Leukocyte Suspensions Research Articles

Pathomorphological Features of The Cornea in Secondary Posttraumatic Dystrophy (Clinical Case)

Secondary corneal dystrophy is a group of diseases characterized by an inflammatory–dystrophic process in the cornea of the eye. This pathology is accompanied by a pronounced pain syndrome and leads to an irreversible decrease in vision, up to complete blindness. To date, many aspects of pathomorphological changes of the cornea in secondary dystrophies remain insufficiently studied due to the impossibility of conducting an intravital study of the cornea tissue. The article presents a case of morphological analysis of a tissue fragment of a pathologically altered cornea in secondary posttraumatic corneal dystrophy. Destructive changes in corneal tissue revealed during light microscopy indicate the presence of signs of fibrinoid swelling in the shell stroma with gross disorganization of the base substance and neovascularization. The study made it possible to correlate the data of pathomorphological changes and the clinical picture, as well as to evaluate the effectiveness of the chosen method of treatment of this pathology (intrastromal injection of a suspension of autologous mononuclear leukocytes of blood into the stroma of the damaged cornea).

Features of oxygen-independent metabolism of neutrophils in the animals sensitized with non-agglutinogenic Brucella strain

The results of evaluation of the peculiarities of functioning of non-enzymatic systems of neutrophils of peripheral blood of guinea pigs before immunization and on the 7th, 14th, 21st, 28th, 42nd, 55th, 69th, 125th days after immunization with Brucella R-strain are presented. Two groups of agouti guinea pigs were formed for the study: control and experimental. The experimental animals were immunized with a suspension of Brucella abortus strain 16/4 at a dose of 1 billion CFU/ml subcutaneously; the control pigs were intact to brucellosis. The activity of antimicrobial systems was evaluated by determining the indices of cationic proteins by photometric method in spontaneous and induced variants. Experimental samples of brucellosis antigens were used as specific inducers in the reaction procedure. It was found that the use of Brucella disintegrans at a concentration of 50 μg/ml and corpuscular antigens at a concentration of 100 μg/ml by protein in the test with cationic proteins as inducers of disintegrants has a stimulating effect on the intensity of cationic protein production in the cell suspension of leukocytes. Stimulation indices were calculated at all observation periods as the ratio of the index of the stimulated sample to the index of the sample without antigen load. Two peaks of stimulating activity of cationic proteins were observed in the course of the study: the maximum rise of stimulation indices was observed on the 14th day after immunization and a less pronounced rise on the 28th day. The peak of agglutinating antibody production was on the 21st day after immunization, complement-binding antibodies – on the 21st-28th day, less intensive rise in titers of agglutinating and complement-binding antibodies was noted on the 69th day. In these terms, a decrease in the stimulated activity of neutrophil cationic proteins was observed. The peak activity of non-enzymatic cationic proteins of neutrophils was found to be ahead of the peak of humoral immune response.

Platelet-associated NETosis in monitoring inflammation in rheumatoid arthritis and systemic lupus erythematosus

Objective. To assess the possibility of using platelet-associated NETosis parameters to monitor inflammatory activity in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE).Materials and methods. 50 patients with RA and 29 patients with SLE were examined. The parameters of early and late platelet-associated NETosis were assessed in autologous mixed cultures containing a leukocyte suspension and intact and ADP-stimulated platelets.Results. In patients with RA and SLE in the acute stage, rates of platelet-associated NETosis were increased compared to healthy individuals (p = 0,00001-0,001). During the period of clinical remission, NETosis parameters decreased in comparison with the exacerbation stage (NET-Рst: p=0,017-0,04), in some cases reaching control values. Based on regression analysis, threshold values for platelet-associated NETosis parameters were determined to assess the activity of the inflammatory process in patients with RA and SLE.Conclusion. Parameters of platelet-associated NETosis are informative laboratory markers of the inflammatory process in patients with RA and SLE.

Creation of a biosample collection from patients after myocardial infarction in the Khanty­ Mansi Autonomous Okrug — Yugra

Aim. Creation of a sample collection of whole blood, leukocyte suspension, genomic deoxyribonucleic acid from patients with myocardial infarction for fundamental and applied research in medicine.Materials and methods. The collection, transportation and processing of biomaterial samples in the biobank of the Medical Institute of Surgut State University were carried out according to standard methods using recommendations, standard operating procedures and national guidelines for biobanking.Results. As of September 1, 2023, the collection has collected 744 aliquots of whole blood and leukocyte suspension and isolated 108 samples of genomic deoxyribonucleic acid from 108 donors after myocardial infarction. Aliquots of samples from the biobank collection are actively used for a number of molecular genetic studies (genotyping, massively parallel sequencing).Conclusion. This biosample collection on the basis of Surgut State University provides current and future research projects in cardiology with high-quality biomaterial.

Identification of functional lncRNAs in atrial fibrillation based on RNA sequencing

BackgroundAtrial fibrillation (AF) is one of the most common arrhythmia contributing to serious conditions such as stroke and heart failure. Recent studies demonstrated that long noncoding RNAs (lncRNAs) were related to cardiovascular disease. However, the molecular mechanisms of AF are not fully clear. This study intended to discover lncRNAs that are differentially expressed in AF compared with controls and evaluate the potential functions of these lncRNAs.MethodsNinety-seven patients (49 patients with AF and 48 patients without AF) were included in this study. Among these patients, leucocyte suspensions of 3 AF patients and 3 controls were sent for RNA-seq analysis to select differentially expressed lncRNA and mRNA. Different lncRNA expressions were validated in another samples (46 AF patients and 45 controls). Gene ontology (GO) enrichment analysis was conducted to annotate the function of selected mRNAs. Alternative splicing (AS) analysis was performed and a lncRNA-mRNA network was also constructed. The receiver operating characteristics (ROC) curve was used to evaluate diagnostic values. Logistic regression analysis was utilized to assess the risk or protective factor of AF.ResultsA total of 223 mRNAs and 105 lncRNAs were detected in AF patients compared with controls. Total 4 lncRNAs (LINC01781, AC009509.2, AL662844.3, AL662844.4) associated with AF were picked out for validation in another samples by quantitative real-time PCR (qRT-PCR), detecting that upregulated AC009509.2 and downregulated LINC01781 in AF patients. Multivariate logistic regression analysis illustrated that left atrial diameter (OR 1.201; 95% CI 1.093-1.320; P=0.000) and AC009509.2 (OR 1.732; 95% CI 1.092-2.747; P=0.020) were related to AF respectively. ROC curve showed that AC009509.2, LINC01781 and left atrial diameter (LAD) were predictors of AF. For LINC01781, the area under the curve (AUC) was 0.654 (95% CI 0.541-0.767, P=0.0113). For AC009509.2, the AUC was 0.710 (95% CI 0.599-0.822, P=0.0005). Bioinformatic methods (GO enrichment, AS analysis and lncRNA-mRNA network construction) were performed to reveal the role of lncRNAs.ConclusionsThis study discussed differentially expressed lncRNA and their potential interaction with mRNA in AF. LncRNA AC009509.2 could be a new potential biomarker for AF prediction.

The study of the pharmacological activity of a cream with a white willow bark extract on the model of contact non-allergic dermatitis

Despite the achievements of medicine, dermatological skin diseases are an urgent problem today as the incidence rate increases annually. A promising direction of modern pharmacotherapy of dermatoses is the creation of innovative etiotropic topical agents on a plant basis. Aim. To study the pharmacological activity of a combined cream with a white willow (Salix alba L.) bark extract and zinc perithionate under the conditional name “Pyrisalix” against the background of the experimental non-allergic dermatitis induced by turpentine oil. Materials and methods. As the study object, a cream made with the active substances of a thick extract of white willow bark and zinc perithionate on a cream base was used; it corresponded to the pharmacotechnological indicators by colloidal, thermal stability and the description of oil/water (20 % of the oil phase). The model of alternative chronic inflammation, non-allergic contact dermatitis, was induced in white non-linear rats by daily application of turpentine oil to the depilated area. The effectiveness of the “Pyrisalix” cream was evaluated based on the total leukocyte count, the peripheral blood leukocyte composition, erythrocyte sedimentation rate (ESR), including the leukocyte-ESR ratio index (LI-ESR), the skinfold thickness, and the skin cover condition. In our experiment, the phagocytic activity of granulocytic neutrophils in a leukocyte suspension obtained from heparinized blood was determined to assess the activity of nonspecific immune factors. Results. The emulsion base of the cream has been determined, according to which the product developed has thermal and colloidal stability. In the course of studying the pharmacological activity of the “Pyrisalix” cream on themodel of non-allergic skin inflammation based on the dynamics of local manifestations of contact dermatitis, hematological, and immunological parameters, it has been found that the “Pyrisalix” cream has a pronounced preventive and therapeutic effect due to its anti-inflammatory and immunological properties. In the experimental conditions, the anti-alterative action of the cream on the emulsion oil/water base studied was equal to or even significantly exceeded the activity of the reference drugs – “Fladex” ointment and “Psorikap” cream by the intensity of inhibition of inflammatory processes in the skin, reduction in total leukocyte count, and normalization of the functional activity of granulocytic neutrophils. Conclusions. The combined cream “Pyrisalix” on the emulsion oil/water base demonstrates a significant antiinflammatory activity on the model of non-allergic contact dermatitis. The combined cream “Pyrisalix” is a promising agent for further study in the treatment of dermatoses.

Polymorphonuclear phenotypical expression of CD18, at baseline and after in vitro activation, in several clinical disorders: Revision of our case series.

In relation to the different and important roles of the beta2 integrins, we have revisited the expression of polymorphonuclear leukocyte CD18 in several clinical disorders, at baseline and after in vitro activation. we have examined subjects with type 1 diabetes mellitus, vascular atherosclerotic disease, type 2 diabetes mellitus without and with macrovascular complications, chronic renal failure on conservative treatment, essential hypertension, deep venous thrombosis, acute ischemic stroke and subjects with venous leg ulcers. unfractioned leukocyte suspension was prepared according to the Mikita's method, while the leukocyte were separated into mononuclear and polymorphonuclear cells with a Ficoll-Hypaque medium. Using specific monoclonal antibody, the CD18 expression was evaluated with cytofluorimetric analysis, using FACScan (Becton Dickinson) be Cellquest software; the activation in vitro with PMA was effected according to modified Yasui and Masuda methods. in type 1 diabetes mellitus, at baseline CD18 is under expressed in comparison with normal control, and not changes after PMA activation were observed; in subjects with vascular atherosclerotic disease, in type 2 diabetes mellitus CD18 is over expressed at baseline but does not vary after activation; in subjects with chronic renal failure, essential hypertension and in subjects with acute ischemic stroke the CD18 up-regulate at baseline compared to normal control, and it increases further after activation; in subjects with deep venous thrombosis the CD18 expression is not different from control group at baseline, but it increases after activation; finally, in subjects with venous leg ulcers the CD18 is normally expressed at baseline, and it does not change after PMA activation. in the different clinical disorders, the trend of this integrin subunit provides some specific information, useful to select the best therapeutic strategy in clinical practice.

Molecular genetic diagnosis in the group of hemophilia A patients in Belarus: 12 new allelic variants in the <i>F8</i> gene

Hemophilia A is the most common severe bleeding disorder caused by various genetic changes in the F8 gene, leading to coagulation factor VIII deficiency. Hemophilia A is characterized by high heterogeneity of genetic defects. The severity of hemophilia A varies depending on the type of genetic defects in the F8 gene. More than 3000 unique variants of the F8 gene are associated with the hemophilia A. Approximately 30% of genetic defects occur de novo. The aim of this study is to determine the spectrum of genetic defects in the F8 gene in children with hemophilia A in Belarus. The study was approved by the Independent Ethics Committee and the Scientific Council of the Belarusian Research Center for Pediatric Oncology, Hematology and Immunology (the Republic of Belarus). The study included 98 patients with hemophilia A, who had been treated or followed up at the Belarusian Research Center for Pediatric Oncology, Hematology and Immunology (the Republic of Belarus). Patients were categorized into 3 groups based on the severity of their disease: severe (n = 82), moderate (n = 3), and mild (n = 13). Twenty (20.4%) patients had a history of inhibitors to factor VIII. For our study, we used venous blood samples. Genomic DNA was isolated from leukocyte suspension (obtained from the whole blood samples) using phenol-chloroform extraction. All severe hemophilia A patients were prescreened for intron 22 and 1 inversions in the F8 gene using inverse and multiplex polymerase chain reaction assays, respectively. Sequencing of F8 coding regions was carried out by next generation sequencing. All clinically relevant variants were confirmed by Sanger sequencing. Genetic testing revealed that 99% of the patients with hemophilia A (n = 97) had pathogenic variants in the F8 gene. Intron 22 and intron 1 inversion mutations within the F8 gene were detected in 45.1% (n = 37) and 1.2% (n = 1) patients with severe hemophilia A, respectively. Two patients had an abnormal pattern of intron 1 inversion, not previously described in the literature. A total of 48 different variants in the F8 gene were detected in 57 patients using next generation sequencing. Eleven of the 48 genetic variants identified have not been previously reported.

A sample-preparation-free, point-of-care testing system for in situ detection of bovine mastitis.

We present a highly integrated point-of-care testing (POCT) device capable of immediately and accurately screening bovine mastitis infection based on somatic cell counting (SCC). The system primarily consists of a homemade cell-counting chamber and a miniature fluorescent microscope. The cell-counting chamber is pre-embedded with acridine orange (AO) in advance, which is simple and practical. And then SCC is directly identified by microscopic imaging analysis to evaluate the bovine mastitis infection. Only 4 μL of raw bovine milk is required for a simple sample testing and accurate SCC. The entire assay process from sampling to result in presentation is completed quickly within 6min, enabling instant "sample-in and answer-out." Under laboratory conditions, we mixed bovine leukocyte suspension with whole milk and achieved a detection limit as low as 2.12 × 104 cells/mL on the system, which is capable of screening various types of clinical standards of bovine milk. The fitting degrees of the proposed POCT system with manual fluorescence microscopy were generally consistent (R2 > 0.99). As a proof of concept, four fresh milk samples were used in the test. The average accuracy of somatic cell counts was 98.0%, which was able to successfully differentiate diseased cows from healthy ones. The POCT system is user-friendly and low-cost, making it a potential tool for on-site diagnosis of bovine mastitis in resource-limited areas.

Evaluation of the Cell Concentration in Suspensions of Human Leukocytes by Ultrasound Imaging: The Influence of Size Dispersion and Cell Type.

This work focuses on the use of ultrasound imaging to evaluate the cell concentration of dilute leukocyte suspensions in the range of 10-3000 cells/µL. First, numerical simulations were used to study the influence of the size dispersion and the leukocyte type on the performance of the concentration estimation algorithms, which were developed in previous works assuming single-sized scatterers. From this analysis, corrections to the mentioned algorithms were proposed and then the performance of these corrections was evaluated from experiments. For this, ultrasound images were captured from suspensions of lymphocytes, granulocytes, and their mixtures. These images were obtained using a 20 MHz single-channel scanning system. Results confirmed that concentration estimates provided by conventional algorithms were affected by the size dispersion of cells, leading to a remarkable underestimation of results. The proposed correction to compensate for cell size dispersion obtained from simulations improved the concentration estimation of these algorithms, for the cell suspensions tested, approaching the results to the reference optical characterization. Moreover, it was shown that these models provided a total leukocyte concentration from the ultrasound images which was independent of the relative populations of different white blood cell types.

Pharmacological activity cream of extract Salix alba and zinc for allergic contact dermatitis

Allergic dermatitis is the most common skin disease, manifested by classic factors in response to the external environment. Solving the problem of global prevalence and intensive growth of the frequency of allergic dermatitis requires improving the medical supply of the population of Ukraine with drugs for the effective treatment of allergic skin diseases. According to the literature, preparations based on white willow bark have a wide range of biological effects, which exhibit anti-inflammatory, anti-allergic, anti-pruritic, analgesic activity and have a positive effect on the immune system and are promising for the treatment and prevention of allergic dermatitis. Zinc takes part in metabolism and stabilization of cell membranes, is part of the main enzymes that take part in antioxidant biochemical reactions. The trace element has a regenerating effect, ensures the transmission of nerve impulses, promotes the synthesis of cortisol. 
 The purpose of our work was to study the effect of a cream based on white willow and Zn antigen-specific link of immune protection of animals on 3 experimental allergic dermatitis. 
 The study was conducted on сavia porcellus. Allergic contact dermatitis was modeled using 2,4-dinitrochlorobenzene. Phagocytic activity of granulocytic neutrophils in a suspension of leukocytes is determined for assessment of the activity of factors of non-specific immunity.
 It is important to determine the status of antigen-specific factors of immune protection, which are directly involved in all doses of the immune response. To assess the activity of factors of nonspecific immunity, the phagocytic activity of granulocytic neutrophils was determined in the suspension of leukocytes obtained from heparinized blood.
 The obtained data on the increase of F1 and F4 indicators in animals against the background of experimental dermatitis indicate the presence of a natural protective defense aimed at neutralizing the allergen and its subsequent removal from the body. In comparison with reference drugs it was found distinct antigen-specific effect, which exceeds Psoricap and is equal Fladex.
 On the model of allergic contact dermatitis in Cavia porcellus, the researched cream showed a pronounced immunotropic activity relative to a decrease in the phagocytic activity of cell.

Effect of toxic air pollutants on the cytokine release by leukocytes in patients with chronic obstructive pulmonary disease

Chronic inflammation in obstructive pulmonary disease develops in genetically predisposed individuals with prolonged or massive exposure to allergens or toxic air pollutants. This effect leads to hyperactivation of immune system and development of uncontrolled inflammatory response. The aim of the study was to determine the level of cytokines in the supernatant of leukocytes from the patients with COPD and asthma following incubation with air toxicants, i.e., a solution of cigarette smoke, an extract of cigarette tobacco, or a solution of exhaust combustion gases. The cytokines were determined by ELISA in the supernatants following exposure of peripheral venous blood leukocytes to the toxicants. To perform the assays, 10-mL samples of peripheral venous blood from the patients were taken into the test tubes with heparin (20 U/mL) in the morning time, not earlier than 2 days after therapeutic infusions of glucocorticosteroids. After gravity sedimentation, the leukocyte-rich was removed, centrifuged at 1500 rpm, then the liquid was discarded, and the leukocyte pellets were diluted with buffered saline (2 106 cells/mL). Individual leukocyte suspensions were divided into 4 wells of an immunological plate, 100 μL each. Equal volumes of test solutions simulating the effect of toxicants were added to three wells. The fourth well contained sterile isotonic sodium chloride solution (negative control). The mixtures in plates were exposed for 45 min at 37 °C followed by centrifugation for 10 min at 1500 rpm. From each well, 50 μL of the supernatant was taken and transferred to the plate for ELISA assays (under the same number). As a result, we have found that the solutions of cigarette smoke and exhaust gases caused release of IL-1β by leukocytes in the patients with asthma and COPD, but not in the samples from control group of healthy volunteers. Spontaneous increase in the IL-1β level was registered in the patients with asthma. The cigarette extracts caused an increased release of TNFα in the supernatant fluid of the patients with COPD. Upon exposure to a solution of exhaust gases, an increased level of TGF-β was revealed in patients with asthma compared to spontaneous cell activation (p < 0.05), as well as an increase in IFNγ contents in the patients with COPD as compared with control group (p < 0.05). No statistically significant changes were revealed for the levels of IL-6, IL-2, IL-4, IL-12, IFNα upon exposure of air toxicants on the leukocytes of patients with COPD, asthma or healthy volunteers.

Clinical and Functional Results of the Use of Suspension of Autologous Mononuclear Leucocytes in the Surgical Treatment of Bullous Keratopathy

The purpose: to explore the clinical effectiveness of a new cell-based surgical method for the treatment of bullous keratopathy that includes intrastromal injection of autologous mononuclear leukocytes into the posterior third of the cornea stroma.Material and methods. The study included 58 patients with diagnosis of bullous kerathopathy. They were divided into 2 groups — the main group received surgical treatment by the presented method; and the comparison group received a course of conservative therapy.Results. In 10 days the patients in the main group had a normal structure of the cornea epithelium, slight edema of the stroma, thin folds of the Descemet’s membrane and moderate edema of the endothelium. Similar results in the comparison group were achieved in 1 month, and it was the best condition of the cornea. Later corneal edema increased. In 3 months the patients of the main group had a normal structure of corneal epithelium, single thin folds of the Descemet’s membrane, slight edema of the posterior third of the stroma only in the optical zone, and slight edema of the endothelium. The achieved results were preserved for 12 months. The best correct visual acuity (BCVA) in the main group was achieved by the 6th month. It increased 10 times from the initial level, and the corneal thickness decreased by 27 % from the initial level by the 9th month. In the comparison group, the BCVA was achieved by the 1st month. It increased 2.7 times from the initial level; the maximum decrease in the index of pachymetry by 6.7 % was achieved by the 3rd month from the initial level, and then these parameters increased.Conclusion. The using of a surgical method for the treatment of bullous keratopathy, including intrastromal injection of suspension of autologous mononuclear leukocytes, contributes to the relief of corneal syndrome, increases the transparency of the cornea and restores its normal thickness. These changes are accompanied by increase of BCVA in the postoperative period compared to the course of conservative therapy.

Effect of High-Volume Leukapheresis on Hematological Indexes of Patients with Hyperleukocytic Leukemia

To improve the collection efficiency of leukapheresis, explore relatively scientific and objective evaluation indicators for collection effect, and observe the effect of high-volume leukapheresis on blood cells and coagulation function. A total of 158 times of high-volume leukapheresis were performed on 93 patients with hyperleukocytic leukemia by using continuous flow centrifugal blood component separator. 1/5-1/4 of total blood volume of the patients was taken as the target value of leukocyte suspension for single treatment. In addition, the total number of white blood cells (WBCs) subtracted, value of WBCs reduction, rate of WBCs reduction, decrease value of WBCs count, decrease rate of WBCs count, amount of hemoglobin (Hb) lost, value of Hb lost, decreased value of Hb, total number of platelet (PLT) lost, the value of PLT loss, and decrease value of PLT count were used to comprehensively evaluate the collection effect of leukapheresis and influence on Hb level and PLT count of the patients. The prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), and fibrinogen (Fib) concentration were detected before and after treatment, and the effect of leukapheresis on coagulation function of the patients was observed. The volume of leukocyte suspension collected in a single treatment was 793.01±214.23 ml, the total number of WBCs subtracted was 353.25 (241.99-547.28)×109, the value of WBCs reduction was 86.98 (63.05-143.43)×109/L, the rate of WBCs reduction was 44.24 (28.37-70.48)%, decrease value of WBCs count was 65.73 (37.17-103.97)×109/L, decrease rate of WBCs count was (35.67±23.08)%, the amount of Hb lost was 17.36 (12.12-24.94) g, the value of Hb lost was 4.31 (3.01-6.12) g/L, decreased value of Hb was 4.80 (-1.25-9.33) g/L, total number of PLT lost was 222.79 (67.03-578.31)×109, the value of PLT loss was 54.45 (17.29-139.08)×109/L, and decrease value of PLT count was 26.00 (8.38-62.50)×109/L. Before and after a single treatment, the PT was 14.80 (13.20-16.98) s and 15.20 (13.08-16.90) s (z=-1.520, P>0.05), the aPTT was 35.20 (28.68-39.75) s and 35.40 (28.00-39.75) s (z=-2.058, P<0.05), the TT was 17.50 (16.30-18.80) s and 17.70 (16.70-19.10) s (z=-3.928, P<0.001), and the Fib concentration was 2.87±1.13 g/L and 2.64±1.03 g/L (t=7.151, P<0.001), respectively. High-volume leukapheresis can improve the efficiency of leukapheresis while maintaining the relative stability of the patients' circulating blood volume. The degree of influence on the patients' Hb level, PLT count, Fib concentration, and comprehensive coagulation indicators reflecting the patients' intrinsic and cxtrinsic coagulation activity is within the body's compensation range.

Effect of lidocaine on oxidative activity of peripheral blood phagocytes

BACKGROUND: Excessive production of reactive oxygen species (ROS) by leukocytes can cause damage to intrinsic tissues. The pathogenesis of sepsis is based on an excessive inflammatory response of the body. Several studies have reported the inhibitory effect of lidocaine on neutrophilic granulocytes.&#x0D; AIM: This study aimed to analyze the effect of lidocaine on the oxidative activity of phagocytes.&#x0D; MATERIALS AND METHODS: Blood from 16 healthy donors was used in this study. Leukocyte mass was extracted using spontaneous sedimentation. Half of the leukocyte samples were incubated in buffered physiological saline with lidocaine. The other half of the leukocyte samples were incubated in physiological saline without lidocaine. The generation of ROS was studied using two methods. Method 1 included a nitro blue tetrazolium (NBT) test), which is based on the ability of ROS to reduce NBT to insoluble diformazan. Method 2 was based on the chemiluminescence reaction. A culture of S. Aureus was used to induce the production of ROS.&#x0D; RESULTS: NBT test revealed a decrease in the oxidative activity of leukocytes in the presence of lidocaine by 18% (p 0.05). The study of luminol-dependent chemiluminescence of leukocyte suspension in the presence of lidocaine revealed a significant 2-fold decrease in both spontaneous and stimulated respiratory activity of cells.&#x0D; CONCLUSIONS: After incubation with lidocaine, phagocytes generated ROS to a significantly lower extent. However, their complete blockade was not recorded. This property of lidocaine may be used in clinical practice to treat an excessive inflammatory response in sepsis.

Tailoring freshwater diets towards boosted immunity and pancreas disease infection robustness in Atlantic salmon post smolts

The aim of the current study was to investigate how freshwater diets impact on immunity in Atlantic salmon smolts in freshwater, during transfer to seawater and in post smolts during the seawater stage with and without pancreas disease (PD) infection. Three specific freshwater diets were prepared: (i) A diet similar in composition to commercial salmon freshwater diets (Standard diet); (ii) A diet composed of vegetable oils (rapeseed, palm and linseed oils) mimicking the fat composition in aquatic insects – the natural diet of wild salmon in freshwater (Fatty acid diet); (iii) A diet enriched with possible immune modulating amino acids including dl-methionine, l-lysine, l-threonine and taurine (Amino acid diet). After seawater transfer, all fish were fed the same commercial diet. Head kidneys were extracted, and their leukocytes isolated from smolts right before transfer to seawater, from post smolts one and six weeks after transfer to seawater, and from post smolts in seawater after 8 weeks of ongoing PD infection. In addition, to provoke bacterial or virus induced inflammation in vitro, the individual leukocyte suspension from all fish were stimulated by lipopolysaccharide (LPS) or polyinosinic acid: polycytidylic acid (PIC).The transfer of smolts from fresh-to seawater changed the transcription of several types of genes. Particularly in isolates from fish fed the Standard or Fatty acid diet in freshwater, overall gene transcription (IL-1β, CD83, INF-γ, cox2, cd36, MGAT2, catalase) declined. However, the Amino acid diet stimulated the LPS induced gene transcription of IL-1β, CD83, Cox2, and INF-γ at this stage. In freshwater smolts, PIC stimulated leukocytes showed higher transcription level of Mx and viperin in the Fatty acid and Amino acid diet groups compared to the Standard diet group. In seawater post smolts, Mx and viperin responded similarly to PIC challenge in all diet groups. Furthermore, leukocytes isolated from PD infected fish, continued responding to PIC, regardless of freshwater diet.

Oral Intake of Semi-refined Carrageenan by Rats Affects Apoptosis of Lymphocytes

Introduction. The safety of generally recognized as safe food additives E407 and E407a is under rigorous debate. The aim of our research was to evaluate the effects of the orally administered food additive E407a (semi-refined carrageenan) on the viability of lymphocytes and their cell death modes. Methods. Totally, 16 adult WAG rats were divided into two equal groups (experimental – oral intake of 140 mg/kg of E407a during 2 weeks; control – oral consumption of drinking water instead). Blood samples were used to obtain leukocyte suspensions stained with Annexin V-FITC and 7-aminoactinomycin D (7-AAD). The region of lymphocytes was analyzed after collecting data using a BD FACSCanto™ II flow cytometer. Results. Oral administration of E407a led to a decrease in the amount of viable (Annexin V-, 7-AAD-) circulating lymphocytes. Furthermore, exposure to semi-refined carrageenan resulted in an elevated number of early apoptotic (Annexin V+, 7-AAD-) lymphocytes. Their percentage was approximately 4 times higher in rats exposed to E407a compared with the control group. Conclusion. Our findings indicate that dietary intake of E407a promotes apoptosis of circulating lymphocytes. Keywords: processed Eucheuma seaweed, annexin V, 7-aminoactinomycin D, flow cytometry.

Semi-refined carrageenan promotes generation of reactive oxygen species in leukocytes of rats upon oral exposure but not in vitro.

To assess the ability of the common food additive E407a (semi-refined carrageenan) to enter leukocytes in vitro and generate reactive oxygen species (ROS) in leukocytes as awhole and granulocytes in particular, both during incubation and in experimental animals. ROS production was assessed in leukocytes incubated with E407afor 2 h at the final concentrations of 5 and 10 g/L using the dye 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), as well as in cells isolated from rats orally exposed to E407a (140 mg/kg of weight) during 2weeks (n = 8) and control rats (n = 8), by flow cytometry. Carrageenan uptake by leukocytes was estimated by confocal microscopy using incubation of rhodamineB isothiocyanate-labelled carrageenan with leukocyte suspensions. Uptake of carrageenan by viable neutrophils, monocytes, and lymphocytes was confirmed. Oral administration of the food additive E407awas associated with excessive ROS formation by viable leukocytes (CD45+, 7‑aminoactinomycin D- cells) and especially in granulocytes. Unexpectedly, adirect impact of semi-refined carrageenan during incubation for 2 h did not affect ROS production in leukocytes, evidenced by statistically insignificant differences in mean fluorescence intensity values of 2',7'-dichlorofluorescein, which is aROS-sensitive product of intracellular H2DCFDA conversion. Oral intake of E407a and direct exposure of leukocyte suspensions to it decreased the viability of leukocytes. Food-grade carrageenan can enter leukocytes without affecting ROS generation as aresult of incubation for 2 h with leukocyte suspensions. On the contrary, oral exposure to E407a is accompanied by ROS overproduction by white blood cells, suggesting an indirect mechanism for the stimulation of ROS synthesis in vivo. E407a promotes cell death of leukocytes both in vivo and in vitro.

Multinucleated giant cells within the in vivo implantation bed of a collagen-based biomaterial determine its degradation pattern

ObjectivesThe aim of the present study was to characterize the cellular reaction to a xenogeneic resorbable collagen membrane of porcine origin using a subcutaneous implantation model in Wistar rats over 30 days.Materials and methodsEx vivo, liquid platelet-rich fibrin (PRF), a leukocyte and platelet-rich cell suspension, was used to evaluate the blood cell membrane interaction. The material was implanted subcutaneously in rats. Sham-operated rats without biomaterial displayed physiological wound healing (control group). Histological, immunohistological, and histomorphometric analyses were focused on the inflammatory pattern, vascularization rate, and degradation pattern.ResultsThe membrane induced a large number of mononuclear cells over the observation period, including lymphocytes, macrophages, and fibroblasts. After 15 days, multinucleated giant cells (MNGCs) were observed on the biomaterial surface. Their number increased significantly, and they proceeded to the center of the biomaterial on day 30. These cells highly expressed CD-68, calcitonin receptor, and MMP-9, but not TRAP or integrin-ß3. Thus, the membrane lost its integrity and underwent disintegration as a consequence of the induction of MNGCs. The significant increase in MNGC number correlated with a high rate of vascularization, which was significantly higher than the control group. Physiological wound healing in the control group did not induce any MNGCs at any time point. Ex vivo blood cells from liquid-PRF did not penetrate the membrane.ConclusionThe present study suggests a potential role for MNGCs in biomaterial degradation and questions whether it is beneficial to accept them in clinically approved biomaterials or focus on biomaterials that induce only mononuclear cells. Thus, further studies are necessary to identify the function of biomaterial-induced MNGCs.Clinical relevanceUnderstanding the cellular reaction to biomaterials is essential to assess their suitability for specific clinical indications and outline the potential benefit of specific group of biomaterials in the respective clinical indications.

Formation of eyeball orbital stump using titanium nickelide tissue-engineered construct and autologous blood mononuclear leukocytes

Objective : to study the morphological features of formation of the eyeball orbital stump using a titanium nickelide tissue-engineered construct and a suspension of autologous blood mononuclear leukocytes in vivo . Materials and methods . Experiments were performed on 54 sexually mature Wistar rats weighing 200–250 g. The animals were divided into 3 groups, depending on type of surgical intervention: group 1 (n = 18) consisted of animals in which eyeball orbital stump was formed after evisceroenucleation through implantation of a titanium nickelide tissue-engineered construct and a suspension of autologous blood mononuclear leukocytes in the scleral sac; group 2 (n = 18) – the eyeball orbital stump was formed through implantation of titanium nickelide tissue-engineered construct in the scleral sac; group 3 (n = 18) – orbital stump was formed using an Alloplant implant. Results . It was established that in group 1 rats, on day 7 following surgery, the specific volume of connective tissue was 7.9 times (р U = 0.048) higher than in group 2 rats and 15.8 times (р U = 0.039) higher than in group 3 rats. On day 14 after surgery, the volume of connective tissue in the eyeball orbital stump of group 1 rats reached the highest value compared to that in the other groups. The numerical density of newly formed vessels in the eyeball orbital stump of group 1 rats, starting from day 14 after surgery up to the end of experiment (day 21), was statistically significantly higher than that in the other groups. Moreover, on day 21, this indicator was 4.0 times (р U = 0.001) higher in group 1 rats than in group 2 rats and 9.8 times (р U = 0,0003) higher than in group 3 rats. Conclusion . Implantation of titanium nickelide tissue-engineered construct and a suspension of autologous blood mononuclear leukocytes into the scleral sac after evisceroenucleation in an in vivo experiment leads to accelerated maturation of the connective tissue and intensive vascularization in the eyeball orbital stump. This ensures strong fixation of the implant and reduces risk of rejection.