Ischemic preconditioning attenuates capillary no-reflow and leukocyte adherence in postischemic pancreatitis

Background and aims Ischemic preconditioning (IPC) has been shown to protect several organs from ischemia–reperfusion injury. Postischemic microvascular dysfunction is considered to be the key mechanism of early graft pancreatitis after transplantation. The aim of the study was to determine whether brief ischemia and reperfusion before prolonged ischemia followed by reperfusion is protective in respect to microcirculatory derangement in postischemic pancreatitis.Methods In an in-situ model of ischemia–reperfusion was induced in the isolated pancreatic tail segment. Wistar rats were randomized to one group (n=7/group) with 2-h ischemia and reperfusion (I/R) and another group with 10-min ischemia and 10-min reperfusion (IPC) before the prolonged ischemia time. Microcirculation was observed for 2 h by intravital-fluorescence microscopy that analyzed functional capillary density and leukocyte adherence in postcapillary venules. Histological damage was quantified by a semiquantitative score (edema, vacuolization, PMN infiltration, necrosis).Results IPC resulted in a significant improvement of functional capillary density (248±20 vs 372±8 cm^–1, P<0.001), a significant reduction in leukocyte adherence in postcapillary venules (476±79 vs 179±15 cells/mm², P<0.001) and in significantly lower histological damage (score 9±0.8 vs 5±1.4, P<0.001), when compared with the ischemia–reperfusion group.Conclusion IPC reduces pancreatic inflammatory reaction by preservation of postischemic microcirculation. Therefore, it might become a useful procedure before organ procurement in pancreas transplantation.     

Hypertonic saline attenuates end-organ damage in an experimental model of acute pancreatitis

BACKGROUND: Hypertonic saline (HTS) has been noted previously to reduce neutrophil activation. The aim of this study was to elucidate the effect of hypertonic resuscitation on the development of end-organ damage in an animal model of pancreatitis. METHODS: Pancreatitis was induced in Sprague-Dawley rats by intraperitoneal injection of 20 per cent L-arginine. Animals were randomized into four groups (each n = 8): controls; pancreatitis without intervention; pancreatitis plus intravenous resuscitation with normal saline (0.9 per cent sodium chloride 2 ml/kg) at 24 and 48 h; or HTS (7.5 per cent sodium chloride 2 ml/kg) at these time points. Pulmonary endothelial leakage was assessed by measurement of lung wet : dry ratios, bronchoalveolar lavage protein and myeloperoxidase activity. RESULTS: Animals that received HTS showed less pancreatic damage than those resuscitated with normal saline (1.0 versus 3.0; P = 0.04). Lung injury scores were also significantly diminished in the HTS group (1.0 versus 3.5; P = 0.03). Pulmonary neutrophil sequestration (myeloperoxidase activity 1.80 units/g) and increased endothelial permeability (bronchoalveolar lavage protein content 1287 microgram/ml) were evident in animals resuscitated with normal saline compared with HTS (1.22 units/g and 277 microgram/ml respectively; P < 0.02). CONCLUSION: HTS resuscitation results in a significant attenuation of end-organ injury following a systemic inflammatory response to severe pancreatitis.     

Interleukin-10 attenuates proinflammatory cytokine production and improves survival in lethal pancreatitis

Given that interleukin (IL)-10 (IL-10) serves as a potent down-regulator of specific proinflammatory cytokines we reasoned that its administration should improve outcome in situations in which the biological response to a severe inflammatory challenge is the critical determinant of survival. To test our hypothesis we administered IL-10 in the setting of lethal pancreatitis to determine its effect on proinflammatory cytokine production and survival. We divided Sprague-Dawley rats into three groups. Controls (Group 1, n = 5) received a sham laparotomy. We induced pancreatitis in Group 2 (n = 9) and Group 3 (n = 9) via laparotomy and intrapancreatic infiltration of one mL of 5 per cent sodium taurocholate. Group 2 was treated only with saline, whereas Group 3 was treated with 10,000 units of IL-10 (in saline) at 30 minutes, 3.5 hours, and 6.5 hours after induction of pancreatitis. Serial blood samples were obtained at 6.5 hours for measurement of amylase, IL-1, and IL-6. The Kaplan-Meier method, Wilcoxon test, and Student's t test were used for analysis. Seven-day survival was 100, 0, and 45 per cent in Groups 1, 2, and 3, respectively. Production of amylase, IL-1, and IL-6 was lower in the IL-10-treated group (Group 3) compared with the group treated with saline alone (Group 2, P < 0.05). We conclude that administration of IL-10 in the setting of otherwise 100 per cent lethal experimental pancreatitis significantly reduces production of amylase, IL-1, and IL-6 and improves survival.     

Effective ablation of pancreatic cancer cells in SCID mice using systemic adenoviral RIP-TK/GCV gene therapy

BACKGROUND: Studies have demonstrated that adenovirus subtype 5 mediated rat insulin promoter directed thymidine kinase (A-5-RIP-TK)/ganciclovir (GCV) gene therapy resulted in significant enhanced cytotoxicity to both PANC-1 and MIA PaCa2 pancreatic cancer cells in vitro. However, little is known about the effect in vivo. In this study we examine the in vivo safety and efficacy of intravenous A-5-RIP-TK/GCV gene therapy. MATERIALS AND METHODS: 1 x 10(6) Mia PaCa2 cells were injected intraperitoneally (i.p.) into SCID mice to create a mouse model of human pancreatic cancer. A-5-RIP-TK gene construct was administered intravenously (i.v.), followed by i.p. GCV administration. Intravenous injection of A-5-RIP-lacZ reporter gene constructs was used for evaluation of Ad-RIP-gene expression in tumors and other tissues. Optimal adenoviral and GCV doses and treatment duration were determined. Tumor volume, serum insulin, and glucose levels were measured. Immunohistochemical staining of pancreata and tumors were performed to assess morphology and hormone expression and apoptotic rates were determined. RESULTS: All A-5-RIP-TK/GCV-treated mice had reduced tumor volume compared with controls, but maximal tumor volume reduction was observed with 10(8) vp followed by GCV treatment for 4 wk. A-5-RIP-TK/GCV gene therapy contributed to significant survival advantage in MIA PaCa2 bearing mice, and the greatest survival benefit was observed with 10(8) vp and was not affected by length of treatment of GCV. A-5-RIP-TK/GCV therapy increased PDX-1 expression and tumor cells apoptosis, and altered islet morphology. However, A-5-RIP-TK/GCV gene therapy caused diabetes associated with islet cell apoptosis, increased delta-cells and reduced pancreatic polypeptide (PP)-cell numbers. CONCLUSIONS: Systemically administered A-5-RIP-TK/GCV is an effective treatment of pancreatic cancer. A-5-RIP-TK/GCV cytotoxicity to malignant cells varies with adenoviral dose and length of GCV treatment. However, A-5-RIP-TK/GCV is associated with islet cell toxicity and diabetogenesis. The type of diabetes observed is distinct from Types 1 and 2 and is associated with islet cell apoptosis and reduced delta- and PP-cells.     

区域动脉灌注5-FU改善大鼠重症急性胰腺炎相关的肺损伤

目的 研究区域动脉灌注（regional arterial infusion,RAI）5-氟尿嘧啶（5-fluorouracil,5-FU）对大鼠重症急性胰腺炎（severe acute pancreatitis,SAP）相关的急性肺损伤（acute lung injury, ALI）的作用及其可能的机制。方法 36只健康成年雄性SD大鼠随机分成3组：对照组（C组）、胰腺炎组（SAP组）、区域动脉灌注5-FU治疗组（5-FU组）。逆行胰胆管注射5%牛磺胆酸钠（1 mL/kg）建立SAP模型。5-FU组在诱导SAP模型后立即行区域动脉灌注5-FU（40 mg/kg）治疗,C组和SAP组区域动脉灌注等量的生理盐水。建模成功后分别于12、24 h取标本并处死大鼠,胰腺和肺脏组织送病理学检查,检测肺组织湿干比,肺组织髓过氧化物酶（myeloperoxidase,MPO）活性,血淀粉酶和血中TNF-α、IL-1β、IL-6的含量。结果 与C组比较,SAP组与5-FU组血淀粉酶、血中促炎细胞因子（TNF-α、IL-1β、IL-6）、肺组织湿干比、肺组织MPO均显著升高（P＜0.05）;与SAP组比较,5-FU组上述指标均显著下降（P＜0．05）。光镜下可见SAP组出现明显的肺损伤,5-FU区域灌注治疗后肺损伤减轻,病理学评分下降。结论 区域动脉灌注5-FU对大鼠重症急性胰腺炎相关的急性肺损伤有改善作用,其作用机制可能与抑制促炎细胞因子的过度表达有关。     

In vitro and in vivo suppression of osteoclast function by adenovirus vector-induced csk gene.

The proto-oncogene c-src, which encodes a non-receptor-type tyrosine kinase c-Src, has been shown to be essential for osteoclastic bone resorption by the finding that the targeted disruption of the c-src gene induced osteopetrosis in mice. The csk (C-terminal Src family kinase) gene encodes a cytoplasmic protein-tyrosine kinase that specifically phosphorylates the negative regulatory site of c-Src (Tyr-527), thereby inhibiting its kinase activity. To regulate osteoclast function by modulating the kinase activity of c-Src, we constructed an adenovirus vector that carries this gene. The recombinant adenovirus vector carrying csk cDNA induced Csk expression in mouse osteoclast-like cells formed in vitro and clearly reduced c-Src kinase activity in a dose-dependent manner. The expression of Csk caused cytoskeletal disorganization of osteoclast-like cells and strongly suppressed pit-forming activity of the cells in vitro. In addition, the viral vector carrying csk gene dramatically suppressed interleukin-1 alpha-induced bone resorption in vivo. Conversely, kinase-inactive Csk caused an increase in c-Src kinase activity and bone resorbing activity of the cells both in vitro and in vivo, acting as a dominant negative molecule against intrinsic Csk. These findings indicate that the inhibition of c-Src activity by adenovirus vector-mediated csk expression offers an efficient means for inhibiting pathological bone resorption by suppressing osteoclast function.     

Macrophage elimination increases bacterial translocation and gut-origin septicemia but attenuates symptoms and mortality rate in a model of systemic inflammation.

OBJECTIVE: The central question tested in this study was whether dichloromethylene-diphosphonate (CL2MDP) liposome-mediated elimination of hepatic and splenic macrophages would influence zymosan-induced bacterial translocation and the zymosan-induced generalized inflammatory response. SUMMARY BACKGROUND DATA: Both an uncontrolled activation of macrophages and the loss of intestinal barrier function have been implicated in the development of adult respiratory distress syndrome and multiple organ failure. METHODS: Macrophage elimination was accomplished by intravenous injection of 200 microL of CL2MDP-liposome suspension. Control mice received an intravenous injection of 200 microL of phosphate-buffered saline. Two days later, the animals were challenged intraperitoneally with zymosan suspended in paraffin to determine a dose-response curve (0.1, 0.5, or 1.0 mg/g body weight). Twenty-four hours after zymosan challenge, signs of systemic stress were determined, and bacterial translocation to the mesenteric lymph node, liver, spleen, and blood was measured. A separate mortality study was performed with a dose of 1.0 mg/g of zymosan suspension. RESULTS: The incidence of the systemic spread of bacteria was significantly increased in the macrophage-depleted mice. Although systemic bacterial translocation was promoted by macrophage elimination, the systemic toxic response was significantly decreased in all macrophage-depleted groups (p < or = 0.01). The 12-day mortality rate was 0% in the macrophage-depleted groups and 27% in the control group (p = 0.05). CONCLUSIONS: The lethal and toxic effects of zymosan appear to be related more to the excessive activation of macrophages than to the systemic spread of bacteria.     

Molecular biology of adenoviral vectors

Gene therapy is based on the use of DNA as a therapeutic material as an alternative therapeutic tool for treatment of human diseases. All proteins are codified into the DNA and several diseases result from the absence or aberrant expression of one or related genes, absence of expression of functional proteins, and alterations for regulation process in transport and degradation mechanisms. In this regard, several diseases could be potentially treated through the expression of the normal form of the involved protein. However, the main objective is to achieve a successful genetic material delivery into the target site and avoid the destruction of DNA or the selected vehicle before arrival at the final destination. Several efficient viral gene transfer systems have been developed. Viral-mediated gene delivery for experimental models has been designed from herpes virus (HV), adenovirus (adenovirous), adeno-associated virus (AAV) and retroviruses (lentiviral vectors). In this review we will discuss the specific biological and cloning properties of adenoviral vectors as a gene transfer tool and potential medical implications for gene therapy.     

Oncolytic adenoviral therapy in gallbladder carcinoma

BACKGROUND: Oncolytic adenoviral therapy is a promising new approach for cancer treatment. The aim of this study was to improve the conditionally replicative adenoviruses (CRAds) for gallbladder cancer therapy by modifying the fiber-knob region for infectivity enhancement and by incorporating tumor-specific promoters (TSPs) for enhanced specificity. METHODS: For promoter-controlled replication, in vitro efficacy of eight TSPs was investigated in two gallbladder cancer cell lines (NOZ and OCUG-1). Infectivity enhancement was analyzed by two different fiber modifications: Arg-Gly-Asp (RGD) incorporation into the HI loop (RGD modification) and a chimeric construct with a serotype 5 shaft and a serotype 3 knob (5/3 fiber modification). Comparisons were made by infectivity analysis and cytotoxicity assays in vitro, followed by tumor suppressive effects tested in vivo. RESULTS: Among TSPs, highest potency was exhibited by the cyclooxygenase-2 (COX-2), Midkine, and vascular endothelial growth factor promoters in both cell lines tested. Fiber chimera (Ad5/3Luc1) conferred significant enhancement of Ad infectivity in comparison with unmodified and RGD-modified vectors. COX-2 CRAds demonstrated selective cytocidal effect in gallbladder cancer cells in vitro. COX-2 promoter-based Ad5/3 CRAds showed significantly enhanced tumor-suppressive effect compared with nonreplicative and RGD-modified CRAd vectors in vivo. CONCLUSIONS: The 5/3 fiber-modified, COX-2 promoter-driven CRAds may prove to be a new agent for the treatment of gallbladder carcinoma.     

Blockade of Macrophage Colony-Stimulating Factor Reduces Macrophage Proliferation and Accumulation in Renal Allograft Rejection

Macrophage accumulation within an acutely rejecting allograft occurs by recruitment and local proliferation. To determine the importance of M-CSF in driving macrophage proliferation during acute rejection, we blocked the M-CSF receptor, c-fms, in a mouse model of acute renal allograft rejection. C57BL/6 mouse kidneys (allografts, n = 20) or BALB/c kidneys (isografts, n = 5) were transplanted into BALB/c mice. Anti-c-fms antibody (AFS98) or control Ig (50 mg/kg/day, i.p.) was given daily to allografts from days 0-5. All mice were killed day 6 postoperatively. Expression of the M-CSF receptor, c-fms, was restricted to infiltrating CD68+ macrophages. Blockade of c-fms reduced proliferating (CD68+/BrdU+) macrophages by 82% (1.1 v 6.2%, p < 0.001), interstitial CD68+ macrophage accumulation by 53% (595 v 1270/mm2, p < 0.001), and glomerular CD68+ macrophage accumulation by 71% (0.73 V 2.48 CD68+ cells per glomerulus, p < 0.001). Parameters of T-cell involvement (intragraft CD4+, CD8+ and CD25+ lymphocyte numbers) were not affected. The severity of tubulointerstitial rejection was reduced in the treatment group as shown by decreased tubulitis and tubular cell proliferation. Macrophage proliferation during acute allograft rejection is dependent on the interaction of M-CSF with its receptor c-fms. This pathway plays a significant and specific role in the accumulation of macrophages within a rejecting renal allograft.