P2Y1 and cysteinyl leukotriene receptors mediate purine and cysteinyl leukotriene co-release in primary cultures of rat microglia.

Inflammation is widely recognized as contributing to the pathology of acute and chronic neurodegenerative conditions. Microglial cells are pathologic sensors in the brain and activated microglia have been viewed as detrimental. Leukotriene, including cysteinyl leukotrienes (CysLTs) are suggested to be involved in brain inflammation and neurological diseases and ATP, by its receptors is a candidate for microglia activation. A23187 (10 microM) stimulated microglia to co-release CysLTs and [3H] adenine based purines ([3H] ABPs), mainly ATP. The biosynthetic production of CysLTs was abolished by 10 microM MK-886, an inhibitor of 5-lipoxygenase-activating protein activity. RT-PCR analysis showed that microglia expressed both CysLT1 / CysLT2 receptors, P2Y1ATP receptors and several members of the ATP binding cassette (ABC) transporters including MRP1, MRP4 and Pgp. The increase in [Ca2+]i elicited by LTD4 (0.1 microM) and 2MeSATP (100 microM), agonists for CysLT- and P2Y1-receptors, was abolished by the respective antagonists, BAYu9773 (0.5 microM) and suramin (50 microM). The stimulation of both receptor subtypes, induced a concomitant increase in the release of both [3H] ABPs and CysLTs that was blocked by the antagonists and significantly reduced by a cocktail of ABC transporter inhibitors, BAPTA/AM (intracellular Ca2+ chelator) and staurosporine (0.1 microM, PKC blocker). P2Y antagonist was unable to antagonise the effects of LTD4 and BAYu9773 did not reduce the effects of 2MeSATP. These data suggest that: i) the efflux of purines and cysteinyl-leukotrienes is specifically and independently controlled by the two receptor types, ii) calcium, PKC and the ABC transporter system can reasonably be considered common mechanisms underlying the release of ABPs and CysLTs from microglia. The blockade of P2Y1 or CysLT1/CysLT2 receptors by specific antagonists that abolished the raise in [Ca2+]i and drastically reduced the concomitant efflux of both compounds, as well as the effects of BAPTA and staurosporine support this hypothesis. In conclusion, the data of the present study suggest a cross talk between the purine and leukotriene systems in a possible autocrine/paracrine control of the microglia-mediated initiation and progression of an inflammatory response.

Treatment of diabetic microangiopathy and edema with total triterpenic fraction of Centella asiatica: a prospective, placebo-controlled randomized study.

The aim of this study was to demonstrate in a prospective, placebo-controlled, randomized study, whether total triterpenic fraction of Centella asiatica (TTFCA) is effective in improving the microcirculation in diabetic microangiopathy and neuropathy, Patients with severe diabetic microangiopathy, neuropathy, and edema; patients with microangiopathy without neuropathy; and healthy subjects were included. Microangiopathy was defined by laser Doppler and capillary filtration (rate on ankle swelling). Inclusion criteria were increase in resting flux and rate of ankle swelling; decrease in venoarteriolar response (VAR) and alteration in flux increase with temperature. Patients were randomized: the treatment group received TTFCA (tablets, 60 mg twice daily for 12 months); those in the placebo group received similar tablets. Healthy controls were followed up as a reference. Groups were comparable; there were no dropouts. There were no differences in the treatment and placebo groups at inclusion. Treatment was well tolerated; no side effects were reported. No variations were observed in normals at 12 months. In the neuropathy A-group, decreases (p<0.05) in RF and RAS were observed in the two treatment groups. The decrease in RAS was associated with a decrease in edema (p<0.05) in both treatment groups. The differences in flux (38%) and in VAR (38%) were associated with a decrease (28%) in the rate of ankle swelling (p<0.05). In patients without neuropathy (B-group) the decrease in flux was 22%, the VAR increased 22.7%, and the RAS decreased 9.5% at 12 months. The variations in normals and the progressive deterioration observed in untreated patients in both groups indicates the difference between treatment and placebo. In conclusion, the decrease in capillary filtration and edema is associated with symptomatic improvement. The action on edema is beneficial for the evolution of neuropathy. The effects of TTFCA on flux, RAS, and edema are important in early stages of microangiopathy to avoid progression to clinical stages.

Actividad bactericida in vitro de miel sobre Escherichia coli y Staphylococcus aureus: Comparación con la actividad de cefalosporinas

Se realizó un estudio comparativo entre la actividad antibacteriana de miel y cefalexina sobre una cepa de Escherichia coli y de miel y cefquinoma sobre una cepa de Staphylococcus aureus mediante ensayos de curva de muerte modificada. En todos los ensayos, la máxima actividad antibacteriana de la miel se observó a una dilución del 50% v/v. Respecto de la eficacia comparativa entre la miel y los antibióticos, se observó que sobre E. coli, cefalexina logró una reducción del conteo de bacterias viables compatible con un efecto bactericida (< 500 ufc/mL), mientras que la miel no logró superar este punto de corte. Lo opuesto se observó para S. aureus, donde la miel logró una reducción del conteo de bacterias viables compatible con un efecto de erradicación bacteriana (< 50 ufc/mL) respecto del efecto bactericida obtenido con cefquinoma (< 500 ufc/mL). Estos resultados preliminares, corroboran la necesidad de revalorar la actividad antibacteriana de productos naturales, cuya eficacia -aunque de manera empírica- ya era conocida desde la antigüedad y que fuera olvidada a partir de la aparición de los antibióticos.

A comparative study was conducted between the antibacterial activity of honey and cephalexin against a strain of Escherichia coli and honey and cefquinome against a strain of Staphylococcus aureus by modified time-kill-curves essays. In all trials, the maximum antibacterial activity of honey was observed at a dilution of 50% v/v. Regarding the comparative efficacy between honey and antibiotics, it was observed that against E. coli, cephalexin achieved a reduction in viable bacteria count compatible with a bactericidal effect (<500 cfu/mL), while honey did not overcome this breakpoint. The opposite was observed for S. aureus, where honey achieved a reduction in the viable bacteria count compatible with a bacterial eradication effect (<50 cfu/mL) with respect to the bactericidal effect obtained with cefquinome (<500 cfu/mL). These preliminary results corroborate the need to revalue the antibacterial activity of natural products, whose efficacy - although empirically - was already known since antiquity and was forgotten after the appearance of antibiotics.

Efecto de la persistencia bacteriana sobre la eficacia de la enrofloxacina y ciprofloxacina frente a una cepa de Escherichia coli

En este trabajo se evaluó el efecto de las bacterias persistentes presentes en un inóculo de alta densidad de una cepa autóctona de Escherichia coli sobre la eficacia de enrofloxacina y ciprofloxacina mediante ensayos in vitro de curvas de muerte bacteriana y de determinación de la concentración preventiva de mutantes. En las curvas de muerte realizadas sobre inóculos de alta densidad, ningún antibiótico presentó actividad bactericida y solo permitieron la sobrevida de bacterias persistentes. En el ensayo para determinar la concentración preventiva de mutantes, sobre la superficie del agar de las placas con elevadas concentraciones de enrofloxacina y ciprofloxacina, las bacterias persistentes permanecieron viables sin desarrollar colonias y adoptando morfología filamentosa como una forma de adaptación y supervivencia. Se discute la utilidad clínica de las concentraciones preventivas de mutantes de enrofloxacina y ciprofloxacina sobre E. coli ya que, estas elevadas concentraciones permitirían la sobrevida de una sub-población de bacterias persistentes originando un reservorio biológico que podría dar origen a infecciones crónicas y a favorecer la emergencia de mutantes resistentes.

This work evaluated the effect of persister cells present in a high inocula size of a wild strain of Escherichia coli on the efficacy of enrofloxacin and ciprofloxacin by in vitro time-kill curve assays and mutant prevention concentration testing. In time-kill curves performed with high inocula size, no antibiotics showed bactericidal activity, but only allowed the survival of persister cells. In the assay to determine the mutant prevention concentration, on the surface of agar plates containing high enrofloxacin and ciprofloxacin concentrations, persister cells remained viable and without bacterial colonies development and adopting filamentous morphology as a form of adaptation and survival. The clinical usefulness of mutant prevention concentrations of enrofloxacin and ciproflocxacin against Escherichia coli is discussed, as these high concentrations would allow the survival of a sub-population of persister cells originating a biological reservoir that could give rise to chronic infections and favor the emergence of resistant mutants.

Caracterización y comparación de la actividad antibacteriana intrínseca del suero de búfalo y del suero de bovino

La actividad antibacteriana de los sueros de bovino y de búfalo fue evaluada in vitro de manera indirecta con dos reacciones de inmunohemólisis: (i) un ensayo de hemólisis-hemoaglutinación sobre eritrocitos no sensibilizados de conejo y (ii) un ensayo de cinética de eritrocitos no sensibilizados de conejo frente a concentraciones crecientes de suero. La actividad bactericida de los sueros fue evaluada in vitro cuantificando la reducción de desarrollo de un inóculo de Escherichia coli en medio de cultivo enriquecido con suero de ambas especies. En el ensayo de hemólisis-hemoaglutinación, la hemólisis máxima se observó hasta la dilución 1:16 del suero de ambas especies. El mayor porcentaje de hemólisis se obtuvo con el suero de búfalo (84,7 ± 9,71%) respecto del suero bovino (71,0 ± 5,05%). Sin embargo no se hallaron diferencias en las diluciones de suero necesarias para obtener el 50% de la hemólisis total, siendo estas de 10,2 ± 2,48 para el suero de bovinos y de 11,8 ± 2,18 para el suero de búfalo. El suero de búfalo redujo el desarrollo bacteriano en un 69,8% respecto del 47,2% obtenido por el suero de bovino. No obstante estos resultados, se necesitarán estudios adicionales para corroborar estos hallazgos in vivo.

The antibacterial activity of serum of bovine and buffaloes was evaluated in vitro indirectly by two immunohemolysis assays: (i) an unsensitized rabbit blood cells hemolysis-hemagglutination assay and (ii) and a hemolytic-kinetic of unsensitized rabbit blood cells in function of increasing serum concentrations. The serum bactericidal activity was evaluated in vitro by quantifying the reduction of the development of an Escherichia coli inoculum in a culture medium enriched with serum of both species. In the hemolysis-hemagglutination assay, the máximum haemolysis was observed until a 1:16 serum dilution for both species. The highest percentage of hemolysis was obtained with buffalo serum (84.7 ± 9.71%) with respect to bovine serum (71.0 ± 5.05%). However, no differences were found in the serum dilutions necessary to obtain 50% of the total hemolysis, being these 10.2 ± 2.48 for the serum of cattle and 11.8 ± 2.18 for the serum of buffalo. Buffalo serum reduced bacterial growth by 69.8% compared to 47.2% obtained by bovine serum. Despite these results, additional studies will be needed to corroborate these findings in vivo.

Actividad antibacteriana in vitro de Ciprofloxacina sobre una cepa autóctona de Escherichia coli: efecto del pH sobre su potencia y efecto de la persistencia bacteriana sobre su modo de acción

En este trabajo se evaluó in vitro: (i) el efecto del pH sobre la actividad bactericida de ciprofloxacina (CFX) frente a una cepa autóctona de Escherichia coli y (ii) el efecto de las bacterias persistentes sobre el modo de acción concentración dependiente de CFX. La actividad antibacteriana de CFX disminuyó a causa del descenso del pH, por lo que los valores de concentración inhibitoria mínima (CIM), concentración bactericida mínima (CBM) y concentración de erradicación bacteriana mínima (CEBM) se incrementaron cuando el pH del medio de cultivo descendió de 7,4 a valores de 6,5 y 5,5. La cinética de eliminación bacteriana de CFX fue bifásica a causa de la selección de una sub-población de bacterias persistentes que presentaron una velocidad de eliminación más lenta. Por lo tanto la actividad bactericida de CFX fue definida por su concentración en relación a la CIM y el tiempo durante el cual se mantuvo la exposición de las bacterias a ésta.

In this in vitro assay was evaluated: (i) the effect of pH on the bactericidal activity of ciprofloxacin (CFX) against a native strain of Escherichia coli, (ii) the effect of persister bacteria on the concentration-dependent mode of action of CFX. The antibacterial activity of CFX decreased with reductions of pH, so the values of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum eradication bacterial concentration (MEBC) were increased when the pH of the culture medium decreased from 7.4 to 6.5 and 5.5. The kinetics of bacterial elimination of CFX presented a biphasic pattern because of the selection of a sub-population of persistent bacteria which presented a slower elimination rate. Therefore, the antibacterial activity of CFX was determined by its concentration in reference to MIC values and the time during which the exposure of the bacteria was maintained.