RESUMO

Amphotericin B is the "gold standard" agent in the management of serious systemic fungal infections. However, this drug can cause nephrotoxicity, which contributes up to 25% of all acute kidney injuries in critically ill patients. Cyclic adenosine monophosphate can protect kidney cells from death due to injury or drug exposure in some cases. Hence, the objective of this work was to evaluate if cAMP could prevent cell death that occurs in renal cell lines subjected to AmB treatment and, if so, to assess the involvement of PKA in the transduction of this signal. Two different renal cell lines (LLC-PK1 and MDCK) were used in this study. MTT and flow cytometry assays showed increased cell survival when cells were exposed to cAMP in a PKA-independent manner, which was confirmed by western blot. This finding suggests that cAMP (db-cAMP) may prevent cell death caused by exposure to AmB. This is the first time this effect has been identified when renal cells are exposed to AmB's nephrotoxic potential.

Assuntos

Anfotericina B/toxicidade , Antifúngicos/toxicidade , AMP Cíclico/administração & dosagem , Rim/efeitos dos fármacos , Animais , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Cães , Citometria de Fluxo , Rim/patologia , Células LLC-PK1 , Células Madin Darby de Rim Canino , Transdução de Sinais/efeitos dos fármacos , Suínos

RESUMO

Cyclosporine is an important immunosuppressive agent; however, nephrotoxicity is one of the main adverse effects. The purpose of this study was to evaluate the effect of inhibiting the protein kinase A (PKA) signaling pathway in nephrotoxicity caused by cyclosporine from the assessment of cell viability, pro-inflammatory cytokines, and nitric oxide (NO) production in LLC-PK1 and MDCK cell lines. Cyclosporine proved to be cytotoxic for both cell lines, as assessed by the mitochondrial enzyme activity assay (MTT), caused DNA fragmentation, determined by flow cytometry using the propidium iodide dye, and activated the PKA pathway (western blot assay). In MDCK cells, the inhibition of the PKA signaling pathway (H89 inhibitor) caused a significant reduction in DNA fragmentation. In both cell lines, the production of IL-6 proved to be a dependent PKA pathway, while TNF-α was not influenced by the inhibition of the PKA pathway. The NO production was increased when cells were pre-incubated with H89 followed by cyclosporine, and this production was dependent on the PKA pathway in LLC-PK1 and MDCK cells lines. Therefore, considering the present study's results, it can be concluded that the inhibition of PKA signaling pathway can aid in reducing the degree of nephrotoxicity caused by cyclosporine.

Assuntos

Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ciclosporina/toxicidade , Rim/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Western Blotting , Linhagem Celular , Sobrevivência Celular , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/genética , Fragmentação do DNA , Cães , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Imunossupressores/toxicidade , Isoquinolinas/farmacologia , Rim/citologia , Óxido Nítrico/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Sulfonamidas/farmacologia , Suínos

RESUMO

Amphotericin B is one of the most effective antifungal agents; however, its use is often limited owing to adverse effects, especially nephrotoxicity. The purpose of this study was to evaluate the effect of inhibiting the PKA signaling pathway in nephrotoxicity using Amphotericin B from the assessment of cell viability, pro-inflammatory cytokines and nitric oxide (NO) production in LLC-PK1 and MDCK cell lines. Amphotericin B proved to be cytotoxic for both cell lines, as assessed by the mitochondrial enzyme activity (MTT) assay; caused DNA fragmentation, determined by flow cytometry using the propidium iodide (PI) dye; and activated the PKA pathway (western blot assay). In MDCK cells, the inhibition of the PKA signaling pathway (using the H89 inhibitor) caused a significant reduction in DNA fragmentation. In both cells lines the production of interleukin-6 (IL)-6 proved to be a dependent PKA pathway, whereas tumor necrosis factor-alpha (TNF-α) was not influenced by the inhibition of the PKA pathway. The NO production was increased when cells were pre-incubated with H89 followed by Amphotericin B, and this production produced a dependent PKA pathway in LLC-PK1 and MDCK cells lines. Therefore, considering the present study's results as a whole, it can be concluded that the inhibition of the PKA signaling pathway can aid in reducing the degree of nephrotoxicity caused by Amphotericin B.

Assuntos

Anfotericina B/toxicidade , Antifúngicos/toxicidade , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citocinas/biossíntese , Rim/efeitos dos fármacos , Óxido Nítrico/biossíntese , Animais , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Fragmentação do DNA/efeitos dos fármacos , Cães , Interleucina-6/biossíntese , Rim/enzimologia , Rim/imunologia , Rim/patologia , Células LLC-PK1 , Células Madin Darby de Rim Canino , Transdução de Sinais , Suínos , Fator de Necrose Tumoral alfa/biossíntese

RESUMO

Seminal plasma removal, an indispensable step in equine semen cryopreservation, is usually done by centrifugation, but this might cause mechanical damage to sperm. A new method for seminal plasma removal from stallion semen, namely a filter composed of a synthetic hydrophilic membrane (Sperm Filter, BotuPharma, Botucatu, Sao Paulo, Brazil), was recently proposed. The objective of this study was to test the use of the Sperm Filter in the removal of seminal plasma before freezing stallion semen. Ejaculates from 31 stallions were divided into two groups and cryopreserved. In group 1 (G1), seminal plasma was removed with the Sperm Filter, and in group 2 (G2), seminal plasma was removed by centrifugation (600×g for 10 minutes). There were no differences (P < 0.05) between G1 and G2 in sperm kinetic parameters or plasma membrane integrity before or after cryopreservation. However, sperm recovery rate was higher (P < 0.05) for G1 versus G2 (mean ± SD, 89.4 ± 7.4% vs. 80.9 ± 5.5%). Therefore, the Sperm Filter was as efficient as centrifugation in removing seminal plasma from the stallion ejaculate. However, filtering was more practical and had significantly fewer sperm lost than the centrifugation technique.

Assuntos

Criopreservação/veterinária , Cavalos , Preservação do Sêmen/veterinária , Sêmen , Animais , Criopreservação/métodos , Filtração/instrumentação , Filtração/métodos , Filtração/veterinária , Masculino , Preservação do Sêmen/métodos

RESUMO

A possible explanation for endometritis in mares is ascendant contamination from the vagina. The presence of Lactobacillus spp. is considered to be important in women for a healthy vaginal environment; however, there are few studies in mares related to the presence of Lactobacillus in the vaginal flora of healthy mares. The present work aims to determine the occurrence of Lactobacillus spp. in the vaginal micro-environment of mares. A total of 35 crossbred multiparous mares, aged between 4 and 12 years, with no history of reproductive problems and with healthy reproductive tracts, were used. Two vaginal swabs were obtained from the mares during estrus for Lactobacillus isolation and PCR evaluation. Ten human female volunteers, aged between 24 and 35 years, sexually active, with no history of gynecological diseases and treatments in the past two years were used. Lactobacillus spp. were isolated from 5.7% of the mares vaginal samples and from 90% of the womens vaginal samples. Lactobacillus DNA was detected by PCR in 22.9% of the mares vaginal samples and in all of the vaginal samples from the healthy women. The primers used here were demonstrated to have in silico specificity for the detection of L. equi (AB425924.1), L. pantheris (DQ471798.1) and L. mucosae (DQ471799.1), but they did not anneal on Enterococcus faecalis (EU887827.1) or E. faecium (EU887814.1). In conclusion, this study showed a low occurrence of Lactobacillus spp. in mares, suggesting that this bacterium may not play a fundamental role in the equilibrium of the vaginal micro-environment of normal mares.(AU)

Assuntos

Animais , Benchmarking , Lactobacillus/metabolismo , Transferência Embrionária , Equidae , Mulheres

RESUMO

A possible explanation for endometritis in mares is ascendant contamination from the vagina. The presence of Lactobacillus spp. is considered to be important in women for a healthy vaginal environment; however, there are few studies in mares related to the presence of Lactobacillus in the vaginal flora of healthy mares. The present work aims to determine the occurrence of Lactobacillus spp. in the vaginal micro-environment of mares. A total of 35 crossbred multiparous mares, aged between 4 and 12 years, with no history of reproductive problems and with healthy reproductive tracts, were used. Two vaginal swabs were obtained from the mares during estrus for Lactobacillus isolation and PCR evaluation. Ten human female volunteers, aged between 24 and 35 years, sexually active, with no history of gynecological diseases and treatments in the past two years were used. Lactobacillus spp. were isolated from 5.7% of the mares’ vaginal samples and from 90% of the women’s vaginal samples. Lactobacillus DNA was detected by PCR in 22.9% of the mares’ vaginal samples and in all of the vaginal samples from the healthy women. The primers used here were demonstrated to have in silico specificity for the detection of L. equi (AB425924.1), L. pantheris (DQ471798.1) and L. mucosae (DQ471799.1), but they did not anneal on Enterococcus faecalis (EU887827.1) or E. faecium (EU887814.1). In conclusion, this study showed a low occurrence of Lactobacillus spp. in mares, suggesting that this bacterium may not play a fundamental role in the equilibrium of the vaginal micro-environment of normal mares.

Assuntos

Animais , Benchmarking , Lactobacillus/metabolismo , Transferência Embrionária , Equidae , Mulheres

RESUMO

AIM: The present study investigates the interaction of TLR4 and RAGE with their respective ligands as inducers of the inflammatory markers IL-6 and TNF-α. Also, the reactivity of peripheral blood mononuclear cells (PBMNC) from type 2 diabetic (T2D) patients and non-diabetic healthy controls (ND) were comparatively studied. METHODS: Concentrations of IL-6 and TNF-α were measured by sandwich Elisa, using kits supplied by Assay Designs (Ann Arbor, MI, USA). PBMNC from T2D and ND were incubated in the presence or absence of LPS, anti-TLR4 or anti-RAGE for 72 hours at 37°C under 5% CO(2). The final volume was adjusted to 300 µL in DMEM supplemented with 10% fetal bovine serum. After incubation, the cells were centrifuged, the supernatant collected and the cytokines measured. RESULTS: PBMNC from T2D were more sensitive to innate immune stimulation with LPS and monoclonal agonist anti-TLR4 than were cells from ND. The actions of LPS, anti-TLR4 and anti-RAGE potentiated the production of IL-6 and TNF-α in both groups. The simultaneous activation of monoclonal anti-RAGE and anti-TLR4 suggests that both antibodies used different receptors on the cell surface, but converged on the same PBMNC signaling metabolic pathways. This simultaneous activation induced a higher production of IL-6 and TNF-α in PBMNC from the T2D patients than from the ND subjects. CONCLUSION: Our results clearly show an exacerbation of innate immunity in PBMNC with T2D that was possibly hyperglycaemia-induced. These data, when analyzed together, suggest the importance of innate immunity in the pathogenesis of T2D.

Assuntos

Diabetes Mellitus Tipo 2/metabolismo , Inflamação/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Receptor 4 Toll-Like/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/sangue , Humanos , Interleucina-6/biossíntese , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/farmacologia , Pessoa de Meia-Idade , Receptor para Produtos Finais de Glicação Avançada/imunologia , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/biossíntese

RESUMO

BACKGROUND AND PURPOSE: The two longest C-termini of the purinergic P2X receptors occur in the P2X2 and P2X7 receptors and are thought to interact with multiple cytoplasmic proteins, among which are members of the cytoskeleton, including microtubules. In this work we asked whether disrupting the microtubule cytoskeleton might affect the functions of these receptors. EXPERIMENTAL APPROACH: Functions of heterologously expressed P2X2 and P2X7 receptors were evaluated with electrophysiology and dye uptake following ATP application. Permeabilization and secretion of pro-inflammatory agents were quantified from fresh or cultured peritoneal mouse macrophages, treated in vitro or in vivo with colchicine. KEY RESULTS: Disrupting the microtubule network with colchicine did not affect currents generated by ATP in P2X2 and P2X7 receptor-expressing cells but inhibited uptake of the dye Yo-Pro-1 in Xenopus oocytes and HEK293 cells expressing these channels. Peritoneal mouse macrophages showed less ATP-induced permeabilization to ethidium bromide in the presence of colchicine, and less reactive oxygen species (ROS) formation, nitric oxide (NO) and interleukin (IL)-1ß release. Colchicine treatment did not affect ATP-evoked currents in macrophages. Finally, in vivo assays with mice inoculated with lipopolysaccharide and ATP showed diminished ROS, IL-1ß, interferon-γ and NO production after colchicine treatment. CONCLUSIONS AND IMPLICATIONS: Colchicine has known anti-inflammatory actions and is used to treat several conditions involving innate immunity, including gout and familial Mediterranean fever. Here we propose a new mechanism of action - inhibition of pore formation induced by activation of P2X receptors - which could explain some of the anti-inflammatory effects of colchicine.

Assuntos

Trifosfato de Adenosina/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Colchicina/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Receptores Purinérgicos P2X2/fisiologia , Receptores Purinérgicos P2X7/fisiologia , Trifosfato de Adenosina/farmacologia , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Morte Celular/efeitos dos fármacos , Colchicina/uso terapêutico , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Feminino , Corantes Fluorescentes/farmacocinética , Células HEK293 , Humanos , Interferon gama/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/ultraestrutura , Masculino , Camundongos , Microscopia de Fluorescência , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Óxido Nítrico/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Oócitos/ultraestrutura , Ratos , Espécies Reativas de Oxigênio/metabolismo , Receptores Purinérgicos P2X2/genética , Receptores Purinérgicos P2X7/genética , Transfecção , Xenopus laevis

RESUMO

*The effects of drought on the Amazon rainforest are potentially large but remain poorly understood. Here, carbon (C) cycling after 5 yr of a large-scale through-fall exclusion (TFE) experiment excluding about 50% of incident rainfall from an eastern Amazon rainforest was compared with a nearby control plot. *Principal C stocks and fluxes were intensively measured in 2005. Additional minor components were either quantified in later site measurements or derived from the available literature. *Total ecosystem respiration (R(eco)) and total plant C expenditure (PCE, the sum of net primary productivity (NPP) and autotrophic respiration (R(auto))), were elevated on the TFE plot relative to the control. The increase in PCE and R(eco) was mainly caused by a rise in R(auto) from foliage and roots. Heterotrophic respiration did not differ substantially between plots. NPP was 2.4 +/- 1.4 t C ha(-1) yr(-1) lower on the TFE than the control. Ecosystem carbon use efficiency, the proportion of PCE invested in NPP, was lower in the TFE plot (0.24 +/- 0.04) than in the control (0.32 +/- 0.04). *Drought caused by the TFE treatment appeared to drive fundamental shifts in ecosystem C cycling with potentially important consequences for long-term forest C storage.

Assuntos

Carbono/metabolismo , Secas , Árvores/metabolismo , Bactérias/metabolismo , Brasil , Dióxido de Carbono/metabolismo , Respiração Celular , Ecossistema , Solo , Fatores de Tempo

RESUMO

OBJECTIVE: To compare the role of Akt/PKB signaling pathway in the modulation of reactive oxygen species (ROS) production by autologous plasma in peripheral blood mononuclear cells (PBMNC) from type 2 diabetic patients and healthy subjects. MATERIALS AND METHODS: This study was approved by Santa Casa Ethical Committee and has included patients diagnosed with diabetes type 2 (DM2) and control group (non-diabetic) (ND). PBMNC were purified utilizing Ficoll-hypaque gradient. ROS was quantified by luminol-dependent chemiluminescence. The Akt/PKB phosphorylation was measured using a CASE kit. Statistical analyses were made with t Student test and chi-square (chi(2)). p<0.05 was considered significant. RESULTS: 12, 13-Phorbol dibutyrate (PDB) stimulated the production of higher levels of ROS in PBMNC from type 2 diabetic patients than that from healthy subjects. Autologous plasma, however, inhibited induced or not ROS production in PBMNC in both groups. The inhibition of PBMNC-ROS derived by autologous plasma from healthy subjects was higher than that from type 2 diabetic patients. Plasma phosphorylated (activated) Akt/PKB. The percentage of phosphorylation induced by autologous plasma in PBMNC from patients and healthy control were 14% and 93%, respectively. Inhibition of ROS production in PBMNC from DM2 were similar for PBMNC+plasma; PBMNC+Akti; and PBMNC+plasma+Akti. However, in ND control, plasma showed a higher ROS inhibition than Akti or plasma plus Akti. CONCLUSIONS: Our results suggest that the low antioxidant capacity observed in autologous plasma from DM2 patients in conjunction with the decreased activation of PKB may cause an imbalance in the oxidizing/reducing responses, possible inducing an oxidative stress state, which could be associated with tissular damage.

Assuntos

Diabetes Mellitus Tipo 2/metabolismo , Leucócitos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Leucócitos/efeitos dos fármacos , Pessoa de Meia-Idade , NADPH Oxidases/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores

RESUMO

BACKGROUND: Granulocytes from healthy subjects and from patients suffering from diabetes mellitus present differences in reactivity to stimulation with cyclic nucleotide-elevating agents. The production of reactive oxygen species (ROS) is inhibited in cells from non-diabetic subjects following such stimulation, but activated through a PKA-independent signaling pathway in granulocytes from type 1 and type 2 diabetic patients. The aim of the present study was to understand better the changes in signaling mechanisms induced by the disease. METHODS: ROS production in granulocytes from healthy subjects and from type 1 and type 2 diabetic patients was measured using a luminol-dependent chemiluminescence assay. Granulocytes were stimulated by the addition of the cAMP-elevating agent dibutyryl cAMP. In some experiments, granulocytes were pre-treated with an inhibitor of PKA or Akt/PKB prior to cAMP stimulation. RESULTS: Intracellular elevation of cAMP induced a PKA-dependent and Akt/PKB-independent inhibition of ROS production in granulocytes from healthy subjects, but a significant activation in cells from both type 1 and type 2 diabetic patients. Most significantly, activation of ROS generation in cells from diabetic patients was shown to be Akt/PKB-dependent and PKA-independent. CONCLUSIONS: These results suggest that chronic hyperglycaemia could induce metabolic adaptation in cAMP-related signaling mechanisms. Epac (exchange protein directly activated by cAMP) is a novel cAMP receptor besides PKA involved in different signaling pathways. The cAMP-stimulated inverse ROS response in granulocytes from type 1 and type 2 diabetic patients may be due to a change in signaling pathways from cAMP/PKA to cAMP/Epac/Akt/PKB. These preliminary results require further studies in order to evaluate their consequences on innate immunity and pathogenesis of diabetes mellitus.

Assuntos

Bucladesina/farmacologia , AMP Cíclico/sangue , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 2/sangue , Granulócitos/metabolismo , Proteínas Proto-Oncogênicas c-akt/sangue , Espécies Reativas de Oxigênio/sangue , Adulto , Feminino , Granulócitos/efeitos dos fármacos , Humanos , Masculino , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Valores de Referência

RESUMO

UNLABELLED: SUMMARY-BACKGROUND: The present study investigates the hypothesis that cells from ill patients and from healthy subjects may have different reactivity under metabolic stimulation as a consequence of an disease-induced metabolic adaptation. METHODS: Granulocytes either from healthy subjects or from type II-Non Insulin Dependent Diabetes Mellitus (NIDDM) patients were compared in their capacities to generate Reactive Oxygen Species (ROS). The ROS generation was comparatively determined in a chemiluminescence assay, luminol-dependent, after cell incubation in the presence of either cyclic AMP - elevating agents or Interleukin 10. In some experiments the cells were pretreated with H89 compound (a PKA inhibitor) or with diphenylene iodonium (DPI), a NADPH-oxidase inhibitor. RESULTS: Our results showed an increased ROS generation in granulocytes from diabetic patients in absence of cyclic AMP-elevating agents or IL-10. In the presence of cyclic AMP-elevating agents was observed an inverse metabolic response in granulocytes from diabetic patients in comparison to cells from healthy subjects. The granulocytes were pre-incubated in the presence of cyclic AMP-elevating agents--amminophylline (AMF) or dibutyryl cyclic AMP (dbcAMP)--or interleukin 10 (IL-10). The AMF, dbcAMP and IL-10 inhibited ROS production by granulocytes from healthy subjects. By contrast, AMF and dbcAMP activated cells from diabetic patients while IL-10 had no effect. The inhibition of ROS induced by AMF, dbcAMP or IL-10 was promptly abolished by the pretreatment of the cells with either PKA H89 inhibitor or NADPH-oxidase inhibitor (DPI) in granulocytes from healthy subjects. In relation to the granulocytes from type 2 diabetics patients, the activation of ROS generation mediated by AMF and dbcAMP was fully abolished by NADPH-oxidase DPI-inhibitor, but not by PKA H89 inhibitor. CONCLUSIONS: Our present results reinforce the hypothesis that cells from ill patients (type II diabetic) when compared to cells from healthy subjects have different reactivity under metabolic stimulation. ROS production by human granulocytes was modulated by cyclic AMP elevating agents and IL-10. The inhibition of the ROS production in cells from healthy subjects was PKA-dependent while the activation in granulocytes from patients was PKA-independent. This inverse metabolic response, in cells from patients, suggests the use of an alternative metabolic pathway PKA-independent, possible cAMP/Epac/PKB-dependent. The correlation between activation of ROS production in granulocytes from diabetic patients and pathogenesis of diabetes can be suggested, however, further and extensive studies are needed for demonstrating this suggestion.

Assuntos

Proteínas Quinases Dependentes de AMP Cíclico/sangue , AMP Cíclico/metabolismo , Diabetes Mellitus Tipo 2/sangue , Granulócitos/metabolismo , Interleucina-10/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Sulfonamidas , Idoso , Aminofilina/farmacologia , Bucladesina/farmacologia , Proteína Quinase Tipo II Dependente de AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Feminino , Granulócitos/efeitos dos fármacos , Granulócitos/imunologia , Humanos , Isoquinolinas/farmacologia , Masculino , Pessoa de Meia-Idade , Valores de Referência

RESUMO

BACKGROUND: The present study was designed to investigate the hypothesis that cells from ill patients and from healthy subjects may have different reactivities under metabolic stimulation. METHODS: The study was performed with granulocytes from non-diabetic subjects and from type II -Non Insulin Dependent Diabetes mellitus (NIDDM) patients. The nitric oxide (NO) generation was comparatively determined by the nitrite concentration (micromolar of nitrite) after cell incubation in the presence of cyclic nucleotide-elevating agents. RESULTS: Our results showed an inverse reactivity for granulocytes from diabetic patients when compared to non-diabetic subjects. Granulocytes were incubated in the presence of drugs that elevate the intracellular level of cyclic AMP aminophylline (AMF), dibutyryl cyclic AMP (dbcAMP)], cyclic GMP [8.Br. cyclic GMP(8.Br.cGMP) or levamisole (LEV)]. The cyclic AMP-elevating agents (AMF and d bcAMP) inhibited NO production by granulocytes from non-diabetic subjects and activated cells from diabetic patients. By contrast, cyclic GMP-elevating agents (8.Br.cGMP and LEV) activated cells from non-diabetic subjects and inhibited granulocytes from diabetic patients. The activation of NO generation by cyclic nucleotides was blocked by pretreatment of granulocytes with L-NAME. CONCLUSION: The authors describe for the first time that both cyclic AMP and cyclic GMP were able to modulate nitric oxide production in human granulocytes and that cell reactivity in ill patients (diabetic) showed altered and inverse response in comparison to granulocytes from healthy subjects. This inverse reactivity possibly reflects a disease-induced adapted metabolic response. The consequences of this altered metabolic response on host defense and inflammation may be speculated, but further experiments are needed to confirm this hypothesis.

Assuntos

8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , AMP Cíclico/sangue , GMP Cíclico/análogos & derivados , GMP Cíclico/sangue , GMP Cíclico/farmacologia , Diabetes Mellitus Tipo 2/sangue , Granulócitos/metabolismo , Óxido Nítrico/sangue , Idoso , Feminino , Granulócitos/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , NG-Nitroarginina Metil Éster/farmacologia , Nitritos/sangue , Valores de Referência

RESUMO

The biochemical processes linked up to cyclic nucleotides related to the phenomenon of phagocytosis in Biomphalaria glabrata hemocytes were studied. In hemocytes the results suggest the presence of a phagocytic capacity similar to that observed in human cells related to regulation by cyclic adenylate monophosphate (cAMP), but not by cyclic guanylate monophosphate (cGMP). This similarity and differences in the metabolic process of phagocytary capacity between human phagocytary cells and hemocytes could represent an interesting model aimed at studying the phylogenetic evolution of enzymatic complexes.

Assuntos

Biomphalaria/imunologia , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Granulócitos/imunologia , Hemócitos/imunologia , Fagocitose , Aminofilina/farmacologia , Animais , Biomphalaria/parasitologia , AMP Cíclico/farmacologia , GMP Cíclico/farmacologia , Granulócitos/metabolismo , Hemócitos/metabolismo , Humanos , Schistosoma mansoni/imunologia , Zimosan/farmacologia

RESUMO

The respiratory burst reaction has been studied in human granulocytes from normal subjects divided in four age groups: (I) 20-29, (II) 30-39, (III) 40-49, and (IV) 50-59 years old. Zimosan opsonized particles (OZ) were used to evaluate, simultaneously, the reducing power and the oxidizing species generation. Our results showed a strong parallelism and a direct correlation between oxidizing species generation and reducing power in the groups (I) and (II), in presence or in the absence of opsonized zimosan. However, the age groups (III) and (IV) showed an increase in the free radicals generation and a significant decrease in the cellular reducing power. This inverse correlation observed between oxidizing/reducing power in the (III) and (IV) age groups may suggest a metabolic cellular disequilibrium.

Assuntos

Envelhecimento/metabolismo , Granulócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Adulto , Envelhecimento/imunologia , Estudos de Avaliação como Assunto , Humanos , Pessoa de Meia-Idade , Fagócitos/imunologia

RESUMO

Entre os fatores determinantes na resistencia e suscetibilidade da Biomphalaria ao Schistosoma mansoni, os hemocitos desempenharam importante papel. Com o objetivo de estudar as interacoes S. mansoni/Biomphalaria relativas aos hemocitos o primeiro passo e certamente relacionado a padronizacao desta populacao de celulas em Biomphalaria nao infectada. Desta forma a quantificacao desta populacao de celulas na hemolinfa bem como sua capacidade fagocitaria foi determinada pela primeira vez. Alem disso, usando cepas de B. glabrata e B. tenagophila suscetiveis e resistentes, o hemocitograma e a capacidade fagocitaria dos hemocitos, apos infeccao com S. mansoni, foram tambem determinados. Cepas de B. glabrata (BA e BH respectivamente) resistentes e suscetiveis bem como cepas de B. tenagophila (TAIM e CF respectivamente) foram infectadas com 10 miracidios das cepas LE e SJ de S. mansoni, respectivamente. Estes caramujos infectados e respectivos controles nao infectados foram avaliados em relacao ao numero de hemocitos circulantes e alteracao da capacidade fagocitaria, usando Zimozan e MTT...

Assuntos

Biomphalaria/classificação , Schistosoma mansoni/parasitologia , Esquistossomose mansoni/parasitologia , Contagem de Células , Fagócitos/metabolismo , Fagócitos/parasitologia , Espectrofotometria

RESUMO

Among the determinant factors in the resistance and susceptibility of Biomphalaria to Schistosoma mansoni, hemocytes play an important role. Aiming at studying S. mansoni/Biomphalaria interactions related to hemocytes, the first step is certainly connected with the standardization of this cell population in uninfected Biomphalaria. In this way, quantification of this cell population in hemolymph, as well as its phagocitary capacity, have been determined for the first time. Furthermore, using susceptible and resistant strains of B. glabrata and B. tenagophila, the hemocytegram and phagocytary capacity of hemocytes after infection with S. mansoni were determined too. Resistant and susceptible strains of B. glabrata (BA and BH, respectively), as well as resistant and susceptible strains of B. tenagophila (TAIM and CF, respectively) were infected with 10 miracidia of the LE and SJ strains of S. mansoni, respectively. These infected snails and respective uninfected controls were assessed in relation to the number of circulating hemocytes and alteration in the phagocytary capacity, by using Zymozan and MTT. Reading was taken by means of a spectrophotometer at 5 hours and 1, 2, 5, 10, 20 and 30 days after infection. The results showed a decrease in population of the circulating phagocytary cells, 5 hours after infection. One day post-infection, the circulating cells of the susceptible snails showed an increased metabolic activity, but the same event could not be observed in the resistant strains. In the subsequent observation periods, significant differences among the strains studied could not be observed until the end of the experiment.

Assuntos

Biomphalaria/parasitologia , Hemócitos/fisiologia , Fagocitose , Schistosoma mansoni , Animais , Contagem de Células , Hemolinfa

RESUMO

O envolvimento cardíaco ocorre em 25 'por cento' a 50 'por cento' dos pacientes com S.I.D.A., quando analisados por ecocardiografia, biópsia miocárdica ou necrópsia. As manifestaçöes clínicas afetam 10 'por cento' dos pacientes, sendo a insuficiência cardíaca a mais frequente. Descrevemos a evolução de um paciente que tinha teste positivo para H.I.V. e miocardite como a primeira manifestação clínica da doença - a miocardite estava associada com arritmias complexas e bloqueio atrioventricular, havendo necessidade de implante de um marcapasso definitivo.

Assuntos

Humanos , Masculino , Adulto , Miocardite/complicações , Miocardite/diagnóstico , Síndrome da Imunodeficiência Adquirida , Albuterol/administração & dosagem , Homossexualidade Masculina/história , Metilprednisolona/administração & dosagem

RESUMO

We evaluated the levels of inositolmono-(IP1), di(IP2), tri-(IP3) and tetraphosphates (IP4) in human neutrophils (N) stimulated with gamma interferon (IFN-gamma) (200 microliters from a pool of cell culture supernatant obtained from 1 x 10(7) PHA-primed peripheral blood mononuclear cells (30-60 min at 37 degrees C, 5% CO2)) in the presence of in the absence of interleukin 10 (IL-10) (10 micrograms/10 microliters). The results, reported as mean +/- SEM cpm, showed that IFN-gamma induced a significant increase only in the IP3 level (N + medium = 1,413 +/- 172 and N + IFN-gamma = 8,875 +/- 832). However, this activation mediated by IFN-gamma was blocked partially in the presence of IL-10 (N + IFN-gamma + IL-10 = 2,430 +/- 239) (P < 0.05). Interleukin 10 alone did not induce significant alterations in the content of IP1 (1,203 +/- 123), IP2 (1,880 +/- 163), IP3 (938 +/- 102) or IP4 (2,403 +/- 345) when compared to the respective controls in the absence of IL-10 (IP1 = 1,625 +/- 132; IP2 = 1,343 +/- 149; P3 = 1,413 +/- 172 and IP4 = 3,281 +/- 234). We also demonstrated the inhibitory effect of IL-10 of chemoluminescence generation by human neutrophils during phagocytosis of opsonized particles (OZ). Chemoluminescence generation was enhanced by IFN-gamma (N = OZ = 42.8 +/- 3.9 and N + OZ + IFN-gamma = 66.5 +/- 4.3) and this effect was reduced by IL-10 (N + OZ + IFN-gamma + IL-10 = 37.6 +/- 5.1). These data suggest that IL-10 modulates the neutrophil response and may be important for the development of new treatments of inflammatory injury.

Assuntos

Interferon gama/imunologia , Interleucina-10/imunologia , Neutrófilos/metabolismo , Fosfatidilinositóis/metabolismo , Humanos , Fagocitose/imunologia

RESUMO

We evaluated the levels of inositolmono-(IP1), di-(IP2), tri- (IP3) and tetraphosphates (IP4) in human neutrophils (N) stimulated with gamma interferon (IFN-gamma) (200 mul from a pool of cell culture supernatant obtained from 1 x 10(7) PHA-primed peripheral blood mononuclear cells (30-60 min at 37 degrees Celsius, 5 per cent CO2) in the presence or in the absence of interleukin 10 (IL-10) (10 mug/10mul). The results, reported as mean + SEM cpm, showed that IFN-gamma induced a significant increase only in the IP3 level (N + medium = 1.413 + 172 and N + IFN-gamma = 8,875 + 832). However, this activation mediated by IFN-gamma was blocked partially in the presence of IL-10 (N + IFN-gamma + IL-10 = 2,430 + 239) (P<0.05). Interleukin 10 alone did not induce significant alterations in the content of IP1 (1,203 + 123), IP2 (1,880 + 163), IP3 (938 + 102) or IP4 (2,403 + 345) when compared to the respective controls in the absence of IL-10 (IP1 = 1,625 + 132; IP2 = 1,343 + 149; IP3 = 1,413 + 172 and IP4 = 3,281 + 234). We also demonstrated the inhibitory effect of IL-10 on chemoluminescence generation by human neutrophils during phagocytosis of opsonized particles (OZ). Chemoluminescence generation was enhanced by IFN-gamma (N + OZ = 42.8 + 3.9 and N + OZ + IFN-gamma = 66.5 + 4.3) and this effect was reduced by IL-10 (N + OZ + IFN-gamma + IL-10 = 37.6 + 5.1). These data suggest that IL-10 modulates the neutrophil response and may be important for the development of new treatments of inflammatory injury.

Assuntos

Humanos , Técnicas In Vitro , Interferon gama/imunologia , Interleucina-10/imunologia , Neutrófilos/imunologia , Fosfatidilinositóis/química , Fagocitose/imunologia