CARACTERIZACIÓN Y EVALUACIÓN in vivo E in vitro DEL LIPOPOLISACÁRIDO DE Aeromonas hydrophila / In Vitro/In Vivo Characterization And Evaluation Of Aeromonas hydrophila Lipopolysacharides (LPS)

A partir de una cepa de A. hydrophila aislada de un brote de enfermedad septicémica en Tilapia nilótica (Piaractus brachypomusoreochromis niloticus), se obtuvieron extractos de lipopolisacárido (LPS) crudo (29,5 mg/ml) y semipurificado (106,5 mg/ml) mediante la técnica fenol-agua caliente descrita por Westphal, Jann (1965). La presencia de proteína fue del 2,3% para el extracto crudo y de 0,1% para el semipurificado; la concentración de polisacáridos osciló entre el 15 y 26%. En electroforesis (SDS-PAGE) se observaron bandas de 14 Kd correspondientes al oligosacárido central y al lípido A del LPS. Tres ratones de 25-35 g fueron inoculados intraperitonealmente con 25 mg/Kg de LPS cru-do, a partir de la primera hora todos los animales mostraron erizamiento, taquipnea e inapetencia; microscópicamente se detectó congestión hepática y pulmonar, hemorragias pulmonares y renales, marginación leucocitaria en hígado y pulmón con predominio de polimorfo-nucleares neutrófilos (PMN) en todos los animales, mostrando un mayor efecto que el control inoculado con LPS de E. coli (Sigma®) a la misma concentración. In vitro el LPS crudo a concentración de 10, 20 y 30 µg/ml indujo proliferación de células mono-nucleares murinas (2 x 10 5 en 200 µl de medio DMEM) por incorporación de timidina tritiada; tanto el LPS control (E. coli), como el LPS crudo de A. hydrophila mostraron cuentas por minuto (CPM ) ascendentes de manera dosis dependiente, el LPS de A. hydrophila desencadeno una proliferación muy similar a la inducida por el control.

Aeromonas hydrophila isolated from a septicemic disease outbreak in tilapia (Piaractus brachypomusoreochromis niloticus) was used to prepare crude (29.5 mg/ml) and semipurified (106.5 mg/ml) lipopolysacharide extracts (LPS) based on the phenol-hot water protocol (Westphal and Jann, 1965). Protein contents were 2.3% and 0.1% for the crude and the semipurified extracts, respectively, while the polysacharides ranged from 15 to 26%. SDS-PAGE showed 14 Kd bands for the central oligosacharide and lipid A of the LPS. Mice (n=3) (25 ~ 35 g) that were intraperitoneally injected (25 mg crude LPS) showed after the first hour bristled fur, tachypnea and loss of appetite. Congestion (liver, lung), hemorrhages (kidney, lung), leukocytes margination -mainly PMN neutrophils-(liver, lung) were the most remarkable microscopic features. These effects were more evident than those found in controls injected with E. coli LPS (Sigma®). Crude LPS at 10, 20 and 30 mg/ml induced In vitro proliferation of murine mononuclear cells (2 x 105 in 200 ml DMEM) by use of tritiated thymidine. Both the crude A. hydrophila and the control E. coli LPS extracts showed dosed-dependent increasing counts per minute. The A. hydrophila LPS elicited a proliferation very similar to the one induced by the control.

Farmacocinética y penetración en líquido tisular de cefepime administrado en conejos hipertérmicos / Pharmacokinetics and penetration into tissue fluid of cefepime administered to hyperthermic rabbits

La farmacocinética sérica y tisular de cefepime administrado por vía endovenosa (20 mg/kg de peso) fue determinada en conejos sanos, con hipertermia producida por lipopolisacáridos de E. coli e implantados en tejido subcutáneo con cajas para recolección de líquido tisular. Diez conejos adultos fueron utilizados en dos experiencias (E1 y E2). Las concentraciones de cefepime en suero (S) y líquido tisular (LT) fueron determinadas por método biológico. Para el análisis cinético se utilizó un modelo no compartimental. Los resultados farmacocinéticos (medias ± error estándar) fueron: tiempo medio de eliminación [t1/2 (E1 S)=1,5±0,2 y (E2 S)=2,0±0,2 h] (p<0,05), área bajo la curva [ABC (E1 S)=181,6±17,5 y (E2 S)=192,3±18,5 (µg/mL/h]; Volumen de distribución en estado estacionario [Vss (E1S)=0,31±0,05 y (E2 S)=0,69±0,28 L/kg] (p<0,05); aclaramiento sérico [CL(E1 S)=118,3±17,7 y (E2 S) =93,1±19,9 (mL/h)kg] (p<0,05); Concentración máxima [Cmax (E1 LT)= 23,5±3,4 y (E2 LT)= 27,6±3,6 (µg/mL]; tiempo en el que se logra la Cmax [t max (E1 LT)=2,3±0,4 y (E2 LT)=1,7±0,4 h)]; t1/2 (E1 LT)= 2,4±0,3 y (E2 LT)=3,4±0,3 h (p<0,05); ABC (E1 LT)=122,0±12,7 y (E2 LT)=156,9±13,6 (µg/mL/h). y penetración [P (E1 LT)=67,3±8,7 y (E2 LT)=88,5±8,7%].

The pharmacokinetic characteristics in serum and tissue of cefepime given intravenously (20 mg/kg body weight) were assessed in healthy rabbits and in rabbits with hyperthermia induced by lipopolysacharide of E. coli, with tissue fluid cages implanted subcutaneously. Ten adult rabbits were used in two trials (E1 and E2). Cefepime concentrations in serum (S) and tissue cage fluid (LT) were determined by biological methods. The kinetic analysis was performed by means of a noncompartmental model. Pharmacokinetic results (means ± standard error): half life of elimination [t1/2 (E1 S)=1.5±0.2 and (E2 S)=2.0±0.2 h] (p<0.05), area under the curve [ABC (E1 S)=181.6±17.5 and (E2 S)= 192.3±18.5 (µg/mL/h]; volume of distribution at steady-state [Vss (E1 S)=0.31±0.05 and (E2 S)=0.69±0.28 L/kg] (p<0.05); total serum clearance [CL (E1 S)=118.3±17.7 and (E2 S)=93.1±19.9 (mL/h)kg] (p<0.05); maximum concentration [Cmax (E1 LT)=23.5±3.4 and (E2 LT)=27.6±3.6 (µg/mL]; time to reach Cmax [t max (E1 LT) =2.3±0.4 and (E2 LT)=1.7±0.4 h)]; t1/2 (E1 LT)=2.4±0.3 and (E2 LT)=3.4±0.3 h (p<0.05); ABC (E1 LT)=122.0±12.7 and (E2 LT)=156.9±13.6 (µg/mL/h); penetration [P (E1 LT)=67.3±8.7 and (E2 LT)=88.5±8.7%]. In conclusion, the pharmacokinetic changes of cefepime observed in rabbits with hyperthermia induced by lipopolysacharide, could be clinically significant if not taken into account when designing the dosing regimens.

Endotoxin Induced Uveitis Model

Endotoxin-induced uveitis model was produced in Lewis rats by footpad injection of Salmonella endotoxin(lipopolysaccharide, LPS). The clinical course and histological examination were observed at intervals of two hours. Immunohistochemical staining of intercellular adhesion molecule-1(ICAM-1) and lymphoyte function associated antigen-1(LFA-1) were also peformed. Initial intraocular inflammatory signs were observed at 8-10 hours after injection. Clinical and histological abnormalities peaked at 24-48 hours and were resolving after 72 hours. Histological changes were limited to anterior uvea. ICAM-1 expression was first noted on cells of iris and ciliary body and LFA-1 was expressed on infiltring inflammatory cells 8 hours after the injection and increased by 24 hours and disappeared after 72 hours. LPS induced uveitis in the rat provides a simple, reproducible model for anterior uveitis. ICAM-1 and LFA-1 expression in uveal tract are increased during early phase of uveitis and may enhance the adherence of inflammatory cells with the subsequent initiation of inflammation.