Helicobacter pylori inactivation and virulence gene damage using a supported sensitiser for photodynamic therapy.

About half of the world's population is currently infected with Helicobacter pylori, which is involved in the development of several gastro-duodenal pathologies. The increasing number of antibiotic resistance reduces the effectiveness of the first-line therapy, so new strategies to improve the H. pylori eradication rates are needed. Antimicrobial Photodynamic Therapy (APDT) benefits from photogenerated reactive oxygen species, such as singlet oxygen, which inactivate microorganisms by means of photosensitising dyes and visible light. Therefore, it could be a suitable alternative for H. pylori eradication in the gastro-duodenal tract, particularly in patients infected with antibiotic resistant strains. We evaluated APDT against H. pylori, in vitro, using a new photosensitising material (PSM) based on a ruthenium(II) complex covalently bound to micrometric glass beads. Five H. pylori isolates (classified according to cagA genotype, and metronidazole-clarithromycin resistance) were used. Bacteria were mixed with the PSM and incubated in the dark or illuminated by blue light. Aliquots (min 1', 2', 5', 15' and 30') were cultured and colonies were counted after 2-3 days. A 99.99999% decrease was detected in the number of colonies in the irradiated wells where the bacterium was mixed with the PSM, compared to non-illuminated wells or with irradiated wells without PSM. It was also confirmed that DNA is a molecular target for oxidant species released during APDT (evaluated by alkaline gel electrophoresis after endonuclease III incubation, ureC and cagA RT-PCR, and bacterial fingerprint). Results were independent of cagA gene and antibiotic resistances.

H. pylori and mitochondrial changes in epithelial cells. The role of oxidative stress.

Infection with H. pylori plays a role in the pathogenesis of gastritis, peptic ulcer, gastric carcinoma, and gastric lymphoma, but mechanisms leading to the various clinical manifestations remain obscure and are the primary focus of research in this field. Proliferation and apoptosis are essential in the maintenance of gastric tissue homeostasis, and changes seen in their balance may condition gastric mucosal changes during infection. Thus, excessive apoptosis or proliferation inhibition will result in cell mass loss, which is observed in gastric ulcers. On the other hand, accelerated epithelial cell turnover is characteristic of carcinogenic mucosas. There is also scientific evidence that demonstrates an association between H. pylori infection and exacerbated synthesis of free radicals, the latter being well known as a primary cause of cell death. A thorough review of the literature and the results of our experimental research lead to conclude that H. pylori-induced oxidative stress activates the intrinsic pathway of apoptosis. Structural and functional changes caused by this process on mitochondrial organelles lie at the origin of gastric mucosal toxicity, and lead to the development of the various manifestations associated with this infection. Based on these data we suggest that therapy with antioxidants should prove beneficial for the clinical management of patients with H. pylori infection.

H. pylori and mitochondrial changes in epithelial cells. The role of oxidative stress

Infection with H. pylori plays a role in the pathogenesis of gastritis,peptic ulcer, gastric carcinoma, and gastric lymphoma, butmechanisms leading to the various clinical manifestations remainobscure and are the primary focus of research in this field.Proliferation and apoptosis are essential in the maintenance ofgastric tissue homeostasis, and changes seen in their balance maycondition gastric mucosal changes during infection. Thus, excessiveapoptosis or proliferation inhibition will result in cell massloss, which is observed in gastric ulcers. On the other hand, acceleratedepithelial cell turnover is characteristic of carcinogenic mucosas.There is also scientific evidence that demonstrates an associationbetween H. pylori infection and exacerbated synthesis offree radicals, the latter being well known as a primary cause of celldeath.A thorough review of the literature and the results of our experimentalresearch lead to conclude that H. pylori-induced oxidativestress activates the intrinsic pathway of apoptosis. Structuraland functional changes caused by this process on mitochondrialorganelles lie at the origin of gastric mucosal toxicity, and lead tothe development of the various manifestations associated with thisinfection. Based on these data we suggest that therapy with antioxidantsshould prove beneficial for the clinical management ofpatients with H. pylori infection(AU)

Regulación de la expresión del co-receptor CCR5 en sujetos con virus de la inmunodeficiencia humana / Expression regulation of CCR5 co-receptor in human immunodeficiency virus subjects

Objetivos Estudiar la expresión diferencial del co-receptor CCR5, en superficie e intracelularmente, en T4 y T8 de pacientes VIH frente a controles. Evaluar causas y mecanismos de regulación de esa expresión en sujetos VIH. Pacientes Se analizaron muestras de 11 controles y 14 de VIH en terapia antirretroviral. Resultados La expresión de superficie del CCR5 en T4 y T8 depacientes VIH disminuyó un 38 y 53% respectivamentefr ente a controles, observándose diferencias estadísticamente significativas. El CCR5 intracelular en T8 fue similar al de controles, sinembargo, en T4 de VIH la proteína intracelular fue más abundante y, como consecuencia, la cantidad total de CCR5 similar a la total de controles. Conclusiones Existe una regulación diferencial en la expresión del CCR5 en T4 y T8 de VIH en relación a controles. En ambas subpoblaciones, la expresión de superficie se halla disminuida, hecho que parece responder a razones diferentes: en T4 se produce internalización de CCR5 quizá como consecuencia de ser arrastrado al interior celular en el proceso de infección por el virus, pero su expresión global es similar a la de controles, en T8 en cambio, no hay internalización sino regulación negativa del receptor en superficie, probablemente consecuencia de la sobreexpresión de interleuquinas ligando del co-receptor que se observaen estos pacientes. Son necesarios estudios que evalúen y expliquen el diferente comportamiento de estas subpoblaciones en la regulación del CCR5 para ampliar el conocimiento sobre la fisiopatología de la enfermedad y facilitar la elección de terapias más efectivas y menos agresiva

Objectives To study the differential expression of CCR5 co-receptor, in surface and intracellularly, in T4 and T8 cells of HIV patients respect to controls. To evaluate causes and mechanisms of that expression regulation in HIV subjects. Patients II controls and 14 HN patients with antirretroviral therapy were studied. Results CCR5 surface expression of HN, both in T4 and in T8, was diminished 38 and 53% respectively respect to controls with significant differences. CCR5 intracellular in T8 was similar to controls, never the less, the intracellular protein in T4 was more abundant and as a consequence, the total amount of CCR5 was similar to the total (surface+intracellular)in controls. Conclusions It exists a diferential regulation in the expression of CCR5 in T4 and T8 of HIV regard to controls. In both subpopulations, the surface expression is diminished,lact that seems to respond to different reasons: In T4 was produced a CCR5 internalization, perhaps as aresult 01 being dragged to the citosol in the virus infection process, but its global expression was similar to controls. However, in T8 there was not internalization but negative regulation of surface receptor, probably due to the sobreexpression of interleukins binding of the co-receptor, what is characteristic in these patients. They are necessary studies that evaluate and explain the different behavior of these subpopulations in the CCR5 regulation to extend the knowledge on the physiopathology of the disease and to facilitate the election of more effective and less aggressive therapies

Flow cytometric analysis of the in situ hybridization of cyclooxygenase isoforms in mesangial cells treated with cyclosporine A.

BACKGROUND: Cyclosporine A increases oxidative stress in kidney and we hypothesized that cyclooxygenase (COX) may be involved in this effect. MATERIAL AND METHODS: Mesangial cells of Cyclosporine A-treated (4, 7 or 10 days) rats were obtained to evaluate mRNA expression of COX-isoforms (COX-1, constitutive and COX-2, inducible) by "in situ" hybridization. Probes were labelled using "Gene Image Random Prime Labelling Protocol" and COX expression was measured by flow cytometry. RESULTS AND DISCUSSION: "In situ" hybridization by flow cytometry is an useful method to detect mRNA. We observed an increased COX-2 expression in a time-dependent manner in parallel with Reactive Oxygen Species synthesis. COX-1 expression increased only at 10 days.