Oxidases and oxygenases in regulation of neutrophil redox pathways in Behçet’s disease patients

This study aimed to determine plasma and neutrophil oxidase activities that may contribute to vascular inflammation in Behçet’s disease (BD) patients. Cyclooxygenase (COX), NADPH oxidase and myeloperoxidase (MPO) activity was determined in neutrophils isolated from BD patients and healthy controls. Functional assay of NADPH oxidase was significantly increased in BD patients, both at basal conditions and in response to fMLP stimulation. There was a significant increase in plasma MPO activity in the disease group as compared to controls. Total COX activity was significantly increased in BD neutrophils. The increase in total COX activity was accompanied with enhanced activity of COX-2, differentiated by using the COX-1 isoform-specific inhibitor SC-560. Neutrophil nitrate/nitrite levels showed no significant difference in BD; however, plasma nitrate/nitrite contents in BD patients were significantly greater compared to controls. In conclusion, increased plasma MPO, neutrophil NADPH and COX activities may contribute to intravascular inflammation documented in BD patients.     

Neutrophil gelatinase-associated lipocalin after off pump versus on pump coronary artery surgery

Abstract

Context: Cardiac surgery.

Objective: To compare plasma and urinary neutrophil gelatinase-associated lipocalin (P-/U-NGAL) in on-pump (n?=?43) versus off-pump (n?=?40) surgery.

Materials and methods: We obtained perioperative P-/U-NGAL and outcome data.

Results: P-/U-NGAL increased after surgery. P-NGAL was higher post-surgery in on pump patients (139 versus 67?µg L^?1; p?<?0.001), but not at 24?h. There were no differences in U-NGAL. Correlation between P-/U-NGAL and plasma creatinine was weak.

Discussion: P-NGAL acts like a neutrophil activation biomarker and U-NGAL like a tubular injury marker.

Conclusion: On-pump patients had greater neutrophil activation. On- versus off-pump surgery had similar impact on tubular cells.     

Recombinant human thioredoxin-1 becomes oxidized in circulation and suppresses bleomycin-induced neutrophil recruitment in the rat airway

Thioredoxin-1 (TRX) is a redox-active protein with anti-inflammatory effects. This study investigated the optimal delivery method and the mechanisms of recombinant human TRX (rhTRX) to suppress neutrophil recruitment in a rat bleomycin (BLM)-induced sustained acute lung injury model. In male Wister rats intratracheally administered with 0.125 mg/kg BLM, 8 mg/kg/day rhTRX was intravenously administered on days 3–6 using one of three protocols: daily bolus injection, 3 h daily infusion or continuous infusion for 96 h. Only the continuous-infusion of rhTRX significantly reduced the neutrophil infiltration compared with the other two methods. The BLM-induced down-regulation of l-selectin expression on circulating neutrophils was inhibited by rhTRX. Oxidized rhTRX showed a comparable effect with reduced rhTRX and rhTRX incubated with plasma or circulating in plasma was more than 99% oxidized. These results suggest that rhTRX becomes oxidized in circulation and continuous infusion of rhTRX suppresses neutrophil recruitment in the airway.     

PEGylated human plasma fibronectin is proteolytically stable,supports cell adhesion,cell migration,focal adhesion assembly,and fibronectin fibrillogenesis

Delayed wound healing in many chronic wounds has been linked to the degradation of fibronectin (FN) by abnormally high protease levels. We sought to develop a proteolytically stable and functionally active form of FN. For this purpose, we conjugated 3.35 kDa polyethylene glycol diacrylate (PEGDA) to human plasma fibronectin (HPFN). Conjugation of PEGDA to HPFN or HPFN PEGylation was characterized by an increase of approximately 16 kDa in the average molecular weight of PEGylated HPFN compared to native HPFN in SDS‐PAGE gels. PEGylated HPFN was more resistant to α chymotrypsin or neutrophil elastase digestion than native HPFN: after 30 min incubation with α chymotrypsin, 56 and 90% of native and PEGylated HPFN respectively remained intact. PEGylated HPFN and native HPFN supported NIH 3T3 mouse fibroblast adhesion and spreading, migration and focal adhesion formation in a similar manner. Fluorescence microscopy showed that both native and PEGylated HPFN in the culture media were assembled into extracellular matrix (ECM) fibrils. Interestingly, when coated on surfaces, native but not PEGylated HPFN was assembled into the ECM of fibroblasts. The proteolytically stable PEGylated HPFN developed herein could be used to replenish FN levels in the chronic wound bed and promote tissue repair. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 493–504, 2013     

Up-regulation of blood arachidonate (20:4) levels in patients with chronic obstructive pulmonary disease

Context and objective: Plasma arachidonate (20:4) levels in patients with chronic obstructive pulmonary disease (COPD) were investigated.

Methods: Plasma was extracted and free fatty acids (FFAs) were separated using column chromatography and measured by fluorescence. Plasma 20:4 levels and its percentage relative to total FFA levels (%20:4) were measured in COPD (n = 18) and control (n = 20) subjects.

Results and conclusions: FFA levels were lower in COPD compared with normals. However, there was a significant increase in %20:4 levels in COPD patients (GOLD stage I/II 0.9 ± 0.4%; GOLD stage III/IV 1.1 ± 0.1%) compared with control subjects (0.6 ± 0.1, p < 0.05). %20:4 is a potential biomarker for COPD.     

Urinary L-FABP and its combination with urinary NGAL in early diagnosis of acute kidney injury after cardiac surgery in adult patients

Background/Aim: The early detection of acute kidney injury (AKI) may be become possible by several promising early biomarkers which may facilitate the early detection, differentiation and prognosis prediction of AKI. In this study, we investigated the value of urinary liver-type fatty acid-binding protein (L-FABP), neutrophil gelatinase-associated lipocalin (NGAL) and their combination in predicting the occurrence and the severity of AKI following cardiac surgery.

Methods: We prospectively followed 109 patients undergoing open heart surgery and identified 26 that developed AKI, defined as an increase in serum creatinine of ≥0.3?mg/dl or ≥150% of baseline creatinine. Serum creatinine (SCr), urinary L-FABP, and NGAL corrected by urine creatinine were tested pre-operation, at 0 hour and 2 hours post-operation. Each marker was assessed at each time point between patients with and without AKI. Receiver operating characteristic (ROC) curves and area under curves (AUC) were used to evaluate the diagnostic accuracy of urinary L-FABP, NGAL and their combination for predicting AKI.

Results: Patients were aged 63.0?±?11.3 years, 66.1% were male and baseline SCr was 70.5?±?19.1 umol/L. Of 109 patients, 26(23.9%) developed AKI (AKIN stage I, II and III were 46.2%, 34.6% and 19.2% separately). The levels of urinary L-FABP and NGAL were significantly higher in AKI patients than non-AKI patients at 0 hour and 2 hours postoperative. AUCs for L-FABP was 0.844 (sensitivity (ST) 0.846, specificity (SP) 0.819, cut-off (CO) 2226.50 μg/g Ucr) at 0 hours and 0.832 at 2 hours (ST 0.808, SP 0.747, CO 673.09 μg/g Ucr) while 0.866 for NGAL at 0 hours (ST 0.769, SP 0.819, CO 131.12 μg/g Ucr) and 0.871 at 2 hours (ST 0.808, SP 0.831, CO 33.73 μg/g Ucr) to predict AKI occurrence. Using a combination of L-FABP and NGAL analyzed at the same timepoint as above, we were able to obtain an AUC of 0.911–0.927, p < 0.001. Similar AUCs of 0.81–0.87 were found to predict AKI stage II–III.

Conclusions: Urinary L-FABP and NGAL increased at an early stage after cardiac surgery. The combination of the two biomarkers enhanced the accuracy of the early detection of postoperative AKI after cardiac surgery before a rise in SCr.     

Therapeutic level of functional human alpha 1 antitrypsin (hAAT) secreted from murine muscle transduced by adeno-associated virus (rAAV1) vector

Alpha 1 antitrypsin (AAT) is a serine proteinase inhibitor (serpin). One well-known function of this protein is to inactivate neutrophil elastase and other neutrophil-derived proteinases, and prevent the destruction of pulmonary extracellular matrix. Deficiency of AAT can cause emphysema due to degradation of interstitial elastin by elastase. The majority of circulating AAT is secreted from the liver. Muscle-directed gene therapy using recombinant adeno-associated virus 2 (rAAV2) vectors has been tested to increase the serum levels of AAT. However, inefficient transduction of rAAV2 vector makes it difficult to reach therapeutic levels of AAT in clinical trials and it remains unclear as to whether muscle-secreted AAT is functional. In the present study, we evaluated five serotypes (1, 2, 3, 4, and 5) of rAAV vectors for transduction efficiency in mouse muscle. Results from these studies showed that rAAV1 is the most efficient vector among these serotypes and mediated at least 100-fold higher levels of AAT secretion than the rAAV2 vector. Western blot analysis showed that this murine muscle-secreted human AAT (hAAT) formed a complex with human neutrophil elastase in a dose-dependent manner. An anti-elastase activity assay showed that murine muscle-secreted hAAT inhibited elastase with equal capacity as hAAT purified from plasma. These results provide strong support for the functionality of AAT in ongoing clinical studies of muscle-directed AAT gene therapy.     

Calpain裂解Atg5决定中性粒细胞发生自噬还是凋亡，以及C5a在其中的可能作用

通过阐明C5a、calpain和Atg5相互作用,为开展新的研究寻找方向．中性粒细胞凋亡控制炎症反应及其强度,多种疾病和中性粒细胞凋亡失调有关,但其发生机制尚未阐明．C5a为补体片段,有多种功能,如诱导中性粒细胞趋化、呼吸爆发、增强吞噬、颗粒酶释放和延迟凋亡．已知calpain涉及中性粒细胞功能及凋亡调节并对该凋亡发生具有特异性．不同刺激因素可通过不同路径调节不同calpain亚型的活性． 已有报道C5a可以通过调节calpain亚型活性而调节中性粒细胞的趋化反应．另外,自噬是真核细胞中广泛存在的生物过程,具有细胞保护作用,Atg5对于自噬体形成必不可少．Calpain可裂解Atg5为24 ku tAtg5,使其失去形成自噬体的功能并介导凋亡．Atg5参与了自噬和凋亡的转换．     

Effects of weight reduction on neutrophil phagocytic activity and oxidative burst activity in female judoists

This study investigated the effects of short‐term weight reduction on neutrophil functions in female judoists. Sixteen actively competing female judoists were divided into two groups. Eight who required weight reduction were de?ned as the weight reduction group, and the remaining eight were used as the control. Blood samples were taken before, immediately after and 8 days after the match. Phagocytic activity and oxidative burst activity of neutrophils were measured by ?ow cytometry. In the weight reduction group, the phagocytic activity per cell decreased signi?cantly at the end of weight reduction compared with the control group. The rate of neutrophils producing reactive oxygen species and the oxidative burst activity per cell increased signi?cantly at the end of weight reduction in both the control and the weight reduction groups. We concluded that weight reduction, consisting of both intense exercise and energy restriction, might possibly cause both an increase in oxidative burst activity and decrease in neutrophil phagocytic activity in female judoists. However, although exercise increased oxidative burst activity, it did not affect neutrophil phagocytic activity alone. Therefore, to avoid this problem, female judoists are recommended to keep their weight within those limits determined by their class, and which can be reduced by exercise. Copyright © 2003 John Wiley & Sons, Ltd.