FEATURES OF NEUTROPHIL CHEMILUMINESCENCE IN THE PATIENTS WITH ADVANCED RECTAL CANCER

Colorectal cancer is one of the most common malignant diseases in Russia worldwide making up 5-6% of all human malignant tumors. Neutrophilic granulocytes are actively involved in development of antitumor response. A key role in tumor regression is assigned to active forms of oxygen produced by neutrophils. In connection with these pre-requisites, our goal was to study functional characteristics of spontaneous and induced chemiluminescent activity of neutrophil granulocytes in patients with rectal cancer before starting pathogenetic therapy and in subsequent dynamics. The paper presents some laboratory results, i.e., functional indices of neutrophilic granulocytes’ activity in 36 patients with rectal cancer being at different stages of oncological process. The control group consisted of 112 practically healthy volunteers, comparable in sex and age to the group of patients under study. To perform the study venous blood was taken from patients to vacuum test tubes with lithium heparin in the morning time before surgical treatment, and on day 7 after the surgical intervention. Evaluation of spontaneous and induced chemiluminescence was performed for 90 minutes in a 36-channel “CL 3606” chemiluminescence analyzer (Russia). The following characteristics were determined: time of the curve transition to maximal chemiluminescence intensity (Tmax), maximal value of chemiluminescence intensity (Imax), integral area under the chemiluminescence curve (S). Luminol was used as the chemiluminescence enhancer. Opsonized zymosan was used to induce the respiratory explosion. Chemiluminescence amplification induced by opsonized zymosan was evaluated by the ratio of induced-tospontaneous chemiluminescence (Sind/spont) designated as an activation index.Analysis of chemiluminescence activity in neutrophilic granulocytes showed a significant increase in spontaneous chemiluminescence activity at the stages III and IV of the disease. The production of active oxygen forms induced in neutrophilic granulocytes by opsonized zymosan increased in all the study groups, relative to control parameters. The area under the curves of spontaneous and induced chemiluminescence in patients with colorectal cancer at all stages of the oncological process is less, as compared to the control group, which, despite high indices of maximal chemiluminescence activity, may indicate insufficient total production of reactive oxygen species. The time-to-peak values of the chemiluminescence curves in patients with rectal cancer at all stages of the disease did not show statistically significant differences from the control group.

Peroxiredoxin 6 (Prdx6) Is Associated with Changes in Autophagy in Myeloid and Non-Myeloid Cells

Introduction: Prdx6, a member of a new family of peroxidases, is found in neutrophil cytosol associated with p67phox. This protein reduces H2O2 with a conserved cysteine (C47) and also expresses PLA2 activity. In human neutrophils, Prdx6 translocates to plasma membrane after activation and enhances Nox2 activity in a cell-free system of oxidase activity. In transgenic K562 cells and PLB-985 cells, suppression of Prdx6 by siRNA or shRNA decreases Nox2 activity. In these studies, we investigated the association of Prdx6with autophagy in PLB-985 and HEK-293 cells. Methods: PLB-985 and HEK-293 cells were cultured under standard conditions and were transfected with a plasmid producing stable expression of an shRNA targeting Prdx6. PLB-985 cells were matured with the addition of 1.3% DM50 for 4 days. DNA for wild type (WT) Prdx6 and mutants for the Prdx6 active site (C47S) and PLA2active sites (H26A, S32A, S140A) were cloned into pcDNA3.1 which also had silent mutations making all corresponding mRNAs resistant to shRNA targeting. Prdx6 knockdown and vector control cells had stable re-expression of the various proteins when cultured with appropriate antibiotics. Superoxide anion (O2-) was determined by SOD inhibitable chemilluminescence with Digenes (National Diagnosistics). Proteins from lysates of various cell lines were separated by 10-12% SDS-PAGE and blotted onto nitrocellulose. Specific proteins were detected standard antibodies and a chemilluminescent technique with quantitation completed using Image J platform. Autophagy in PLB-985 cells with or without suppression of Prdx6 was completed by expression of LC3-II by Western blot after stimulation with serum opsonized zymsoan (5mg/mL). In HEK-293 cells, autophagy was determined by quantification of LC3-II (western blot) under based conditions with the addition of 10µM Chloraquine or starvation conditions with minimal media. Results: Stable suppression of Prdx6 by shRNA was achieved in both PLB-985 cells (down to 30%) and HEK cells (25%). Re-expression of WT and Prdx6 mutant proteins re-established normal levels. O2-production in response to lµM FMLP was decreased by 44% in KD PLB-985 cells compared to control and re-establishment of oxidase activity depended on PLA2, not Prdx activity. In PLB-985 cells, LC3-II levels increased (seven fold) over the first 30 minutes after exposure to opsonized zymosan, decreasing to baseline at 60 minutes. Prdx6 suppressed PLB-985 cells showed no change in LC3-II after opsonized zymosan. In HEK-293 cells lacking an active oxidase, suppression of Prdx6 resulted in increased autophagy under basal and starvation conditions. Furthermore, in HEK-293 cells, reintroduction of empty vector or Prdx6 mutated at the Prdx active site (C47S) did not suppress autophagy under basal conditions. However, re-introduction of WT and mutant Prdx6 at the PLA2 active site (D140A) did suppress autophagy demonstrating that this effect was associated with Prdx activity. Similar results were obtained under starvation conditions. Conclusions: In neutrophil like cells Prdx6 enhanced Nox2 activity through its PLA2 activity and suppression of Prdx6 decreased autophagy as measured by LC3-II accumulation after exposure to a phagocytic stimulus. In non-myeloid HEK-293 cells, suppression of Prdx6 was associated with increased autophagy under basal and starvation conditions, and this effect was dependent on Prdx activity. Prdx6, then, plays a critical role in neutrophil functions such as Nox2 activity and autophagy. Prdx6 may also be involved in autophagy in non-myeloid cells, and the mechanism for its involvement in autophagy may relate to different activities expressed by this protein. Disclosures No relevant conflicts of interest to declare.

Regulation of 5-oxo-ETE synthesis by nitric oxide in human polymorphonuclear leucocytes upon their interaction with zymosan and Salmonella typhimurium

Nitric oxide significantly increased 5-oxo-ETE formation in neutrophils. 5-oxo-ETE is a key 5-lipoxygenase metabolite in human polymorphonuclear leucocytes exposed to NO upon interaction with opsonized zymosan or Salmonella typhimurium.

Activity of Neutrophil β-Glucuronidase in Diabetic and Nondiabetic Patients With Chronic Generalized Periodontitis and Healthy Subjects

Objective. The aim of the study was to establish the dynamics of β-glucuronidase activity in subjects suffering from type 1 diabetes and chronic untreated generalized periodontitis, subjects suffering from chronic untreated generalized periodontitis only, and control subjects not suffering from generic diseases with healthy periodontal tissue. Material and Methods. The study involved 165 19–50-year-old subjects who were divided into three groups: healthy subjects (n=55), subjects with chronic untreated generalized periodontitis (n=55), and subjects with type 1 diabetes and chronic untreated generalized periodontitis (n=55). Neutrophilic leukocytes of peripheral venous blood were exposed to bacterial stimuli: opsonized zymosan, nonopsonized Staphylococcus aureus, and prodigiosan. The activity of β-glucuronidase was determined by the spectrofluorimetry method. Results. The diagnostic value of changes in β-glucuronidase activity of neutrophilic leukocytes markedly increased in all study groups after stimulation of neutrophilic leukocytes by opsonized zymosan, nonopsonized Staphylococcus aureus, and prodigiosan as compared to control media not exposed to any stimulus (P<0.001). The strongest relationship (canonical correlation coefficient eta, 0.993) between the intensity of periodontal pathology markers and the activity of β-glucuronidase of neutrophilic leukocytes in incubated media in patients with type 1 diabetes mellitus and periodontitis was found under the effect of nonopsonized Staphylococcus aureus. Conclusions. If periodontal impairment is severe, diabetes mellitus possibly causes a faster destruction of the periodontal tissue and presents a higher risk of periodontitis for patients with diabetes.

Effect of disodiumN,N-dialanyl protoporphyrinate, diarginine protoporphyrinate and diarginineN,N-dialanyl protoporphyrinate on respiratory burst of human neutrophilsin vitro

The effect of three amino acid protoporphyrin IX (PPIX) derivatives evaluated as sensitizers in photodynamic therapy — disodium N,N -dialanyl protoporphyrinate ( PP(Ala)2Na2), diarginine protoporphyrinate (PPArg2) and diarginine N,N -dialanyl protoporphyrinate (PP(Ala)2Arg2) – non-irradiated and pre-irradiated with UV-A, on respiratory burst of non-stimulated and opsonized zymosan stimulated neutrophils was studied. A potential synergistic effect of diazepam (7-chloro-1-methyl-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one) was also examined. PP(Ala)2Na2showed strong pro-oxidant effect towards non-stimulated neutrophils, while PPArg2and PP(Ala)2Arg2revealed no significant effect. In the case of stimulated neutrophils all studied porphyrins showed antioxidant effect, although for PP(Ala)2Na2this effect was significantly weaker than that of PPArg2and PP(Ala)2Arg2. After pre-irradiation with UV-A (λ = 365 nm, fluence 2.0 J.cm-2, fluence rate 6.7 mW.cm-2) the antioxidant activity of all studied sensitizers towards non-stimulated granulocytes did not change significantly when compared to effects of non-irradiated porphyrins, while in the case of stimulated cells, only PPArg2caused significant decrease of respiratory burst. Non-irradiated diazepam showed significant antioxidant effect and enhanced antioxidant effect of all studied porphyrins towards stimulated neutrophils, while after UV-A pre-irradiation it revealed no significant antioxidant effect on non-stimulated and stimulated neutrophils, both alone and in combination with porphyrin sensitizers.