Nafamostat mesilate inhibits high-mobility group box 1 by lipopolysaccharide stimulation in murine macrophage RAW 264.7

In recent investigations, high-mobility group box 1 (HMGB1) has been recognized to be an important factor in the development of sepsis. On the other hand, a serine protease inhibitor, nafamostat mesilate (NM) inhibits the enzyme activities of various protease and coagulation factors. We examined whether NM could inhibit HMGB1 in a rat sepsis model and thus could potentially be useful for treating sepsis. We administered NM to rats before administering lipopolysaccharide and thereafter measured the HMGB1 levels of the serum and lung tissue. We used a mouse macrophage cell line and we performed lipopolysaccharide stimulation under NM administration and thereafter measured the quantity of HMGB1 and cytokines in the supernatant, and cell signal in the cells. We were thereby able to reduce the degree of injury to pulmonary tissue by administering NM. The HMGB1 levels of the serum and lung tissue were thus found to be inhibited. This action was confirmed at the cell level, and the release of HMGB1 and cytokines from the cell decreased. Regarding the cell signal in each cell, we observed the inhibition of the phosphorylation of IkappaB. We thus concluded that it is possible to prevent the occurrence of pulmonary disorders in an endotoxic shock model by administering NM, however, this also inhibits the cell signal in a cell, mainly by the phosphorylation of IkappaB, thereby inhibiting the HMGB1 levels. Our findings thus suggest that the administration of NM may therefore potentially improve the condition of patients who have septic shock.     

Systemic involvement of high-mobility group box 1 protein and therapeutic effect of anti-high-mobility group box 1 protein antibody in a rat model of crush injury

Patients with crush injury often present systemic inflammatory response syndrome and fall into multiple organ failure. The mechanism by which the local tissue damage induces distant organ failure is still unclear. We focused on high-mobility group box 1 protein (HMGB1) as one of the damage-associated molecular pattern molecules that cause systemic inflammation in crush injury. We investigated involvement of HMGB1 and the effects of treatment with anti-HMGB1 antibody in a rat model of crush injury. Both hindlimbs of rats were compressed for 6 h and then released. In the crush injury group, the level of serum HMGB1 peaked at 3 h after releasing compression, followed by the increasing in the serum levels of interleukin 6 and tumor necrosis factor α. Hematoxylin-eosin staining showed substantial damage in the lung 24 h after the crush injury, with upregulation of the expression of receptor for advanced glycation end products, as revealed by immunohistochemical analysis. Intravenous administration of anti-HMGB1 antibody improved survival (n = 20 each group) and significantly suppressed serum levels of HMGB1, interleukin 6, and tumor necrosis factor α compared with the untreated crush injury group (n = 6-9 each group). Histological findings of lung damage were ameliorated, and the expression of receptor for advanced glycation end products was hampered by the treatment. These results indicate that HMGB1 is released in response to damage immediately after crush injury and acts as a proinflammatory mediator. Administration of anti-HMGB1 antibody reduced inflammatory reactions and improved survival by blocking extracellular HMGB1. Thus, HMGB1 appears to be a therapeutic target, and anti-HMGB1 antibody may become a promising novel therapy against crush injury to prevent the progression to multiple organ failure.     

对高迁移率族蛋白B1作用的新认识

高迁移率族蛋白B1(high mobility grouo box 1 protein，HMGB1)是存在于真核生物细胞内的一类非组蛋白染色体结合蛋白，曾经作为一种转录N子和促生长因子而被广泛研究。新近研究发现。HMGB1可分泌到胞浆乃至胞外，并可与白细胞介素-1(IL-1)、IL-6、肿瘤坏死因子-α(TNF-α)等重要炎症因子相互诱生。循环HMGB1在脓毒症后期升高，有可能作为一种“晚期”炎症介质参与脓毒症的发生与发展过程。     

Recombinant bactericidal/permeability-increasing protein (rBPI21) in combination with sulfadiazine is active against Toxoplasma gondii

The activity of recombinant bactericidal/permeability-increasing protein (rBPI21), alone or in combination with sulfadiazine, on the intracellular replication of Toxoplasma gondii was assessed in vitro and in mice with acute toxoplasmosis. rBPI21 markedly inhibited the intracellular growth of T. gondii in human foreskin fibroblasts (HFFs). Following 72 h of exposure, the 50% inhibitory concentration of rBPI21 for T. gondii was 2.6 micrograms/ml, whereas only slight cytotoxicity for HFF cells was observed at the concentrations tested. Subsequent mathematical analyses revealed that the combination of rBPI21 with sulfadiazine yielded slight to moderate synergistic effects against T. gondii in vitro. Infection of mice orally with C56 cysts or intraperitoneally (i.p.) with RH tachyzoites resulted in 100% mortality, whereas prolongation of the time to death or significant survival (P = 0.002) was noted for those animals treated with 5 to 20 mg of rBPI21 per kg of body weight per day. Treatment with rBPI21 in combination with sulfadiazine resulted in significant (P = 0.0001) survival of mice infected i.p. with tachyzoites but not of mice infected orally with T. gondii cysts. These results indicate that rBPI21 is active in vitro and in vivo against T. gondii and that its activity is significantly enhanced when it is used in combination with sulfadiazine. To our knowledge, this is the first report of the activity of rBPI21 against a protozoan parasite.     

血必净注射液对脓毒症大鼠高迁移率族蛋白B1的影响

脓毒症是目前临床危重患者的主要死因之一。既往认为，肿瘤坏死因子-α（TNF—α）、白细胞介素-1（IL-1）等早期致炎细胞因子在引起机体失控性炎症反应与组织损害中发挥关键作用。研究表明，高迁移率族蛋白B1（HMGB1）作为新的晚期炎症因子参与了内毒素的致病过程和脓毒症的致死效应，并与脓毒症严重程度和预后密切相关。据报道，HMGB1与术后多器官功能障碍综合征（MODS）的发生有密切关系，术后并发MODS患者的血浆HMGB1水平显著升高；严重创伤患者伤后HMGB1水平明显增加。     

Autoantibodies against bactericidal/permeability-increasing protein in patients with cystic fibrosis

Cystic fibrosis (CF), a genetic disorder, is characterized by chronic pulmonary infection/inflammation which leads to respiratory failure. The presence of anti-neutrophil cytoplasmic autoantibodies (ANCA) has previously been observed in the sera of patients with CF. In view of the known relationship of ANCA with primary vasculitis and of their putative pathogenetic role in these disorders, we studied the presence, specificity and isotype of ANCA and their clinical associations in 66 adult CF patients. None of the 66 CF samples had autoantibodies to the major ANCA antigens, proteinase 3 or myeloperoxidase. However, 60/66 (91%) CF samples contained IgG and 55/66 (83%) IgA, autoantibodies to bactericidal/permeability increasing protein (BPI), a recently characterized ANCA specificity. All the IgA anti-BPI-positive samples were also IgG anti-BPI-positive. The autoantibody specificity was confirmed by inhibition assay and immunoblotting of CF sera against a neutrophil granule preparation. Furthermore, in this cross-sectional study, anti-BPI levels were inversely correlated with the observed reductions in FEV1 and FVC (IgA anti-BPI and FEV1: r = 0.508, <it>p</it> &lt; 0.0001), and both IgG and IgA anti-BPI levels were higher in CF patients with secondary vasculitis (<it>n</it> = 6) than in those without (<it>p</it> &lt; 0.05). ANCA with specificity for BPI were present in the majority of CF sera in this study and autoimmune processes may be associated with the development of pulmonary injury in CF.      

杀菌蛋白rBPI23与haFGF融合基因及真核表达质粒的构建

目的利用克隆人杀菌／通透性增加蛋白（rBPI23）和人酸性成纤维细胞生长因子（haFGF）cDNA,构建BF融合基因和真核表达载体,为能制备既能杀菌又能促进创伤愈合的双功能多肽创造条件。方法从人中性粒细胞和人胎脑组织中提取mRNA,经逆转录聚合酶链式反应（RT—PCR）分别获得rBPI23和haFGF编码序列的eDNA,运用重组PCR技术将两段基因通过一疏水性多肽接头（Gly4Ser）3DNA序列进行体外基因重组,合成融合基因,并将其梅建于真核表达载体peDNA3．1（＋）中。结果所扩增的rBPI23和haFGFCDNA序列与Genebank中报道的rBPI23、haFGFCD-NA序列一致。构建的BF融合基因及真核表达质粒DNA序列,经测序分析,与设计序列符合率为100％。结论本研究结果为进一步探讨BF融合蛋白的表达、生物学活性和特性奠定了基础。     

Circulating high-mobility group box 1 (HMGB1) concentrations are elevated in both uncomplicated pneumonia and pneumonia with severe sepsis

OBJECTIVE: High-mobility group box 1 (HMGB1) has been proposed as a late mediator of sepsis, but human data are sparse and conflicting. We describe plasma HMGB1 concentrations in humans with community-acquired pneumonia (CAP), the most common cause of severe sepsis, and test the hypotheses that HMGB1 levels are higher in CAP than healthy controls, higher in CAP with severe sepsis than CAP without severe sepsis, and higher in severe sepsis nonsurvivors than survivors. DESIGN: Random, outcome-stratified sample from a prospective study of 1,895 subjects hospitalized with CAP. SETTING: Twenty-eight U.S. teaching and community hospitals. PATIENTS: There were 122 CAP subjects (43 never developed severe sepsis, 49 developed severe sepsis and survived hospitalization, and 30 developed severe sepsis and died) and 38 healthy controls. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Median day of onset of severe sepsis was day of admission. HMGB1 was measured daily for the first week and analyzed using repeated-measures models with and without multivariable adjustment for baseline characteristics. HMGB1 concentrations were higher in CAP subjects compared with controls (median concentration on day of admission vs. controls, 190 vs. 0 ng/mL, p = .0001; 93.7% of all CAP measurements were elevated). HMGB1 remained elevated throughout the hospital course with no significant trend (p = .64) and did not differ between those with and without severe sepsis (p = .30). HMGB1 concentrations were higher in severe sepsis nonsurvivors than survivors (p = .001). HMGB1 concentrations remained elevated at discharge (median final HMGB1 measure, 176 ng/mL). Findings persisted in multivariable models and were robust to sensitivity analyses using alternative definitions of severe sepsis. CONCLUSIONS: HMGB1 is elevated in almost all CAP subjects, and higher circulating HMGB1 is associated with mortality. But immunodetectable HMGB1 levels were also persistently elevated in those patients who fared well. Thus, additional work is needed to understand the biological activities of serum HMGB1 in sepsis.     

Protection against endotoxic shock by bactericidal/permeability-increasing protein in rats.

Bactericidal/permeability-increasing protein (BPI) is a neutrophil primary granule protein that inhibits effects of LPS in vitro. The current study examined the effects of BPI on hemodynamics, mortality, and circulating endotoxin and cytokines in conscious rats with endotoxic shock. Catheters were implanted into the right femoral artery and vein. 1 d later, human recombinant BPI (10 mg/kg) or vehicle was intravenously injected immediately, 30 min, or 2 h after intravenous injection of LPS (7.5 mg/kg). Mean arterial pressure (MAP) and heart rate were monitored and blood was collected before and after injection. BPI given immediately or 30 min after LPS prevented the LPS-induced reduction in MAP at 4-8 h and markedly reduced mortality. BPI given 2 h after LPS injection had no protective effect. BPI treated immediately after LPS reduced the circulating levels of endotoxin and IL-6 but increased the circulating levels of TNF. We propose that BPI exerts its protective effect through a TNF-independent mechanism, by inhibiting endotoxin-stimulated production of IL-6.     

The antiangiogenic properties of bactericidal/permeability-increasing protein (BPI)

Inhibition of angiogenesis is regarded as a promising tool in the treatment of diseases such as cancer, arthritis and atherosclerosis. This fact has led to the search for novel endogenous or synthetic angiogenesis inhibitors. Recently, antiangiogenic properties were ascribed to an endogenous molecule that until only recently was known for its antibacterial effects. This molecule, bactericidal/permeability-increasing protein (BPI), that was discovered as a bacterial permeabilizing and lipopolysaccharide-neutralizing protein, was found to inhibit angiogenesis by specific induction of apoptosis in endothelial cells. This paper gives a short introduction on angiogenesis and reviews the current knowledge on BPI as an angiogenesis inhibitor. In addition, the issue of commonality between antibacterial and antiangiogenic functions will be addressed.     

The antiangiogenic properties of bactericidal/permeability-increasing protein (BPI)

Inhibition of angiogenesis is regarded as a promising tool in the treatment of diseases such as cancer, arthritis and atherosclerosis. This fact has led to the search for novel endogenous or synthetic angiogenesis inhibitors. Recently, antiangiogenic properties were ascribed to an endogenous molecule that until only recently was known for its anti-bacterial effects. This molecule, bactericidal/permeability-increasing protein (BPI), that was discovered as a bacterial permeabilizing and lipopolysaccharide-neutralizing protein, was found to inhibit angiogenesis by specific induction of apoptosis in endothelial cells. This paper gives a short introduction on angiogenesis and reviews the current knowledge on BPI as an angiogenesis inhibitor. In addition, the issue of commonality between antibacterial and anti-angiogenic functions will be addressed.     

Value of blood tests including serum group IIA phospholipase A2 and bactericidal/permeability-increasing protein in Crohn's disease

OBJECTIVE: Determination of the activity of Crohn's disease at a defined time-point is a challenging task since only endoscopy guidelines are given and secondary clinical findings, subjective symptoms and non-specific laboratory tests have therefore to be relied on. The purpose of the current study was to investigate the ability of blood tests to differentiate patient groups with different clinical disease activity and different clinical outcomes during follow-up in Crohn's disease. MATERIAL AND METHODS: During a visit to hospital, 73 outpatients with Crohn's disease were examined, a clinical score was calculated and blood samples were collected for 22 laboratory tests. The patients were also grouped according to clinical outcome during a 6-year follow-up. RESULTS: Serum group IIA phospholipase A2 and alpha-1-antitrypsin values were outside the reference interval more frequently (62% and 42%, respectively) than the other tests in active Crohn's disease. Only weak correlations were found between the clinical score and the test values, and the best correlation was found with serum lysozyme (r = 0.40). In a logistic regression model, the best prediction of disease activity at entry to the study was reached with a model including serum orosomucoid and serum lysozyme and the best prediction of clinical outcome during follow-up was reached using a model including serum albumin. CONCLUSIONS: Serum group IIA phospholipase A2 appeared to be the most sensitive marker of inflammation in Crohn's disease among the 22 blood tests compared. No reliable predictions of disease activity at the time of blood sampling or clinical outcome later during follow-up could be made from the blood tests studied.     

Clinical role of autoantibody against bactericidal/permeability-increasing protein in chronic airway infection

Chronic airway infections often have persistent colonization of mucoid-derivative and/or biofilm-formed bacteria on the small airway surface. Such long-term colonization by bacteria sometimes induces harmful immune reactions in the host. Bactericidal/permeability-increasing protein (BPI) is one of the important agents in the phagocyte activity of neutrophils. An antineutrophil cytoplasmic autoantibody against BPI (BPI-ANCA) was recently detected. The clinical role of BPI-ANCA in 66 patients with chronic airway infections was investigated. Corresponding antigens in neutrophils against the antibody in their sera were observed by the indirect immunofluorescence method. After confiriming the antigen to be BPI, the presence of BPI-ANCA in their serum samples was determined by the enzyme-linked immunosorbent assay method. The titers were analyzed in relation to clinical symptoms, prognosis, and bacteriologic aspects, including in vitro examinations. Thirty-five of 66 samples (53%) were positive for cytoplasmic ANCA, and 48 cases (73%) were positive for serum BPI-ANCA. There were no positive cases among the sera samples of 7 healthy individuals. High titers of BPI-ANCA and a high frequency of BPI-positive indications were seen in cases with persistent colonization of gram-negative bacteria (P<0.05), and they also correlated with clinical symptoms and prognosis. In in vitro examinations, BPI-ANCA dose-dependently suppressed BPI that was induced by the interaction of neutrophils and gram-negative bacteria. BPI-ANCA is characteristically found in cronic infections, and it suppesses the BPI activity of neutrophils. An autoimmune manifestation such as this might increase the intractability of chronic airway infections.     

Elevated high-mobility group box 1 levels in patients with cerebral and myocardial ischemia

The plasticity of bone marrow-derived stem cells (BMSCs) has resulted in positive remodeling and the regeneration of viable tissues. However, BMSC release of growth factors, which limit apoptosis and inflammation, may play an important role in conferring organ protection. Recent studies also indicate that those patients with higher circulating BMSC counts may be more resistant to septic and traumatic insults. There are clear sex differences in response to such insults. Within the population of BMSC, mesenchymal stem cells (MSCs) may have clinical advantages. Therefore, we hypothesize that sex differences in the MSC paracrine response to acute injury exist.Mesenchymal stem cells were obtained from male and female mice. One million MSCs per well (triplicate wells per group) were stressed by hypoxia and increasing doses of endotoxin (lipopolysaccharide [LPS]) and hydrogen peroxide. Mesenchymal stem cell activation was determined by measuring vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha production by enzyme-linked immunosorbent assay. Differences were considered significant if P < 0.05. RESULTS: Lipopolysaccharide resulted in significant activation of both male and female MSCs. However, LPS provoked significantly more VEGF production in female MSCs versus male MSCs at all LPS doses. Hypoxia of 1 h and hydrogen pyroxide exposure also caused significantly more VEGF production in female MSCs versus male MSCs. Female MSCs expressed significantly less tumor necrosis factor alpha than male MSCs after acute LPS and hypoxia. CONCLUSION: This study constitutes the first demonstration that sex differences exist in activated MSC function. Sex differences in progenitor cell function may have important implications in understanding the observed sex differences in the host's response to injury.     

磁刺激诱导下高迁移率族蛋白B1对星形胶质细胞迁移能力的影响

目的 探讨磁刺激诱导星形胶质细胞迁移的机制及高迁移率族蛋白B1（HMGB1）对星形胶质细胞迁移的影响。 方法 取新生2～3d SD大鼠大脑皮质星形胶质细胞,用细胞免疫荧光法检测细胞纯度;将细胞分为磁刺激组和对照组,磁刺激组给予磁刺激,对照组在相同环境下,不给予磁刺激。将细胞分为实验组和控制组,实验组用10μmol/ml U0126抑制剂预刺激30min,控制组不加入U0126抑制剂,以探讨细胞外信号调节激酶（ERK1/2）与HMGB1的关系;将细胞分为siRNA转染组和HMGB1-siRNA转染组,SiRNA组给予HMGB1 siRNA处理,以探讨HMGB1对细胞迁移能力的影响。用Western blot检测磁刺激对HMGB1和ERK1/2的影响,用划痕实验检测磁刺激对星形胶质细胞迁移能力的影响。 结果 10Hz磁刺激可促进ERK磷酸化,增强星形胶质细胞的迁移能力,并可增加HMGB1的表达水平;用U0126试剂处理后,HMGB1的表达量明显下降;HMGB1 siRNA转染后HMGB1表达量明显下降,且星形胶质细胞的迁移能力显著下降。 结论 星形胶质细胞在磁刺激作用下,通过激活ERK通道,促进ERK磷酸化,增加下游HMGB1的表达水平,且HMGB1以自分泌的形式促进自身迁移。     

The bactericidal/permeability-increasing protein (BPI) in infection and inflammatory disease

Gram-negative bacteria (GNB) and their endotoxin present a constant environmental challenge. Endotoxins can potently signal mobilization of host defenses against invading GNB but also potentially induce severe pathophysiology, necessitating controlled initiation and resolution of endotoxin-induced inflammation to maintain host integrity. The bactericidal/permeability-increasing protein (BPI) is a pluripotent protein expressed, in humans, mainly neutrophils. BPI exhibits strong antimicrobial activity against GNB and potent endotoxin-neutralizing activity. BPI mobilized with neutrophils in response to invading GNB can promote intracellular and extracellular bacterial killing, endotoxin neutralization and clearance, and delivery of GNB outer membrane antigens to dendritic cells. Tissue expression by dermal fibroblasts and epithelia could further amplify local levels of BPI and local interaction with GNB and endotoxin, helping to constrain local tissue infection and inflammation and prevent systemic infection and systemic inflammation. This review article focuses on the structural and functional properties of BPI with respect to its contribution to host defense during GNB infections and endotoxin-induced inflammation and the genesis of autoantibodies against BPI that can blunt BPI activity and potentially contribute to chronic inflammatory disease.