracanal a base de hidróxido de cálcio associada a restaurações coronais muito bem adaptadas, para se evitar infiltração, é fundamental em tratamentos endodônticos. No entanto, os efeitos do estresse alcalino, nos Enterococcus alcalino-resistentes, podem prejudicar sua fagocitose, contribuindo para sua persistência na doença endodôntica.(AU)


Enterococcus faecalis (E. faecalis) is an microorganism present in persistent endodontic lesions, with greater resistance than other bacteria to the calcium hydroxide, an alkaline intracanal dressing which eliminate several bacterial species during endodontic treatment. The objectives of this study were: (a) to evaluate the response of E. faecalis, isolated from root canal, under alkaline-stress, starvation, antimicrobial resistance/susceptibility and biofilm formation on dentin disks; (b) to evaluate the phagocytic ability and the nitric oxide (NO) concentration of human macrophages against root canal E. faecalis isolates submitted to alkaline stress; (c) to evaluate the intensity of TLR2 and CD14 expression on the surface of macrophages challenged with the different bacterial strains. The bacterial strains used were: ATCC 4083 (CANAL 1) and a clinical strain, obtained by us, from a primary endodontic lesion (CANAL 2), both isolated from pulpless teeth; and ATCC29212, isolated from urine (URINE), was a reference for comparison. All strains were inoculated in alkaline-BHI broth for 4, 24, 48 and 72 hours. The alkalineresistant bacteria were seeded in agar and quantified by CFU/mL. Antimicrobial susceptibility of bacterial strains, stressed or not (control) was determined by the Etest and the biovolume after biofilm formation was quantified by microscopy. To evaluate the phagocytic ability, macrophages obtained by culture of peripheral blood monocyte, were challenged with bacterial strains, stressed or not in BHI-alkaline for 30 minutes at 5:1 ratio (bacteria/macrophages) and stained with Acridine Orange. The total of macrophages with internalized bacteria and also the number of internalized bacteria per cell (<5 and =5) were counted. The NO concentration in the supernatants was measured by Griess reaction and the intensity of TLR2 and CD14 expression on the surface of macrophages was also analyzed by flow cytometry. Results shows less resistance to alkaline stress in root canal strains and less resistance to tested antibiotics when compared with urine enterococci. The lack of nutrient was a determining factor for the bacterial growth in all enterococci strains. The biovolume of biofilm formed by all strains were similar, and were not altered after exposure to an alkaline-BHI. In the presence of alkaline-stressed bacteria, there was a smaller number of macrophages with internalized bacteria, when compared to the control. The NO production or the TLR2 and CD14 expression were not altered. Regardless of the strain or alkaline environment, the number of macrophages that showed =5 internalized bacteria per cell was higher. Without an alkaline-stress the NO production results higher in the urine strain, when compared with the root canal strains, however, was not modificated after the exposure of bacteria to alkalinestress. We conclude that root-canal strains have different features when compared with urine enterococci, with the main differences being evident in their resistance/susceptibility to antibiotics; thus, we suggest that researches with aims directed to interpreting responses to endodontic treatment should be conducted with strains from root-canals. Besides, an alkaline environment associated to a starvation condition can reduce bacterial growth. Additionally, alterations in the structure of bacterial cell wall after alkali-stressing possibly made their recognition difficult, reducing their ability to be phagocytized, but not their ability to activate NO production. Therefore, intracanal medication with calcium hidroxyde dressing and coronal restorations, to prevent infiltration, should be critical in treatments of endodontics infections. However, the impact of alkaline stress, in alkaline-resistant enterococci, can impair the phagocytosis, contributing to their persistence in endodontic disease.(AU)


Subject(s)
Humans , Enterococcus faecalis/drug effects , Enterococcus faecalis/physiology , Macrophages/microbiology , Macrophages/physiology , Phagocytosis/physiology , Biofilms/growth & development , Cell Survival , Cells, Cultured , Colony Count, Microbial , Dental Pulp Cavity/microbiology , Nitric Oxide/metabolism , Time Factors , Urine/microbiology
6.
Rev. bras. enferm ; 68(2): 320-324, Mar-Apr/2015. tab
Article in Portuguese | LILACS, BDENF | ID: lil-752520

ABSTRACT

RESUMO Objetivo: investigar a presença de micro-organismos nas narinas dos profissionais de enfermagem de um hospital de ensino brasileiro. Método: estudo transversal, em duas unidades de internação especializadas em HIV/aids. Foram coletadas amostras de secreção nasal de profissionais de enfermagem no período de um mês. As amostras foram processadas no laboratório de microbiologia da instituição e a análise dos dados resultantes por meio do software Statistical Package for the Social Sciences (SPSS) versão 19.0. Os aspectos éticos foram contemplados. Resultados: dos 73 profissionais de enfermagem do serviço, foram coletadas amostras de secreção nasal de 61 (80,2%). Foram isolados seis tipos de micro-organismos em 22 (41,0%) culturas positivas. Destaca-se que o Staphylococcus aureus representou 22,9%, sendo quatro resistentes à oxacilina (MRSA). Conclusão: o Staphylococcus aureus foi o micro-organismo de maior prevalência nos indivíduos deste estudo. .


RESUMEN Objetivo: investigar la presencia de microorganismos en las fosas nasales del personal de enfermería de un hospital universitario brasileño. Método: estudio transversal en dos unidades de hospitalización especializados en VIH/SIDA. Muestras de secreción nasal de enfermeras fueron recolectados durante un mes. Las muestras fueron procesadas en el laboratorio de microbiología de la institución y se analizaron con el paquete estadístico para el software de Ciencias Sociales (SPSS) versión 19.0. Los aspectos éticos fueron cubiertos. Resultados: 73 de los profesionales de enfermería, se recogieron muestras de las secreciones nasales de 61 (80,2%). Se aislaron seis tipos de microorganismos en 22 (41,0%) cultivos positivos. Es de destacar que el Staphylococcus aureus representó el 22,9%, cuatro oxacilina-resistente (MRSA). Conclusión: Staphylococcus aureus fue la prevalencia más microorganismo en los individuos de este estudio. .


ABSTRACT Objective: to investigate the presence of microorganisms in the nostrils of the nursing professionals of a Brazilian teaching hospital. Method: cross-sectional study in two inpatient units specialized in HIV/AIDS. Nasal secretion samples of nursing professionals were collected in one month. The samples were processed at the microbiology laboratory of the institution and analyzed using the Statistical Package for the Social Sciences (SPSS) software, version 19.0. Ethical aspects were abided. Results: from the 73 members of the nursing staff, samples of nasal secretions were collected from 61 (80.2%). Six types of microorganisms were isolated in 22 (41.0%) positive cultures. It is noteworthy that Staphylococcus aureus accounted for 22.9%, four of them oxacillin-resistant (MRSA). Conclusion: Staphylococcus aureus microorganism accounted for the largest prevalence in individuals of this study. .


Subject(s)
Humans , Animals , Mice , Biomarkers/metabolism , Gonorrhea/immunology , Inflammation Mediators/metabolism , Inflammation/etiology , Neisseria gonorrhoeae/pathogenicity , Neutrophils/immunology , Bacterial Adhesion , Carcinoembryonic Antigen/genetics , Carcinoembryonic Antigen/metabolism , Cytokines/metabolism , Enzyme-Linked Immunosorbent Assay , Gene Expression Profiling , Gonorrhea/metabolism , Gonorrhea/microbiology , Inflammation/metabolism , Inflammation/pathology , Intracellular Signaling Peptides and Proteins/metabolism , Mice, Transgenic , Neisseria gonorrhoeae/immunology , Neutrophils/microbiology , Oligonucleotide Array Sequence Analysis , Oxidative Stress , Phagocytosis/physiology , Protein-Tyrosine Kinases/metabolism , Signal Transduction
7.
Acta cir. bras ; 29(12): 781-786, 12/2014. tab, graf
Article in English | LILACS | ID: lil-731023

ABSTRACT

PURPOSE: To investigate whether there are differences between the phagocytic function of the remaining lower spleen pole after subtotal splenectomy and autogenous splenic implants. METHODS: Thirty-six male Wistar rats, weighting 364 ± 60g were used. They were subjected to subtotal splenectomy preserving the lower spleen pole and to autogenous splenic implant in the greater omentum. Its viability was assessed microscopically. Phagocytic function was assessed by splenic uptake of the radioisotope-labeled colloid and by macrophages counting. RESULTS: The viability of the autogenous splenic implant and of the lower spleen pole was found in 33 animals, with no difference between them. The weight of the implants was higher than the lower pole of animals from groups G1, G7, G30, G60 and G120. The implants phagocytic function by radioisotope uptake was higher than the lower pole in G7 and G120 groups and it did not differ from the other groups. The number of macrophages was higher in G1, G60, G90 and G120 and did not differ from the other groups. CONCLUSION: Until the 16th week, the phagocytic function was more pronounced in autogenous splenic implants when compared with the lower spleen pole, but it became similar thereafter. .


Subject(s)
Animals , Male , Autografts/physiology , Macrophages/physiology , Phagocytosis/physiology , Splenectomy , Spleen/physiology , Autografts/anatomy & histology , Cell Count/methods , Follow-Up Studies , Models, Animal , Omentum , Postoperative Period , Rats, Wistar , Spleen/anatomy & histology , Spleen/surgery , Spleen/transplantation
8.
Arq. bras. endocrinol. metab ; 57(8): 594-602, Nov. 2013. graf, tab
Article in English | LILACS | ID: lil-696898

ABSTRACT

OBJECTIVE: This study investigated the effect of interval training on blood biochemistry and immune parameters in type 1 diabetic rats. MATERIALS AND METHODS: Male Wistar rats were divided into four groups: sedentary (SE, n = 15), interval training (IT, n = 17), diabetic sedentary (DSE, n = 17), diabetic interval training (DIT, n = 17). Diabetes was induced by i.v. injection of streptozotocin (60 mg/kg). Swimming Interval Training consisted of 30-s exercise with 30-s rest, for 30 minutes, during 6 weeks, four times a week, with an overload of 15% of body mass. Plasma glucose, lactate, triacylglycerol and total cholesterol concentrations, phagocytic capacity, cationic vesicle content, and superoxide anion and hydrogen peroxide production by blood neutrophils and peritoneal macrophages were evaluated. Proliferation of mesenteric lymphocytes was also estimated. RESULTS: Interval training resulted in attenuation of the resting hyperglycemic state and decreased blood lipids in the DIT group. Diabetes increased the functionality of blood neutrophils and peritoneal macrophages in the DSE group. Interval training increased all functionality parameters of peritoneal macrophages in the IT group. Interval training also led to a twofold increase in the proliferation of mesenteric lymphocytes after 6 weeks of exercise in the DIT group. CONCLUSION: Low-volume high-intensity physical exercise attenuates hyperglycemia and dislipidemia induced by type 1 diabetes, and induces changes in the functionality of innate and acquired immunity.


OBJETIVO: Este estudo investigou os efeitos do treinamento intervalado sobre parâmetros bioquímicos e imunológicos em ratos diabéticos do tipo 1. MATERIAIS E MÉTODOS: Ratos Wistar machos foram divididos em quatro grupos: sedentário (SE, n = 15), treinamento intervalado (TI, n = 17), sedentário diabético (SED, n = 17) e treinamento intervalado diabético (TID, n = 17). O diabetes foi induzido por uma injeção intravenosa de estreptozotocina (60 mg/kg). O treinamento intervalado de natação consistiu de 30s de exercício com 30s de recuperação, 30 minutos, durante 6 semanas, 4 vezes por semana, com sobrecarga de 15% da massa corporal. Foram avaliados glicemia, lactato sanguíneo, concentração de triacilglicerol e colesterol total, capacidade fagocítica, conteúdo de vesículas catiô­nicas, produção de ânion superóxido e peróxido de hidrogênio por neutrófilos sanguíneos e macrófagos peritoneais. A proliferação de linfócitos mesentéricos também foi avaliada. RESULTADOS: O treinamento intervalado resultou em atenuação do estado hiperglicêmico e diminuiu os lipídeos sanguíneos no grupo TID. O diabetes aumentou a funcionalidade dos neutrófilos sanguíneos e macrófagos peritoneais do grupo SED. O treinamento intervalado aumentou todos os parâmetros funcionais dos macrófagos peritoneais do grupo TI. O treinamento intervalado também aumentou duas vezes a proliferação dos linfócitos mesentéricos após seis semanas de exercício do grupo TID. CONCLUSÃO: O treinamento intervalado atenua a hiperglicemia e a dislipidemia induzida pelo diabetes do tipo 1 e induz mudanças na funcionalidade da imunidade inata e adquirida.


Subject(s)
Animals , Male , Diabetes Mellitus, Experimental/chemically induced , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Type 1/metabolism , Dyslipidemias/etiology , Hyperglycemia/etiology , Physical Conditioning, Animal/methods , Biomarkers , Blood Glucose/metabolism , Cell Proliferation , Disease Models, Animal , Diabetes Mellitus, Type 1/complications , Hydrogen Peroxide/metabolism , Neutrophils/metabolism , Phagocytosis/physiology , Rats, Wistar , Sedentary Behavior , Streptozocin/pharmacology , Superoxides/metabolism
9.
São Paulo; s.n; s.n; set. 2013. 115 p. tab, graf, ilus.
Thesis in Portuguese | LILACS | ID: biblio-837009

ABSTRACT

Sporothrix shenckii é o agente etiológico da esporotricose, micose de carater crônico e de ampla distribuição mundial. No Brasil, vem crescendo o número de casos da doença, bem como a incidência de formas clínicas graves ou atípicas. Nosso grupo de pesquisa desenvolveu um anticorpo monoclonal contra o componente antigênico proteíco de 70 kDa, secretado pelas células leveduriformes de S. schenckii, denominado anticorpo monoclonal (AcMo) P6E7. Este AcMo apresentou atividade profilática e terapêutica na esporotricose experimental, no entanto, este anticorpo possui origem murina, o que pode gerar uma resposta imunogênica quando administrado em humanos, impossibilitando sua utilização por tempo prolongado. Visando sua possível utilização no tratamento da esporotricose humana, a nossa proposta foi a humanização do AcMo P6E7 na forma de FvFc (Fv-linker- Fc) através de engenharia genética. Inicialmente construimos duas versões uma humanizada e outra quimérica do AcMo contra a fração de 70 kDa do antigeno de S. schenckii. Os anticorpos foram expressos em vetores de expressão dicistrônicos e produzidos com eficiência e estabilidade estrutural em células de mamíferos da linhagem CHO. Posteriormente, esses anticorpos foram purificados por cromatografia de afinidade e analisados quanto a sua capacidade de ligação ao fungo e função efetora. Verificamos que os FvFcs construídos foram capazes de se ligar a porção de 70 kDa do antígeno de S. schenckii. Através de ensaios de fagocitose, constatamos que os fragmentos FvFc do P6E7 humanizado e quimérico foram capazes de opsonizar as leveduras de S. schenckii aumentando, assim, o índice fagocítico em macrófagos humanos. Esses dados em conjunto, sugerem a possível utilização do anticorpo construído no tratamento da esporotricose humana


Sporothrix shenckii is the etiological agent of sporotrichosis, a chronical fungal infection that shows a worldwide distribution. In Brazil, there is a growing number of cases of sporotrichosis, as well as the incidence of severe or atypical clinical forms. Our research group developed a monoclonal antibody (mAb) against the fungal antigenic component a protein of 70 kDa, secreted by S. schenckii yeasts called P6E7. This mAb showed prophylactic and therapeutic activity in experimental sporotrichosis, however, this antibody has murine origin, which can generate an immune response when administered to humans, precluding their use for prolonged time. For its possible use in the treatment of human sporotrichosis, our proposal is the humanization of mAb P6E7 through genetic engineering. Initially, we built two versions of the original antibody: an humanized version and a chimeric antibody both against the 70 kDa fraction from S. schenckii antigen. The antibodies were expressed in dicistronic expression vectors and were efficiently produced in mammalian cells CHO strain, showing good structural stability. Subsequently, these antibodies were purified by affinity chromatography and assayed for their binding ability to the fungus and effector function. We found that the built os FvFcs (Fv-linker- Fc) fragments were capable of binding to the 70 kDa portion of S.schenckii antigen. Through phagocytosis assays, we found that the FvFc fragments from the humanized and chimeric P6E7 were able to opsonize S. schenckii yeasts, thus increasing the phagocytic index in human macrophages. Together, These data suggest the potential use of the antibodies constructed in the treatment of human sporotrichosis


Subject(s)
Antibodies, Monoclonal, Humanized/adverse effects , Antibodies/classification , Sporotrichosis/prevention & control , ZAP-70 Protein-Tyrosine Kinase , Humanization of Assistance , Phagocytosis/physiology
10.
Egyptian Journal of Medical Human Genetics [The]. 2013; 14 (1): 95-101
in English | IMEMR | ID: emr-150727

ABSTRACT

The prevalence of obesity, insulin resistance and type 2 diabetes has steadily increased in the last decades. In addition to the genetic and environmental factors, gut microbiota may play an important role in the modulation of intermediary phenotypes leading to metabolic disease. Infection is an important cause of morbidity and mortality in diabetic patients. Chronic hyperglycemia impairs host defense mechanism such as cell mediated immunity, polymorphonuclear leukocyte function, and antibody formation. So we aimed to study the association between intestinal microflora [Lactobacilles acidophilus] count and phagocytic activity of polymorphonuclear leukocytes in humans with type 2 diabetes. The study included 20 type 2 diabetic patients with good glycemic control and 20 type 2 diabetic patients with poor glycemic control. In addition, 20 normal healthy subjects were included as normal controls. The fecal composition of L. acidophilus was detected using de Man Rogosa Sharp agar followed by further confirmation using the polymerase chain reaction technique. Phagocytic function of polymorphonuclear leukocytes was assessed using the phagocytosis index%. Fecal L. acidophilus count was significantly increased among uncontrolled diabetic patients, while the phagocytosis index% was significantly reduced among the same patients. In uncontrolled diabetics, a significant positive correlation was observed between fecal L. acidophilus count and HbA1c and a significant negative correlation between phagocytic activity and L. acidophilus count. In conclusion, type 2 diabetes is associated with compositional changes in fecal L. acidophilus especially in the uncontrolled diabetes. The levels of glucose tolerance or severity of diabetes should be considered while linking the level of intestinal microbiota with a phagocytosis index of leukocytes


Subject(s)
Humans , Male , Female , Phagocytosis/physiology , Lactobacillus acidophilus , Polymerase Chain Reaction/methods , Neutrophils/physiology
11.
J. appl. oral sci ; 20(5): 503-509, Sept.-Oct. 2012. graf, tab
Article in English | LILACS | ID: lil-654912

ABSTRACT

Phagocytosis by neutrophils and monocytes constitutes the main defense mechanism against bacterial challenges in periodontitis. Phagocytosis by neutrophils has already been evaluated, whereas phagocytic function of monocytes has hardly been addressed so far. Objectives: The aim of this study was to assess phagocytosis by neutrophils and monocytes in periodontitis. Material and Methods: The sample included 30 subjects with severe periodontitis and 27 control subjects without periodontal disease. The phagocytic index (PhI) was calculated as the mean number of adhered/ingested Saccharomyces cerevisiae per phagocytozing monocyte or neutrophil multiplied by the percentage of phagocytes involved in phagocytosis. Results: A significant reduction in phagocyte functions was observed in individuals with periodontitis. The median of PhI of neutrophils using nonsensitized S. cerevisiae was 3 for the control group, and 1.5 for the periodontitis group (p=0.01, Mann-Whitney test). The median of PhI of monocytes with non-sensitized S. cerevisiae was 26.13 for the control group, and 13.23 for the periodontitis group (p=0.03, Mann Whitney test). The median of PhI of monocytes assessed with sensitized S. cerevisiae was 97.92 for the control group and 60.1 for the periodontitis group (p=0.005, t-test). Conclusion: The data demonstrated a reduction in the function of phagocytes, suggesting a decrease in immune defenses in periodontitis.


Subject(s)
Adult , Female , Humans , Male , Middle Aged , Young Adult , Monocytes/physiology , Neutrophils/physiology , Periodontitis/immunology , Phagocytosis/physiology , Case-Control Studies , Immunity, Cellular/physiology , Periodontitis/blood , Statistics, Nonparametric , Saccharomyces cerevisiae/cytology
12.
Yonsei Medical Journal ; : 654-657, 2012.
Article in English | WPRIM | ID: wpr-22414

ABSTRACT

Phagocytosis or endocytosis by macrophages is critical to the uptake of fine particles, including nanoparticles, in order to initiate toxic effects in cells. Here, our data enhance the understanding of the process of internalization of silver nanoparticles by macrophages. When macrophages were pre-treated with inhibitors to phagocytosis, caveolin-mediated endocytosis, or clathrin-mediated endocytosis, prior to exposure to silver nanoparticles, Interleukin-8 (IL-8) production was inhibited. Although cell death was not reduced, the inflammatory response by macrophages was compromised by phagocytosis and endocytosis inhibitors.


Subject(s)
Cell Line , Cell Survival/drug effects , Endocytosis/physiology , Humans , Interleukin-8/metabolism , Macrophages/drug effects , Metal Nanoparticles/chemistry , Phagocytosis/physiology , Silver/chemistry
13.
Rev. Inst. Med. Trop. Säo Paulo ; 53(5): 247-253, Sept.-Oct. 2011. ilus, graf, tab
Article in English | LILACS | ID: lil-602359

ABSTRACT

Twelve strains of Trypanosoma cruzi isolated from wild reservoirs, triatomines, and chronic chagasic patients in the state of Paraná, southern Brazil, and classified as T. cruzi I and II, were used to test the correlation between genetic and biological diversity. The Phagocytic Index (PI) and nitric-oxide (NO) production in vitro were used as biological parameters. The PI of the T. cruzi I and II strains did not differ significantly, nor did the PI of the T. cruzi strains isolated from humans, triatomines, or wild reservoirs. There was a statistical difference in the inhibition of NO production between T. cruzi I and II and between parasites isolated from humans and the strains isolated from triatomines and wild reservoirs, but there was no correlation between genetics and biology when the strains were analyzed independently of the lineages or hosts from which the strains were isolated. There were significant correlations for Randomly Amplified Polymorphic Deoxyribonucleic acid (RAPD) and biological parameters for T. cruzi I and II, and for humans or wild reservoirs when the lineages or hosts were considered individually.


Doze cepas de Trypanosoma cruzi isoladas de reservatórios silvestres, triatomíneos e de pacientes chagásicos crônicos do Estado do Paraná, Brasil, classificadas como Tc I e II foram usadas para avaliar a correlação entre genética e diversidade biológica. Índice fagocítico (IF) e produção de óxido nítrico (ON) in vitro foram os parâmetros biológicos utilizados. O IF de cepas T. cruzi I e II não diferiram significativamente assim como o IF de cepas isoladas de humanos, triatomíneos ou de reservatórios silvestres. Há diferença estatística na inibição da produção de ON entre T. cruzi I e II e entre parasitos isolados de humanos e de cepas isoladas de triatomíneos e reservatórios silvestres, mas não foi observada correlação entre genética e biologia quando as cepas foram analisadas independentemente da linhagem ou hospedeiros das quais elas foram isoladas. Observou-se correlação significativa para amplificação aleatória do DNA polimórfico e parâmetros biológicos de Tc I ou II e para os seres humanos ou reservatório silvestre quando linhagens ou hospedeiros são consideradas separadamente.


Subject(s)
Animals , Female , Humans , Mice , Genetic Variation/genetics , Macrophages, Peritoneal/parasitology , Nitric Oxide/biosynthesis , Phagocytosis/physiology , Trypanosoma cruzi/genetics , Disease Reservoirs/parasitology , Host-Parasite Interactions , Insect Vectors/parasitology , Mice, Inbred BALB C , Macrophages, Peritoneal/cytology , Triatominae/parasitology , Trypanosoma cruzi/classification , Trypanosoma cruzi/physiology
14.
J. appl. oral sci ; 18(1): 100-104, Jan.-Feb. 2010. ilus
Article in English | LILACS | ID: lil-545033

ABSTRACT

Little attention has been paid to the toxicity of silver amalgam fillings, which have been used over the centuries in Dentistry. Amalgam particles may accidentally and/or traumatically be embedded into the submucosal tissue during placement of a restoration and perpetuate in such area. This article presents a case of amalgam tattoo and investigates whether it is related to the patient's repeated episodes of sinusitis. The patient was a 46-year-old woman with a 2 mm diameter radiopaque lesion in the right oral mucosa detected on a panoramic radiograph and presented as a black macula clinically. A complete surgical resection was carried out. The histopathological examination revealed deposits of dark-brownish pigments lining the submucosal tissue with adjacent lymphocytic inflammatory infiltrate and multinucleated giant cells phagocyting pigments. There was a negative staining for both iron and melanin. One year after lesion removal, the patient reported that the sinusitis crises had ceased after repeated episodes for years. It may be speculated that the inflammatory process related to amalgam tattoo seems to lead to a local immune response that causes sinusitis because it enhances the human leukocyte antigen DR (HLA-DR) tissue expression.


Subject(s)
Female , Humans , Middle Aged , Dental Amalgam/adverse effects , Mouth Diseases/complications , Pigmentation Disorders/complications , Sinusitis/etiology , Chronic Disease , Foreign Bodies/complications , Foreign Bodies/pathology , Giant Cells/pathology , HLA-DR Antigens/analysis , Lymphocytes/pathology , Mouth Diseases/diagnosis , Phagocytosis/physiology , Pigmentation Disorders/diagnosis
15.
Clinics ; 65(11): 1167-1173, 2010. graf
Article in English | LILACS | ID: lil-571441

ABSTRACT

OBJECTIVE: To analyze the effects of exhausting long-duration physical exercise (swimming) sessions of different durations and intensities on the number and phagocytic capacity of macrophages and neutrophils in sedentary rats. INTRODUCTION: Exercise intensity, duration and frequency are important factors in determining immune response to physical effort. Thus, the effects of exhausting long-duration exercise are unclear. METHODS: Wistar rats were divided into two groups: an untreated group (macrophage study) and oyster glycogen-treated rats (neutrophil study). In each group, the animals were subdivided into five groups (10 rats per group): unexercised controls, an unadapted low-intensity exercise group, an unadapted moderate-intensity exercise group, a preadapted low-intensity exercise group and a preadapted moderate-intensity exercise group. All exercises were performed to exhaustion, and preadaptation consisted of 5, 15, 30 and 45 min sessions. RESULTS: Macrophage study: the number of peritoneal macrophages significantly decreased (9.22 ± 1.78 x 10(6)) after unadapted exercise but increased (21.50 ± 0.63 x 10(6)) after preadapted low-intensity exercise, with no changes in the moderate-intensity exercise group. Phagocytic capacity, however, increased by more than 80 percent in all exercise groups (low/moderate, unadapted/preadapted). Neutrophil study: the number of peritoneal neutrophils significantly decreased after unadapted (29.20 ± 3.34 x 10(6)) and preadapted (50.00 ± 3.53 x 10(6)) low-intensity exercise but increased after unadapted (127.60 ± 5.14 x 10(6)) and preadapted (221.80 ± 14.85 x 10(6)) moderate exercise. Neutrophil phagocytic capacity decreased by 63 percent after unadapted moderate exercise but increased by 90 percent after corresponding preadapted sessions, with no changes in the low-intensity exercise groups. CONCLUSION: Neutrophils and macrophages of sedentary rats respond differently to exercise-induced stress. Adaptation sessions reduce exercise-induced stress on the immune system.


Subject(s)
Animals , Male , Rats , Macrophages, Peritoneal/physiology , Neutrophils/physiology , Phagocytosis/physiology , Physical Conditioning, Animal/physiology , Swimming/physiology , Glycogen/pharmacology , Models, Animal , Macrophages, Peritoneal/drug effects , Neutrophils/drug effects , Physical Exertion/physiology , Rats, Wistar , Time Factors
16.
Braz. j. biol ; 69(3): 899-905, Aug. 2009. ilus, graf, tab
Article in English | LILACS | ID: lil-527160

ABSTRACT

Chelonia mydas is a sea turtle that feeds and nests on the Brazilian coast and a disease called fibropapillomatosis is a threat to this species. Because of this, it is extremely necessary to determine a methodology that would enable the analysis of blood leukocyte function in these sea turtles. In order to achieve this aim, blood samples were collected from C. mydas with or without fibropapillomas captured on the São Paulo north coast. Blood samples were placed in tubes containing sodium heparin and were transported under refrigeration to the laboratory in sterile RPMI 1640 cell culture medium. Leukocytes were separated by density gradient using Ficoll-PaqueTM Plus, Amershan Biociences®. The following stimuli were applied in the assessment of leukocyte function: Phorbol Miristate-Acetate (PMA) for oxidative burst activity evaluation and Zymosan A (Saccharomyces cerevisiae) Bio Particles®, Alexa Fluor® 594 conjugate for phagocytosis evaluation. Three cell populations were identified: heterophils, monocytes and lymphocytes. Monocytes were the cells responsible for phagocytosis and oxidative burst.


Chelonia mydas é uma tartaruga marinha que freqüenta o litoral brasileiro para alimentação e nidificação e uma doença denominada fibropapilomatose é uma das mais importantes ameaças à sobrevivência dessa espécie. Desta forma, a definição de uma metodologia que permita analisar a função dos leucócitos sangüíneos torna-se extremamente necessária. Foram utilizadas amostras sangüíneas de C. mydas com e sem fibropapilomas capturadas no litoral norte do estado de São Paulo. As amostras sangüíneas foram colocadas em tubos contendo heparina sódica e transportadas em meio de cultura celular RPMI 1640 estéril e sob refrigeração. Os leucócitos foram obtidos por gradiente de densidade usando Ficoll-PaqueTM Plus, Amershan Biociences®. Os estímulos aplicados foram Miristato Acetato de Phorbol (PMA) para avaliação de burst oxidativo e Zymosan A (Saccharomyces cerevisiae) Bio Particles®, Alexa Fluor® 594 conjugate para avaliação de fagocitose. Foram identificadas três populações celulares: heterófilos, monócitos e linfócitos. Os monócitos foram as células responsáveis pela fagocitose e pelo burst oxidativo.


Subject(s)
Animals , Flow Cytometry/veterinary , Leukocytes/physiology , Papilloma/veterinary , Phagocytosis/physiology , Respiratory Burst/physiology , Turtles/blood , Flow Cytometry/methods , Papilloma/blood , Papilloma/physiopathology
17.
Braz. j. med. biol. res ; 42(7): 599-605, July 2009. graf
Article in English | LILACS | ID: lil-517795

ABSTRACT

To determine the effects of saturated and unsaturated fatty acids in phosphatidylcholine (PC) on macrophage activity, peritoneal lavage cells were cultured in the presence of phosphatidylcholine rich in saturated or unsaturated fatty acids (sat PC and unsatPC, respectively), both used at concentrations of 32 and 64 ìM. The treatment of peritoneal macrophages with 64 ìM unsat PC increased the production of hydrogen peroxide by 48.3% compared to control (148.3 ± 16.3 vs 100.0 ± 1.8%, N = 15), and both doses of unsat PC increased adhesion capacity by nearly 50%. Moreover, 64 ìM unsat PC decreased neutral red uptake by lysosomes by 32.5% compared to the untreated group (67.5 ± 6.8 vs 100.0 ± 5.5%, N = 15), while both 32 and 64 ìM unsat PC decreased the production of lipopolysaccharide-elicited nitric oxide by 30.4% (13.5 ± 2.6 vs 19.4 ± 2.5 ìM) and 46.4% (10.4 ± 3.1 vs 19.4 ± 2.5 ìM), respectively. Unsat PC did not affect anion production in non-stimulated cells or phagocytosis of unopsonized zymosan particles. A different result pattern was obtained for macrophages treated with sat PC. Phorbol 12-miristate 13-acetate-elicited superoxide production and neutral red uptake were decreased by nearly 25% by 32 and 64 ìM sat PC, respectively. Sat PC did not affect nitric oxide or hydrogen peroxide production, adhesion capacity or zymosan phagocytosis. Thus, PC modifies macrophage activity, but this effect depends on cell activation state, fatty acid saturation and esterification to PC molecule and PC concentration. Taken together, these results indicate that the fatty acid moiety of PC modulates macrophage activity and, consequently, is likely to affect immune system regulation in vivo.


Subject(s)
Animals , Male , Rats , Linoleic Acids/pharmacology , Macrophages, Peritoneal/drug effects , Phagocytosis/drug effects , Phosphatidylcholines/pharmacology , Cell Adhesion/drug effects , Cell Adhesion/physiology , Hydrogen Peroxide/metabolism , Macrophages, Peritoneal/physiology , Nitric Oxide Synthase/metabolism , Nitric Oxide/metabolism , Phagocytosis/physiology , Rats, Wistar , Reactive Oxygen Species/metabolism
18.
Rev. Soc. Bras. Med. Trop ; 42(2): 103-106, Mar.-Apr. 2009. ilus
Article in English | LILACS | ID: lil-512909

ABSTRACT

Monocytes/macrophages play a critical role in the defense mechanisms against malaria parasites, and are the main cells responsible for the elimination of malaria parasites from the blood circulation. We carried out a microscope-aided evaluation of the stages of in vitro phagocytosis of Plasmodium falciparum-infected erythrocytes, by human monocytes. These cells were obtained from healthy adult individuals by means of centrifugation through a cushion of Percoll density medium and were incubated with erythrocytes infected with Plasmodium falciparum that had previously been incubated with a pool of anti-plasmodial immune serum. We described the stages of phagocytosis, starting from adherence of infected erythrocytes to the phagocyte membrane and ending with their destruction within the phagolisosomes of the monocytes. We observed that the different erythrocytic forms of the parasite were ingested by monocytes, and that the process of phagocytosis may be completed in around 30 minutes. Furthermore, we showed that phagocytosis may occur continuously, such that different phases of the process were observed in the same phagocyte.


Monócitos/macrófagos desempenham uma função crítica nos mecanismos de defesa antiplasmódio e constituem as principais células responsáveis pela eliminação das formas eritrocitárias do plasmódio da circulação sangüínea. Realizamos uma avaliação microscópica dos estágios da fagocitose in vitro de eritrócitos infectados por Plasmodium falciparum por monócitos humanos. Essas células foram obtidas de indivíduos adultos sadios por centrifugação em Percoll e incubadas com eritrócitos infectados por Plasmodium falciparum previamente incubados com um pool de soro imune contra plasmódio. Descrevemos os estágios da fagocitose, desde a aderência dos eritrócitos infectados até sua destruição nos fagolisossomas dos monócitos. Observou-se que eritrócitos infectados por todos os diferentes estágios assexuados do parasito foram ingeridos pelos monócitos e que o processo de fagocitose pode se completar em cerca de 30 minutos. Além disso, mostramos que a fagocitose pode ocorrer de uma forma contínua, de tal maneira que diferentes fases do processo foram observadas no mesmo fagócito.


Subject(s)
Adult , Animals , Humans , Erythrocytes/parasitology , Monocytes/physiology , Plasmodium falciparum , Phagocytosis/physiology , Cells, Cultured
19.
Int. j. morphol ; 26(3): 557-562, Sept. 2008. ilus
Article in English | LILACS | ID: lil-556713

ABSTRACT

Whether bovine haemal nodes are involved in turnover of red blood cells has been a subject of some controversy In this study, fluorescent and conventional optical microscopy of conventionally or immunohistochemically stained node sections, together with transmission electron microscopy, showed the presence of erythrocyte precursors and megakaryocytes, and evidence of active involvement in the destruction and replacement of old or degenerate red cells and the platelets.


Si nodos linfáticos hemales bovinos están involucrados en la cantidad de volumen de glóbulos rojos ha sido objeto de cierta controversia. En este estudio, secciones de nodos linfáticos teñidas convencionalmente o inmunohistoquimicamente fueron analizadas con microscopía óptica fluorescente y convencional, junto con microscopio electrónico de transmisión, los que revelaron la presencia de precursores eritrocíticos y megacariocitos, y la evidencia de participación activa en la destrucción y sustitución de glóbulos rojos viejos o degenerados y plaquetas.


Subject(s)
Male , Adult , Cattle , Animals , Child , Cattle/anatomy & histology , Cattle/blood , Erythropoiesis/physiology , Erythropoiesis/genetics , Phagocytosis/physiology , Lymph Nodes/anatomy & histology , Lymph Nodes/growth & development , Lymph Nodes/ultrastructure , Microscopy, Electron, Transmission/veterinary , Microscopy, Fluorescence/veterinary
20.
Article in English | IMSEAR | ID: sea-51615

ABSTRACT

Papillon- Lefèvre Syndrome (PLS) is a rare autosomal recessive trait, which is transmitted with an estimated frequency of one to four per million individuals. It is characterized by palmar plantar keratosis and severe early-onset periodontitis affecting both deciduous and permanent dentition. In this report, we present clinical, microbiological and leukocyte function test findings of a thirty-five year-old patient with symptoms typical of Papillon-Lefèvre Syndrome except for premature loss of deciduous and permanent dentition. The patient exhibited palmar plantar keratosis and an isolated, moderately deep periodontal pocket in the third quadrant. No anaerobic bacteria were isolated from the plaque culture. The neutrophil function test revealed defective chemotaxis and phagocytosis while intracellular killing and respiratory burst were normal.


Subject(s)
Adult , Chemotaxis, Leukocyte/physiology , Humans , Keratoderma, Palmoplantar/pathology , Male , Neutrophils/physiology , Papillon-Lefevre Disease/diagnosis , Penetrance , Periodontal Pocket/pathology , Periodontitis/pathology , Phagocytosis/physiology , Tooth Exfoliation/pathology , Tooth, Deciduous/pathology
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