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Braz. j. infect. dis ; 21(6): 620-626, Nov.-Dec. 2017. graf
Article in English | LILACS | ID: biblio-888922


ABSTRACT Objectives: The plague, which is an infectious disease caused by Yersinia pestis, still threatens many populations in several countries. The worldwide increase in human plague cases and the potential use of the bacteria as a biological weapon reinforce the need to study the immunity that is induced by potential vaccine candidates. To determine the immunogenicity of antigenic preparations based on the F1 protein and the total extract from Y. pestis, we assessed the role of these antigens in inducing an immune response. Methods: The immunogenicity of antigenic preparations based on the Y. pestis (YP) total extract and the Y. pestis fraction 1 capsular antigen protein (F1) was determined in Swiss-Webster mice immunized with 40 µg or 20 µg for each preparation. Immunophenotyping was performed by flow cytometry. Results: Animals immunized with the YP total extract did not elicit detectable anti-F1 antibodies (Ab) in the hemaglutination/inhibition (HA/HI) test. Animals immunized with 40 µg or 20 µg of the F1 protein produced anti-F1 Abs, with titres ranging from 1/16 to 1/8132. The average of CD3+-CD4+ and CD3+-CD8+ T cells did not differ significantly between the groups. Neither YP total extract nor F1 protein induced a significant expression of IFN-γ and IL-10 in CD4+ T lymphocytes. In addition, F1 failed to induce IFN-γ expression in CD8+ T cells, unlike the YP total extract. Conclusion: The results showed that F1 protein is not an immunogenic T cell antigen, although the YP total extract (40 µg dose) favoured CD8+ T cell-mediated cellular immunity.

Animals , Female , Rats , Spleen/immunology , Yersinia pestis/immunology , Plague Vaccine/immunology , Immunogenicity, Vaccine , Antigens, Bacterial/immunology , Plague/prevention & control , Spleen/cytology , CD4-Positive T-Lymphocytes/immunology , Immunophenotyping , Interferon-gamma/immunology , Interleukin-10/immunology , CD4-CD8 Ratio , CD8-Positive T-Lymphocytes , Flow Cytometry , Immunity, Cellular
ABCD arq. bras. cir. dig ; 30(2): 108-113, Apr.-June 2017. graf
Article in English | LILACS | ID: biblio-885701


ABSTRACT Background: Extramedullary hematopoiesis depends on complex pathophysiological mechanisms linked to hematopoietic stem cells and the proteins considered mediators of the inflammation. The identification of hematopoietic cells outside bone marrow in the adult is an occurrence that can occasionally follows the inflammatory response, was considered a secondary occurrence, but current biomolecular studies have changed that concept. Aim: Describe the presence of clusters of precursor cells of platelets (megakaryocytes), and cells of the inflammatory response in the abdominal wall and spleen of rats with experimentally induced incisional hernias and repaired with different synthetic prostheses. Methods: Twenty-five rats with incisional hernias previously performed, were divided into groups of five animals each: Group 1, repair of the hernia defect without prosthetic implant; Group 2, repair with polypropylene prosthesis; Group 3, repair using polypropylene with low weight; Group 4, the use of polypropylene and polyglecaprone prosthesis; Group 5, of polypropylene and polyglactin prosthesis. All prostheses were cut in rhombus format with area 2,625 cm². The animals were reoperated after 10 days, the abdominal walls were removed with the viscera attached to them and the material was processed for histological study. Results: Megakaryocyte niches in the abdominal wall and spleen, occasionally removed together with the adhesions produced in animals with implantation of prostheses and significant inflammatory reaction. Conclusion: The intense inflammatory reaction due to the prostheses with polypropylene in their composition was disproportionate to the expected response, indicating that further studies should be accomplished including immunophenotyping evaluation and specific panels of monoclonal antibodies to better understand the findings.

RESUMO Racional: A hematopoiese extramedular depende de mecanismos fisiopatológicos complexos, havendo relação destas células-tronco hematopoiéticas com proteínas mediadoras da inflamação. A identificação de células hematopoiéticas fora da medula óssea no adulto, situação que ocasionalmente pode acompanhar a resposta inflamatória era considerada ocorrência secundária, mas estudos biomoleculares modificaram este conceito. Objetivo: Descrever agrupamentos de células precursoras das plaquetas (megacariócitos) e células da resposta inflamatória, na parede abdominal e no baço de ratos com hérnias incisionais induzidas experimentalmente e reparadas com diferentes próteses sintéticas. Métodos: Vinte e cinco ratos com hérnias incisionais previamente realizadas foram distribuídos em grupos com cinco animais: Grupo 1, reparo do defeito herniário sem implante de prótese; Grupo 2, reparo com prótese de polipropileno; Grupo 3, reparo empregando polipropileno com baixa gramatura; Grupo 4, utilização de prótese de polipropileno e poliglecaprone; Grupo 5, prótese de polipropileno e poliglactina. Todas as próteses foram recortadas na forma de losangos com área de 2,625 cm². Os animais foram reoperados após 10 dias, as paredes abdominais foram retiradas em bloco com as vísceras a elas aderidas e o material foi processado em rotina histológica. Resultados: Foram evidenciados nichos de megacariócitos na parede abdominal e no baço coletado juntamente com as aderências em animais com implante de próteses, além de reação inflamatória significativa. Conclusão: A intensa reação inflamatória, local e sistêmica em relação às próteses com polipropileno em sua composição, foi desproporcional à resposta esperada, requerendo aprofundamento do estudo com avaliação da imunofenotipagem e painéis específicos de anticorpos monoclonais para melhor esclarecimento.

Animals , Rats , Spleen/cytology , Blood Platelets , Abdominal Wall , Incisional Hernia/surgery , Incisional Hernia/immunology , Inflammation/etiology , Polymers , Prosthesis Design , Stem Cells , Surgical Mesh/adverse effects , Rats, Wistar , Inflammation/immunology
J. appl. oral sci ; 25(1): 90-100, Jan.-Feb. 2017. tab, graf
Article in English | LILACS | ID: biblio-841165


Abstract IL-10 expressing regulatory B cells (B10) play a key role in immune system balance by limiting excessive inflammatory responses. Effects of toll-like receptor signaling and co-stimulatory molecules on B10 activity during innate and adaptive immune responses are not fully understood. Objective This study is to determine the effects of P. gingivalis LPS and CpG on B10 cell expansion and IL-10 competency in vitro. Material and Methods Spleen B cells were isolated from C57BL/6J mice with or without formalin-fixed P. gingivalis immunization. B cells were cultured for 48 hours under the following conditions: CD40L, CD40L+LPS, CD40L+CpG, and CD40L+LPS+CpG in the presence or absence of fixed P. gingivalis. Percentages of CD1dhiCD5+ B cells were measured by flow cytometry. IL-10 mRNA expression and secreted IL-10 were measured by real-time quantitative PCR and by ELISA respectively. Results P. gingivalis LPS plus CD40L significantly increased CD1dhiCD5+ B cell percentages and secreted IL-10 levels in both immunized and non-immunized mice B cells in the presence or absence of P. gingivalis, compared with control group. Secreted IL-10 levels were significantly increased in CD40L+LPS treated group compared with CD40L treatment group in the absence of P. gingivalis. CpG plus CD40L significantly decreased CD1dhiCD5+ B cell percentages, but greatly elevated secreted IL-10 levels in immunized and non-immunized mice B cells in the absence of P. gingivalis, compared with CD40L treatment group. Conclusions P. gingivalis LPS and CpG differentially enhance IL-10 secretion and expansion of mouse B10 cells during innate and adaptive immune responses.

Animals , Lipopolysaccharides/physiology , Interleukin-10/immunology , Porphyromonas gingivalis/physiology , CD40 Ligand/physiology , Toll-Like Receptor 9/agonists , Toll-Like Receptor 4/agonists , B-Lymphocytes, Regulatory/immunology , Spleen/cytology , Time Factors , RNA, Messenger/analysis , Enzyme-Linked Immunosorbent Assay , Random Allocation , Cells, Cultured , Interleukin-10/analysis , Interleukin-10/metabolism , Toll-Like Receptor 9/physiology , Toll-Like Receptor 4/physiology , Real-Time Polymerase Chain Reaction , Immunity, Innate , Mice, Inbred C57BL
Braz. j. med. biol. res ; 48(12): 1095-1100, Dec. 2015. graf
Article in English | LILACS | ID: lil-762920


In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43−) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.

Animals , Male , Mice , B-Lymphocytes/immunology , Heat-Shock Proteins/immunology , Immunomodulation/genetics , /genetics , RNA, Messenger/immunology , T-Lymphocyte Subsets/immunology , B-Lymphocytes/metabolism , Flow Cytometry , Gene Expression/genetics , Heat-Shock Proteins/therapeutic use , Immunologic Memory/physiology , Immunophenotyping/classification , Inflammation Mediators/analysis , Interferon-gamma/analysis , /immunology , /analysis , Mice, Knockout , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , RNA, Messenger/genetics , Spleen/cytology , Spleen/immunology , T-Lymphocyte Subsets/classification , Vaccines, DNA/immunology , Vaccines, DNA/therapeutic use
Indian J Exp Biol ; 2015 Mar; 53(3): 158-163
Article in English | IMSEAR | ID: sea-158406


Chyawanprash is an ayurvedic formulation used in Indian traditional medicinal system for its beneficial effect on human health. We investigated the immunostimulatory effects of Chyawanprash (CHY) using in vitro assays evaluating the secretion of cytokines such as Tumor Necrosis Factor-alpha (TNF-α), Interleukin-1beta (IL-1β) and Macrophage Inflammatory Protein-1-alpha (MIP-1-α) from murine bone marrow derived Dendritic Cells (DC) which play pivotal role in immunostimulation. The effects of CHY on phagocytosis in murine macrophages (RAW264.7) and Natural Killer (NK) cell activity were also investigated. At non-cytotoxic concentrations (20–500 µg/ml), CHY enhanced the secretion of all the three cytokines from DC. CHY also stimulated both, macrophage (RAW264.7) as well as NK cell activity, in vitro. In conclusion, the data substantiates the immunoprotective role of CHY at cellular level mediated by immunostimulation in key immune cells viz. dendritic Cells, macrophages and NK cells.

Adjuvants, Immunologic/pharmacology , Animals , Cell Line , Cytokines/analysis , Cytotoxicity, Immunologic/drug effects , Dendritic Cells/drug effects , Drug Evaluation, Preclinical , In Vitro Techniques , Killer Cells, Natural/drug effects , Macrophages/drug effects , Male , Medicine, Ayurvedic , Mice , Mice, Inbred C57BL , Phagocytosis/drug effects , Plant Preparations/pharmacology , Specific Pathogen-Free Organisms , Spleen/cytology , Zymosan
Article in English | WPRIM | ID: wpr-190706


Analysis of the T-cell receptor (TCR) repertoire of innate CD4+ T cells selected by major histocompatibility complex (MHC) class II-dependent thymocyte-thymocyte (T-T) interaction (T-T CD4+ T cells) is essential for predicting the characteristics of the antigens that bind to these T cells and for distinguishing T-T CD4+ T cells from other types of innate T cells. Using the TCRmini Tg mouse model, we show that the repertoire of TCRalpha chains in T-T CD4+ T cells was extremely diverse, in contrast to the repertoires previously described for other types of innate T cells. The TCRalpha chain sequences significantly overlapped between T-T CD4+ T cells and conventional CD4+ T cells in the thymus and spleen. However, the diversity of the TCRalpha repertoire of T-T CD4+ T cells seemed to be restricted compared with that of conventional CD4+ T cells. Interestingly, the frequency of the parental OT-II TCRalpha chains was significantly reduced in the process of T-T interaction. This diverse and shifted repertoire in T-T CD4+ T cells has biological relevance in terms of defense against diverse pathogens and a possible regulatory role during peripheral T-T interaction.

Amino Acid Sequence , Animals , Antigens, Surface/metabolism , CD4-Positive T-Lymphocytes/cytology , Cell Communication , Cell Differentiation/genetics , Clonal Evolution , Histocompatibility Antigens Class II/immunology , Immunity, Innate , Immunophenotyping , Lymphocyte Count , Mice , Mice, Knockout , Mice, Transgenic , Peptide Fragments/chemistry , Phenotype , Receptors, Antigen, T-Cell/chemistry , Receptors, Antigen, T-Cell, alpha-beta/chemistry , Spleen/cytology , Thymocytes/cytology
Salud pública Méx ; 56(5): 465-472, sep.-oct. 2014. ilus, tab
Article in Spanish | LILACS | ID: lil-733320


Objetivo. Describir la tendencia de las tasas de incidencia y mortalidad por cáncer oral (CaO) en Cali, Colombia durante el periodo 1962-2007. Material y métodos. Se obtuvieron las tasas estandarizadas por edad (población mundial) de incidencia (TIEE) y mortalidad (TMEE) por CaO con información del Registro Poblacional de Cáncer en Cali-Colombia (RPCC) y de la Secretaría de Salud Pública Municipal de Cali (SSPM), respectivamente. Se utilizó el porcentaje de cambio anual (APC) para describir la tendencia de las mismas. Resultados. Se registraron 1637 casos nuevos de CaO y la edad promedio al diagnóstico fue de 60 años. Las TIEE disminuyeron entre 1962-2007 en hombres APC= -1.3 (IC95%:-2.0; -0.6) y mujeres, APC= -1.0 (IC95%: -1.7; -0.4). Las TMEE disminuyeron entre 1984-2001 sólo en los hombres, APC= -2.8 (IC95%: -4.1; -1.5). Conclusión. La morbilidad y mortalidad por CaO ha disminuido de manera significativa en Cali, Colombia. El tipo de tumor asociado con estos cambios fue el carcinoma de células escamosas.

Objective. To describe the time trends of the incidence and mortality rates of oral cancer (OC) in Cali, Colombia between 1962-2007. Materials and methods. Age-standardized (Segi's world population) incidence (ASIR) and mortality (ASMR) rates for oral cancer were estimated using data from the Population-based Cancer Registry of Cali, Colombia and from the database of the Municipal Secretary of Public Health (MSPH) respectively. Annual percentage change (APC) was used to measure the changes in rates over time. Results. 1637 new cases of oral cancer were registered in the CPCR and the mean age upon diagnosis was 60 years. The ASIR decreased from 1962-2007 in men APC= 1.3 (IC95%:-2.0; -0.6) and women APC= -1.0 (IC95%: -1.7; -0.4).The ASMR decreased from 1984-2001 only in men, APC=2.8 (IC95%: -4.1; -1.5). Conclusions. There was a significant decrease in the incidence and mortality rates for OC in Cali, Colombia. The type of tumor associated to these changes was the squamous cell carcinoma.

Animals , Male , Mice , Rats , Chromosome Aberrations , Epoxy Compounds/toxicity , Mutagens/toxicity , Sister Chromatid Exchange , Cells, Cultured , Cell Cycle/drug effects , Species Specificity , Spleen/cytology , Spleen/drug effects
Rev. bras. ter. intensiva ; 26(3): 287-291, Jul-Sep/2014. tab, graf
Article in Portuguese | LILACS | ID: lil-723280


Objetivo: Para desenvolver modelos experimentais de transfusão de hemácias, o primeiro passo é assegurar a viabilidade dos eritrócitos transfundidos. Avaliamos a viabilidade de eritrócitos transfundidos com validação in vitro e in vivo de eritrócitos suínos homólogos armazenados por 14 dias. Métodos: Neste estudo piloto, o sangue coletado de um suíno Agroceres® foi estocado em duas unidades de hemácias. A validação in vivo foi realizada pela marcação dos eritrócitos com Na2 51CrO4 e recuperação dos eritrócitos viáveis após 24 horas da infusão em um animal autólogo e quatro homólogos. A validação in vitro foi realizada na avaliação basal e após 14 dias, pela mensuração da hemoglobina, hematócrito, índice de hemólise e hemoglobina livre em seis unidades de hemácias. Foi realizada uma esplenectomia post-mortem para avaliar o sequestro esplênico de eritrócitos, e a radioatividade das amostras de sobrenadante foi contada para avaliar a hemólise intravascular. Resultados: Após 14 dias de estocagem, as unidades de hemácias tinham volumes menores e concentração total de hemoglobina equivalente em comparação aos padrões humanos. A concentração de hemoglobina livre aumentou de 31,0±9,3 para 112,4±31,4mg/dL (p<0,001) e o índice de hemólise aumentou de 0,1±0,1 para 0,5±0,1% (p<0,001). Entretanto, esses testes se encontravam dentro da faixa aceitável para os padrões humanos. A percentagem de radioatividade nas amostras de sobrenadante foi similar na avaliação basal e após 24 horas, afastando, assim, a presença de hemólise significante. Não se encontraram evidências de sequestro esplênico de eritrócitos radioativos. ...

Objective: To develop experimental models of erythrocyte transfusion, the first step is to ensure the viability of the red blood cells transfused. In this pilot study, we assessed the viability of transfused red blood cells with validation in vitro and in vivo of homologous swine erythrocytes stored for 14 days. Methods: Blood collected from one Agroceres® swine was stored in two red blood cell units. In vivo validation was performed by labeling the red blood cells with Na2 51CrO4 and recovering the viable erythrocytes after 24 hours of infusion in one autologous and four homologous animals. In vitro validation was performed at baseline and after 14 days in sixteen red blood cell units by measuring hemoglobin, hematocrit, hemolysis index and free hemoglobin. A post-mortem splenectomy was performed to evaluate the splenic sequestration of erythrocytes, and the radioactivity of the supernatant samples was counted to evaluate intravascular hemolysis. Results: After 14 days of storage, the red blood cell units had lower volumes and equivalent total concentrations of hemoglobin and hematocrit compared to human standards. The free hemoglobin concentration increased from 31.0±9.3 to 112.4±31.4mg/dL (p<0.001), and the hemolysis index increased from 0.1±0.1 to 0.5±0.1% (p<0.001). However, these tests were within the acceptable range for human standards. The percentage of radioactivity in supernatant samples was similar at baseline and after 24 hours, thus excluding significant hemolysis. No evidence of splenic sequestration of radioactive erythrocytes was found. Conclusion: Swine red blood cells stored for 14 days are viable and can be used in experimental studies of transfusion. These validation experiments are important to aid investigators in establishing experimental models of transfusion. .

Animals , Humans , Male , Erythrocyte Transfusion/methods , Erythrocytes/cytology , Blood Preservation/methods , Cell Survival/physiology , Hematocrit , Hemoglobins/metabolism , Hemolysis/physiology , Models, Animal , Pilot Projects , Species Specificity , Swine , Spleen/cytology , Time Factors
Article in English | WPRIM | ID: wpr-194863


Kinetin (Kn) is a cytokinin growth factor that exerts several anti-aging and antioxidant effects on cells and organs. To investigate the mechanism underlying apoptotic events in aging cells induced by D-galactose (D-gal), we examined the effect of Kn delivered via nuchal subcutaneous injection on D-gal-induced aging and apoptosis in rats. Our results showed that interleukin (IL)-2 levels and mitochondrial membrane potential (DeltaPsim) were decreased by Kn in aging rats while IL-6 production and apoptosis increased. In addition, the expression of anti-apoptotic Bcl-2 was low while that of Bax was high in the aging group. After treated with Kn, compared with aging group, there showed obvious difference in Kn group with elevated IL-2, proliferation index, Bcl-2, DeltaPsim and decreased IL-6 and Bax in splenic lymphocyte. Based on these results, we concluded that Kn can effectively protect the rat spleen from aging, apoptosis, and atrophy.

Aging/drug effects , Animals , Apoptosis/drug effects , Female , Galactose/pharmacology , Interleukin-6/physiology , Interleukins/physiology , Kinetin/pharmacology , Male , Membrane Potential, Mitochondrial/drug effects , Rats , Spleen/cytology
West Indian med. j ; 62(9): 787-792, Dec. 2013. ilus, graf
Article in English | LILACS | ID: biblio-1045757


OBJECTIVE: This study aims to explore the chemokine receptor 7 (CCR7) expression of spleen dendritic cells (DCs) and their role in the changes of migration and activity of spleen DCs in multiple-organ dysfunction syndrome (MODS). METHODS: The MODS model of mice was reproduced. The mice were randomly assigned to the following groups: normal, three-hour to six-hour, 24-hour to 48-hour, and 10-day to 12-day postzymosan injection. CD11c and CD205 were analysed by immunohistochemistry; the expressions of CD86 and CCR7 of DCs were studied using flow cytometry analyses. RESULTS: In normal mice, many DCs were found at the margin between the red and white pulp. In the three-hour to six-hour and 24- to 48-hour group, DC effectively upregulated CD86 and CCR7, and they were distributed in T-cell areas. In the 10-day to 12-day group, DCs were distributed at the margin by the immature form. CONCLUSION: The CCR7 expression level of DCs had close correlations with the migration of DCs. Chemokine receptor 7 can be used to evaluate the migration and functional activity of DCs in MODS.

OBJETIVO: Este estudio persigue explorar la expresión del receptor de la quimiocina 7 (CCR7) de células dendríticas del bazo (CD), y su papel en los cambios de la migración y la actividad del las células DC del bazo en el síndrome de disfunción orgánica múltiple (SDOM). MÉTODOS: Se reprodujo el modelo SDOM de los ratones. Los ratones fueron asignados aleatoriamente a los siguientes grupos de inyección de post-zymosan: hora normal, tres a seis horas, 24 horas a 48 horas, y de 10 a 12 días. CD11c y CD205 fueron analizados mediante inmunohistoquímica. Las expresiones de CD86 y CCR7 de CD se estudiaron mediante análisis de citometría de flujo. RESULTADOS: En los ratones normales, muchas células CD fueron encontradas en el margen entre la pulpa roja y la blanca. En el grupo de tres a seis horas y el grupo de 24 a 48 horas, CD86y CCR7 fueron efectivamente sobre-regulados en CD, y distribuidos en las áreas de células T. En el grupo de 10 a 12 días, las CDs fueron distribuidas en el margen por la forma inmadura. CONCLUSIÓN: El nivel de expresión CCR7 de las CDs tuvo estrecha correlación con la migración de las CDs. El receptor de la quimiocina de tipo 7 puede utilizarse para evaluar la migración y la actividad funcional de las CDs en SDOM.

Animals , Male , Mice , Spleen/cytology , Dendritic Cells/immunology , Receptors, Chemokine/immunology , Multiple Organ Failure/pathology , Immunohistochemistry , Cell Movement , Disease Models, Animal , Mice, Inbred C57BL , Multiple Organ Failure/immunology
Braz. j. med. biol. res ; 46(5): 417-425, maio 2013. tab, graf
Article in English | LILACS | ID: lil-675669


We evaluated changes in levels by comparing serum proteins in senescence-accelerated mouse-prone 8 (SAMP8) mice at 2, 6, 12, and 15 months of age (SAMP8-2 m, -6 m, -12 m, -15 m) to age-matched SAM-resistant 1 (SAMR1) mice. Mice were sacrificed, and blood was analyzed by 2-dimensional electrophoresis combined with mass spectrometry. Five protein spots were present in all SAMP8 serum samples, but only appeared in SAMR1 samples at 15 months of age except for spot 3, which also showed a slight expression in SAMR1-12 m sera. Two proteins decreased in the sera from SAMP8-2 m, -6 m, and -12 m mice, and divided into 2 spots each in SAMP8-15 m sera. Thus, the total number of altered spots in SAMP8 sera was 7; of these, 4 were identified as Ig kappa chain V region (M-T413), chain A of an activity suppressing Fab fragment to cytochrome P450 aromatase (32C2_A), alpha-fetoprotein, and apolipoprotein A-II. M-T413 is a monoclonal CD4 antibody, which inhibits T cell proliferation. We found that M-T413 RNA level was significantly enhanced in splenocytes from SAMP8-2 m mice. This agreed with serum M-T413 protein alterations and a strikingly lower blood CD4+ T cell count in SAMP8 mice when compared to the age-matched SAMR1 mice, with the latter negatively correlating with serum M-T413 protein volume. Age-related changes in serum proteins favored an increase in autoantibodies and alpha-fetoprotein and a decrease of apolipoprotein A-II, which occurred in SAMP8 mice at 2 months of age and onwards. These proteins may serve as candidate biomarkers for early aging.

Animals , Male , Mice , Aging/blood , Apolipoprotein A-II/blood , Autoantibodies/blood , Oxidative Stress/genetics , alpha-Fetoproteins/metabolism , Aging/genetics , Apolipoprotein A-II/genetics , Autoantibodies/genetics , Biomarkers/blood , Biomarkers/metabolism , Oxidation-Reduction , Proteomics , Spleen/cytology , alpha-Fetoproteins/genetics
Bol. latinoam. Caribe plantas med. aromát ; 12(3): 313-321, mayo 2013. ilus, tab
Article in Spanish | LILACS | ID: lil-723577


Lepidium meyenii Walp, Brassicaceae (Maca) is a plant native to Peru to conferring immunostimulatory activity. The objective was to evaluate the immunomodulatory effect of the aqueous extract (EAc) of yellow ecotype on gene expression of three hematopoietic cytokines (IL-3, GM-CSF and IL-7) in splenocytes from Balb/c mice immunosuppressed with cyclophosphamide. Levels of mRNA were measured by RT-PCR. Two days after immunosuppression (IS), in splenocytes from mice treated with EAc increased expression of mRNA was demonstrated for IL-3, GM-CSF e IL-7 (p < 0.05) compared to the untreated group. Five days after IS, in mice treated with EAc found higher cell counts in bone marrow, peripheral blood and endogenous colonies formed units in spleen compared to the untreated group. It is concluded that administration of EAc in immunocompromised mice can reverse the suppressive effects of cyclophosphamide.

Lepidium meyenii Walp., Brassicaceae (Maca) es una planta oriunda del Perú a la que se atribuye actividad inmunoestimuladora. El objetivo fue evaluar el efecto inmunomodulador del extracto acuoso (EAc) del ecotipo amarillo sobre la expresión génica de tres citoquinas hematopoyéticas (IL-3, GM-CSF e IL-7) en esplenocitos de ratones Balb/c inmunosuprimidos con ciclofosfamida. Los niveles de mRNA se midieron por RT-PCR. Dos días después de la inmunosupresión (IS), en los esplenocitos de los ratones tratados con EAc se evidenció mayor expresión de mRNA para IL-3, GM-CSF e IL-7 (p<0.05) respecto al grupo no tratado. Cinco días después de la IS, en los ratones tratados con EAc se encontró mayor recuento de células en la médula ósea, sangre periférica y unidades formadoras de colonias endógenas en el bazo respecto al grupo no tratado. Se concluye que la administración de EAc a ratones inmunocomprometidos puede revertir los efectos supresores de la ciclofosfamida.

Animals , Female , Mice , Plant Extracts/pharmacology , Immunologic Factors/pharmacology , Immunocompromised Host , Lepidium/chemistry , Adjuvants, Immunologic/pharmacology , Spleen/cytology , Granulocyte-Macrophage Colony-Stimulating Factor , Mice, Inbred BALB C , MicroRNAs/analysis , Peru , Real-Time Polymerase Chain Reaction
Int. j. morphol ; 30(4): 1585-1589, dic. 2012. ilus
Article in English | LILACS | ID: lil-670183


Gumboro disease is caused by the infectious bursal disease virus (IBDV) which rapidly destroys immature B-lymphocytes of bursa of Fabricious, and causes immune suppression and high mortality in commercial broiler farms in Bangladesh. To investigate the possible effect of IBDV on lymphocytes and its distribution in the major lymphoid organs, bursa of Fabricious including spleen and thymus of naturally Gumboro-infected broilers, a research was conducted in the Department of Anatomy and Histology, collaboration with the Department of Pathology, Bangladesh Agricultural University, Bangladesh. Bursa of Fabricious, spleen and thymus of 21-days-old Gumboro-infected and non-infected broilers of same age (control) were routinely processed and stained by hematoxylin and eosin to examine the distribution of lymphocytes in the major lymphatic organs as well as quantified the number of lymphocytes under high power magnification field and compared with those of control. The number of lymphocytes in bursa of Fabricious, spleen and thymus of Gumboro-infected broilers were 27.20 ± 1.53, 66.50 ± 2.70 and 79.30 ± 3.92 whereas 121 ± 3.82, 89.90 ± 2.09 and 106.30 ± 4.07 were in non-infected control respectively. The numbers of lymphocytes were significantly (p < 0.05) lower in all lymphatic organs of Gumboro-infected broilers than those of non-infected control. The significant numbers of lymphocytes decrease in spleen and thymus suggest that IBVD not only destroy lymphocytes in bursa of Fabricious, but also in spleen and thymus and thus may severely suppress the immune response of IBVD affected broilers.

La enfermedad de Gumboro es causada por el virus de la bursitis infecciosa (VBI), que destruye rápidamente los linfocitos B inmaduros de la bolsa de Fabricio, y causa supresión inmune y la elevada mortalidad en las granjas comerciales de pollos de engorde en Bangladesh. Para investigar el posible efecto del VBI en los linfocitos y su distribución en los órganos linfoides principales, la bolsa de Fabricio, incluyendo el bazo y el timo de pollos de engorde naturalmente infectados con Gumboro, se realizó una investigación en el Departamento de Anatomía e Histología, y el Departamento de Patología, Universidad Agrícola de Bangladesh, Bangladesh. Tanto la bolsa de Fabricio, bazo y el timo de pollos de engorde con 21 días de edad infectados con Gumboro y no infectados de la misma edad (control) se procesaron de forma rutinaria y se tiñeron con H & E para examinar la distribución de los linfocitos en los órganos linfáticos principales, así cuantificar el número de linfocitos bajo campo de alta magnificación y compararlos con los de control. El número de linfocitos en la bolsa de Fabricio, bazo y timo de pollos infectados con Gumboro fue 27,20 ± 1,53, 66,50 ± 2,70 y 79,30 ± 3,92, respectivamente, mientras que en los controles no infectados fue 121 ± 3,82, 89,90 ± 2,09 y 106,30 ± 4,07 respectivamente. El número de linfocitos fue significativamente (p < 0,05) más bajo en todos los órganos linfáticos de pollos de engorde infectados con Gumboro que los no infectados. La disminuición significativa de linfocitos en el bazo y timo, sugiere que el VBI no sólo destruye linfocitos en la bolsa de Fabricio, sino también en el bazo y el timo y, por tanto, puede suprimir severamente la respuesta inmune de pollos de engorde afectados por VBI.

Animals , Poultry Diseases , Lymphocytes , Infectious bursal disease virus , Lymphoid Tissue/cytology , Poultry , Spleen/cytology , Thymus Gland/cytology , Bursa of Fabricius/cytology , Chickens , Lymphoid Tissue/immunology
Invest. clín ; 53(3): 237-249, sep. 2012. ilus, tab
Article in Spanish | LILACS | ID: lil-676475


Las células dendríticas (CDs) son esenciales en el desarrollo y regulación la respuesta inmunitaria (RI). Existen controversias en cuanto al potencial de inducción de la RI por las CDs en el período neonatal. Se ha propuesto que la RI específica de un neonato depende de la relación cuantitativa CD/linfocito T, y del momento, etapa neonatal o adulta, del encuentro con el antígeno, lo que parece influir sobre las propiedades fenotípicas y biológicas de las CDs, modificando su comportamiento. Por tal motivo, nos planteamos evaluar el efecto de un antígeno, Leishmania mexicana (L. mexicana) y de las citoquinas TNFa y RANTES sobre las características fenotípicas y propiedades migratorias, in vitro, de las CDs esplénicas provenientes de ratones BALB/c neonatos y adultos, usando citometría de flujo y la cámara de Boyden. Las CDs de ratones neonatos y adultos, en condiciones basales, expresan de manera similar, las moléculas CD40, CD86, CMHII y CD54. Este mismo fenómeno se observó al incubar dichas células con el Ag (L. mexicana) a excepción de la molécula CD40 cuya intensidad de expresión se elevó significativamente (P<0,05) en ambos grupos de estudio. El índice de migración de las CDs en presencia de medio de cultivo condicionado de L. mexicana, RANTES y TNFa fue mayor en adultos que en neonatos. Estos resultados muestran que las CDs neonatales son fenotípicamente similares a las adultas. Ante los mismos estímulos se comportan de manera diferente, sugiriendo la existencia de otros factores, que pudieran explicar la mayor susceptibilidad a infecciones en la etapa neonatal.

Dendritic cells (DCs) are essential in the development and regulation of the immune response (IR). The inherent potential of DCs to induce a specific immune response in the neonatal period is controversial. It has been suggested that the specific IR in neonates depends on the quantitative relation of DC/T lymphocytes, as well as on the neonatal or adult age at which the interaction antigen/DC/T lymphocytes occurs. This suggests that this contact has an influence on the phenotypic and/or biological properties of DCs, which modifies its behavior. Therefore, the effects of Leishmania mexicana (L. mexicana) and of TNFa and RANTES cytokines on immunophenotypical characteristics were evaluated on spleen DCs, from neonate and adult BALB/c mice, by using flow cytometry and in vitro migratory properties with a Boyden Chamber. In basal conditions, neonate and adult DCs express the same molecules (CD40, CD86, MHCII and CD54). When the DCs interact with the antigen L. mexicana, the expression of these molecules are similar in adults and in neonates, with the exception of CD40 whose intensity of expression was raised (P<0,05) in both groups. The rate of migration of the DCs in a culture medium conditioned of L. mexicana, RANTES and TNFa was higher in adults than in newborn mice. These observations suggest that neonatal and adult mice DCs have similar phenotypic characteristics. Under the effect of the same stimulus they respond differently; suggesting that other factors are involved in the higher susceptibility that newborns have to infections.

Animals , Female , Humans , Mice , Cell Movement , /physiology , Dendritic Cells/parasitology , Dendritic Cells/physiology , Leishmania mexicana/physiology , Spleen/cytology , Tumor Necrosis Factor-alpha/physiology , Age Factors , Animals, Newborn , Mice, Inbred BALB C , Phenotype
Braz. j. pharm. sci ; 47(1): 175-183, Jan.-Mar. 2011. graf, tab
Article in English | LILACS | ID: lil-586538


Cyclosporine A (CsA) is widely used as an immunosuppressant for the treatment of autoimmune diseases and immune regulation in transplant patients. Due to its wide applicability, studies of unwanted side effects of CsA are imperative. It has been found that not all patients treated with CsA display the same types/patterns of adverse effects. To ascertain the bases for these differential responses, potential differing effects of CsA on B-lymphocytes were analyzed. This entailed an assessment of changes in CsA viability and mitotic activity within splenocyte populations from BALB/c and ICR mice. These particular strains were examined because: (1) in each of them, previously have been shown that differed in the respond to biological response modifiers, such as bacterial agents, and/or immunogens; (2) our own earlier studies showing strain-associated differences in ex vivo splenocyte/lymphocyte responses to other drug; and, (3) a potential immunomodulatory effect of any agent should be studied in at least two different strains during a broad toxicological evaluation. Splenocytes from each strain were treated with 200 μg/mL CsA, and CD4+, CD8+, and CD19+ cell viabilities were monitored at various time points during the exposure period. In general, there appeared to be a trend toward greater decreases in viability among BALB/c B-lymphocytes than their ICR counterparts as incubation progressed. Differences related with T-lymphocyte sensitivity to drug associated to strains was not observed, because it was uniformly lethal throughout. With regard to mitotic activity, cells from ICR mice were more susceptible to inhibition of spontaneous cell division at low concentrations of CsA (relative to the rates of blastogenesis by BALB/c counterparts). At higher concentrations of the drug however, there were no differences in the sensitivity of each strain. This work provides new insight into the mechanism of action of CsA and illustrates the need for at least two different strains of mice/rodents for the evaluation of the overall toxicological potential of any test agent.

Ciclosporina A (CsA) é amplamente usada como imunossupressor para o tratamento de doenças autoimunes e regulação imune nos pacientes transplantados. Devido à alta aplicabilidade, são imperativos os estudos sobre seus efeitos colaterais indesejáveis. Descobriu-se que nem todos os pacientes tratados com CsA apresentam os mesmos tipos/padrões de efeitos adversos. Para averiguar as bases dessas respostas diferentes, analisaram-se efeitos potenciais diferentes da CsA nos linfócitos B. Isto envolveu a avaliação de alterações na viabilidade da CsA e da atividade mitótica dentro das populações de esplenócitos de camundongos BALB/c e ICR. Essas espécies, em particular, foram examinadas porque: (1) cada uma delas mostrou, previamente, respostas diferentes a modificadores de respostas biológicas, tais como agentes bacterianos e/ou imunogênicos; (2) nossos estudos anteriores mostraram diferenças associadas às espécies em respostas ex vivo de esplenócitos/linfócitos a outro fármaco e (3) qualquer efeito imunomodulatório potencial de um agente em teste deveria ser estudado, no mínimo, em duas espécies diferentes durante a avaliação toxicológica ampla. Esplenócitos de cada espécie foram tratados com 200 μg/mL de CSA e a viabilidade das células CD4+, CD8+ e CD19+ foi monitorada em vários tempos durante o período de exposição. No geral, parece haver uma tendência em relação a aumentos maiores na viabilidade entre os linfócitos B de BALB/c do que no de ICR, à medida que a incubação progride. Não se observou diferenças na sensibilidade do linfócito T, uma vez que o fármaco foi uniformemente letal. Com relação à atividade mitótica, as células de camundongos ICR se mostraram mais suscetíveis à inibição da divisão celular espontânea em baixas concentrações de CsA (relativamente às taxas de blastogênese de BALB/c). Em concentrações maiores do fármaco, entretanto, não houve diferenças na sensibilidade em cada uma das espécies. Este trabalho propicia nova visão do mecanismo de ação de CsA e ilustra a necessidade de, pelo menos, duas espécies diferentes de camundongos/roedores para a avaliação da toxicidade potencial de qualquer agente em teste.

Animals , Female , Adult , Mice , Spleen/cytology , Spleen , Cyclosporine/immunology , Immunologic Factors/analysis , Immunotoxins/analysis , Immunotoxins/adverse effects , Mice, Inbred BALB C , Mice, Inbred ICR , Lymphocyte Activation , B-Lymphocytes
Rev. bras. parasitol. vet ; 20(1): 71-74, jan.-mar. 2011. tab
Article in English | LILACS | ID: lil-608259


Canine ehrlichiosis is caused by the bacterium Ehrlichia canis and is characterized by a systemic febrile disease of unknown pathogenesis. This study evaluated the expression of cytokines TNF-α, IL-10, IFN-γ, in splenic cells and blood leukocytes during the acute phase of ehrlichiosis and after treatment with doxycycline hyclate in dogs experimentally infected with the E. canis Jaboticabal strain. The study results showed a significant expression of TNF-α 18 days post-inoculation, reducing by approximately 70 percent after treatment. There was a unique peak of expression of IL-10 and IFN-γ 18 and 30 days post-inoculation, respectively. This study suggests that TNF-α plays a role in the pathogenesis of the acute phase of canine ehrlichiosis and that treatment with doxycycline hyclate reduces the systemic effects of this cytokine, possibly by reducing or eliminating parasitemia.

A erliquiose canina é causada pela bactéria Ehrlichia canis, que desencadeia no hospedeiro uma doença febril e sistêmica, de patogênese pouco conhecida. O presente estudo avaliou a expressão das citocinas TNF-α, IL-10, IFN-γ, em células esplênicas e em leucócitos sanguíneos, durante a fase aguda da erliquiose e após o tratamento com hiclato de doxiciclina, em cães experimentalmente infectados com a amostra E. canis Jaboticabal. Os resultados mostraram expressão significativa de TNF-α 18 dias após a inoculação, reduzindo aproximadante 70 por cento após o tratamento. Houve um único pico de expressão de IL-10 e de IFN-γ entre 18 e 30 dias após a inoculação, respectivamente. Este estudo sugere que o TNF-α participa da patogenia da fase aguda da erliquiose canina, e que o tratamento com hiclato de doxiciclina reduz os efeitos sistêmicos dessa citocina, possivelmente por reduzir ou eliminar a parasitemia.

Animals , Dogs , Dog Diseases/immunology , Dog Diseases/microbiology , Ehrlichia canis/physiology , Ehrlichiosis/veterinary , Leukocytes/immunology , Spleen/cytology , Spleen/immunology , Tumor Necrosis Factor-alpha/biosynthesis , Ehrlichia canis/classification , Ehrlichiosis/immunology
Mem. Inst. Oswaldo Cruz ; 105(3): 263-268, May 2010. ilus, graf, tab
Article in English | LILACS | ID: lil-547296


Experimental autoimmune encephalomyelitis (EAE) is mediated by CD4+ Th1 cells that mainly secrete IFN-γ and TNF-α, important cytokines in the pathophysiology of the disease. Spontaneous remission is, in part, attributed to the down regulation of IFN-γ and TNF-α by TGF-β. In the current paper, we compared weight, histopathology and immunological parameters during the acute and recovery phases of EAE to establish the best biomarker for clinical remission. Female Lewis rats were immunised with myelin basic protein (MBP) emulsified with complete Freund's adjuvant. Animals were evaluated daily for clinical score and weight prior to euthanisation. All immunised animals developed the expected characteristics of EAE during the acute phase, including significant weight loss and high clinical scores. Disease remission was associated with a significant reduction in clinical scores, although immunised rats did not regain their initial weight values. Brain inflammatory infiltrates were higher during the acute phase. During the remission phase, anti-myelin antibody levels increased, whereas TNF-α and IFN-γ production by lymph node cells cultured with MBP or concanavalin A, respectively, decreased. The most significant difference observed between the acute and recovery phases was in the induction of TNF-α levels in MBP-stimulated cultures. Therefore, the in vitro production of this cytokine could be used as a biomarker for EAE remission.

Animals , Female , Rats , Encephalomyelitis, Autoimmune, Experimental/immunology , Interferon-gamma/biosynthesis , Lymph Nodes/metabolism , Spleen/immunology , Tumor Necrosis Factor-alpha/biosynthesis , Acute Disease , Biomarkers/analysis , Encephalomyelitis, Autoimmune, Experimental/pathology , Lymph Nodes/cytology , Lymph Nodes/immunology , Myelin Basic Protein , Rats, Inbred Lew , Spleen/cytology , Time Factors , Weight Loss
Article in English | WPRIM | ID: wpr-197698


The objective of this study was to explore the immunomodulatory effects of betulinic acid (BA) extracted from the bark of white birch on mice. Female mice were orally administered BA for 14 days in doses of 0, 0.25, 0.5, and 1 mg/kg body weight. We found that BA significantly enhanced the thymus and spleen indices, and stimulated lymphocyte proliferation induced by Concanavalin A and lipopolysaccharide as shown by MTT assay. Flow cytometry revealed that BA increased the percentage of CD4+ cells in thymus as well as the percentage of CD19+ and the ratios of CD4+/CD8+ in spleen. BA increased the number of plaque-forming cell and macrophage phagocytic activity as indicated by a neutral red dye uptake assay, and the peritoneal macrophages levels of TNF-alpha were also increased. In contrast, serum levels of IgG and IgM and serum concentrations of IL-2 and IL-6 were significantly decreased in BA-treated mice compared to the control as assayed by haemagglutination tests and ELISA, respectively. Taken together, these results suggest that BA enhances mouse cellular immunity, humoral immunity, and activity of macrophages. Thus, BA is a potential immune stimulator and may strengthen the immune response of its host.

Adaptive Immunity/drug effects , Animals , Betula/chemistry , Cell Proliferation/drug effects , Cytokines/blood , Female , Immunity, Innate/drug effects , Immunologic Factors/pharmacology , Macrophages/drug effects , Mice , Phagocytosis/drug effects , Random Allocation , Spleen/cytology , Thymus Gland/cytology , Triterpenes/pharmacology
Article in English | IMSEAR | ID: sea-22237


BACKGROUND & OBJECTIVE: The antigen H present on the surface of red cells in varying concentration, is maximum in O group red cells, but absent in Bombay phenotype individuals. This differentiation is generally detected by seed extracts of Ulex europaeus. The titre of such an extract is usually low and is subjected to batch variation. Hence, we carried out this study to raise potent murine monoclonal antibody against H antigen. METHODS: Spleen cells of female BALB/c mice immunized with O group red cells were fused in presence of polyethylene glycol (PEG) 1500 with a mouse myeloma cell line Sp2/0 Ag14 in hypoxanthine aminopterine thymidine (HAT) selective medium and incubated at 37 degrees, 5 per cent CO(2) and 95 per cent humidity for a week. RESULTS: The culture supernatants showing anti-H activity, were further subcloned and two clones 3E8A10 and 3E8A11 generated which showed a good potency, avidity and specificity. INTERPRETATION & CONCLUSION: The anti-H clones thus produced indigenously provided a potent reagent in distinguishing normal O group from Bombay phenotype individuals. The unlimited availability makes this reagent cost-effective to ensure a constant supply of hybrid clones with the similar specificities.

ABO Blood-Group System/blood , ABO Blood-Group System/immunology , Animals , Antibodies, Monoclonal/immunology , Cell Fusion , Cell Line, Tumor , Culture Media , Female , Mice , Mice, Inbred BALB C , Spleen/cytology