Aggregatibacter actinomycetemcomitans induces detachment and death of human gingival epithelial cells and fibroblasts via elastase release following leukotoxin‐dependent neutrophil lysis

Aggregatibacter actinomycetemcomitans is considered to be associated with periodontitis. Leukotoxin (LtxA), which destroys leukocytes in humans, is one of this bacterium's major virulence factors. Amounts of neutrophil elastase (NE), which is normally localized in the cytoplasm of neutrophils, are reportedly increased in the saliva of patients with periodontitis. However, the mechanism by which NE is released from human neutrophils and the role of NE in periodontitis is unclear. In the present study, it was hypothesized that LtxA induces NE release from human neutrophils, which subsequently causes the breakdown of periodontal tissues. LtxA‐treatment did not induce significant cytotoxicity against human gingival epithelial cells (HGECs) or human gingival fibroblasts (HGFs). However, it did induce significant cytotoxicity against human neutrophils, leading to NE release. Furthermore, NE and the supernatant from LtxA‐treated human neutrophils induced detachment and death of HGECs and HGFs, these effects being inhibited by administration of an NE inhibitor, sivelestat. The present results suggest that LtxA mediates human neutrophil lysis and induces the subsequent release of NE, which eventually results in detachment and death of HGECs and HGFs. Thus, LtxA‐induced release of NE could cause breakdown of periodontal tissue and thereby exacerbate periodontitis.     

Roles of phospholipase D in phorbol myristate acetate‐stimulated neutrophil respiratory burst

The phorbol myristate acetate (PMA) stimulated nutrophil respiratory burst has been considered to simply involve the activation of protein kinase C (PKC). However, the PLD activity was also increased by 10‐fold in human neutrophils stimulated with 100 nM PMA. Unexpectedly, U73122, an inhibitor of phospholipase C, was found to significantly inhibit PMA‐stimulated respiratory burst in human neutrophils. U73122 at the concentrations, which were sufficient to inhibit the respiratory burst completely, caused partial inhibition of the PLD activity but no inhibition on PKC translocation and activation, suggesting that PLD activity is also required in PMA‐stimulated respiratory burst. Using 1‐butanol, a PLD substrate, to block phosphatidic acid (PA) generation, the PMA‐stimulated neutrophil respiratory burst was also partially inhibited, further indicating that PLD activation, possibly its hydrolytic product PA and diacylglycerol (DAG), is involved in PMA‐stimulated respiratory burst. Since GF109203X, an inhibitor of PKC that could completely inhibit the respiratory burst in PMA‐stimulated neutrophils, also caused certain suppression of PLD activation, it may suggest that PLD activation in PMA‐stimulated neutrophils might be, to some extent, PKC dependent. To further study whether PLD contributes to the PMA stimulated respiratory burst through itself or its hydrolytic product, 1,2‐dioctanoyl‐sn‐glycerol, an analogue of DAG , was used to prime cells at low concentration, and it reversed the inhibition of PMA‐stimulated respiratory burst by U73122. The results indicate that U73122 may act as an inhibitor of PLD, and PLD activation is required in PMA‐stimulated respiratory burst.     

Coupling of polyphosphoinositide breakdown with calcium efflux in formyl-methionyl-leucyl-phenylalanine-stimulated rabbit neutrophils

Exposure of rabbit neutrophils to formyl-methionyl-leucyl-phenylalanine (FMLP) induced the efflux of 45Ca2+ from pre-labeled cells which was almost complete within 30 s. On the other hand, FMLP-induced 45Ca2+ influx did not become apparent until 60 s after stimulation. When [3H]arachidonic acid-labeled neutrophils were stimulated with FMLP, the radioactivities in phosphatidylinositol 4,5-biphosphate (TPI) and phosphatidylinositol 4-phosphate (DPI) significantly decreased in parallel with the induction of 45Ca2+ efflux. In contrast, degradation of polyphosphoinositides in [3H]glycerol-labeled neutrophils was not significant until 60 s. Taken together, these results indicate that the early degradation of polyphosphoinositides, especially of those rich in arachidonic acid is closely associated with the initial efflux of calcium in FMLP-stimulated rabbit neutrophils. The study of resynthesis of polyphosphoinositides by measuring 32Pi incorporation into these lipids is also presented.     

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the β₂ integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both β₂ integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.     

人中性粒细胞多肽HNP1，3体外抗单纯疱疹病毒Ⅰ型的作用

体外观察人中性粒细胞多肽1,3(Humanneutrophilpeptide,HNP1,3)及阿昔洛韦(Acyclovir,ACV)对单纯疱疹病毒-Ⅰ型(Herpessimplexvirus1,HSV-1)的抑制作用。以Vero细胞为靶细胞,用各种浓度HNP1,3与游离病毒颗粒(直接失活组)及感染病毒后的靶细胞(复制抑制组)进行相互作用,镜下观察各药物对HSV-1致细胞病变效应的抑制作用,并采用ELISA法测定感染48h后药物对HSV-1囊膜糖蛋白分泌的抑制作用。MTT法检测各药物对细胞的毒性作用。结果显示直接失活组中,HNP1,3可使HSV-1的致细胞病变效应减轻,对HSV-1直接失活的50%有效浓度(EC50)为8.1μg/mL、10.03μg/mL;复制抑制组中,ACV使HSV-1的致细胞病变效应减轻,EC50为0.68μg/mL。MTT检测结果表明HNP1,3在治疗浓度范围内无明显细胞毒性。以上结果表明HNP1,3除具有较强的抗菌作用和抗人类免疫缺陷病毒Ⅰ型(Humanimmunodeficiencyvirus1,HIV-1)活性外,还能失活HSV-1病毒颗粒,从而逆转病毒及其蛋白的病毒效应(致细胞病变)和抑制病毒蛋白质的合成。     

Foreword: bacterial homologues of eukaryotic membrane proteins

Abstract

Human lipocalin-1 interacting membrane receptor (LIMR) was the first lipocalin receptor to be identified, as a specific receptor for lipocalin-1 (Lcn1). Subsequently LIMR has been reported to interact with other ligands as well, notably with the bovine lipocalin β-lactoglobulin (BLG) and with the unrelated secretoglobin uteroglobin (UG). To study the ligand-binding behaviour of this prototypic lipocalin receptor in more detail, a system was developed for the recombinant expression of LIMR in Drosophila Schneider 2 (S2) cells, and for the subsequent solubilization and purification of the protein. The receptor forms dimers or larger oligomers when solubilized in n-dodecyl β-D-maltoside (DDM). The full-length, functional receptor was captured onto a surface plasmon resonance (SPR) chip via an α-V5 antibody, and the binding of various potential ligands was followed in time. In this way, LIMR was shown to be highly specific for Lcn1, binding the lipocalin with low micromolar to high nanomolar affinity. No interactions with any of the other putative ligands could be detected, raising doubts about the physiological relevance of the reported binding of BLG and UG to the receptor.     

Clinical implication of pretreatment neutrophil to lymphocyte ratio in soft tissue sarcoma

Introduction: Elevated neutrophil to lymphocyte ratio has been identified as a prognostic indicator in malignancies whereas; its association with extremity and trunk soft tissue sarcoma remain unclear. The aim of this study is to determine the utility of full blood neutrophil lymphocyte ratio (NLR) in preoperative diagnosis and its predictive value for survival in patients managed for soft tissue sarcoma of the trunk and extremities.

Method: 223 patients who presented with a soft tissue tumor were retrospectively reviewed. The study period was from January 2002–December 2009. Preoperative NLR as well as demographics, clinical and histopathological data were analysed.

Results: Full blood NLR was significantly higher in patient with a soft tissue sarcoma compared to benign soft tissue tumors (p < 0.001). Cox regression analysis demonstrated that elevated NLR >5 (p < 0.05) may be an adverse prognostic factor for Overall Survival.

Conclusion: The preoperative NLR is a simple, investigation predicting the preoperative diagnosis of a soft tissue sarcoma and a predictor of worse overall survival for patient with a soft tissue sarcoma.