RESUMEN
BACKGROUND: The Argentinean program was initiated more than a decade ago as the first experience of systematic translational research focused on NCL in Latin America. The aim was to overcome misdiagnoses and underdiagnoses in the region. SUBJECTS: 216 NCL suspected individuals from 8 different countries and their direct family members. METHODS: Clinical assessment, enzyme testing, electron microscopy, and DNA screening. RESULTS AND DISCUSSION: 1) The study confirmed NCL disease in 122 subjects. Phenotypic studies comprised epileptic seizures and movement disorders, ophthalmology, neurophysiology, image analysis, rating scales, enzyme testing, and electron microscopy, carried out under a consensus algorithm; 2) DNA screening and validation of mutations in genes PPT1 (CLN1), TPP1 (CLN2), CLN3, CLN5, CLN6, MFSD8 (CLN7), and CLN8: characterization of variant types, novel/known mutations and polymorphisms; 3) Progress of the epidemiological picture in Latin America; and 4) NCL-like pathology studies in progress. The Translational Research Program was highly efficient in addressing the misdiagnosis/underdiagnosis in the NCL disorders. The study of "orphan diseases" in a public administrated hospital should be adopted by the health systems, as it positively impacts upon the family's quality of life, the collection of epidemiological data, and triggers research advances. This article is part of a Special Issue entitled: "Current Research on the Neuronal Ceroid Lipofuscinoses (Batten Disease)".
RESUMEN
Trypanosoma cruzi invasion and replication in cardiomyocytes and other tissues induce cellular injuries and cytotoxic reactions, with the production of inflammatory cytokines and nitric oxide, both sources of reactive oxygen species. The myocyte response to oxidative stress involves the progression of cellular changes primarily targeting mitochondria. Similar alterations could be taking place in mitochondria from the skeletal muscle; if that is the case, a simple skeletal muscle biopsy would give information about the cardiac energetic production that could be used as a predictor of the chagasic cardiopathy evolution. Therefore, in the present paper we studied skeletal muscle mitochondrial structure and the enzymatic activity of citrate synthase and respiratory chain complexes I to IV (CI-CIV), in Albino Swiss mice infected with T. cruzi, Tulahuen strain and SGO Z12 and Lucky isolates, along the infection. Changes in the mitochondrial structure were detected in 100% of the mitochondria analyzed from the infected groups: they all presented at least 1 significant abnormality such as increase in their matrix or disorganization of their cristae, which are probably related to the enzymatic dysfunction. When we studied the Krebs cycle functionality through the measurement of the specific citrate synthase activity, we found it to be significantly diminished during the acute phase of the infection in Tulahuen and SGO Z12 infected groups with respect to the control one; citrate synthase activity from the Lucky group was significantly increased (p<0.05). The activity of this enzyme was reduced in all the infected groups during the chronic asymptomatic phase (p<0.001) and return to normal values (Tulahuen and SGO Z12) or increased its activity (Lucky) by day 365 post-infection (p.i.). When the mitochondrial respiratory chain was analyzed from the acute to the chronic phase of the infection through the measurement of the activity of complexes I to IV, the activity of CI remained similar to control in Tulahuen and Lucky groups, but was significantly augmented in the SGO Z12 one in the acute and chronic phases (p<0.05). CII increased its activity in Tulahuen and Lucky groups by day 75 p.i. and in SGO Z12 by day 365 p.i. (p<0.05). CIII showed a similar behavior in the 3 infected groups, remaining similar to control values in the first two stages of the infection and significantly increasing later on (p<0.0001). CIV showed an increase in its activity in Lucky throughout all stages of infection (p<0.0001) and an increase in Tulahuen by day 365days p.i. (p<0.0001); SGO Z12 on the other hand, showed a decreased CIV activity at the same time. The structural changes in skeletal muscle mitochondria and their altered enzyme activity began in the acute phase of infection, probably modifying the ability of mitochondria to generate energy; these changes were not compensated in the rest of the phases of the infection. Chagas is a systemic disease, which produces not only heart damage but also permanent skeletal muscle alterations.
Asunto(s)
Enfermedad de Chagas/metabolismo , Mitocondrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Animales , Enfermedad de Chagas/patología , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Femenino , Masculino , Ratones , Mitocondrias Musculares/ultraestructura , Músculo Esquelético/ultraestructuraRESUMEN
Chagasic cardiopathy has become one of the most frequent causes of heart failure and sudden death, as well as one of the most common causes of cardio-embolic stroke in Latin America. The myocyte response to oxidative stress involves the progression of cellular changes, primarily targeting the mitochondria and modifying therefore the energy supply. In this paper we analysed the effect of the infection of mice with 2 different strains of Trypanosoma cruzi (Tulahuen and SGO Z12) in the chronic indeterminate stage (75 days post-infection), upon the structure and function of cardiac mitochondria. The structural results showed that 83% of the mitochondria from the Tulahuen-infected mice presented an increase in their matrix and 91% of the mitochondria from the SGO Z12-infected group showed a reduction in their diameter (P < 0.05). When the Krebs cycle and mitochondrial respiratory chain functionality was analysed through the measurement of the citrate synthase and complexes I to IV activity, it showed that their activity was altered in all cases in a similar manner in both infected groups. In this paper we have demonstrated that the chronic indeterminate phase is not 'silent' and that cardiac mitochondria are clearly involved in the genesis and progression to the chronic chagasic cardiopathy when different factors alter the host-parasite equilibrium.
Asunto(s)
Cardiomiopatía Chagásica/patología , Cardiomiopatía Chagásica/fisiopatología , Corazón/parasitología , Interacciones Huésped-Parásitos , Mitocondrias/enzimología , Mitocondrias/parasitología , Trypanosoma cruzi/patogenicidad , Animales , Cardiomiopatía Chagásica/parasitología , Enfermedad de Chagas/parasitología , Enfermedad de Chagas/patología , Enfermedad de Chagas/fisiopatología , Enfermedad Crónica , Citrato (si)-Sintasa/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Electrocardiografía , Femenino , Corazón/fisiopatología , Humanos , Masculino , Ratones , Mitocondrias/patología , Miocardio/metabolismo , Miocardio/patología , Parasitemia/parasitología , Parasitemia/fisiopatología , Especificidad de la Especie , Trypanosoma cruzi/clasificaciónRESUMEN
This research paper explores the significant transformative potential of Mixed Reality (MR) technology as enabler of the metaverse, specifically aimed at enhancing mental health therapies. The emerging world of the metaverse, a multiuser, adaptive, three-dimensional digital space, paired with the interactive and immersive benefits of MR technology, promises a paradigm shift in how mental health support is delivered. Unlike traditional platforms, MR allows for therapy within the comfort of the user's familiar surroundings, while incorporating the benefits of social collaboration and interactions. The metaverse environment fosters heightened personalization and deeper user engagement, thereby offering a more tailored approach to computerized therapy. Beyond its immersive capabilities, MR offers potential for real-time, smart adaptations to the users' psycho-physiological state, targeting unique patients' needs on a diverse spectrum of therapeutic techniques, thus broadening the scope of mental health support. Furthermore, it opens avenues for continuous emotional support in everyday life situations. This research discusses the benefits and potentials of integrating MR within a mental health metaverse, highlighting how this innovative approach could significantly complement traditional therapeutic methods, fostering improved treatment efficacy, focusing on social and collective experiences, and increasing patient engagement.
RESUMEN
The objective was to assess risk of hospitalization and mortality of comorbidities using divisive hierarchical risk clustering to advice clinical interventions. Subjects and Methods: Data from the EHR of a general population, 3799885 adults, followed by 5 years. Model were performed using Spark and Scikit-learn and accuracy for the models was analyzed. Results: The number of models generated depends in part on the number of chronic diseases included (ex testing a sample of six diseases, a total number of 397 models for all-cause mortality and 431 models for hospitalization). The estimated models offered an ordered selection for the relevant clinical variables and their estimated risk as a group and for the individual patient in the group. Accuracy was assessed according to age, sex and the cardinality of the comorbid groups. A mobile version and dashboard were developed. Conclusion: The software developed stratified hospital admission and mortality risk in clusters of chronic diseases, and for a given patient, it could advise intensifying treatment or reallocating the patient risk.
Asunto(s)
Atención a la Salud , Hospitalización , Adulto , Humanos , Comorbilidad , Enfermedad Crónica , Análisis por ConglomeradosRESUMEN
Congestive heart failure (CHF) would be associated with mitochondrial abnormalities and increased of reactive species of oxygen (ROS). To clarify these issues we studied the structure, function of the mitochondrial enzyme nitro oxide synthase inducible (iNOS) and lipoperoxidation of membranes, one of their products through the peroxide nitrite ion (ONOO-), in the heart muscle of patients with heart failure congestive (ICC) grade III and IV (according to New York Heart Association). We included 25 patients who underwent cardiovascular surgery to biopsies of the heart muscle. They were stratified into a group with CHF (n = 18) and control group (n = 7). In di-chas biopsies analyzed the enzymatic activity of mitochondrial complex III spectrophotometrically, which was measured in mM.ubiquinona-1.mg prot, while the mitochondrial morphology was analyzed by the Zeiss electron microscope, the areas were quantified with program Axionvision 4.6. Lipoperoxidation of membranes was measured by the presence of ONOO-by immunohistochemistry against primary antibody against 3-nitrotyrosine was used lab kit system biogenic steptobidin biotin peroxidase (SBA) and coloring triamiobencidina (TAB), it is made with semicuantificacion intensity SCORE test. The statistical test used was ANOVA. The heart muscle of patients with CHF showed that the mitochondrial area was reduced by 78% compared with the control (160.37 µm2 ± 9.87) (936.81 µm2 ± 78.48) p 0.0001. There was also a 70% reduction in complex III activity compared to control (1.9 10-2 mM ubiq.mim-prot 1.mg ± 12.6) (5.79 10-2mM ubiq.mim prot-1.mg ± 36.6) p . The presence of ONOO-was significantly increased in patients with CHF. Alterations ultraestructutural and functional mitochondria found in patients with CHF and increased ROS are involved in the measures of physiopathology CCI and whites should be taken into account for future therapies of this condition.
Asunto(s)
Insuficiencia Cardíaca/fisiopatología , Mitocondrias Cardíacas/fisiología , Especies Reactivas de Oxígeno/metabolismo , Adulto , Estudios de Casos y Controles , Progresión de la Enfermedad , Femenino , Insuficiencia Cardíaca/enzimología , Insuficiencia Cardíaca/patología , Humanos , Inmunohistoquímica , Masculino , Microscopía Electrónica de Transmisión , Mitocondrias Cardíacas/ultraestructura , Óxido Nítrico Sintasa de Tipo II/metabolismo , Índice de Severidad de la Enfermedad , Tirosina/análogos & derivados , Tirosina/biosíntesisRESUMEN
INTRODUCTION: We studied plasmatic TNF-alpha, nitric oxide (NO) and citrulline behaviors and probable morphological mitochondrial alterations in aortic smooth muscle cells, in rats with atherogenesis induced by hyperfibrinogenemia in: A) control, B) multiple injured for 30 days and C) multiple injured for 60 days. MATERIAL AND METHODS: Hyperfibrinogenemia induction: adrenaline injection (0,1 mg/rat/day). TNF-alpha (pg/dL) was determined by Elisa and NO (microM) and citrulline (mM) by spectrophotometry. Morphological mitochondrial alterations were studied by electronic microscopy. Variables were analized: ANOVA, r coefficient and chi2 test. RESULTS: We observed a significant increment of TNF-alpha in multiple injured for 30 days (B) (50.05 +/- 2.29) as well as in multiple injured for 60 days (C) (74.99 +/- 2.82) related to control (A) (33.01 +/- 1.49) (p<0.001 in both groups). Citrulline presented a significant increased in (B) (5.56 +/- 0.20) and (C) (6.84 +/- 0.13) when compared to (A) (4.41 +/- 0.23) (p<0.001 in both situations). Mean while NO biodisponibility diminished significantly in (B) (8.97 +/- 0.70) and in (C) (5.32 +/- 0.68) when compared to (A) (21.65 +/- 1.74) (p<0.001 in both situations). CONCLUSIONS: Hyperfibrinogenemia could modify the NO physiopathological pathway and produced morphological mitochondrial alterations in aortic smooth muscle cells, probably producing ischemic lesions in the vascular wall and altering the vasodilatation response.
Asunto(s)
Aterosclerosis/etiología , Citrulina/sangre , Fibrinógeno/análisis , Enfermedades Metabólicas/sangre , Óxido Nítrico/sangre , Estrés Oxidativo , Factor de Necrosis Tumoral alfa/sangre , Animales , Aterosclerosis/sangre , Aterosclerosis/patología , Biomarcadores/sangre , Perros , RatasRESUMEN
Several research efforts have been focused on finding newer and more efficient photosensitizers for photodynamic therapy (PDT). Although, it was demonstrated that riboflavin is an efficient photosensitizer for PDT, the effect of its ester derivate, riboflavin 2',3',4',5'-tetraacetate (RFTA), which has higher cellular uptake, has not been well defined. To evaluate the cell death generated by applying RFTA as the photosensitizer in PDT in a human cancer cell line of squamous carcinoma (SCC-13), these cells were incubated with riboflavin and its ester derivate, RFTA followed by irradiation with different blue light doses. Cell viability was evaluated using neutral red uptake assay and cell death was evaluated using transmission electron microscopy, TUNEL assay and annexin V-PE/7AAD double staining. The expression of caspase-3, Bax, Bcl-2, ERK 1/2 and p38(MAPK) was evaluated by Western blotting and generation of intracellular ROS and changes in anion superoxide levels were analyzed using 2',7'-dichlorofluorescein-diacetate and dihydroethidium dye, respectively. RFTA-PDT generated a decrease in cancer cell viability in a light dose-response. Treated SCC-13 cells exhibited chromatin condensation, formation of apoptotic bodies, increases in TUNEL-positive cells, phosphatidylserine externalization and decreased procaspase-3 and Bcl-2 protein expression and increment of ERK 1/2 phosphorylation. Moreover, trolox abolished the effect of PDT on cell viability linking the increase in intracellular ROS levels with the cell death observed, whereas that the pre-treatment with MEK inhibitor did not induce changes in SCC-13 cell survival. These findings demonstrate the effects of RFTA in triggering apoptosis induced by ROS (\O2(-)) production after visible light irradiation of squamous carcinoma cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Fármacos Fotosensibilizantes/farmacología , Riboflavina/análogos & derivados , Riboflavina/farmacología , Apoptosis/efectos de la radiación , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Caspasa 3/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Fragmentación del ADN/efectos de los fármacos , Fragmentación del ADN/efectos de la radiación , Humanos , Luz , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fotoquimioterapia , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/uso terapéutico , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Riboflavina/química , Riboflavina/uso terapéutico , Proteína X Asociada a bcl-2/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
In an experimental model of atherogenesis induced by hyperfibrinogenemia (HF), the pharmacological response of vitamin E was studied in order to assess its antioxidant effect on the mitochondrial morphofunctional alterations in aortic smooth muscle cells. Three groups of male rats were used: (Ctr) control, (AI) atherogenesis induced for 120 days, and (AIE) atherogenesis induced for 120 days and treated with vitamin E. HF was induced by adrenalin injection (0.1 mg/day/rat) for 120 days. AIE group was treated with the administration of 3.42 mg/day/rat of vitamin E for 105 days after the first induction. Mitochondria morphology was analyzed by electronic microscopy (EM) and mitochondrial complexes (MC) by spectrophotometry. In group AI the total and mean number of mitochondria reduced significantly, the intermembranous matrix increased, and swelling was observed with respect to Ctr and AIE (P < 0.01). These damages were related to a significant decrease in the activity of citrate synthase and complexes I, II, III, and IV in group AI in comparison to Ctr (P < 0.001). Similar behavior was presented by group AI compared to AIE (P < 0.001). These results show that vitamin E produces a significative regression of inflammatory and oxidative stress process and it resolved the morphofunctional mitochondrial alterations in this experimental model of atherogenic disease.
RESUMEN
Introducción. La enfermedad CLN8 es uno de los 13 tipos genéticos reconocidos de lipofuscinosis neuronal ceroidea, un grupo de trastornos neurodegenerativos de acumulación lisosómica, los más frecuentes en la infancia. La causan mutaciones en la proteína transmembrana CLN8 de 286 aminoácidos, cuya función se desconoce. Las variantes patológicas en el gen CLN8 se asociaron con dos fenotipos diferentes: la variante infantil tardía en individuos de diversos países alrededor del mundo, y la epilepsia progresiva con retraso mental, que aparece en pacientes finlandeses y turcos. Caso clínico. Niña que mostró retraso psicomotor y demencia desde el nacimiento, convulsiones tonicoclónicas, mioclonía, ataxia con atrofia cerebelosa y muerte temprana a los 12 años. La microscopia electrónica de la piel mostró una mezcla de citosomas con patrones de depósitos osmiofílicos granulares, curvilíneos y de «huella digital», y mitocondrias hipertrofiadas. Se encontraron dos variantes patológicas de ADN en el gen CLN8 (exón 2 c.1A>G; p.?/ exón 3 c.792C>G; p.Asn264Lys), lo que confirmó un genotipo heterocigoto compuesto. Conclusión. Éste es el caso índice en América Latina para el nuevo fenotipo congénito de la enfermedad CLN8. La sospecha de esta patología debería sustentarse genéticamente en casos de síndrome neurodegenerativo con retraso psicomotor desde el nacimiento, dificultad del habla y convulsiones. El curso clínico incluye ataxia, atrofia cerebelosa y muerte temprana
Introduction. CLN8 disease is one of the thirteen recognized genetic types of neuronal ceroid lipofuscinosis, a group of neurodegenerative lysosomal storage disorders, most frequent in childhood. A putative 286 amino acids transmembrane CLN8 protein with unknown function is affected. Pathological variants in the CLN8 gene were associated with two different phenotypes: variant late-infantile in individuals from many countries worldwide, and epilepsy progressive with mental retardation, appearing in Finnish and Turkish subjects. Case report. The girl showed psychomotor delay and dementia since birth, tonic-clonic seizures, myoclonus, ataxia with cerebellar atrophy, and early death at 12 years old. Electron microscopy of the skin showed mixed GROD, curvilinear, fingerprint cytosomes and mitochondrial hypertrophy. Two pathological DNA variants in the CLN8 gene (exon 2 c.1A>G; p.?/ exon 3 c.792C>G; p.Asn264Lys) were found confirming a compound heterozygous genotype. Conclusion. This case is the Latin American index for a new congenital phenotype of the CLN8 disease. The congenital phenotype has to be added to the clinical spectrum of the CLN8 disease. The suspicion of CLN8 disease should be genetically sustained in challenging cases of a neurodegenerative syndrome with psychomotor delay since birth, speech difficulty and seizures. The course includes ataxia, cerebellar atrophy, and early death
Asunto(s)
Humanos , Femenino , Niño , Lipofuscinosis Ceroideas Neuronales/diagnóstico por imagen , Lipofuscinosis Ceroideas Neuronales/genética , Transporte de Proteínas/genética , Insuficiencia de Crecimiento/genética , Imagen por Resonancia Magnética/métodos , Microscopía Electrónica/métodosRESUMEN
In this work we have analyzed the involvement of cell death pathways during the process of follicular atresia in the hematophagous insect vector Dipetalogaster maxima. Standardized insect rearing conditions were established to induce a gradual follicular degeneration stage by depriving females of blood meal during post-vitellogenesis. We first characterized the morpho-histological and ultrastructural changes of the ovarian tissue at early and late follicular atresia by light and transmission electron microscopy. Apoptosis was investigated by DAPI nuclear staining, TUNEL labeling and the detection of active caspase-3 by immunofluorescence. Autophagy was assessed by the measurement of acid phosphatase activity in ovarian homogenates and monitored by the detection of the specific marker of autophagic compartments, LC3. High levels of acid phosphatase activity were detected at all atretic stages. However, follicular cells of follicles undergoing incipient degeneration in early atresia exhibited features of apoptosis such as chromatin condensation, DNA fragmentation and the presence of active caspase-3. The ultrastructural findings and the increased levels of LC3-II found at late follicular atresia supported the relevance of autophagy at this atretic stage, although the extent of autophagosome formation demonstrated that this cell death pathway also occurred at early atresia. In late atresia, follicular cells also displayed more drastic changes compatible with necrosis. Taken together, results showed that apoptosis, autophagy and necrosis were operative during follicular atresia in D. maxima. Moreover, it was shown that the relevance of these cell death mechanisms correlates with the time elapsed since the onset of the degenerative process.
Asunto(s)
Muerte Celular , Atresia Folicular , Insectos Vectores/fisiología , Reduviidae/fisiología , Animales , Enfermedad de Chagas/transmisión , Femenino , Insectos Vectores/ultraestructura , Masculino , Folículo Ovárico/ultraestructura , Reduviidae/ultraestructuraRESUMEN
Tripeptidyl-peptidase 1 (TPP1) null or residual activity occurs in neuronal ceroid lipofuscinosis (NCL) with underlying TPP1/CLN2 mutations. A survey of 25 South American CLN2 affected individuals enabled the differentiation of two phenotypes: classical late-infantile and variant juvenile, each in approximately 50% of patients, with residual TPP1 activity occurring in approximately 32%. Each individual was assigned to one of three subgroups: (I) n=11, null TPP1 activity in leukocytes; (II) n=8, residual TPP1 activity of 0.60-15.85 nmol/h/mg (nr 110-476); (III) n=6, activity not measured in leukocytes. Curvilinear bodies (CB) appeared in almost all studied CLN2 subjects; the only exceptions occurred in cases of subgroup II: two individuals had combined CBs/fingerprints (FPs), and one case had pure FPs. There were 15 mutations (4 first published in this paper, 3 previously observed in South America by our group, and 8 previously observed by others). In subgroup I, mutations were either missense or nonsense; in subgroups II and III, mutations prevailed at the non-conserved intronic site, c.887-10A>G (intron 7), and to a lesser extent at c.89+5G>C (intron 2), in heterozygous combinations. Grouping phenotypically and genetically known individuals on the basis of TPP1 activity supported the concept that residual enzyme activity underlies a protracted disease course. The prevalence of intronic mutations at non-conserved sites in subgroup II individuals indicates that some alternative splicing might allow some residual TPP1 activity.
Asunto(s)
Aminopeptidasas/genética , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/genética , Lipofuscinosis Ceroideas Neuronales/enzimología , Lipofuscinosis Ceroideas Neuronales/genética , Fenotipo , Serina Proteasas/genética , Adolescente , Adulto , Alelos , Empalme Alternativo , Aminopeptidasas/metabolismo , Argentina , Niño , Preescolar , Biología Computacional , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Femenino , Humanos , Intrones , Masculino , Microscopía Electrónica de Transmisión , Mutación , Lipofuscinosis Ceroideas Neuronales/patología , Linaje , Estudios Prospectivos , Reproducibilidad de los Resultados , Estudios Retrospectivos , Serina Proteasas/metabolismo , América del Sur , Tripeptidil Peptidasa 1 , Adulto JovenRESUMEN
Photodynamic inactivation of Candida albicans produced by 5-(4-trifluorophenyl)-10,15,20-tris(4-N,N,N-trimethylammoniumphenyl)porphyrin (TFAP(3+)), 5,10,15,20-tetrakis(4-N,N,N-trimethylammoniumphenyl)porphyrin (TMAP(4+)) and 5,10,15,20-tetrakis(4-N-methylpyridyl)porphyrin (TMPyP(4+)) was investigated to obtain insight about the mechanism of cellular damage. In solution, absorption spectroscopic studies showed that these cationic porphyrins interact strongly with calf thymus DNA. The electrophoretic analysis indicated that photocleavage of DNA induced by TFAP(3+) took place after long irradiation periods (>5 h). In contrast, TMAP(4+) produced a marked reduction in DNA band after 1 h irradiation. In C. albicans, these cationic porphyrins produced a â¼3.5 log decrease in survival when the cell suspensions (10(7) cells/mL) were incubated with 5 µM photosensitizer and irradiated for 30 min with visible light (fluence 162 J/cm(2)). After this treatment, modifications of genomic DNA isolated from C. albicans cells were not found by electrophoresis. Furthermore, transmission electron microscopy showed structural changes with appearance of low density areas into the cells and irregularities in cell barriers. However, the photodamage to the cell envelope was insufficient to cause the release of intracellular biopolymers. Therefore, modifications in the cytoplasmic biomolecules and alteration in the cell barriers could be mainly involved in C. albicans photoinactivation.
Asunto(s)
Candida albicans/efectos de los fármacos , Luz , Fármacos Fotosensibilizantes/farmacología , Porfirinas/farmacología , Animales , Candida albicans/citología , Candida albicans/genética , Cationes/síntesis química , Cationes/química , Cationes/farmacología , Bovinos , ADN/química , ADN de Hongos/genética , ADN de Hongos/aislamiento & purificación , Fármacos Fotosensibilizantes/síntesis química , Fármacos Fotosensibilizantes/química , Porfirinas/síntesis química , Porfirinas/química , Relación Estructura-ActividadRESUMEN
We report the enhanced bactericidal activity of ofloxacin in drug-containing Eudragit E100(®) dispersions (EuCl-OFX) against Pseudomonas aeruginosa and the effect of the cationic polymer on bacterial membrane. Organisms treated with EuCl-OFX showed changes in cell morphology, altered outer membrane (OM) and cytoplasm with low electrodensity areas. Zeta potential of bacterial surface was shifted to positive. Sensitization to lytic agents was also observed. A profound effect on bacterial size, granularity and membrane depolarization was found by flow cytometry. Cultures exposed to drug-free polymer also showed some damaged bacterial membranes, but there was no significant cell death. Inhibition of P. aeruginosa by EuCl-OFX may involve surface effect and, to some extent, permeation effect. The cationic polymer act to mitigate the electronegativity of cell surface in the process of disorganizing the OM, rendering it more permeable to antibiotic. In addition, cytoplasmic membrane depolarization turns bacterial cell more vulnerable. The effects on membranes combined with the mechanism of action of quinolone explain the improved bactericidal action exhibited by EuCl-OFX. The behavior described for Eudragit E100(®) against P. aeruginosa may be a useful tool to broaden the spectrum of antibiotics whose clinical use is limited by the impermeability of the bacterial OM.
Asunto(s)
Acrilatos/farmacología , Farmacorresistencia Bacteriana , Ofloxacino/farmacología , Polímeros/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Detergentes/farmacología , Sinergismo Farmacológico , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Pseudomonas aeruginosa/citologíaRESUMEN
The pathogenesis of chronic chagasic cardiopathy is still under discussion; there is considerable evidence that inflammatory infiltrates and their mediators have a direct effect on cardiac cells. Here we studied the structure and function of cardiac mitochondria in chronic chagasic myocardiopathy. Cardiac mitochondrial structure and enzyme activity of citrate synthase and complexes I to IV of the respiratory chain were studied in albino Swiss mice infected with Trypanosoma cruzi (Tulahuen strain or SGO Z12 isolate) on 365 days post-infection (dpi). The presence of parasites in cardiac and skeletal muscle was also investigated. The activity of complexes I to IV was altered in different ways, according to the strain employed (P<0.0001), in relation to the cristae disorganisation and the parasite persistence found in the Tulahuen group, and the chronic inflammatory process described in both groups; citrate synthase activity also increased in both infected groups. Changes in mitochondrial structure were detected in 89% of Tulahuen- and 58% of SGO Z12-infected mice. In this paper we demonstrate that parasite persistence and inflammation are likely to be involved in the structural and functional alterations in cardiac mitochondria from chronically T. cruzi-infected mice, demonstrating that the parasite strain determines different mitochondrial changes in chagasic cardiopathy.
Asunto(s)
Cardiomiopatía Chagásica/parasitología , Mitocondrias Cardíacas/parasitología , Trypanosoma cruzi , Animales , Respiración de la Célula/fisiología , Cardiomiopatía Chagásica/metabolismo , Cardiomiopatía Chagásica/fisiopatología , Enfermedad Crónica , Progresión de la Enfermedad , Femenino , Corazón/parasitología , Masculino , Ratones , Mitocondrias Cardíacas/fisiología , Músculo Esquelético/parasitología , Tasa de SupervivenciaRESUMEN
Photodynamic inactivation (PDI) of Escherichia coli has been studied in cultures treated with zinc(II) 2,9,16,23-tetrakis[4-(N-methylpyridyloxy)]phthalocyanine (ZnPPc(+4)) to obtain insight about the mechanism of damage. This phthalocyanine is rapidly bound to cells, reaching a value of approximately 0.8 nmol/10(6) cells when the cultures were incubated with 2 microM sensitizer. After 30 min of irradiation, a 4 log decrease of E. coli survival was observed. The photocytotoxic action was investigated in plasmid and genomic DNA by electrophoretic analysis. Absorption spectroscopic studies showed that this cationic phthalocyanine interacts strongly with DNA (K(DNA)=4.7 x 10(6)M(-1)). Photocleavage of calf thymus DNA sensitized by ZnPPc(+)4 was not found even after long irradiation periods. Similar results were also observed in genomic DNA extracted from E. coli cells after PDI treatment. Modifications of plasmid DNA isolated from bacteria were only observed after long irradiation periods. However, under these conditions transmission electron microscopy of the PDI bacteria revealed an aggregation of cytoplasmic macromolecules and irregularities in cell barriers. Also, scanning electron microscopy showed a shrunken appearance in cells after PDI. Even so, release of intracellular biopolymers was not detected by absorption. On the other hand, outer and inner membranes permeabilization assays showed an increase in the permeability. Consequently, alterations in the cell membrane functionality induced by ZnPPc(+4) appear to be the major cause of E. coli inactivation upon PDI.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Escherichia coli/efectos de los fármacos , Escherichia coli/efectos de la radiación , Indoles/administración & dosificación , Compuestos Organometálicos/administración & dosificación , Cationes , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Indoles/química , Isoindoles , Luz , Compuestos Organometálicos/química , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/administración & dosificación , Fármacos Fotosensibilizantes/química , Dosis de Radiación , Compuestos de ZincRESUMEN
The mechanistic aspects of Escherichia coli photodynamic inactivation (PDI) have been investigated in bacteria treated with 5,10,15-tris[4-(3-N,N,N-trimethylammoniumpropoxy)phenyl]-20-(4-trifluoromethylphenyl)porphyrin iodide (A3B3+) and visible light. The photosensitization activity of A3B3+ porphyrin was compared with that of 5,10,15,20-tetra(4-N,N,N-trimethylammonium phenyl)porphyrin p-tosylate (TMAP4+), which is an active tetracationic sensitizer to eradicate bacteria. The PDI damages on plasmid and genomic DNA were analyzed by electrophoresis. DNA photocleavage was observed after a long period of irradiation, when the bacterial cells are largely photoinactivated. Transmission electron microscopy (TEM) revealed structural changes with appearance of low density areas into the cells and irregularities in cell barriers, which could affect the normal cell membrane functionality. Also, damages on the cell-wall were not detected by scanning electron microscopy (SEM) and release of intracellular biopolymers was not found after PDI. These results indicate that the photodynamic activity of these cationic porphyrins produces DNA photodamage after a long period of irradiation. Therefore, an interference with membrane functions could be the main cause of E. coli photoinactivation upon short PDI treatments.
Asunto(s)
Escherichia coli/efectos de los fármacos , Escherichia coli/efectos de la radiación , Interacciones Hidrofóbicas e Hidrofílicas , Porfirinas/química , Porfirinas/farmacología , Compuestos de Amonio Cuaternario/química , Agua/química , Cationes/química , Membrana Celular/efectos de los fármacos , Membrana Celular/efectos de la radiación , Escherichia coli/ultraestructura , Metilación , Viabilidad Microbiana/efectos de la radiación , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Estructura Molecular , FotoquímicaRESUMEN
INTRODUCTION: We studied plasmatic TNF-alpha, nitric oxide (NO) and citrulline behaviors and probable morphological mitochondrial alterations in aortic smooth muscle cells, in rats with atherogenesis induced by hyperfibrinogenemia in: A) control, B) multiple injured for 30 days and C) multiple injured for 60 days. MATERIAL AND METHODS: Hyperfibrinogenemia induction: adrenaline injection (0,1 mg/rat/day). TNF-alpha (pg/dL) was determined by Elisa and NO (microM) and citrulline (mM) by spectrophotometry. Morphological mitochondrial alterations were studied by electronic microscopy. Variables were analized: ANOVA, r coefficient and chi2 test. RESULTS: We observed a significant increment of TNF-alpha in multiple injured for 30 days (B) (50.05 +/- 2.29) as well as in multiple injured for 60 days (C) (74.99 +/- 2.82) related to control (A) (33.01 +/- 1.49) (p<0.001 in both groups). Citrulline presented a significant increased in (B) (5.56 +/- 0.20) and (C) (6.84 +/- 0.13) when compared to (A) (4.41 +/- 0.23) (p<0.001 in both situations). Mean while NO biodisponibility diminished significantly in (B) (8.97 +/- 0.70) and in (C) (5.32 +/- 0.68) when compared to (A) (21.65 +/- 1.74) (p<0.001 in both situations). CONCLUSIONS: Hyperfibrinogenemia could modify the NO physiopathological pathway and produced morphological mitochondrial alterations in aortic smooth muscle cells, probably producing ischemic lesions in the vascular wall and altering the vasodilatation response.
Asunto(s)
Animales , Perros , Ratas , Aterosclerosis , Citrulina/sangre , Fibrinógeno , Enfermedades Metabólicas/sangre , Óxido Nítrico/sangre , Estrés Oxidativo , Factor de Necrosis Tumoral alfa/sangre , Aterosclerosis/sangre , Aterosclerosis/patología , Biomarcadores/sangreRESUMEN
La Terapia Fotodinámica (PDT) es un tratamiento del cáncer basado en la acumulación específica de una droga fotosensible en el tejido maligno. Su posterior radiación con una longitud de onda apropiada, induce la producción de singuletes de oxígeno responsable de la peroxidación de las organelas y la muerte de las células neoplásicas. En el Centro de Microscopía Electrónica de la Universidad Nacional de Córdoba se diseñó y construyó un prototipo de fuente de irradiación no coherente de 630nm el que posibilitó la aplicación de PDT por primera vez en nuestro país. Este prototipo ha sido aplicado satisfactoriamente en el tratamiento de la queratosis actínica. Fueron tratadas 100 lesiones en 27 pacientes utilizando como fotosensibilizador al ácido d amino levulínico (ALA) al 20 por ciento La activación lumínica duró de 5 a 20 minutos dependiendo de la extensión y profundidad de la lesión. Los resultados obtenidos fueron los siguientes: Remisión Completa de las lesiones (RC) 84por ciento, Remisión parcial (RP) 10 por ciento, Sin respuesta (SR) 0 por ciento y Sin datos (SD) 6 por ciento. En el último grupo están incluidos aquellos pacientes que no retornaron para su evaluación. La alta efectividad, sumada a la inmejorable respuesta cosmética y la reducida agresividad, hacen de PDT el método de elección en el tratamiento de esta patología. El prototipo utilizado en este estudio demostró ser además de no invasivo y bien tolerado, altamente efectivo.