Granulocyte Activation Research Articles

PECULIARITIES OF THE WOUND PROCESS DEVELOPMENT IN PATIENTS WITH THERMAL INJURY WHEN USED SILVER PREPARATIONS IN BURN WOUNDS SURGICAL TREATMENT

Fundamental research in the field of burn wound development process highlights new pathogenetic mechanisms that underlie the improvement of medical practice, the main purpose of which is to create optimal conditions for early regeneration of the affected skin, as well as to eliminate the development of local and general complications. Irrational general and local treatment of wounds contribute to the slowing down of regenerative processes, thereby worsening the prognosis and consequences of thermal injury.
 It was found out that in patients of both study groups in the first period of the study the characteristics of the cellular composition of the wound impressions of the wound bottom of the area of thermal damage correspond to the degenerative type.
 In patients of the main group in the second term of the study in the area of thermal damage cells with high levels of functional and metabolic activity begin to appear, which increase antimicrobial resistance and reduce the risk of generalization of the infectious process which is the main source of burns.
 In patients of the main group, an increase in the activity of neutrophilic granulocytes in the induced test was found in the second term of the study, which, in our opinion, indicates the involvement of neutrophilic granulocytes with high functional and metabolic activity. Under conditions of significant activation of resident monocytes in response to injury in patients of the main group in the second and third term, the area of thermal damage migrates functionally effective monocytes. In patients of the main group the preservation of the activity of myeloperoxidase - the main enzyme of the oxygen-dependent phase of phagocytosis at the minimum subcompensated level, which leads to the preservation of the oxygen-dependent metabolic reaction of neutrophilic granulocytes in the subsequent study, Increases in the content of PAS-positive substances in neutrophilic granulocytes indicate the preservation of the functional reserve for activation and completion of the oxygen-independent phase of phagocytosis.
 The use of the developed treatment promotes the involvement of functionally active phagocytic cells in the burn wound, reduces the toxic load on monocytes and neutrophils, which helps to maintain their functional activity at the subcompensated level. These trends in patients of the main group reduce the risk of both local and general infectious complications of burn disease.
 It was found that the level of spontaneous functional activity in the HCT test is associated with the activity of resident monocytes. They form the primary barrier that protects the body from infection or harmful macromolecular complexes. Monocytes - "inflammatory", which constantly come from the free pool and their functional activity against microbial antigens is much higher than resident monocytes. An indicator of the activity of monocytes - "inflammatory" is an indicator of functional activity in the induced HCT test. The results of the study indicate that under conditions of significant activation of resident monocytes in response to injury in patients of the main group in the second and third terms of the area of thermal damage migrate functionally effective monocytes.

I CANNOT SEE YOUR FACE! A CASE OF PURTSCHER DISEASE

TOPIC: Critical Care TYPE: Medical Student/Resident Case Reports INTRODUCTION: Purtscher-like retinopathy is an acquired form of retinopathy due to hemorrhagic or vasoocclusive vasculopathy associated with acute pancreatitis and other systemic diseases 1. It is associated with severe vision. Recent studies suggested ophthalmoscopy performed within first 24 hours of developing acute pancreatitis was a good indicator for predictor of disease evolution, outcome and overall poor prognosis 2. We present a rare case of a young African American male who had experienced acute vision loss in the setting of severe alcoholic pancreatitis. CASE PRESENTATION: 34-year-old African American male with a history of alcoholic dependence presents with abdominal pain. His last drink was 36 hours ago. It's described as sharp and radiating to his back. He denies any other ingestions or drug use. On presentation, vital signs show that he is mildly hypertensive, but otherwise within normal limits. Physical exam reveals epigastric tenderness on palpation with positive bowel signs, negative murphy sign, cullens sign and gray turners sign. Labs were significant for an elevated lipase as well as liver enzymes. No significant osmolar gap. Intravenous fluids were provided as well as pain control and patient was placed on a withdrawal assessment scale. Within 12 hours of admission, he started to experience alcoholic hallucinosis with decreased visual acuity and inability to recognize facial features. Fundoscopy revealed retinal whitening bilaterally, Intraretinal flame heme along inferior arcade of the right eye, cherry red spot of the left eye within the macula, consistent with Purtscher-like retinopathy in the setting of acute pancreatitis declaring him legally blind. Patient signed a 302 to receive further inpatient psychiatric care. DISCUSSION: The patient's acute psychosis from alcohol compounded his complaint of acute vision loss. Purtscher retinopathy is a rare complication of acute pancreatitis. The etiology is unclear, however in relation to pancreatitis, the condition may be caused by complement induced granulocyte activity causing cotton wool spots, retinal hemorrhages, and papilledema [2]. It should be suspected if patients voice concern of decreased visual acuity. Previously high dose steroids have been used but there is no evidence that this treatment improves morbidity. Recovery of visual acuity is variable with some patients regaining vision with mild loss of acuity and others remain legally blind. Certainly, a rare and interesting complication of acute pancreatitis. CONCLUSIONS: Evaluation and treatment of patients with acute pancreatitis with visual complaints should raise concern for Purtcher's retinopathy. There are no current evidence-based treatment options. Close follow up with a retinal specialist is required. Further clinical studies are required to investigate treatment options for Purtcher's retinopathy. REFERENCE #1: Bissonnette B. Purtscher Disease. AccessAnesthesiology 2019. REFERENCE #2: C; CDRABFB. [Retinopathy in severe acute pancreatitis]. Oftalmologia (Bucharest, Romania : 1990). REFERENCE #3: Buckley, S. and James, B., 2020. [online] Pmj.bmj.com. Purtschers Retinopathy. [Accessed 7 July 2020]. DISCLOSURES: No relevant relationships by Abdelrhman Abo-zed, source=Web Response No relevant relationships by Melissa Chrites, source=Web Response No relevant relationships by Erica OKane, source=Web Response No relevant relationships by Syed Arsalan Zaidi, source=Web Response

High-dimensional profiling reveals phenotypic heterogeneity and disease-specific alterations of granulocytes in COVID-19

Since the outset of the COVID-19 pandemic, increasing evidence suggests that the innate immune responses play an important role in the disease development. A dysregulated inflammatory state has been proposed as a key driver of clinical complications in COVID-19, with a potential detrimental role of granulocytes. However, a comprehensive phenotypic description of circulating granulocytes in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients is lacking. In this study, we used high-dimensional flow cytometry for granulocyte immunophenotyping in peripheral blood collected from COVID-19 patients during acute and convalescent phases. Severe COVID-19 was associated with increased levels of both mature and immature neutrophils, and decreased counts of eosinophils and basophils. Distinct immunotypes were evident in COVID-19 patients, with altered expression of several receptors involved in activation, adhesion, and migration of granulocytes (e.g., CD62L, CD11a/b, CD69, CD63, CXCR4). Paired sampling revealed recovery and phenotypic restoration of the granulocytic signature in the convalescent phase. The identified granulocyte immunotypes correlated with distinct sets of soluble inflammatory markers, supporting pathophysiologic relevance. Furthermore, clinical features, including multiorgan dysfunction and respiratory function, could be predicted using combined laboratory measurements and immunophenotyping. This study provides a comprehensive granulocyte characterization in COVID-19 and reveals specific immunotypes with potential predictive value for key clinical features associated with COVID-19.

Expression characteristics of interferon-stimulated genes and possible regulatory mechanisms in lupus patients using transcriptomics analyses.

BackgroundType I interferon signature is one of the most important features of systemic lupus erythematosus (SLE), which indicates an active immune response to antigen invasion. Characteristics of type I interferon-stimulated genes (ISGs) in SLE patients have not been well described thus far.MethodsWe analyzed 35,842 cells of PBMC single-cell RNA sequencing data of five SLE patients and three healthy controls. Thereafter, 178 type I ISGs among DEGs of all cell clusters were screened based on the Interferome Database and AUCell package was used for ISGs activity calculation. To determine whether common ISG features exist in PBMCs and kidneys of patients with SLE, we analyzed kidney transcriptomic data from patients with lupus nephritis (LN) from the GEO database. MRL/lpr mice model were used to verify our findings.FindingsWe found that monocytes, B cells, dendritic cells, and granulocytes were significantly increased in SLE patients, while subsets of T cells were significantly decreased. Neutrophils and low-density granulocytes (LDGs) exhibited the highest ISG activity. GO and pathway enrichment analyses showed that DEGs focused on leukocyte activation, cell secretion, and pathogen infection. Thirty-one common ISGs were found expressed in both PBMCs and kidneys; these ISGs were also most active in neutrophils and LDGs. Transcription factors including PLSCR1, TCF4, IRF9 and STAT1 were found to be associated to ISGs expression. Consistently, we found granulocyte infiltration in the kidneys of MRL/lpr mice. Granulocyte inhibitor Avacopan reduced granulocyte infiltration and reversed renal conditions in MRL/lpr mice.InterpretationThis study shows for the first time, the use of the AUCell method to describe ISG activity of granulocytes in SLE patients. Moreover, Avacopan may serve as a granulocyte inhibitor for treatment of lupus patients in the future.

Vitamin C, Hydrocortisone, and the Combination Thereof Significantly Inhibited Two of Nine Inflammatory Markers Induced by Escherichia Coli But Not by Staphylococcus Aureus - When Incubated in Human Whole Blood.

ABSTRACTVitamin C combined with hydrocortisone is increasingly being used to treat septic patients, even though this treatment regimen is based on questionable evidence. When used, a marked effect on key players of innate immunity would be expected, as sepsis is featured by a dysregulated immune response.Here, we explored the effect of vitamin C and hydrocortisone alone and combined, in an ex vivo human whole-blood model of Escherichia coli- or Staphylococcus aureus-induced inflammation. Inflammatory markers for activation of complement (terminal C5b-9 complement complex [TCC]), granulocytes (myeloperoxidase), platelets (β-thromboglobulin), cytokines (tumor necrosis factor [TNF], IL-1β, IL6, and IL-8), and leukocytes (CD11b and oxidative burst) were quantified, by enzyme-linked immunosorbent assay, multiplex technology, and flow cytometry.In E. coli- and S. aureus-stimulated whole blood, a broad dose-titration of vitamin C and hydrocortisone alone did not lead to dose–response effects for the central innate immune mediators TCC and IL-6. Hence, the clinically relevant doses were used further. Compared to the untreated control sample, two of the nine biomarkers induced by E. coli were reduced by hydrocortisone and/or vitamin C. TNF was reduced by hydrocortisone alone (19%, P = 0.01) and by the combination (31%, P = 0.01). The oxidative burst of monocytes and granulocytes was reduced for both drugs alone and their combination, (ranging 8–19%, P < 0.05). Using S. aureus, neither of the drugs, alone nor in combination, had any effects on the nine biomarkers.In conclusion, despite the limitation of the ex vivo model, the effect of vitamin C and hydrocortisone on bacteria-induced inflammatory response in human whole blood is limited and following the clinical data.

Characterization of low-density granulocytes in COVID-19.

Severe COVID-19 is characterized by extensive pulmonary complications, to which host immune responses are believed to play a role. As the major arm of innate immunity, neutrophils are one of the first cells recruited to the site of infection where their excessive activation can contribute to lung pathology. Low-density granulocytes (LDGs) are circulating neutrophils, whose numbers increase in some autoimmune diseases and cancer, but are poorly characterized in acute viral infections. Using flow cytometry, we detected a significant increase of LDGs in the blood of acute COVID-19 patients, compared to healthy controls. Based on their surface marker expression, COVID-19-related LDGs exhibit four different populations, which display distinctive stages of granulocytic development and most likely reflect emergency myelopoiesis. Moreover, COVID-19 LDGs show a link with an elevated recruitment and activation of neutrophils. Functional assays demonstrated the immunosuppressive capacities of these cells, which might contribute to impaired lymphocyte responses during acute disease. Taken together, our data confirms a significant granulocyte activation during COVID-19 and suggests that granulocytes of lower density play a role in disease progression.

A cross-sectional cohort study of the activity and turnover of neutrophil granulocytes in juvenile idiopathic arthritis

BackgroundThe inflammatory process in juvenile idiopathic arthritis (JIA) involves both the innate and the adaptive immune system. The turnover and activity of neutrophil granulocytes may be reflected by proteins secreted from primary or secondary granules and from the cytoplasm of sequestered cells. Our primary aim was to compare the levels of the secondary neutrophil granule protein human neutrophil lipocalin (HNL), in JIA patients and controls, and to explore a possible priming of neutrophils through parallel analyses in plasma and serum. A secondary aim was to relate the levels of HNL to two other well-studied leukocyte proteins, S100A8/A9 and myeloperoxidase (MPO), as well as to clinical aspects of JIA.MethodsThe concentrations of the three biomarkers in serum, two of them also in plasma, were measured using enzyme-linked immunosorbent assay in 37 children with JIA without medical treatment, in high disease activity based on juvenile arthritis disease activity score 27 (JADAS27), 32 children on medical treatment, mainly in lower disease activity, and 16 healthy children. We assessed for differences between two groups using the Mann-Whitney U test, and used the Kruskal-Wallis test for multiple group comparisons. Spearman rank correlation, linear and multiple regression analyses were used for evaluation of associations between biomarker concentrations and clinical scores.ResultsThe concentrations of HNL and MPO in serum were significantly increased in children with JIA (p < 0.001, p = 0.002) compared with healthy children, but we found no difference in the plasma levels of HNL and MPO between children with JIA and controls. The serum concentrations of MPO and HNL were unaffected by medical treatment, but S100A8/A9 was reduced by medical treatment and correlated with JADAS27 in both univariate (r = 0.58, p < 0.001) and multivariate (r = 0.59, p < 0.001) analyses.ConclusionsNeutrophil granulocytes in children with JIA are primed to release primary and secondary granule proteins, without relation to medical treatment, whereas signs of increased turnover and sequestration of neutrophil granulocytes are reduced by treatment. Levels of neutrophil-originating proteins in serum most likely reflect underlying disease activities of JIA.

IMMUNOLOGICAL CRITERIA FOR EFFECTIVE SYSTEMIC USE OF MURAMYL PEPTIDE IMMUNOMODULATOR IN THE TREATMENT OF PATIENTS WITH BURN INJURIES

Abstract. Introduction. The problem of providing effective care to patients with burn injuries remains far from being finally solved. Due to significant immune disorders that occur against the background of thermal damage, the use of immunomodulatory drugs remains promising. Therefore, the aim of this study was to analyze the effectiveness of systemic use of the immunomodulatory drug Liastenum® in the complex treatment of patients with burn injuries.&#x0D; Materials and methods: The study involved 35 patients with burn injuries with the index of damage severity ranging from 30 to 60 units. Patients in the main group (n = 15) in addition to the usual treatment received an immunomodulator Liastenum®, which was administered intramuscularly at a dose of 2 mg once every 3 days (course dose — 10 mg). Treatment of patients in the comparison group (n = 20) did not involve the use of drugs with targeted immunocorrective action. Examination of patients included laboratory immunological examination of venous blood with assessment of various parts of the immune system and determination of the “Neutrophilic granulocytes form factor” indicator, which was performed on the 3rd, 14th, 21st day after the injury.&#x0D; The results of the study and their discussion. The obtained results allowed to confirm the ability of Liastenum® to stimulate the functional activity of neutrophilic granulocytes, restoring the phagocytic reserves of these cells, to normalize the level of lymphocytes and their individual subpopulation forms. In general, the action of the immunomodulatory drug was characterized by a balanced effect on the cellular, humoral parts of the immune system, phagocytosis.&#x0D; Conclusions. The effectiveness of systemic use of immunomodulatory drug Liastenum® in a comprehensive program of care for patients with burn injuries by balanced correction of immune disorders was laboratory established.

Loss of PTPN22 Promotes Intestinal Inflammation by Compromising Granulocyte-mediated Antibacterial Defence.

A single nucleotide polymorphism in protein tyrosine phosphatase non-receptor type 22 [PTPN22] has been associated with the onset of autoimmune disorders, but protects from Crohn's disease. PTPN22 deficiency in mice promotes intestinal inflammation by modulating lymphocyte function. However, the impact of myeloid PTPN22 in colitis development remains unclear. The aim of this study was to investigate the role of PTPN2 in the IL-10 and the T cell transfer colitis models. PTPN22-deficient mice were crossed with IL-10-/- and RAG2-/- mice. Naïve T cells were injected in RAG-/- mice to induce T-cell transfer colitis. Spontaneous colitis in IL-10-/- mice was monitored for up to 200 days. Here, we demonstrate that PTPN22 in non-lymphoid immune cells is required to protect against T cell transfer-mediated and IL-10 knock-out colitis. Analysis of the intestinal immune landscape demonstrated a marked reduction of granulocyte influx into the inflamed colon in PTPN22-deficient mice. On a molecular level, granulocytes were not only reduced by numbers, but also revealed a defective function. In particular, granulocyte activation and granulocyte-mediated bacteria killing was impaired upon loss of PTPN22, resulting in elevated bacterial burden and translocation beyond the intestinal epithelial barrier in PTPN22-deficient mice. Consistently, antibiotic-induced depletion of bacteria reverted the increased colitis susceptibility in PTPN22-deficient mice, whereas granulocyte depletion induced acolitis phenotype in wild-type mice similar to that observed in PTPN22-deficient mice. In conclusion, our data demonstrate that PTPN22 is essential for adequate granulocyte activation and antimicrobial defence to protect the inflamed intestine from bacterial invasion and exacerbated colitis.

Влияние пептида GHK-D-Ala на механизмы врожденного иммунитета и процессы перекисного окисления липидов в условиях инфицированной раны

The objective is to study the effects of Gly-His-Lys-D-Ala (GHK-D-Ala) peptide on mechanisms of innate immunity and lipid peroxidation processes in infected wound. Materials and methods. The experiments were carried out on 80 Wistar male rats. The infected wound was modeled by applying a full-layer wound on the back and performing primary wound surgery with primary suturing 24 hours later. GHK-D-Ala peptide was injected at a dose of 0.5 µg/kg into the wound area every day for 30 days. The phagocytic activity of blood neutrophils was studied in spontaneous and stimulated nitroblue tetrazolium reduction test (NBT-test). The activity of lipid peroxidation (LPO) processes in rats’ blood serum was assessed by the content of malondialdehyde (MDA) and acylhydroperoxides (AHP). Results. GHK-D-Ala administration had a positive effect on both phagocytosis mechanisms and the level of LPO products in comparison with the control group. There was a significant increase in NST-positive neutrophils in the spontaneous test against peptide administration on days 3, 7, 10, and 30 (p˂0.001) and in the stimulated NST test on days 3, 7, and 10 (p &lt; 0.001). The functional neutrophil reserve had been significantly increased by day 10 (p˂0.05) and had been significantly lower by day 30 (p &lt; 0.05). Phagocytic index and phagocytic number were significantly higher on days 3 and 10 (p &lt; 0.01). The phagocyte activity index significantly increased on days 3 and 10 (p &lt; 0.05-0.001).A significant decrease in the MDA concentration was demonstrated on days 3, 7, 10, and 30 (p&lt;0,001). There was a significant decrease in the content of AGP on days 3, 7, 10 and 30 (p &lt; 0.001). Conclusion. The corrective effect of Gly-His-Lys-D-Ala peptide on the phagocytic activity of granulocytes and the processes of lipid peroxidation in infected skin wounds was established.

P316 Human neutrophil elastase derived fragment of calprotectin (S100a9) is a serum biomarker (CPa9-HNE) for monitoring of anti-TNFα treatment response in Crohn’s disease

Abstract Background In patients with inflammatory bowel disease (IBD), up to 30% do not achieve response, 66% do not achieve remission, and 40% experience loss of response to anti-TNFα treatment. In Crohn’s disease (CD), fecal calprotectin is the most widely used fecal biomarker for diagnosis and monitoring of disease activity. Fecal sampling and processing are tedious compared to serum sampling, and biomarker quantification in serum is more efficient, convenient, and also more acceptable for patients. Therefore, there is a need for a robust and reliable serum calprotectin biomarker that performs better than conventional serum biomarkers. The aim of this study was to investigate the performance of a novel serum calprotectin assay, which quantifies a specific human neutrophil elastase (HNE) generated calprotectin (S100a9) fragment (CPa9-HNE), as a measure of true neutrophil granulocyte activity, for monitoring treatment responses in patients with CD. Methods We included 30 CD patients with clinically active (Harvey-Bradshaw Index (HBI)≥5) luminal inflammation scheduled for anti-TNF treatment (adalimumab or infliximab). Serum samples where obtained at baseline (n=30), week 1 (n=29), week 8 (n=28), week 26 (n=15), and week 56 (n=7). CPa9-HNE (Calprotectin, S100a9, specific fragment generated by human neutrophil elastase), CRP and fecal calprotectin were quantified. Remitters (n=11) were defined as patients in clinical remission (HBI&amp;lt;5) at week 8 with no flares at week 26 or week 56. Non-remitters (n=19) were defined by having clinical active disease (HBI≥5) at week 8 or having a flare at week 26 or week 56. Results Patients with severe disease activity (HBI&amp;gt;16) had significantly elevated serum levels of CPa9-HNE compared with patients in remission (p&amp;lt;0.05), with mild (p&amp;lt;0.05) or moderate (p&amp;lt;0.01) disease activity. CPa9-HNE serum levels were significantly suppressed in remitters compared with non-remitters at week 8 (p&amp;lt;0.001), week 26 (p&amp;lt;0.01) and week 56 (p&amp;lt;0.05) (Figure 1). Fecal calprotectin and CRP were significantly suppressed at week 8, week 26 and week 56 compared with baseline levels for remitters and non-remitters, but there was no significant difference between remitters and non-remitters (Figure 1). Non-remitters were more likely to have high levels of CPa9-HNE at baseline (tertile 1,2 vs tertile 3; OR: 6.8 (CI:1.22-36.5), p=0.027) and week 8 (tertile 1,2 vs tertile 3; OR: 24 (CI:2.56-279), p=0.0014). Conclusion CPa9-HNE is a novel non-invasive biomarker for monitoring treatment response to anti-TNFα for CD patients and may be used as a surrogate marker for assessing disease activity and to predict outcome of anti-TNFα treatment.

Quantitative proteomic analyses provide insights into the hyalinocytes and granulocytes phagocytic killing of ivory shell Babylonia areolata in vitro

The molecular mechanisms underlying the phagocytosis of haemocytes remain unclear in Babylonia areolata. In this study, the phagocytic activities of hyalinocytes and granulocytes in B. areolata were examined by flow cytometry combined with electron microscopy and proteomics analyses in vitro. The ultrastructural features of haemocytes that internalised and engulfed Vibrio parahaemolyticus were revealed by electron microscopy. The results of V. parahaemolyticus challenge using electron microscopy showed a process during phagosome formation in hyalinocytes. The phagocytic rates of hyalinocytes and granulocytes that ingested fluorescent microspheres were approximately 31.19 ± 4.79% and 10.66 ± 1.18%, respectively. The phagocytosis rate of hyalinocytes to V. parahaemolyticus was 44.99 ± 5.03%, and that of granulocytes was 49.67 ± 4.06%. After V. parahaemolyticus challenge, differentially expressed proteins were identified by label-free quantitative proteomic analysis. The identified proteins included cathepsins, clathrin, heat shock protein 90 kDa, molecular chaperone HtpG, integrin, collagen, laminin, protein kinase A, Ras-related C3 botulinum toxin substrate 1, Ras-related protein, GTPase, V-type H + -transporting ATPase, and Rab-7A, which were involved in different signalling pathways. Some immune-related KEGG pathways were significantly up-regulated or down-regulated after V. parahaemolyticus challenge, including lysosome, proteasome, NOD-like receptor, PI3K-Akt, JAK-STAT, and Wnt signalling pathways. The results suggest that there are differences in the phagocytotic abilities between hyalinocytes and granulocytes of B. areolata based on changes in morphological, functional, and molecular features.

Alterations of Phagocytic Activity and Capacity in Granulocytes and Monocytes Depend on the Pathogen Strain in Porcine Polytrauma.

Background: Polytraumatized patients undergo a strong immunological stress upon insult. Phagocytes (granulocytes and monocytes) play a substantial role in immunological defense against bacteria, fungi and yeast, and in the clearance of cellular debris after tissue injury. We have reported a reduced monocytes phagocytic activity early after porcine polytrauma before. However, it is unknown if both phagocyte types undergo those functional alterations, and if there is a pathogen-specific phagocytic behavior. We characterized the phagocytic activity and capacity of granulocytes and monocytes after polytrauma.Methods: Eight pigs (Sus scrofa) underwent polytrauma consisting of lung contusion, liver laceration, tibial fracture and hemorrhagic shock with fluid resuscitation and fracture fixation with external fixator. Intensive care treatment including mechanical ventilation for 72 h followed. Phagocytic activity and capacity were investigated using an in vitro ex vivo whole blood stimulation phagocytosis assays before trauma, after surgery, 24, 48, and 72 h after trauma. Blood samples were stimulated with Phorbol-12-myristate-13-acetate and incubated with FITC-labeled E. coli, S. aureus or S. cerevisiae for phagocytosis assessment by flow cytometry.Results: Early polytrauma-induced significant increase of granulocytes and monocytes declined to baseline values within 24 h. Percentage of E. coli-phagocytizing granulocytes significantly decreased after polytrauma and during further intensive care treatment, while their capacity significantly increased. Interestingly, both granulocytic phagocytic activity and capacity of S. aureus significantly decreased after trauma, although a recovery was observed after 24 h and yet was followed by another decrease. The percentage of S. cerevisiae-phagocytizing granulocytes significantly increased after 24 h, while their impaired capacity after surgery and 72 h later was detected. Monocytic E. coli-phagocytizing percentage did not change, while their capacity increased after 24–72 h. After a significant decrease in S. aureus-phagocytizing monocytes after surgery, a significant increase after 24 and 48 h was observed without capacity alterations. No significant changes in S. cerevisiae-phagocytizing monocytes occurred, but their capacity dropped 48 and 72 h.Conclusion: Phagocytic activity and capacity of granulocytes and monocytes follow a different pattern and significantly change within 72 h after polytrauma. Both phagocytic activity and capacity show significantly different alterations depending on the pathogen strain, thus potentially indicating at certain and possibly more relevant infection causes after polytrauma.

Assessment of the Impact of Betulin on the Immune Status of Cows with Leukemia-Associated Infection

This study examined the immunity dynamics of bovine brucellosis-complicated leukemia, as well as the efficacy of treatment with betulin. Blood serum samples were taken from cows with specific brucellosis antibodies and bovine leukemia virus antibodies based on the results of complex diagnostic studies. Animals (n = 5) selected based on serological studies were injected subcutaneously with betulin-PEG three times with an interval of 7 days at a dose of 10 mg/kg. Blood was taken for hematological and immunological studies before the injection, and after the first, second and third injections. It was found that leukemia and brucellosis infections were accompanied by a significant increase in the number of leukocytes and lymphoid cells, as well as strong fluctuations in the leukocyte functional and metabolic activity parameters (from insignificant in some individual animals to significantly increased in others). Immunological restructuring of the body after triplicate administration of betulin-PEG was characterized by the restoration of the number of lymphoid cells to the level of clinically healthy animals, as well as a significant increase in the functional and metabolic activity of the neutrophilic granulocytes, especially their oxygen-dependent systems. Based on the results, the product is recommended as an effective means to restrain the leukemia process that is complicated with associated infection, as well as to use for seronegative animals in order to increase nonspecific resistance to BLV infection.&#x0D; Keywords: leukemia, brucellosis, cattle, immune status, betulin, immunocorrection, associated infection

Biogenic Ferrihydrite Nanoparticles: Synthesis, Properties In Vitro and In Vivo Testing and the Concentration Effect.

Biogenic ferrihydrite nanoparticles were synthesized as a result of the cultivation of Klebsiella oxytoca microorganisms. The distribution of nanoparticles in the body of laboratory animals and the physical properties of the nanoparticles were studied. The synthesized ferrihydrite nanoparticles are superparamagnetic at room temperature, and the characteristic blocking temperature is 23–25 K. The uncompensated moment of ferrihydrite particles was determined to be approximately 200 Bohr magnetons. In vitro testing of different concentrations of ferrihydrite nanoparticles for the functional activity of neutrophilic granulocytes by the chemiluminescence method showed an increase in the release of primary oxygen radicals by blood phagocytes when exposed to a minimum concentration and a decrease in secondary radicals when exposed to a maximum concentration. In vivo testing of ferrihydrite nanoparticles on Wister rats showed that a suspension of ferrihydrite nanoparticles has chronic toxicity, since it causes morphological changes in organs, mainly in the spleen, which are characterized by the accumulation of hemosiderin nanoparticles (stained blue according to Perls). Ferrihydrite can also directly or indirectly stimulate the proliferation and intracellular regeneration of hepatocytes. The partial detection of Perls-positive cells in the liver and kidneys can be explained by the rapid elimination from organs and the high dispersion of the nanomaterial. Thus, it is necessary to carry out studies of these processes at the systemic level, since the introduction of nanoparticles into the body is characterized by adaptive-proliferative processes, accompanied by the development of cell dystrophy and tension of the phagocytic system.

Micromechanical properties and functional activity of granulocytes when simulating exogenous loading with ATP in vitro

The micromechanical properties of leukocytes make a certain contribution to the blood flow velocity in the microcirculatory bed, while the micromechanical properties themselves change under the influence of a complex network of purinergic signals.The aim of the work was to study the micromechanical properties and functional activity of granulocytes in normal conditions and in patients with acute lymphoblastic leukemia when simulating exogenous loading with ATP in vitro.Materials and methods. Leukocytes were isolated from the blood of patients with acute lymphoblastic leukemia and healthy people. Each sample was divided into a test sample and a control sample. In the test samples, the loading with ATP in vitro was simulated. Leukocytes of the control samples were incubated in the culture medium without the addition of ATP. Young’s modulus and adhesion force were measured using an atomic force microscope in the force spectroscopy mode. The cell surface potential was measured in an atomic force microscope in the Kelvin mode. To assess the functional activity of granulocytes, hypoosmotic tests in vitro and determination of migration activity were used.Results. In tests with exogenous ATP, both in samples from healthy people and from patients with acute lymphoblastic leukemia, a decrease in the rigidity and potential of the plasma membrane surface, an increase in the adhesive properties of leukocytes and migration activity were found. At the same time, the responses of granulocytes to the osmotic loading were different: for example, in the group of healthy people, the loading with ATP caused cell contraction and a decrease in the use of the membrane reserve by the cell in a hypotonic environment, and in patients with acute lymphoblastic leukemia, it caused an increase in the volume and more intensive use of the membrane reserve in volume regulation.Conclusion. The revealed effects indicate the leading role of the ATP molecule in the signal transduction mechanisms between blood cells in the microvasculature. The increase in the adhesive properties of the cell surface of granulocytes revealed in the study, in parallel with the increase in their migration activity under the influence of the ATP molecule, can contribute to the development of inflammation in the vessel wall.

Activation of Neutrophil Granulocytes by Platelet-Activating Factor Is Impaired During Experimental Sepsis

Platelet-activating factor (PAF) is an important mediator of the systemic inflammatory response. In the case of sepsis, proper activation and function of neutrophils as the first line of cellular defense are based on a well-balanced physiological response. However, little is known about the role of PAF in cellular changes of neutrophils during sepsis. Therefore, this study investigates the reaction patterns of neutrophils induced by PAF with a focus on membrane potential (MP), intracellular pH, and cellular swelling under physiological and pathophysiological conditions and hypothesizes that the PAF-mediated response of granulocytes is altered during sepsis. The cellular response of granulocytes including MP, intracellular pH, cellular swelling, and other activation markers were analyzed by multiparametric flow cytometry. In addition, the chemotactic activity and the formation of platelet–neutrophil complexes after exposure to PAF were investigated. The changes of the (electro-)physiological response features were translationally verified in a human ex vivo whole blood model of endotoxemia as well as during polymicrobial porcine sepsis. In neutrophils from healthy human donors, PAF elicited a rapid depolarization, an intracellular alkalization, and an increase in cell size in a time- and dose-dependent manner. Mechanistically, the alkalization was dependent on sodium-proton exchanger 1 (NHE1) activity, while the change in cellular shape was sodium flux- but only partially NHE1-dependent. In a pathophysiological altered environment, the PAF-induced response of neutrophils was modulated. Acidifying the extracellular pH in vitro enhanced PAF-mediated depolarization, whereas the increases in cell size and intracellular pH were largely unaffected. Ex vivo exposure of human whole blood to lipopolysaccharide diminished the PAF-induced intracellular alkalization and the change in neutrophil size. During experimental porcine sepsis, depolarization of the MP was significantly impaired. Additionally, there was a trend for increased cellular swelling, whereas intracellular alkalization remained stable. Overall, an impaired (electro-)physiological response of neutrophils to PAF stimulation represents a cellular hallmark of those cells challenged during systemic inflammation. Furthermore, this altered response may be indicative of and causative for the development of neutrophil dysfunction during sepsis.

EFFECT OF EXOGENOUS NITRIC MONOXIDE THERAPY ON IMMUNOLOGICAL PARAMETERS IN BISPHOSPHONATE OSTEONECROSIS OF THE JAWS

Bisphosphonate osteonecrosis of the jaw (BONCH) is a drug-induced osteonecrosis in patients with a history of malignancies. It is characterized by progressive destruction of the jawbone with the development of a chronic inflammatory process in the parotid tissues. For the treatment of the disease, the use of exogenous nitrogen monoxide (NO) synthesized using the Plason device is proposed. The study of immunological parameters in patients with BONCH in the main group (n = 26), who received NO-therapy, and in the comparison group (пи = 27), who received traditional sanitation with 0,05 % chlorhexidine bigluconate. The dynamics of indicators in the main group - a decrease in the activity of the cellular link (activity of phago-quoting granulocytes and monocytes) to normal values, an increase in the intensity of local immunity (IgA) and a decrease in the concentration of humoral factors of acute (IgM) and chronic (IgG) inflammation p &lt; 0,05) - confirms the anti-inflammatory and immunomodulatory effect of exogenous NO therapy. In the case of the comparison group, reducing only one indicator (AFM) to normal values does not allow us to prove the effect of traditional sanitation on immunological parameters in General. Thus, the use of exogenous nitrogen monoxide for patients with BONCH is justified both at the stage of preoperative preparation and as a basic conservative therapy if surgical treatment is not possible.

Inhibition of Arginase 1 Liberates Potent T Cell Immunostimulatory Activity of Human Neutrophil Granulocytes.

Myeloid cell arginase-mediated arginine depletion with consecutive inhibition of T cell functions is a key component of tumor immune escape. Both, granulocytic myeloid-derived suppressor cells (G-MDSC) and conventional mature human polymorphonuclear neutrophil granulocytes (PMN) express high levels of arginase 1 and can act as suppressor cells of adaptive anti-cancer immunity. Here we demonstrate that pharmacological inhibition of PMN-derived arginase 1 not only prevents the suppression of T cell functions but rather leads to a strong hyperactivation of T cells. Human PMN were incubated in cell culture medium in the absence or presence of an arginase inhibitor. T cells from healthy donors were then activated either polyclonally or in an antigen-specific manner in the supernatants of the PMN cultures at different PMN-T cell ratios. T cell proliferation was completely suppressed in these supernatants in the absence of an arginase inhibitor. Arginase inhibition led to a strong hyperinduction of T cell proliferation, which exceeded control activation conditions up to 25-fold. The hyperinduction was correlated with higher PMN-T cell ratios and was only apparent when PMN arginase activity was blocked sufficiently. The T cell stimulatory factor was liberated very early by PMN and was present in the < 3 kDa fraction of the PMN supernatants. Increased T cell production of specific proinflammatory cytokines by PMN supernatant in the presence of arginase inhibitor was apparent. Upon arginase inhibition, downregulation of important T cell membrane activation and costimulation proteins was completely prevented or de novo induction accelerated. Antigen-specific T cell cytotoxicity against tumor cells was enhanced by PMN supernatant itself and could be further increased by PMN arginase blockade. Finally, we analyzed anergic T cells from multiple myeloma patients and noticed a complete reversal of anergy and the induction of strong proliferation upon T cell activation in PMN supernatants by arginase inhibition. In summary, we discovered a potent PMN-mediated hyperactivation of human T cells, which is apparent only when PMN arginase-mediated arginine depletion is concurrently inhibited. Our findings are clearly relevant for the analysis and prevention of human tumor immune escape in conjunction with the application of arginase inhibitors already being developed clinically.

Pathways of Neutrophil Granulocyte Activation in Hereditary Angioedema with C1 Inhibitor Deficiency

Hereditary angioedema (HAE) with C1-inhibitor deficiency belongs to bradykinin-mediated angioedemas. It is characterized by recurrent subcutaneous and/or submucosal swelling episodes (HAE attacks) and erythema marginatum skin rash as a pre-attack (prodromal) phase. HAE attacks were shown to be accompanied by peripheral blood neutrophilia. We aimed to find molecular mechanisms that may explain the distinct role of neutrophil granulocytes in HAE. Plasma levels of blood cells and factors related to neutrophil activation (cytokines, chemokines, chemotactic factors, enzymes, and neutrophil extracellular trap) were measured in plasma samples obtained from patients during symptom-free periods (n = 77), during prodromal phase (n = 8) and attacks (n = 14), during a spontaneously resolved attack (n = 1), and in healthy controls (n = 79). Higher counts of white blood cells, lymphocytes, and neutrophil granulocytes were found in symptom-free patients compared with controls; these cell counts were elevated further during HAE attacks. The level of chemokine (C–C motif) ligand 5, monocyte chemoattractant protein-1, and myeloperoxidase were also higher in the symptom-free patients than in the controls. Levels of monocyte chemoattractant protein-1, leukotriene B4, neutrophil elastase, and myeloperoxidase were elevated during attacks. During erythema marginatum, white blood cells and monocyte count and levels of interleukin 8 were elevated compared with symptom-free period. Similar changes were detected during the attack follow-up. We conclude that the activation of NGs in symptom-free periods and a further increase observed during attacks suggests that NGs may be involved in the pathomechanism of HAE with C1-INH deficiency.