Regulation of extracellular ATP of human erythrocytes treated with α-hemolysin. Effects of cell volume, morphology, rheology and hemolysis.

Alpha-hemolysin (HlyA) of uropathogenic strains of Escherichia coli irreversibly binds to human erythrocytes (RBCs) and triggers activation of ATP release and metabolic changes ultimately leading to hemolysis. We studied the regulation of extracellular ATP (ATPe) of RBCs exposed to HlyA. Luminometry was used to assess ATP release and ATPe hydrolysis, whereas changes in cell volume and morphology were determined by electrical impedance, ektacytometry and aggregometry. Exposure of RBCs to HlyA induced a strong increase of [ATPe] (3-36-fold) and hemolysis (1-44-fold), partially compensated by [ATPe] hydrolysis by ectoATPases and intracellular ATPases released by dead cells. Carbenoxolone, a pannexin 1 inhibitor, partially inhibited ATP release (43-67%). The un-acylated toxin ProHlyA and the deletion analog HlyA∆914-936 were unable to induce ATP release or hemolysis. For HlyA treated RBCs, a data driven mathematical model showed that simultaneous lytic and non-lytic release mainly governed ATPe kinetics, while ATPe hydrolysis became important after prolonged toxin exposure. HlyA induced a 1.5-fold swelling, while blocking this swelling reduced ATP release by 77%. Blocking ATPe activation of purinergic P2X receptors reduced swelling by 60-80%. HlyA-RBCs showed an acute 1.3-2.2-fold increase of Ca2+i, increased crenation and externalization of phosphatidylserine. Perfusion of HlyA-RBCs through adhesion platforms showed strong adhesion to activated HMEC cells, followed by rapid detachment. HlyA exposed RBCs exhibited increased sphericity under osmotic stress, reduced elongation under shear stress, and very low aggregation in viscous media. Overall results showed that HlyA-RBCs displayed activated ATP release, high but weak adhesivity, low deformability and aggregability and high sphericity.

Análisis comparativo de anticuerpos monoclonales en la respuesta inflamatoria pulmonar, en un modelo experimental de ventilación mecánica / Comparative analysis of monoclonal antibodies in the pulmonary inflammatory response, in an experimental model of mechanic ventilation

Objetivo: Evaluar si el pretratamiento con anticuerpos monoclonales antiFNTα y anti-IL-6, administrados de manera independiente, atenúa el daño pulmonar en un modelo experimental de lesión pulmonar inducida por la ventilación mecánica. Materiales y Métodos: Se utilizaron 24 ratas Wistar que fueron separadas en cuatro grupos experimentales: 1) bajo Vt (n = 6): Vt 7 ml/kg, PEEP 5 cmH2O, 2) alto Vt (n = 6): Vt 25 ml/kg, ZEEP (PEEP = 0), 3) anti-IL-6 (n = 6): Vt 25 ml/kg, ZEEP y 30 mg/kg de tocilizumab intraperitoneal 24 h antes de la ventilación mecánica, 4) anti-FNTα (n = 6): Vt 25 ml/kg, ZEEP y 100 ug/kg de adalimumab intraperitoneal 24 h antes de la ventilación mecánica. Se evaluaron el daño histológico cuantificado según el puntaje reportado por Villar et al y la hemodinamia medida con la presión arterial media. Los datos fueron analizados con ANOVA y las pruebas de comparaciones múltiples de Dunn y de Tukey. Resultados: En el grupo tratado con anti-FNTα y en los animales tratados con anti-IL-6, se observó un menor daño histológico pulmonar que en el resto de los grupos. Por otro lado, no se encontraron diferencias en la mecánica pulmonar y en la presión arterial media entre grupos. Conclusiones: Bajo estas condiciones experimentales, los anticuerpos monoclonales anti-FNTα y anti-IL-6 mostraron efectos protectores a nivel pulmonar, lo que postula a estas drogas como estrategias promisorias para atenuar la lesión pulmonar inducida por la ventilación mecánica.(AU)

Objective: To evaluate whether pretreatment with monoclonal antibodies, anti- TNFα and anti-IL-6 administered independently attenuates lung damage in an experimental model of ventilator-induced lung injury. Materials and Methods: Twenty-four Wistar rats were used. Animals were divided into four experimental groups: 1) low Vt (n = 6): Vt 7 mL/kg, PEEP 5 cmH2O; 2) high Vt (n = 6): Vt 25 mL/kg, ZEEP (PEEP = 0); 3) anti-IL-6 (n = 6): Vt 25 mL/kg, ZEEP, and intraperitoneal tocilizumab 30 mg/kg, 24 hours prior to mechanical ventilation; 4) anti-TNFα (n = 6): Vt 25 mL/kg, ZEEP, and intraperitoneal adalimumab 100 µg/kg, 24 hours before the VM. Histological damage measured by Villar score, and hemodynamics measured with mean arterial pressure were evaluated. Data were analyzed using ANOVA, Dunn's multiple comparison test and Tukey's multiple comparison test. Results: In groups treated with anti-TNFα and anti-IL-6, less histological damage was observed in comparison with the rest of groups. On the other hand, no statistically significant differences were found in pulmonary mechanics and mean arterial pressure among groups. Conclusions: Under these experimental conditions, monoclonal antibodies anti-TNFα and anti-IL-6 showed protective effects on lungs, indicating that these drugs are promising strategies to attenuate ventilator-induced lung injury (AU)

Pretreatment Combination Reduces Remote Organ Damage Secondary to Intestinal Reperfusion Injury in Mice: Follow-up Study.

BACKGROUND: Intestinal ischemia-reperfusion injury occurs after different surgical treatments, including intestinal transplantation. This harmful process may have an effect in remote organs, leading to multiple organ dysfunction syndrome and death. Therefore, to establish strategies to attenuate local and remote damage constitutes a challenge for experimental and clinical surgeons in the intestinal surgical field. METHODS: We evaluated the effect of ischemic preconditioning and tacrolimus pretreatment applied alone and in combination against local and remote damage caused by prolonged intestinal ischemia-reperfusion injury in a mouse model of warm ischemia. RESULTS: Ischemic preconditioning applied alone and in combination with tacrolimus decreased histological damage (P < .05), number of apoptotic cells (P < .05), nitrosative stress (P < .01), and serum lactate dehydrogenase activity (P < .05) and lowered uremia (P < .05) compared with untreated post-reperfused intestines. Regarding remote organ damage, combination therapy was the unique condition able to attenuate lung (mainly neutrophil infiltration and hemorrhage), liver (sinusoidal congestion and hepatic vacuolization), and kidney (acute tubular necrosis and hydropic degeneration) histological alterations (P < .05), compared with the untreated group. CONCLUSIONS: These results support the application of these strategies in combination to minimize the impact of ischemia-reperfusion injury in the whole organism as a strategy to prevent multiple organ dysfunction syndromes and minimize the clinical impact.

Ischemic preconditioning and tacrolimus pretreatment as strategies to attenuate intestinal ischemia-reperfusion injury in mice.

The intestine is highly sensitive to ischemia-reperfusion injury (IRI), a phenomenon occurring in different intestinal diseases. Several strategies to mitigate IRI are in experimental stages; unfortunately, no consensus has been reached about the most appropriate one. We report a protocol to study ischemic preconditioning (IPC) evaluation in mice and to combine IPC and tacrolimus (TAC) pretreatment in a warm ischemia model. Mice were divided into treated (IPC, TAC, and IPC + TAC) and untreated groups before intestinal ischemia. IPC, TAC, and IPC + TAC groups were able to decrease postreperfusion nitrites levels (P < .05). IPC-containing groups had a major beneficial effect by preserving the integrity of the intestinal histology (P < .05) and improving animal survival (P < .002) compared with TAC alone or the untreated group. The IPC + TAC group was the only one that showed significant improvement in lung histological analysis (P < .05). The TAC and IPC + TAC groups down-regulated intestinal expression of interleukin (II)-6 and IL1b more than 10-fold compared with the control group. Although IPC and TAC alone reduced intestinal IRI, the used of a combined therapy produced the most significant results in all the local and distant evaluated parameters.

Amelioration of renal damage by administration of anti-thymocyte globulin to potential donors in a brain death rat model.

Brain death (BD), a non-immunological factor of renal injury, triggers an inflammatory process causing pathological signs of cell death in the kidney, such as necrosis and apoptosis. Kidneys from brain dead donors show lower success rates than kidneys from living donors and one strategy to improve transplantation outcome is to precondition the donors. For the first time, anti-rat thymoglobulin (rATG) was administered in an experimental brain death animal model to evaluate if it could ameliorate histopathological damage and improve organ function. Animals were divided into three groups: V (n=5) ventilated for 2h; BD (n=5) brain death and ventilated for 2h; and BD+rATG (n=5) brain death, ventilated for 2h, rATG was administered during brain death (10mg/kg). We observed lower creatinine levels in treatment groups (means): V, 0·88±0·22 mg/dl; BD, 1·37±0·07 mg/dl; and BD+rATG, 0·64±0·02 mg/dl (BD versus BD+rATG, P<0·001). In the BD group there appeared to be a marked increase of ATN, whereas ATN was decreased significantly in the rATG group (V, 2·25±0·5 versus BD, 4·75±0·5, P<0·01; BD+rATG, 2·75±0·5 versus BD 4·75±0·5 P<0·01). Gene expression was evaluated with reverse transcription-polymerase chain reaction; tumour necrosis factor (TNF)-α, interleukin (IL)-6, C3, CD86 showed no significant difference between groups. Increased IL-10 and decreased CCL2 in BD+rATG compared to BD (both cases P<0·01). Myeloperoxidase was increased significantly after the brain death setting (V: 32±7·5 versus BD: 129±18). Findings suggest that rATG administered to potential donors may ameliorate renal damage caused by BD. These findings could contribute in the search for specific cytoprotective interventions to improve the quality and viability of transplanted organs.

Defining the nonreturn time for intestinal ischemia reperfusion injury in mice.

Among the abdominal organs, the intestine is probably the most sensitive to ischemia reperfusion injury (IRI), a phenomenon that occurs in many intestinal disorders. Few studies have reported in detail the impact of intestinal ischemia time in mice. We evaluated the effect of various warm intestinal ischemia times in an intestinal IRI model in mice. Adult male Balb/c mice were divided into 4 groups that differed in intestinal ischemia time: G1, 30; minutes; G2, 35 minutes; G3, 40 minutes; and G4, 45 minutes. Histological evaluation showed average Park scores as follows: G1 0.6 ± 0.55; G2 1.8 ± 0.45; G3 4.8 ± 2.25; and G4 5 ± 1.79. All animals from G1 survived 30 hours. G2 animals showed intermediate behavior with all succumbing between 18 and 30 hours postprocedure. G3 and G4 displayed similar survival results with animals succumbing before 6 hours after intestinal reperfusion. These data showed that Park index scores of 3 or higher were related to early death. We concluded that the 5 minutes between 35 and 40 minutes is the critical limit, after which all mice die after reperfusion. This result may represent a valuable tool for future research in mice.

Preconditioning donor with a combination of tacrolimus and rapamacyn to decrease ischaemia-reperfusion injury in a rat syngenic kidney transplantation model.

Reperfusion injury remains one of the major problems in transplantation. Repair from ischaemic acute renal failure (ARF) involves stimulation of tubular epithelial cell proliferation. The aim of this exploratory study was to evaluate the effects of preconditioning donor animals with rapamycin and tacrolimus to prevent ischaemia-reperfusion (I/R) injury. Twelve hours before nephrectomy, the donor animals received immunosuppressive drugs. The animals were divided into four groups, as follows: group 1 control: no treatment; group 2: rapamycin (2 mg/kg); group 3 FK506 (0, 3 mg/kg); and group 4: FK506 (0, 3 mg/kg) plus rapamycin (2 mg/kg). The left kidney was removed and after 3 h of cold ischaemia, the graft was transplanted. Twenty-four hours after transplant, the kidney was recovered for histological analysis and cytokine expression. Preconditioning treatment with rapamycin or tacrolimus significantly reduced blood urea nitrogen and creatinine compared with control [blood urea nitrogen (BUN): P < 0·001 versus control and creatinine: P < 0·001 versus control]. A further decrease was observed when rapamycin was combined with tacrolimus. Acute tubular necrosis was decreased significantly in donors treated with immunosuppressants compared with the control group (P < 0·001 versus control). Moreover, the number of apoptotic nuclei in the control group was higher compared with the treated groups (P < 0·001 versus control). Surprisingly, only rapamycin preconditioning treatment increased anti-apoptotic Bcl2 levels (P < 0·001). Finally, inflammatory cytokines, such as tumour necrosis factor (TNF)-α and interleukin (IL)-6, showed lower levels in the graft of those animals that had been pretreated with rapamycin or tacrolimus. This exploratory study demonstrates that preconditioning donor animals with rapamycin or tacrolimus improves clinical outcomes and reduce necrosis and apoptosis in kidney I/R injury.