Neutrophil response to Neisseria meningitidis: inhibition of adhesion molecule expression and phagocytosis by recombinant bactericidal/permeability-increasing protein (rBPI21)

Polymorphonuclear neutrophil (PMNL) activation enhances microbial clearance but also contributes to the vascular damage and multiorgan failure associated with severe meningococcal sepsis. By use of a whole blood model of meningococcal bacteremia, loss of PMNL L-selectin and up-regulation of CD11b was observed in response to Neisseria meningitidis serogroups B and C, which is followed by opsonophagocytosis. PMNL priming with either Escherichia coli lipopolysaccharide (LPS) or FMLP prior to meningococcal challenge resulted in enhancement of both PMNL L-selectin shedding (1.5- to 4-fold) and phagocytosis (2- to 3-fold). Blockade of meningococcal LPS lipid A with recombinant bactericidal/permeability-increasing protein (rBPI21) resulted in partial inhibition of the PMNL activation and phagocytosis response to N. meningitidis. The effect of rBPI21 was reversed by excess E. coli LPS or FMLP. It is proposed that PMNL priming by N. meningitidis results in an exaggerated activation and phagocytosis response to the organism.     

Alterations of Notch/Jagged mRNA and protein expression after partial hepatectomy in rats

Objective. To investigate alterations of tumor necrosis factor (TNF)-α, interleukin (IL)-6, the Notch/Jagged signaling pathway, and hepatocyte proliferation, and their correlation in rats subjected to partial hepatectomy. Material and methods. Blood and liver tissues were sampled immediately (0), 5, 15, 30, 60, 180, 360, and 720 min, and 1, 2, 4, and 7 days after partial (70%) hepatectomy to measure plasma levels of TNF-α and IL-6, the expression of proliferating cell nuclear antigen (PCNA), Notch-1 and Jagged-1 protein, and mRNA in liver tissue. Results. Systemic levels of IL-6 significantly correlated with the expression of PCNA in hepatocytes (p<0.05) after partial (70%) hepatectomy. The increased expression of Notch-1 protein was located in the sinusoidal endothelium, while Jagged-1 protein was predominantly located in bile duct cells. Expression of both proteins was increased around the portal vein. mRNA expression of Notch-1 decreased between 6 h and 2 days after partial hepatectomy, while Jagged-1 increased between 3 and 6 h, decreased between 12 h and 2 days, and normalized on day 4 after hepatectomy. Conclusions. Partial hepatectomy-induced hepatocyte proliferation was accompanied by an increase in systemic levels of IL-6, a pro-inflammatory response that plays a part in the proliferative process. The Notch/Jagged signaling pathways are activated and probably represent important contributors to the regenerative process following hepatectomy including both hepatocyte proliferation and bile duct formation and growth.     

Anti-neutrophil cytoplasmic autoantibodies (ANCA) to bactericidal/permeability-increasing (BPI) protein recognize the carboxyl terminal domain.

OBJECTIVES: to identify the region of bactericidal/permeability-increasing protein (BPI) recognized by anti-BPI ANCA. METHODS: sera from 140 patients with a variety of clinical diagnoses (20 systemic vasculitis, 12 cystic fibrosis, 22 bronchiectasis/chronic obstructive airways disease, three diabetes mellitus, 13 chronic renal failure, 12 primary sclerosing cholangitis, eight ulcerative colitis, three Crohn's disease, seven cancer, and 40 other or unknown diagnoses) known to be reactive against native (nBPI), were screened by solid phase enzyme linked immunosorbent assay (ELISA) against a panel of recombinant fusion proteins; holo BPI (rBPI), recombinant lipopolysaccharide binding protein (rLBP), an N-terminal fragment of rBPI (rBPI21 ) and 'fusion' proteins containing the C- or N-terminal ends of BPI spliced with N-or C-ends of LBP, respectively. RESULTS: a strong correlation was seen between the degree of reactivity to rBPI and the BPI C-terminal fusion protein, r=0.69, P < 0.001, as well as between nBPI and rBPI protein, r=0.55, P < 0.001, but not between nBPI and the N-terminal region of BPI (rBPI21), or proteins containing only the N-terminal fragment. Binding to proteins containing the BPI C-terminus was confirmed to be specific by fluid phase inhibition ELISA and Western blot analyses. CONCLUSIONS: together these data suggest that circulating autoantibodies to BPI from patients with different diseases recognize the C-terminal region of BPI.     

Antineutrophil cytoplasm autoantibodies against bactericidal/permeability-increasing protein in inflammatory bowel disease.

BACKGROUND: Bactericidal/permeability-increasing protein (BPI), a constituent of primary neutrophil granules, is a potent natural antibiotic and an antineutrophil cytoplasm antibody (ANCA) antigen in cases of vasculitis in which the target antigen is neither myeloperoxidase (MPO) nor proteinase-3 (PR3). AIM: To investigate BPI as a possible target antigen for ANCAs in inflammatory bowel disease. METHODS: ANCAs were detected by routine immunofluorescence (IIF) and solid phase enzyme linked immunosorbent assay (ELISA) performed for antibodies to the purified neutrophil granule proteins; MPO, PR3, cathepsin-G, lactoferrin, and BPI in serum samples from 88 patients with inflammatory bowel disease (36 with Crohn's disease, 52 with ulcerative colitis). Thirty patients with bacterial enteritis acted as controls. RESULTS: Significantly more patients with ulcerative colitis were ANCA positive by IIF (60%) than patients with Crohn's disease (28%) or infectious enteritis (23%) (p < 0.001). IgG anti-BPI antibodies were present in 29% of patients with ulcerative colitis, 14% of patients with Crohn's disease, and 23% of patients with infectious enteritis, occurring in 44% of those patients with inflammatory bowel disease who were ANCA positive by IIF. Antibodies to other ANCA antigens were rare. The presence of ANCAs was not related to either disease activity or extent; presence of anti-BPI antibodies was significantly related to both a lower serum albumin concentration (p = 0.001) and a higher erythrocyte sedimentation rate (p = 0.02) in patients with ulcerative colitis, and to colonic involvement in patients with Crohn's disease (p = 0.01). CONCLUSION: BPI is a significant minority target antigen for ANCAs in inflammatory bowel disease that seems related to colonic Crohn's disease and disease activity in ulcerative colitis. Anti-BPI antibodies occur in infectious enteritis.     

Provocation of massive hepatic necrosis by endotoxin after partial hepatectomy in rats

When rats received endotoxin 48 hours after two-thirds liver resection, 50% of them died within 12 hours with massive hepatic necrosis at a dose that did not affect sham-operated rats. In the hepatic sinusoids, fibrin deposition and endothelial cell destruction occurred 5 hours after endotoxin administration. When antithrombin III concentrate was infused concomitantly with endotoxin administration, all rats survived 12 hours, and the extent of hepatic necrosis and the deranged serum glutamic pyruvic transaminase values were significantly attenuated at 5 hours compared with those in the control rats. Similar improvements in the incidence of mortality and liver injury were observed after treatment with gum arabic before hepatectomy. The stimulatory state of Kupffer cells based on the ability to produce superoxide anions estimated by formazan deposition after liver perfusion with nitro blue tetrazolium and phorbol myristate acetate was increased between 24 and 72 hours after operation. This increase disappeared after gum arabic treatment. It is concluded that massive hepatic necrosis can occur as a result of sinusoidal fibrin deposition provoked by endotoxin in partially hepatectomized rats. Activated Kupffer cells may contribute to this provocation.     

Liver structural transformation after partial hepatectomy and repeated partial hepatectomy in rats: A renewed view on liver regeneration

BACKGROUND The phenomenon of liver regeneration after partial hepatectomy(PH) is still a subject of considerable interest due to the increasing frequency of half liver transplantation on the one hand, and on the other hand, new surgical approaches which allow removal of massive space-occupying hepatic tumors, which earlier was considered as inoperable. Interestingly, the mechanisms of liver regeneration are extensively studied after PH but less attention is paid to the architectonics of the regenerated organ. Because of this, the question "How does the structure of regenerated liver differ from normal, regular liver?" has not been fully answered yet. Furthermore, almost without any attention is left the liver's structural transformation after repeated hepatectomy(of the re-regenereted liver).To compare the architectonics of the lobules and circulatory bed of normal, regenerated and re-regenerated livers.METHODS The livers of 40 adult, male, albino Wistar rats were studied. 14 rats were subjected to PH-the 1st study group(SG_1); 10 rats underwent repeated PH – the 2nd study group(SG2); 16 rats were subjected to sham operation-control group(CG); The livers were studied after 9 months from PH, and after 6 months from repeated PH. Cytological(Schiff reaction for the determination of DNA concentration), histological(HE, Masson trichrome, CK8 Immunohistochemical marker, transparent slides after Indian Ink injection,), morphometrical(hepatocytes areas, perimeters and ploidy) and Electron Microscopical(Scanning Electron Microscopy of corrosion casts) methods were used.RESULTS In the SG_1 and SG_2, the area of hepatocytes and their perimeter are increased compared to the CG(P 0.05). However, the areas and perimeters of the hepatocytes of the SG_1 and SG_2 groups reveal a lesser difference. In regenerated(SG_1) and re-regenerated(SG_2) livers, the hepatocytes form the remodeled lobules, which size(300-1200 μm) exceeds the sizes of the lobules from CG(300-600 μm). The remodeled lobules(especially the "mega-lobules" with the sizes 1000-1200 μm) contain the transformed meshworks of the sinusoids, the part of which is dilated asymmetrically. This meshwork might have originated from the several portal venules(interlobular and/or inlet). The boundaries between the adjacent lobules(including mega-lobules) are widened and filled by connective tissue fibers, which gives the liver parenchyma a nodular look. In SG_2 the unevenness of sinusoid diameters, as well as the boundaries between the lobules(including the mega-lobules) are more vividly expressed in comparison with SG_1. The liver tissue of both SG_1 and SG_2 is featured by the slightly expressed ductular reaction.CONCLUSION Regenerated and re-regenerated livers in comparison with normal liver contain hypertrophied hepatocytes with increased ploidy which together with transformed sinusoidal and biliary meshworks form the remodeled lobulli.     

Monocyte tissue factor induction by lipopolysaccharide (LPS): dependence on LPS-binding protein and CD14, and inhibition by a recombinant fragment of bactericidal/permeability-increasing protein

Mononuclear phagocytes, stimulated by bacterial lipopolysaccharide (LPS), have been implicated in the activation of coagulation in sepsis and endotoxemia. In monocytes LPS induces the synthesis of tissue factor (TF) which, assembled with factor VII, initiates the blood coagulation cascades. In this study we investigated the mechanism of LPS recognition by monocytes, and the consequent expression of TF mRNA and TF activity. We also studied the inhibition of these effects of LPS by rBPI23, a 23-kD recombinant fragment of bactericidal/permeability increasing protein, which has been shown to antagonize LPS in vitro and in vivo. Human peripheral blood mononuclear cells, or monocytes isolated by adherence, were stimulated with Escherichia coli O113 LPS at physiologically relevant concentrations (> or = 10 pg/mL). The effect of LPS was dependent on the presence of the serum protein LBP (lipopolysaccharide-binding protein), as shown by the potentiating effect of human recombinant LBP or serum. Furthermore, recognition of low amounts of LPS by monocytes was also dependent on CD14 receptors, because monoclonal antibodies against CD14 greatly reduced the LPS sensitivity of monocytes in the presence of serum or rLBP. Induction of TF activity and mRNA expression by LPS were inhibited by rBPI23. The expression of tumor necrosis factor showed qualitatively similar changes. Considering the involvement of LPS-induced TF in the potentially lethal intravascular coagulation in sepsis, inhibition of TF induction by rBPI23 may be of therapeutic benefit.     

Ethanol reduces p38 kinase activation and cyclin D1 protein expression after partial hepatectomy in rats

BACKGROUND/AIMS: Chronic ethanol consumption inhibits liver regeneration. We examined the effects of chronic ethanol consumption on two mitogen-activated protein kinases in relation to induction of cell cycle proteins after partial hepatectomy (PH). METHODS: Male Wistar rats were ethanol-fed (EF) or pair-fed (PF) for 16 weeks before PH. Hepatic activation of extracellular signal regulated kinase (ERK)1/2, p38 kinase and expression of cyclinD1, cyclin-dependent kinase-4 (cdk4) and proliferating cell nuclear antigen (PCNA) were studied. RESULTS: In PF rats, PH-induced p38 activation was evident at 2h and was maximal at 12h. There was a close temporal relationship between p38 activation, cyclin D1 and PCNA expression. Alcohol exposure reduced p38 activation, cyclin D1 and PCNA, each by approximately 50%. ERK1/2 activation occurred during the first 2h post-PH in both EF and PF rats, and there was no later increase in PF rats. In vivo inhibition of p38 suppressed PCNA expression whereas the effect of ERK1/2 inhibition was inconsistent. CONCLUSIONS: p38 kinase activation is linked temporally with cyclin D1 expression after PH and appears to exert cell cycle control in the adult liver. p38 signaling also appears to be a target for the inhibitory effect of chronic alcohol on liver regeneration.     

Amelioration of hepatic ischemia/reperfusion injury in the remnant liver after partial hepatectomy in rats

BACKGROUND AND AIMS: Reactive oxygen species have been implicated in the development of hepatic ischemia/reperfusion (I/R) injury. I/R injury remains an important problem in massive hepatectomy and organ transplantation. The aim of this study was to examine the effect of edaravone, a newly synthesized free radical scavenger, on I/R injury in the remnant liver after partial hepatectomy in rats. METHODS: Partial (70%) hepatic ischemia was induced in rats by occluding the hepatic artery, portal vein, and bile duct to left and median lobes of liver. Total hepatic ischemia (Pringle maneuver) was induced by occluding the hepatoduodenal ligament. Edaravone was intravenously administered to rats just before reperfusion and partial (70%) hepatectomy was performed just after reperfusion. RESULTS: Edaravone significantly reduced the increases in the levels of serum alanine aminotransferase and aspartate aminotransferase in rats with liver injury induced by 90-min of partial ischemia followed by 120-min of reperfusion. Histopathological analysis showed that edaravone prevented inflammatory changes in the livers with I/R injury. Edaravone also decreased the levels of myeloperoxidase activity, which is an index of neutrophil infiltration, and interleukin-6 mRNA, which is a proinflammatory cytokine. Additionally, edaravone improved the survival rate in partial hepatectomy rats with I/R injury induced by the Pringle maneuver. CONCLUSIONS: Edaravone administration prior to reperfusion protected the liver against I/R injury. Edaravone also improved the function of the remnant liver with I/R injury after partial hepatectomy. Therefore, edaravone may have applicability for major hepatectomy and liver transplantation in the clinical setting.     

肝部分切除大鼠肠源性内毒素血症与肝再生的关系

目的:观察肝部分切除大鼠肠源性内毒素血症的动态变化与肝再生的关系. 方法:随机将Wistar大鼠分为正常对照组(NC)、假手术组(SO)和肝大部切除组(PH),测定NC组及SO,PH组术后6, 12,24,36,48,72,120,168h血浆ET浓度,同时动态观察残余肝组织的再生情况. 结果:PH组大鼠血浆ET浓度在PH后6-72 h升高,期间出现2次峰值,第1次在PH后12h,第2次在PH后48h,明显高于NC组及SO组对应时间点(P<0.01),72 h后迅速下降至NC组水平.PH后残余肝质量、肝再生率均出现明显的动态变化,但分别与血浆内毒素水平的变化趋势比较,相关系数分别为-0.408(P>0.05),-0.167(P>0.05);PH后再生肝组织DNA合成S期肝细胞的数量、PCNA的标记指数均出现显著动态改变,但分别与血浆ET水平的变化趋势比较,相关系数分别为0.062(P>0.05),0.058(P>0.05). NC,SO肝组织中上述反映肝再生的指标改变不明显. 结论:PH后残余肝再生全程中IETM的变化与肝再生程度的变化无关,提示IETM对肝再生程度没有产生直接影响, 但不能否定因IETM所引起细胞因子的释放而导致对肝再生的影响作用.