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The emergence of resistant fungal species and the toxicity of currently available antifungal drugs are relevant issues that require special consideration. Cyclodextrins inclusion complexes could optimize the antimicrobial activity of such drugs and create a controlled release system with few side effects. This study aimed to assess the in vitro toxicity and antifungal effectiveness of nystatin (Nys) and chlorhexidine (Chx) complexed or not with ß-cyclodextrin (ßCD). First, a drug toxicity screening was performed through the Artemia salina bioassay. Then, the minimum inhibitory concentrations (MICs) against Candida albicans were determined with the broth microdilution test. After MICs determination, the cytotoxicity of the drugs was evaluated through the methyl-thiazolyl-tetrazolium (MTT) and neutral red (NR) assays and through cell morphology analysis. The PROBIT analysis was used to determine the median lethal concentration (LC50), and the cell viability values were submitted to one-way analysis of variance(ANOVA)/Tukey (α = 0.05). Overall, the ßCD-complexed antifungals were less toxic against A. salina than their raw forms, suggesting that inclusion complexes can reduce the toxicity of drugs. The MICs obtained were as follows: Nys 0.5 mg/L; Nys:ßCD 4 mg/L; Chx 4 mg/L; and Chx:ßCD 8 mg/L. Chx showed significant cytotoxicity (MTT: 12.9 ± 9.6%; NR: 10.6 ± 12.5%) and promoted important morphological changes. Cells exposed to the other drugs showed viability above 70% with no cellular damage. These results suggest that antifungals complexed with ßCD might be a biocompatible option for the treatment of Candida-related infections.
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Antifúngicos , beta-Ciclodextrinas , Antifúngicos/toxicidad , Candida , Nistatina/toxicidad , Candida albicans , Clorhexidina/farmacología , beta-Ciclodextrinas/toxicidadRESUMEN
Introduction. In recent years, the Herbaspirillum genus has emerged as a pathogen in healthcare-related infections and has became stablished as an opportunistic pathogen.Hypothesis/Gap Statement. Little is known about the pathogenesis induced by Herbaspirillum genus.Aim. To evaluate the cytotoxic effects of genus Herbaspirillum, its ability to adhere to lung human cells and the ability of environmental and clinical strains of Herbaspirillum to induce pneumonia in mice.Methodology. Environmental and clinical isolates of Herbaspirillum were examined for their cytotoxic effects on the Calu-3 cell lineage. Cytotoxic activity of secretome was tested using MTT/neutral red assays and cell morphology analysis. Herbaspirillum adhesion on Calu-3 cells was assessed using bright-field microscopy and cell-associated bacteria were counted. A mouse model of acute lung infection was done using a clinical and an environmental strain. Adult male mice were used, and the pneumonia was inducted by intra-tracheal inoculation of 108 or 109 bacteria. Mice weight variations were evaluated at the end of the experiment. Bronchoalveolar lavage was collected and evaluated for total and differential cytology. A histological examination of lungs was performed giving a histological score.Results. The secretomes of all the strains induced morphological alterations in cells, but only H. seropedicae SmR1 were cytotoxic in MTT and neutral red assays. Clinical strains of H. frisingense AU14459 and H. hutttiense subsp. huttiense AU11883 exhibited low adherence to lung cells, while SmR1 was non-adhesive. Following intratracheal inoculation, mice treated with 109 c.f.u. of the SmR1 and AU11883 strains lost 18 and 6% of their weight over 7 days, respectively, and presented moderate clinical signs. Infected mice showed inflammatory cell infiltration in the perivascular and peribroncheal/peribronchiolar spaces. Bronchoalveolar fluid of mice inoculated with SmR1 109 c.f.u. presented an increase in total leucocyte cells and in neutrophils population.Conclusion. These in vivo and in vitro results provide insights into how some Herbaspirillum strains cause infection in humans, providing a basis for the characterization of pathogenesis studies on this emerging infectious agent.
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Exosomas/metabolismo , Infecciones por Bacterias Gramnegativas/microbiología , Herbaspirillum/patogenicidad , Neumonía/microbiología , Animales , Adhesión Bacteriana , Líquido del Lavado Bronquioalveolar/citología , Línea Celular , Supervivencia Celular , Infecciones por Bacterias Gramnegativas/patología , Herbaspirillum/aislamiento & purificación , Herbaspirillum/metabolismo , Humanos , Pulmón/microbiología , Pulmón/patología , Masculino , Ratones , Neumonía/patología , VirulenciaRESUMEN
Abstract Ocotea porosa (Nees & Mart.) Barroso, commonly known as "imbuia", "canela-imbuia" or "imbuia-amarela" in Brazil, is a tree of the Southern Atlantic Forest. The present study investigates the anatomy of leaf and stem, volatile oil chemistry, as well as cytotoxicity and insecticidal activities of the essential oil of O. porosa. Species identification was achieved by anatomy features, mainly due to paracytic and anomocytic stomata; non-glandular trichomes; biconvex midrib and petiole with a collateral open arc vascular bundle; presence of a sclerenchymatous layer, starch grains and crystal sand in the stem; and the presence of phenolic compounds in the epidermis, phloem and xylem of the midrib, petiole and stem. The main volatile components of the essential oil were α-pinene (19.71%), β-pinene (13.86%) and bicyclogermacrene (24.62%). Cytotoxicity against human cancer cell (MCF-7), mouse cancer cell (B16F10) and mouse non-tumoral cell (McCoy) was observed as well as insecticidal activity of the essential oil against susceptible 'Ft. Dix' bed bugs (Cimex lectularius L.) by topical application.
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Chinches , Aceites Volátiles/farmacología , Ocotea/anatomía & histología , Ocotea/química , Insecticidas/farmacología , Pruebas de Toxicidad , Tallos de la Planta/química , Hojas de la Planta/química , HistocitoquímicaRESUMEN
Abstract The gut is the main organ that mediates the contact between antigens with our organism, controlling the immune response against environmental factors, such as microbiota and food. Innate lymphoid cells participate in the gut-associated lymphoid tissue (GALT) maturation during the prenatal and early postnatal periods. After birth, breast milk provides the essential elements for the continuity of development of this tissue, leading to structural changes and healthy microbiota installation. The microbiota participates in the organogenesis of the GALT, as in the formation of intestinal villi, stimulating the proliferation of stem cells and maintaining the integrity of epithelial barrier. Foods are also involved in maturation of the GALT, where the protein source depletion reduced the number of resident lymphocytes. This unique microenvironment present in the intestinal lamina propria (LP) and mesenteric lymph nodes (mLN) induce tolerance to innocuous antigens from the diet, known as Oral Tolerance. Antigens sampled by intestinal epithelium cells are transferred to specialized dendritic cells, residing in the LP, which migrate to the mesenteric lymph nodes where they participate in the induction of regulatory T cells (Treg). Understanding these phenomena may establish the intestinal mucosa as a tool in therapy of inflammatory bowel diseases and immunological disorders.
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Tolerancia Periférica , Microbiota , Sistema Inmunológico , Intestinos/fisiologíaRESUMEN
BACKGROUND:: The aim of this study was to evaluate the effects of the antioxidant allopurinol and ischemic post-conditioning on the deleterious effects of ischemia followed by reperfusion (I/R) in a standardized model of ischemia involving infra-renal aortic occlusion in rats. METHODS:: The animals were randomly divided into five groups: (A) animals not subjected to ischemia; (B) animals subjected to 2 h of ischemia and reperfusion only once; (C) animals given an allopurinol dose by gavage, then subjected to 2 h of ischemia and reperfusion only once; (D) animals subjected to 2 h of ischemia and post-conditioning and (E) animals that received allopurinol, then subjected to 2 h of ischemia and post-conditioning. The blood samples and small intestine segments were harvested for analysis after 3 days. RESULTS:: The protective effects of the use of allopurinol and ischemic post-conditioning were observed by measuring aspartate aminotransferase, alanine aminotransferase and lactate levels. The benefits of post-conditioning were evident from the total antioxidant capacity and creatinine levels, but these could not ascertain any positive effects of allopurinol. The histological analysis of mesentery revealed that both methods were effective in minimizing the harmful effects of the ischemia and reperfusion process. CONCLUSION:: Individual protocols significantly reduced I/R systemic injuries, but no additional protection was observed when the two strategies were combined.
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Alopurinol/farmacología , Antioxidantes/farmacología , Aorta Abdominal/cirugía , Poscondicionamiento Isquémico/métodos , Extremidad Inferior/irrigación sanguínea , Daño por Reperfusión/prevención & control , Animales , Biomarcadores/sangre , Modelos Animales de Enfermedad , Femenino , Ratas Wistar , Flujo Sanguíneo Regional , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Daño por Reperfusión/fisiopatologíaAsunto(s)
Edema/etiología , Inflamación/etiología , Periodontitis/complicaciones , Receptor de Bradiquinina B1/inmunología , Pérdida de Hueso Alveolar/etiología , Pérdida de Hueso Alveolar/inmunología , Pérdida de Hueso Alveolar/patología , Animales , Chondrus , Modelos Animales de Enfermedad , Edema/inducido químicamente , Edema/inmunología , Edema/patología , Extremidades/patología , Inflamación/inmunología , Inflamación/patología , Interleucina-1beta/inmunología , Masculino , Periodontitis/inmunología , Periodontitis/patología , Ratas , Ratas Wistar , Receptor de Bradiquinina B1/análisis , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
PURPOSE: To analyze the effects of allopurinol and of post-conditioning on lung injuries induced by lower-limb ischemia and reperfusion. METHODS: Thirty rats were used. They were divided in 5 groups: (1) group A: abdominal aortic dissection only, (2) group B: ischemia and reperfusion, (3) group C: administered allopurinol (100mg/Kg) a few hours before procedure, (4) group D: post-conditioned and (5) group E: administered allopurinol and post-conditioned. With the exception of group A, all groups were submitted to infrarenal aortic ischemia for 2 hours, and reperfusion for 72 hours. After euthanasia, lungs were removed for histological analysis. They were graded under two scores: pulmonary injury (neutrophil infiltration, interstitial edema, vascular congestion, and destruction of lung architecture) and lymphocytic score (neutrophil infiltration, lymphoid aggregate and secondary follicle). RESULTS: On the pulmonary injury score, the degree of injury was smaller than in groups D and E, when compared to group B, p<0.05. Group C did not obtain the same result (p>0,05). On the lymphocytic score, there was no statistic difference among groups, p>0.05. CONCLUSION: Both post-conditioning and the combination of allopurinol and post-conditioning were effective in remote lung protection induced by lower-limbs I/R. When used in isolation, allopurinol showed no protective effect.
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Alopurinol/uso terapéutico , Antimetabolitos/uso terapéutico , Poscondicionamiento Isquémico , Lesión Pulmonar/prevención & control , Daño por Reperfusión/complicaciones , Animales , Modelos Animales de Enfermedad , Femenino , Lesión Pulmonar/etiología , Masculino , Ratas , Ratas WistarRESUMEN
Purpose: To analyze the effects of allopurinol and of post-conditioning on lung injuries induced by lower-limb ischemia and reperfusion. Methods: Thirty rats were used. They were divided in 5 groups: (1) group A: abdominal aortic dissection only, (2) group B: ischemia and reperfusion, (3) group C: administered allopurinol (100mg/Kg) a few hours before procedure, (4) group D: post-conditioned and (5) group E: administered allopurinol and post-conditioned. With the exception of group A, all groups were submitted to infrarenal aortic ischemia for 2 hours, and reperfusion for 72 hours. After euthanasia, lungs were removed for histological analysis. They were graded under two scores: pulmonary injury (neutrophil infiltration, interstitial edema, vascular congestion, and destruction of lung architecture) and lymphocytic score (neutrophil infiltration, lymphoid aggregate and secondary follicle). Results: On the pulmonary injury score, the degree of injury was smaller than in groups D and E, when compared to group B, p 0.05. Group C did not obtain the same result (p>0,05). On the lymphocytic score, there was no statistic difference among groups, p>0.05. Conclusion: Both post-conditioning and the combination of allopurinol and post-conditioning were effective in remote lung protection induced by lower-limbs I/R. When used in isolation, allopurinol showed no protective effect.(AU)
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Animales , Ratas , Alopurinol/análisis , Alopurinol/uso terapéutico , Lesión Pulmonar/tratamiento farmacológico , Lesión Pulmonar/veterinaria , Ratas Wistar/lesionesRESUMEN
Abstract Purpose: To analyze the effects of allopurinol and of post-conditioning on lung injuries induced by lower-limb ischemia and reperfusion. Methods: Thirty rats were used. They were divided in 5 groups: (1) group A: abdominal aortic dissection only, (2) group B: ischemia and reperfusion, (3) group C: administered allopurinol (100mg/Kg) a few hours before procedure, (4) group D: post-conditioned and (5) group E: administered allopurinol and post-conditioned. With the exception of group A, all groups were submitted to infrarenal aortic ischemia for 2 hours, and reperfusion for 72 hours. After euthanasia, lungs were removed for histological analysis. They were graded under two scores: pulmonary injury (neutrophil infiltration, interstitial edema, vascular congestion, and destruction of lung architecture) and lymphocytic score (neutrophil infiltration, lymphoid aggregate and secondary follicle). Results: On the pulmonary injury score, the degree of injury was smaller than in groups D and E, when compared to group B, p<0.05. Group C did not obtain the same result (p>0,05). On the lymphocytic score, there was no statistic difference among groups, p>0.05. Conclusion: Both post-conditioning and the combination of allopurinol and post-conditioning were effective in remote lung protection induced by lower-limbs I/R. When used in isolation, allopurinol showed no protective effect.
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Animales , Masculino , Femenino , Ratas , Daño por Reperfusión/complicaciones , Alopurinol/uso terapéutico , Lesión Pulmonar/prevención & control , Poscondicionamiento Isquémico , Ratas Wistar , Modelos Animales de Enfermedad , Lesión Pulmonar/etiología , Antimetabolitos/uso terapéuticoRESUMEN
Abstract Sapium belongs to Euphorbiaceae family and comprises 23 species. Sapium glandulosum (L.) Morong is popularly known in Brazil as "pau-leiteiro" and "leitosinha" and it is used in traditional medicine to cicatrisation. Its leaf extracts have shown analgesic, anti-inflammatory and antibacterial activities. The preliminary set of pharmacognostic tools used for quality assessment of medicinal plant parts is macro- and micro-anatomy and S. glandulosum has not anatomical and histochemical description. Thus the aim of this study was to investigate the anatomical and histochemical characteristics of the leaf and stem of S. glandulosum as a means of providing information for quality assessment of herbal industry. The leaves and stems were investigated by employing field emission scanning electron microscopy, light microscopy, and histochemistry techniques. The analysis showed that S. glandulosum had the following anatomical features: dorsiventral and amphistomatic leaves; paracytic stomata; tabular crystal druses; non-articulated and branched laticifers; midrib's biconvex shape with vascular systems in open arc with invaginated ends; petiole with a round shape and slight concavity on the adaxial side; six collateral vascular bundles in U-shaped organisation; a circular stem shape and a sclerenchymatous ring. In the histochemical tests lipophilic components were found in cuticle and in the latex; phenolic compounds were met in the mesophyll and in the latex; starch grains were found in the parenchymatous sheath; lignified elements were met in the sclerenchymatous ring in the cortex and in the perivascular sclerenchymatous caps, beyond in the vessel elements. These features are helpful when conducting a quality control process.
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In this study, resveratrol-loaded nanocapsules were developed and its antitumor activity tested on a melanoma mice model. These nanocapsules were spherically-shaped and presented suitable size, negative charge and high encapsulation efficiency for their use as a modified-release system of resveratrol. Nanoencapsulation leads to the drug amorphization. Resveratrol-loaded nanoparticles reduced cell viability of murine melanoma cells. There was a decrease in tumor volume, an increase in the necrotic area and inflammatory infiltrate of melanoma when resveratrol-loaded nanocapsules were compared to free resveratrol in treated mice. Nanoencapsulation of resveratrol also prevented metastasis and pulmonary hemorrhage. This modified-release technology containing resveratrol can be used as a feasible approach in order to inhibit murine melanoma tumor growth.
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Melanoma Experimental/patología , Nanocápsulas/química , Estilbenos/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ratones Endogámicos C57BL , Nanocápsulas/ultraestructura , Resveratrol , Carga Tumoral/efectos de los fármacos , Difracción de Rayos XRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Euphorbia umbellata latex (sap) has normally been used in folk medicine in southern Brazil to treat different types of cancers. AIM OF STUDY: To carry out a biomonitored investigation of partitioned latex using in vitro assay, to identify the main mechanisms related with the action of the most active fraction as well as to develop a phytochemical study with this material. MATERIALS AND METHODS: Biological screening was performed with hexane, chloroform, ethyl acetate and methanol fractions from the latex of E. umbellata using MTT, trypan blue, and neutral red assays to determine the cytotoxicity against HRT-18, HeLa and Jurkat cells and flow cytometry, DNA quantification, acridine orange and Hoechst 33342 staining to investigate mechanisms of action for the hexane extract. The phytochemical study of the hexane fraction was performed by chromatographic procedures and the substances were identified by NMR analysis. The isolated terpenes were evaluated using MTT to determine the cytotoxicity against Jurkat cells. RESULTS: All the fractions presented concentration and time dependent cytotoxicity. The hexane fraction showed the highest cytotoxicity; whereas the Jurkat cell was the lineage with the highest sensitivity (IC50 1.87µg/mL). Fragmentation of DNA and apoptosis are two mechanisms related with the toxicity of hexane fraction. The hexane fraction arrested the cell cycle in the G0/G1 phase, and the selectivity index was 4.30. Phytochemical study of the hexane fraction led to isolation of euphol (main compound) and germanicol acetate. Both substances demonstrated some slight cytotoxic activity against Jurkat cells after 72h; however the activity was minimal compared to vincristine (anticancer standard drug). CONCLUSION: The current research proves that the fractions of the latex from E. umbellata have a cytotoxic effect against three different cancer cells lines. The hexane fraction showed high in vitro cytotoxic effects against Jurkat cells demonstrating that the effect may be due to non-polar constituents. The two isolated terpenes (euphol and germanicol acetate) showed poor cytotoxic activity indicating that the anticancer properties of the extract may be caused by other substances present in the hexane fraction.
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Citotoxinas/farmacología , Euphorbia/química , Extractos Vegetales/farmacología , Apoptosis/efectos de los fármacos , Brasil , Línea Celular Tumoral , Citotoxinas/química , Fragmentación del ADN/efectos de los fármacos , Fase G1/efectos de los fármacos , Células HeLa , Humanos , Células Jurkat , Medicina Tradicional/métodos , Fitoquímicos/química , Fitoquímicos/farmacología , Extractos Vegetales/química , Fase de Descanso del Ciclo Celular/efectos de los fármacosRESUMEN
The phenomenon of oral tolerance refers to a local and systemic state of tolerance induced in the gut after its exposure to innocuous antigens. Recent findings have shown the interrelationship between cellular and molecular components of oral tolerance, but its representation through a network of interactions has not been investigated. Our work aims at identifying the causal relationship of each element in an oral tolerance network, and also to propose a phenomenological model that's capable of predicting the stochastic behavior of this network when under manipulation. We compared the changes of a "healthy" network caused by "knock-outs" (KOs) in two approaches: an analytical approach by the Perron Frobenius theory; and a computational approach, which we describe within this work in order to find numerical results for the model. Both approaches have shown the most relevant immunological components for this phenomena, that happens to corroborate the empirical results from animal models. Besides explain in a intelligible fashion how the components interacts in a complex manner, we also managed to describe and quantify the importance of KOs that hasn't been empirically tested.
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Tolerancia a Medicamentos/inmunología , Modelos Teóricos , Administración Oral , Animales , Tolerancia Inmunológica , Modelos AnimalesRESUMEN
The aim of this study was to evaluate the probiotic properties of Pediococcus acidilactici B14 and to study its resistance in the gastrointestinal system when combined with Lactobacillus acidophilus ATCC 4356 and used in a potentially symbiotic aerated soy based dessert. P. acidilactici B14 showed some important probiotic characteristics such as survival rate of 45.9% at pH 2.5; 72.4% in 0.3% bile salts and 95.8% after gastrointestinal transit at pH 4.0. Tolerance against the antibiotics cephalexin, neomycin, vancomycin, cefotaxime and penicillin G was also observed. The strain inhibited antagonism against the following cultures: Escherichia coli ATCC 25922, Bacillus cereus ATCC 33018, Staphylococcus aureus ATCC 6538P and Salmonella sp. The mixed culture of P. acidilactici B14 with L. acidophilus ATCC 4356 showed a survival rate of 92.4% after the passage through the gastrointestinal system at pH 4.0. Furthermore, in the presence of the food matrix, an average increase in cell viability, after being subjected to the gastrointestinal system of 9.9% at pH 2.0 and 6.1% at pH 4.0, was observed. This characterized the adequacy of the associated culture as probiotic in the development of a functional food such as soy based aerated symbiotic dessert.
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BACKGROUND: Diarrheagenic Escherichia coli (DEC) strains are important causes of diarrhea. However, they cannot be distinguished from E. coli of the intestinal microbiota by conventional microbiological tests. METHODS: This work presents a two-system multiplex PCR for detection of DEC. Primers for 16S rRNA gene were added as internal amplification control to validate negative reactions. The multiplex-PCR system 1 contains primers for detection of Shiga toxin producing E. coli (STEC; stx1, stx2), enteropathogenic E. coli (EPEC; eae, bfpA), atypical enteropathogenic E. coli (aEPEc; eae), enteroinvasive E. coli (ETEC; lt, st), enteroinvasive E. coli (EIEC; ial), and the internal amplification control 16S rRNA. The system 2 contains primers for EIEC (ipaH), enteroaggregative E. coli (CVD432), diffusely adherent E. coli (daaE), and 16S rRNA. The protocol was tested with E. coli reference strains, and also with cultures of fecal specimens of people with diarrhea and healthy controls. RESULTS: The protocol correctly identified the DEC reference strains. No DEC marker was amplified for negative controls; these results were validated by the amplification of a fragment of the 16S rRNA gene. The frequency of DEC was 7.6% for both patients and healthy controls; two Shigella sonnei strains were detected in the group with diarrhea. The identity of the amplicons was confirmed by DNA sequencing. CONCLUSION: The protocol is specific for DEC Shigella and is suitable for clinical laboratories.
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Escherichia coli Enteropatógena/aislamiento & purificación , Infecciones por Escherichia coli/microbiología , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Escherichia coli Enteropatógena/genética , Infecciones por Escherichia coli/diagnóstico , Heces/microbiología , Humanos , Sensibilidad y EspecificidadRESUMEN
Brown spider (Loxosceles sp.) venom affects the endothelium of vessels and triggers disruptive activity in the subendothelial matrix. The vascular disorders observed after venom exposure include leukocyte and platelet activation, disseminated intravascular coagulation, an increase in vessel permeability and hemorrhage into the dermis. In this study, we report additional evidence regarding the mechanism of endothelial cell cytotoxicity induced by Loxosceles intermedia venom. Exposure to venom led to endothelial cell detachment in a time-dependent manner. Loss of cell anchorage and cell-cell adhesion following venom exposure was accompanied by changes in the distribution of the α5ß1 integrin and VE-cadherin. An ultrastructural analysis of cells treated with venom revealed morphological alterations characteristic of apoptosis. Moreover, after venom exposure, the ratio between Bax and Bcl-2 proteins was disturbed in favor of Bax. In addition, late apoptosis was only observed in cells detached by the action of venom. Accordingly, there was no increase in apoptosis when cells were exposed to L. intermedia venom in suspension, suggesting that the loss of cell anchorage provides the signal to initiate apoptosis. Thus, L. intermedia venom likely triggers endothelial cell death indirectly through an apoptotic mechanism known as anoikis.
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Anoicis/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Venenos de Araña/farmacología , Arañas/metabolismo , Animales , Antígenos CD/metabolismo , Aorta Torácica/citología , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Aorta Torácica/ultraestructura , Proteínas Reguladoras de la Apoptosis/metabolismo , Brasil , Cadherinas/metabolismo , Adhesión Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Endotelio Vascular/ultraestructura , Integrina alfa5beta1/metabolismo , Cinética , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , ConejosRESUMEN
BACKGROUND: Kinins are released during dermal injury and inflammation and seem to contribute to the pathogenesis of cutaneous diseases. OBJECTIVE: Participation of kinins in skin inflammatory process was evaluated using knockout mice and non-peptide kinin receptor antagonists. METHODS: Chronic skin inflammation was induced by multiple applications of TPA in mice ear. RESULTS: The B(2) knockout mice (B(2)(-/-)) showed a significant increase of ear weight (23 ± 10%) and epidermal cellular hyperproliferation and acanthosis formation upon histological analysis when compared with wildtype mice. Also, evaluation of PCNA levels by Western blot and immunohistochemistry confirmed the increase in the epidermis hyperproliferation in the ear skin of B(2)(-/-) mice. In contrast, no modification in these parameters was detected in B(1) knockout mice (B(1)(-/-)). However, mice lacking both kinin receptors (B(1)B(2)(-/-)) presented a considerable reduction of epidermis thickness and in PCNA levels. Following the establishment of skin inflammation (5th day of TPA application) treatment with the non-peptide antagonists SSR 240612 (B(1) receptor antagonist), FR 173657 (B(2) receptor antagonist), or SSR 240612 plus FR 173657 topically applied, caused a significant inhibition of ear weight (20 ± 5%, 34 ± 4% and 32 ± 6%, respectively). In the histological analysis, the antagonists produced a reduction in epidermal hyperplasia and acanthosis formation; but the treatment with a combination of the two antagonists did not increase efficacy. CONCLUSION: Kinin receptors seem to be involved in the control of the keratinocyte hyperproliferative process, and non-peptide kinin receptor antagonists may be useful tools in the treatment of hyperproliferative skin disorders.
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Receptor de Bradiquinina B1/metabolismo , Receptor de Bradiquinina B2/metabolismo , Enfermedades de la Piel/patología , Administración Tópica , Animales , Proliferación Celular , Dioxoles/farmacología , Femenino , Inflamación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Antígeno Nuclear de Célula en Proliferación/biosíntesis , Quinolinas/farmacología , Sulfonamidas/farmacología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Bites from the Loxosceles genus (brown spiders) cause severe clinical symptoms, including dermonecrotic injury, hemorrhage, hemolysis, platelet aggregation and renal failure. Histological findings of dermonecrotic lesions in animals exposed to Loxosceles intermedia venom show numerous vascular alterations. Study of the hemorrhagic consequences of the venom in endothelial cells has demonstrated that the degeneration of blood vessels results not only from degradation of the extracellular matrix molecule or massive leukocyte infiltration, but also from a direct and primary activity of the venom on endothelial cells. Exposure of an endothelial cell line in vitro to L. intermedia venom induce morphological alterations, such as cell retraction and disadhesion to the extracellular matrix. The aim of the present study was to investigate the interaction between the venom toxins and the endothelial cell surface and their possible internalization, in order to illuminate the information about the deleterious effect triggered by venom. After treating endothelial cells with venom toxins, we observed that the venom interacts with cell surface. Venom treatment also can cause a reduction of cell surface glycoconjugates. When cells were permeabilized, it was possible to verify that some venom toxins were internalized by the endothelial cells. The venom internalization involves endocytic vesicles and the venom was detected in the lysosomes. However, no damage to lysosomal integrity was observed, suggesting that the cytotoxic effect evoked by L. intermedia venom on endothelial cells is not mediated by venom internalization.
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Membrana Celular/efectos de los fármacos , Endocitosis , Venenos de Araña/farmacología , Arañas/química , Animales , Línea Celular , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Lisosomas/química , Lisosomas/efectos de los fármacos , Conejos , Venenos de Araña/análisis , Venenos de Araña/químicaRESUMEN
Alpha5beta1 integrin from both wild-type CHO cells (CHO-K1) and deficient in proteoglycan biosynthesis (CHO-745) is post-translationally modified by glycosaminoglycan chains. We demonstrated this using [35S]sulfate metabolic labeling of the cells, enzymatic degradation, immunoprecipitation reaction with monoclonal antibody, fluorescence microscopy, and flow cytometry. The alpha5beta1 integrin heterodimer is a hybrid proteoglycan containing both chondroitin and heparan sulfate chains. Xyloside inhibition of sulfate incorporation into alpha5beta1 integrin also supports that integrin is a proteoglycan. Also, cells grown with xyloside adhered on fibronectin with no alteration in alpha5beta1 integrin expression. However, haptotactic motility on fibronectin declined in cells grown with xyloside or chlorate as compared with controls. Thus, alpha5beta1 integrin is a proteoglycan and the glycosaminoglycan chains of the integrin influence cell motility on fibronectin. Similar glycosylation of alpha5beta1 integrin was observed in other normal and malignant cells, suggesting that this modification is conserved and important in the function of this integrin. Therefore, these glycosaminoglycan chains of alpha5beta1 integrin are involved in cellular migration on fibronectin.
Asunto(s)
Movimiento Celular/fisiología , Fibronectinas/fisiología , Glicosaminoglicanos/química , Integrina alfa5beta1/química , Animales , Células CHO , Cricetinae , Cricetulus , Electroforesis en Gel de Agar , Citometría de Flujo , Inmunoprecipitación , Microscopía FluorescenteRESUMEN
Smooth pufferfish of the family Tetraodontidae had become pure genomic models because of the remarkable compaction of their genome. This trait seems to be the result of DNA loss following its divergence from the sister family Diodontidae, which possess larger genomes. In this study, flow cytometry was used for estimate the genome size of four pufferfish species from the Neotropical region. Cytogenetic data and confocal microscopy were also used attempting to confirm relationships between DNA content and cytological parameters. The haploid genome size was 0.71 + or - 0.03 pg for Sphoeroides greeleyi, 0.34 + or - 0.01 pg for Sphoeroides spengleri, 0.82 + or - 0.03 pg for Sphoeroides testudineus (all Tetraodontidae), and 1.00 + or - 0.03 pg for Chilomycterus spinosus (Diodontidae). These differences are not related with ploidy level, because 46 chromosomes are considered basal for both families. The value for S. spengleri represents the smallest vertebrate genome reported to date. Since erythrocyte cell and nuclear sizes are strongly correlated with genome size, the variation in this last is considered under both adaptive and evolutionary perspectives.