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 , SuínosRESUMO
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 , SuínosRESUMO
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/biossínteseRESUMO
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/métodosRESUMO
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 , MulheresRESUMO
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.
Assuntos
Animais , Benchmarking , Lactobacillus/metabolismo , Transferência Embrionária , Equidae , MulheresRESUMO
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/biossínteseRESUMO
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 laevisRESUMO
*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 TempoRESUMO
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.