Neutrophilic Leukocytes Research Articles

Platelet Indices in Patients With Gram-Negative and Gram-Positive Sepsis: A Retrospective Cross-Sectional Study.

Different inflammatory processes and sepsis can significantly affect the number of platelets and platelet indices. Therefore, in this study, total platelet count (PLT), thrombocrit (Pct), platelet distribution width (PDW), mean platelet volume (MPV), and platelet-large cell ratio (P-LCR) were analyzed in patients with Gram-negative and Gram-positive bacterial sepsis and in sterile blood cultures. Inclusion criteria were an increased number of inflammatory parameters (elevated values ​​of leukocytes,C-reactive protein (CRP), procalcitonin (PCT), andpositive blood culture. Exclusion criteria were patients who did not have elevated values ​​of inflammatory parameters and did not have a positive blood culture.Samples were collected from patients who had sepsis confirmed by blood cultures at the Department of Microbiology and Molecular Diagnostics at University Clinical Hospital Mostar in the period from 2019 to 2022. Three groups were analyzed, patients who had sterile blood cultures, patients with blood cultures with isolated Gram-positive bacteria, and patients with blood cultures with isolated Gram-negative bacteria. Specific infectious agents were identified for each group of patients. In addition to the above, PLT, Pct, MPV, PDW, P-LCR, PCT, CRP, the total number of leukocytes, and the number of neutrophil leukocytes were analyzed in each group. The values of PCT, CRP, and the number of neutrophile leukocytes were significantly higher in patients with Gram-negative sepsis as compared to Gram-positive sepsis and to control group. Patients with sepsis have decreased PLT and Ptc and increased values of MPV, PDW, and P-LCR. In sepsis caused by the Gram-negative bacteria, i.e., Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii, the values of the same parameters were more changed compared to sepsis caused by Gram-positive bacteria, i.e., Streptococcus pneumoniae, Enterococcus spp., and methicillin-resistant Staphylococcus aureus (MRSA). When comparing Gram-negative negative bacteria, PLT was lowest in sepsis caused by Escherichia coli, the PDW value was highest in sepsis caused by Acinetobacter baumannii, and MPV and P-LCR were the highest in sepsis caused by Klebsiella pneumoniae. Our study showed that platelet indices are significantly changed in patients with sepsis. Patients with sepsis have decreased values of PLT and Pct and increased values of MPV, PDW, and P-LCR, indicating an increase in thrombocyte production. Moreover, the results were more prominent in sepsis caused by Gram-negative bacteria compared to sepsis caused by Gram-positive bacteria.

Study of biomarkers to determine severity and lung damages in COVID-19 patients.

Identifying inflammation and lung damage markers is crucial in reducing morbidity and mortality of coronavirus disease 2019 (COVID-19). This study aimed to examine the validity and reliability of severity and post-infection lung damage and analyse their relationship. This was a prospective analysis study at the Airlangga University Hospital, Surabaya, Indonesia, from March to August 2021. The infection`s severity was measured by examining angiotensin-converting enzyme 2 (ACE2) levels and complete blood count. Lung damage was estimated by reviewing Krebs von de Lungen (KL)-6, matrix metalloproteinase (MMP)-9, tissue inhibitor metalloproteinase (TIMP)-1, and MMP-9/TIMP-1. Two-factor confirmatory factor analysis (CFA) and canonical correlation were calculated using Lisrel and SPSS (version 25). The research sample included 76 patients. The t count loading factor values were calculated: ACE2 (6.00), neutrophils (-0.80), lymphocytes (-0.63), neutrophil-lymphocyte ratio (NLR, 1.27), eosinophils (-1.52), basophils (1.72), monocytes (0.05), platelets (0.53), leukocytes (-0.51), platelet-lymphocyte ratio (PLR, -1.15), KL-6 (10.47), MMP-9 (11.91), TIMP-1 (11.79), and MMP-9/TIMP-1 (-0.24). The t values were: neutrophil covariance error (6.11), lymphocytes (6.12), NLR (6.10), eosinophils (6.08), basophils (6.07), monocytes (6.12), platelets (6.12), leukocytes (6.12), PLR (6.10), ACE2 (0.97), KL-6 (5.63), MMP-9 (2.08), TIMP-1 (2.77), and MMP-9/TIMP-1 (6.12). t value canonical correlation of 7.04 (t count > 1.96) indicated a correlation between the severity of the patient and post-infection lung damage. The severity was adequately measured through ACE2, IL-6, IL-10, neutrophils, lymphocytes, leukocytes, and NLR. Lung damage was measured with KL-6, MMP-9, and TIMP-1. There was a correlation between disease severity and lung damage.

The Effect of Montelukast Treatment on Elderly Patients Diagnosed with COVID-19

Abstract Background/Aims: The clinical course in COVID-19 patients can vary from asymptomatic cases to acute respiratory distress syndrome (ARDS), respiratory failure and multiorgan dysfunction. Clinical progression is thought to be mainly due to the release of proinflammatory cytokines. The most common symptoms are fever, cough, malaise, and shortness of breath. Montelukast, which is used in the treatment of seasonal allergic rhinitis and asthma, has brought its use in COVID-19 infection due to its anti-inflammatory and cytokine secretion-reducing effect. There are many studies in the literature that montelukast treatment has a positive effect on the prognosis and mortality of COVID-19. However, there are not enough studies evaluating the efficacy of montelukast treatment in elderly patients. The aim of our study is to evaluate the clinical and laboratory efficacy of montelukast treatment in patients aged 60 and over in COVID-19 disease, and to indicate the differences from the studies in the literature. Methods Our research was planned as a retrospective, single-center, observational study. The medical records of 75 COVID-19 patients aged 60 and over who were hospitalized in the internal medicine clinic of Ankara Bilkent City Hospital between September 2021 and December 2022 were included. Diagnosis of COVID-19 was confirmed with a reverse transcription polymerase chain reaction (RT-PCR) test from nasopharyngeal swab. Results Clinical findings and results were compared between the patients who received montelukast and the control group. There was no statistically significant difference between the two groups in terms of cough, dyspnea, gastroenteritis and oxygen theraphy requirement. There is no significant difference between the two groups in terms of the need for intensive care unit admission and mortality. The length of hospital stay was compared in both groups, it was 10.88±7.24 days in the control group and 10.51±5.44 days in the montelukast group, and there was no statistically significant difference between the groups. The laboratory parameters of the patients in both groups were compared. The neutrophil count and leukocyte count measured before hospitalization were found to be significantly lower in the patient group receiving montelukast (p=0.022, p=0.016). No significant difference was found in other laboratory parameters. Conclusions Although montelukast treatment has positive effects on prognosis in COVID-19 disease in the literature, a similar effect was not observed in the population aged 60 and over in our study. We did not find the positive effect of short-term montelukast treatment on the prognosis of patients aged 60 years and older who were hospitalized due to COVID-19. We thought that this was due to the low efficacy of montelukast in the elderly population. Our study is one of the first to examine montelukast therapy in the geriatric population with COVID-19.

Tumor necrosis factor regulates leukocyte recruitment but not bacterial persistence during Staphylococcus aureus craniotomy infection

BackgroundCraniotomy is a common neurosurgery used to treat intracranial pathologies. Nearly 5% of the 14 million craniotomies performed worldwide each year become infected, most often with Staphylococcus aureus (S. aureus), which forms a biofilm on the surface of the resected bone segment to establish a chronic infection that is recalcitrant to antibiotics and immune-mediated clearance. Tumor necrosis factor (TNF), a prototypical proinflammatory cytokine, has been implicated in generating protective immunity to various infections. Although TNF is elevated during S. aureus craniotomy infection, its functional importance in regulating disease pathogenesis has not been explored.MethodsA mouse model of S. aureus craniotomy infection was used to investigate the functional importance of TNF signaling using TNF, TNFR1, and TNFR2 knockout (KO) mice by quantifying bacterial burden, immune infiltrates, inflammatory mediators, and transcriptional changes by RNA-seq. Complementary experiments examined neutrophil extracellular trap formation, leukocyte apoptosis, phagocytosis, and bactericidal activity.ResultsTNF transiently regulated neutrophil and granulocytic myeloid-derived suppressor cell recruitment to the brain, subcutaneous galea, and bone flap as evident by significant reductions in both cell types between days 7 to 14 post-infection coinciding with significant decreases in several chemokines, which recovered to wild type levels by day 28. Despite these defects, bacterial burdens were similar in TNF KO and WT mice. RNA-seq revealed enhanced lymphotoxin-α (Lta) expression in TNF KO granulocytes. Since both TNF and LTα signal through TNFR1 and TNFR2, KO mice for each receptor were examined to assess potential redundancy; however, neither strain had any impact on S. aureus burden. In vitro studies revealed that TNF loss selectively altered macrophage responses to S. aureus since TNF KO macrophages displayed significant reductions in phagocytosis, apoptosis, IL-6 production, and bactericidal activity in response to live S. aureus, whereas granulocytes were not affected.ConclusionThese findings implicate TNF in modulating granulocyte recruitment during acute craniotomy infection via secondary effects on chemokine production and identify macrophages as a key cellular target of TNF action. However, the lack of changes in bacterial burden in TNF KO animals suggests the involvement of additional signals that dictate S. aureus pathogenesis during craniotomy infection.

The morphofunctional state of the hematoalveolar barrier of the lungs in COVID-19 patients compared to pandemic influenza

Background. It is known that viral infections, such as pandemic influenza A(H1N1) and COVID-19, are characterized by acute respiratory distress syndrome, which in some cases is accompanied by high mortality. It has also been established that diffuse alveolar damage (DAD) is common to both influenza A and COVID-19. However, there are some differences between these infections. In contrast with pandemic flu, the most frequent symptom of COVID-19 is thromboembolic complications. The examination of the autopsy material showed that, despite the large number of conducted studies, the morphofunctional state of the hematoalveolar barrier of the lungs has not been sufficiently studied and some mechanisms of the development of pathological reactions of the оrganism in response to the action of influenza viruses A(H1N1) pdm09 and SARS-CoV-2 remain unclear. But the post-mortem picture of the hematoalveolar barrier can allow us to see all previous links of the viral lung damage process. This, in turn, will help to solve the problem of timely diagnosis of the disease severity, prognosis, prevention of further development of the disease and appropriate methods of therapy. The purpose of the work was to study the features of the SARS-CoV-2 virus effect on the morphofunctional state of the hematoalveolar region of the lungs at autopsy, which allows the pathogenesis of COVID-19 to clarified, critical mechanisms, thedevelopment of which leads to fatal consequences, to be established, and a therapy strategy to be proposed. Materials and methods. 8 samples of autopsy material of the lungs of patients with COVID-19 were studied. The comparison groups were patients with pandemic influenza A caused by the H1N1 virus (3 autopsies) and lung samples obtained during surgery from a patient with traumatic pneumothorax at a distance from the main focus. Lung samples were processed according to standard methods of biological material preparation for electron microscopy. From the obtained blocks, semi-thin sections with a thickness of 1 μm were made. They were stained with methylene blue, and studied under a light microscope at a magnification of 500 times. Digital photographs were obtained from the most informative areas. Ultrathin sections were analyzed in electron microscope after being contrasted in uranium and lead salts. The incidence of thrombosis in the studied groups was calculated. Statistical processing was performed using the Fisher’s non-parametric exact test and the Biostat PC software package. Results. The conducted studies showed that patients with pandemic influenza A had a pronounced lesion of the alveolar part of the lungs with damage to the alveolar walls of the lungs and the appearance of hyaline membranes in them. Desquamated pneumocytes, erythrocytes, neutrophilic leukocytes, macrophages and fibrin clots were found in alveoli filled with serous fluid. Among the features of the influenza virus, the preservation of cellular elements in the alveolar walls, the convergence of the alveolar septums and the formation of large areas with a high level of neutrophilic and macrophage invasion should be noted. Macrophages patrol the fields of damaged alveolar septums, separating them from the surrounding tissue. The subsequent fibrotization indicates the replacement of the destroyed lung tissue. The group of patients with COVID-19, in contrast to the pandemic flu, has significantly higher rates of thrombosis of various calibers. This is the result of disseminated intravascular coagulation, which often leads to fatal consequences. Erythrocytes in vascular thrombi have a tendency to agglutination, which is more pronounced in oncological patients. For all studied cases, epithelial membrane damage is characterized by almost complete loss of epithelial cells, which is accompanied by the denudation of capillaries, in which there is an accumulation of erythrocytes that gives them the look of «blood sausages». Sometimes these vessels are attached to hyaline membranes, sometimes they are arranged in chains or groups. No significant neutrophilic invasion or granulomatous inflammation is observed. Conclusions. The conducted studies showed that patients with pandemic influenza A had a pronounced lesion of the alveolar part of the lungs with a high level of neutrophilic and macrophage invasion. The mechanisms of the pathogenesis of COVID-19 are the direct destructive effect of the virus on the epithelial cells of the hemato-alveolar barrier, which obviously leads to the release of inflammatory cytokines that stimulate the systemic reaction of intravascular coagulation. It has been established that for oncology patients with a history of chemotherapy, the processes of thrombosis and destruction of alveolar walls during COVID-19 were one of the most pronounced lesions among the patients. The obtained results suggest that the fatal consequences of COVID-19 can obviously be prevented if therapeutic measures aimed at destroying the virus, blocking the targets for its penetration into cells, breaking the chain of intravascular blood coagulation with the use of corticosteroids, anticoagulants, cytokine inhibitors, etc. are taken immediately after receiving a positive diagnosis for COVID-19 and carrying out a set of laboratory tests to assess the severity of the disease.

ГЕНЕРАЦІЯ АКТИВНИХ ФОРМ КИСНЮ ЛЕЙКОЦИТАМИ КРОВІ ЩУРІВ ПІСЛЯ ІМПЛАНТАЦІЇ ХІРУРГІЧНОЇ СІТКИ З ПОКРИТТЯМ НА ОСНОВІ ТАНТАЛУ

Introduction. Over the past 20 years, polypropylene has become the main material used in hernioplasty, and has proven itself as an excellent material for the restoration of the abdominal wall during hernia repair. Since polypropylene surgical meshes do not decompose well in the body, they can stimulate the development of an inflammatory process in the surrounding tissues, which subsequently causes adhesions. The development of a postoperative inflammatory process after implantation of polypropylene surgical meshes is observed in 30–40 % of patients. This affects the management of the postoperative period, increases the time of stay of patients in the hospital and their period of convalescence. These data force scientists to continue the search for the optimal surgical mesh, which would suit specialists not only from the side of the physical properties of the surgical mesh, but also from the side of its biocompatible and anti-inflammatory properties. Tantalum is successfully used to produce biocompatible medical implants in surgery, orthopedics and dentistry. In previous studies, we have repeatedly noted its excellent anti-inflammatory and antibacterial properties, indicating the possibility of its use as a coating for mesh implants. One of the typical responses to surgical intervention is the generation of reactive oxygen species by leukocyte neutrophils, which are signaling molecules that damage the endothelium of vessels and promote the migration of cells of the immune system to the center of inflammation. The aim of the study – to determine the generation of reactive oxygen species in leukocytes of rats of the control group and experimental rats with implantation of uncoated and tantalum-based surgical meshes. Materials and Methods. ROS generation was assessed in rat blood leukocytes using the dye 2,7-dichlo­ro­dihydrofluorescein diacetate (H2DCFDA) by flow cytometry 28 days after implantation of uncoated and tantalum-coated surgical meshes. Results and Discussion. Analyzing the obtained results, it was determined that the use of tantalum-based surgical meshes does not cause excessive generation of ROS by leukocytes, in contrast to the use of an implant without a coating. Implantation of uncoated surgical mesh caused excessive production of reactive oxygen species in blood leukocytes of rats, as evidenced by statistically significant differences in the mean fluorescence intensity of 2,7- dichlorodihydrofluorescein diacetate. Conclusions. The use of tantalum-based surgical meshes causes less generation of ROS in leukocytes compared to the use of uncoated surgical meshes, and does not provoke the development of adhesions and purulent-septic processes in the postoperative period, which is confirmed by a morphological study. This determines the possibility of their use in surgical practice to improve the durability and stability of use as biomedical implants and prevention of adhesion formation.

Isolated and combined pneumocystosis of the pulmonary in patients with HIV infection according to the results of pathomorphological study

The most common opportunistic disease in patients with HIV infection is Pneumocystis pneumonia, diagnosis of which is not difficult, and treatment significantly reduces the risk of death. Purpose of the study. To analyze the results of a pathomorphological study of the lungs with isolated pneumocystis and in combination with other lung diseases (bacterial, cytomegalovirus pneumonia and pulmonary tuberculosis) in patients with HIV infection. Materials and methods of research. The results of pathomorphological studies of patients with HIV infection (n=141), who were previously observed in hospitals in the region: patients with pneumocystis pneumonia (n=24), with pneumocystis pneumonia and bacterial pneumonia (n=52), with pneumocystis pneumonia and pulmonary tuberculosis (n=56), with pneumocystis and cytomegalovirus pneumonia (n=9). Results. At the time of death, 72.7% of patients with HIV infection had a viral load level of more than 300000, and 83.7% had a CD4+ lymphocyte level of less than 41 cells/μl. In all patients, treatment with trimethoprim/sulfamethoxazole was started too late. A pathological examination of patients with HIV infection and isolated Pneumocystis pneumonia revealed changes characteristic of this disease. Nosocomial microorganisms were identified in patients with pneumocystosis and bacterial pneumonia. At autopsy in patients with HIV infection with Pneumocystis pneumonia and pulmonary tuberculosis, the picture of tuberculosis was varied, heterogeneous and significantly different from classic granulomatous inflammation. With combined damage to the lungs by pneumocystis and cytomegalovirus, foamy, vacuole-like granular masses (PCS-positive), accumulations of neutrophilic leukocytes were observed in the lumens of the alveoli, and single cells of the “owl’s eye” type were detected. Conclusion. A pathological autopsy confirmed mixed infection in patients with HIV infection.

Reparative osteogenesis in mandible in cases of filling a bone defect with hydroxyapatite-containing osteotropic material and injecting the surrounding soft tissues with thymalin: experimental and morphological study.

Aim of the study was to identify the morphological features of reparative osteogenesis in the lower jaw bone of rats in cases of filling a bone defect with hydroxyapatite-containing osteotropic material (bone graft "Biomin GT") and injecting the surrounding soft tissues with thymalin. Materials and Methods: An experiment was conducted on 48 mature rats of the WAG population weighing 160-180 grams which were divided into four groups. Group 1 included 12 rats with a simulated holey defect in the lower jaw. Group 2 included 12 rats with a simulated holey defect in the lower jaw followed by its closure with hydroxyapatite-containing osteotropic material (bone graft "Biomin GT"). Group 3 included 12 rats with a simulated holey defect in the lower jaw with injecting the surrounding soft tissues with thymalin. Group 4 included 12 rats with a simulated holey defect in the lower jaw followed by its closure with hydroxyapatite-containing osteotropic material (bone graft "Biomin GT") and injecting the surrounding soft tissues with thymalin. The material for the morphological study was a fragment of the lower jaw from the area of the simulated holey defect. Histological, morphometric and statistical research methods were used. Results: In this study, it was shown by the authors an activation of reparative osteogenesis in the lower jaw under conditions of simultaneous filling the bone defect with hydroxyapatite-containing osteotropic material (bone graft "Biomin GT") and injection the surrounding bone defect soft tissue with thymalin. Stimulation of reparative osteogenesis in the lower jaw of rats occurred due to rapid cleaning of the bone defect cavity from necrotic tissues and hematoma fragments; a decrease in the number of neutrophil leukocytes, an increase in the number and morphofunctional state of monocytes, macrophages, lymphocytes, cells of fibroblastic differon; balanced change (increase or decrease) in the number and morphofunctional state of bone forming osteoblasts and bone resorbing osteoclasts depending on the stage of reparative osteogenesis; activation of hematopoietic processes in lamellar bone tissue from the regenerate; activation of bone tissue mineralization processes. Conclusions: Thymalin injection in the soft tissues surrounding the bone defect in the lower jaw, filled with hydroxyapatite-containing osteotropic material (bone graft "Biomin GT"), significantly stimulates the process of reparative osteogenesis, which makes it possible to recommend this technique in dentistry for treatment the patients with mandible bone tissue defects.

Investigation of the effects of pulmonary adenomatosis on oxidative dna damage, inflammation, apoptosis, and autophagy in lung tissues in sheep

Pulmonary adenomatosis infections are an infectious and neoplastic disease that affects the respiratory system in sheep. Although the existence and prevalence of this disease have been revealed in studies, there is no definitive treatment today. Diagnosis of the disease; It can be detected by clinical, macroscopic, microscopic, and immunohistochemical methods. Although there are studies reporting the existence and prevalence of the disease today, the pathogenesis has not been fully elucidated. In this study, the effects of pulmonary adenomatosis (PA) infections on lung tissue oxidative DNA damage, inflammation, apoptosis, and autophagy were investigated in sheep. Care was taken to ensure that the lung samples used as negative controls did not show any macroscopic signs of pneumonia. The presence of tumoral foci was examined in the lung tissues to be used as positive control. Then the samples were examined histopathologically. All samples obtained were examined immunohistochemically for the presence of Jaagsiekte Sheep Retrovirus (JSRV) antibodies. In the study, the expression levels of IL1-β, Tnf-α, 8-OHdG, Bcl-2, BAX, Caspase3, LC3B, and LC3A were examined in 6 negative and 12 positive lung tissue samples for PA. In the lung tissues, proliferation of bronchiolar and alveolar epithelial cells, as well as alveolar macrophages and neutrophil leukocyte cell infiltration in their lumens, were observed. It revealed that severe OPA cases had more severe levels of IL1-β, Tnf-α, 8-OHdG, BAX, Caspase3, LC3A, and LC3B expressions than mild cases did. As a result of the research, in cases where the severity of tumor foci due to PA in lung tissues increased; Oxidative stress, inflammation, apoptosis, and autophagy were found to be significantly increased.

The state of cellular immunity link and steroid hormones in bitches under different conditions of the reproductive system

The purpose of the conducted study was to establish the correlation between indicators of the cellular immunity link and the concentration of steroid hormones in bitches under different conditions of the reproductive system. Seventeen crossbred bitches aged from 2 to 5 years were involved in the experiment. They were divided into three groups depending on the state of the reproductive system: anestrus, sexual cycle, and pyometra. The following immunophysiological indicators were determined in blood plasma: the absolute number of leukocytes, lymphocytes, and their immunoregulatory subpopulations, and the phagocytic activity of neutrophils. The content of progesterone, estradiol, and cortisol was determined in serum. According to the results of the research, it was established that the absolute number of neutrophils, as the most reactogenic cells of the innate link of cellular immunity, was 0.51 G/L higher in the group of animals with pyometra than in the group of animals during the sexual cycle, and by 0.84 G/L in bitches in conditions of anestrus. The absolute number of phagocytic active neutrophils in conditions of pyometra was 2.32 ± 0.58 G/L (Р < 0.001). However, as a percentage of the absolute number of neutrophils, this indicator was 66 %. During the sexual cycle, the absolute number of neutrophils capable of phagocytosis was 2.33 ± 0.51 G/L, which is 78 % of the absolute number of all neutrophilic leukocytes. The indicator of the absolute number of lymphocytes was reliably (P < 0.05) lower in the group of bitches with pyometra – 1.48 ± 0.35 G/L, which is 0.92 G/L less compared to the group of animals in the anestrous period. Analysis of the absolute number of monocytes showed that this indicator was the same in bitches during the pyometra and anestrous periods and was lower by 0,07 G/L during the sexual cycle. The absolute number of B-lymphocytes, as a humoral link of adaptive immunity, in bitches in conditions of the anestrous and estrous period was the same and amounted to 0.30 G/L. In the group of bitches with pyometra, a reliable decrease of this indicator by 33 % was established (Р < 0.05). The population of natural killer (NK) lymphocytes was also the lowest in bitches with pyometra, almost twice as compared to animals during the sexual cycle. Conducted research on determining the absolute quantitative indicators of the cellular immunity link showed their dependence on the functional state of the reproductive system. It was established that the absolute number of neutrophils in pyometra was higher than in animals in anestrus. However, at the same time, their relative number capable of phagocytosis was the lowest. In conditions of pyometra, the lowest number of lymphocytes and their main subpopulations were observed.

PSII-3 Sheep Neutrophil Activation in Response to Garlic in Vitro

Abstract St. Croix hair sheep are known to resist parasitic infection. Parasitic resistance is in part due to the increased number and ability of neutrophil leukocytes to fight pathogens. Dietary supplements such as garlic (Allium sativum L.) are antimicrobial immune modulators that can enhance production. This study sought to evaluate the impact of fresh filtered garlic on sheep neutrophils through In vitro treatment. The study collected blood from the jugular vein of five clinically healthy female St. Croix sheep at NC A&T State University Farm and isolated neutrophils from 10 mL whole blood samples from each animal. The neutrophils were treated with fresh filtered garlic after the total cell count was measured, and 1 million cells were taken for treatment. The total protein concentration in the neutrophil supernatant was measured before and after treatment. Garlic (10 ug/mL PBS) was added to neutrophils, incubated (30 minutes, 37 °C) and centrifuged. Total RNA was extracted from the pellet using the TRIZOL RNA Isolation Protocol (Sigma-Aldrich). The RNA concentration and purity were assessed using a Nanodrop Spectrophotometer (Thermo Scientific Inc.). Pooled RNA from each group was used for cDNA synthesis, followed by qPCR analysis of the expression of 84 test genes related to the innate and adaptive immune response (RT2 profiler array) of the host. Total protein concentration in the supernatant was determined using the BCA Assay (Pierce BCA Protein Assay Kit). In vitro treatment with garlic extract resulted in increased total secreted protein concentrations from treated neutrophils, as well as upregulation of 10 genes involved in immune response and downregulation of 4 genes associated with inflammation and immune regulation. The study findings indicate that garlic stimulates gene transcription and protein secretion in neutrophils from St. Croix sheep in vitro. Studies are needed on the possible impacts on neutrophil function and animal response to infection for sustainable use of garlic in animal production.

Delta Neutrophil Index as a New Marker of Purulent Inflammation in Men With Non-odontogenic Abscesses of the Neck.

Introduction If non-odontogenic abscesses and phlegmons (all purulent inflammations where the etiology is not a diseased tooth) of the neck are not promptly treated, they can lead to serious complications and even end in the death of the affected patient. Classical markers of inflammation such as plasma concentration of leukocytes (WBC), neutrophils (Neu), C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR) are elevated in inflammatory neck diseases, but none of them has been proven as a definite marker in the prediction of this type of pathology. This necessitates the search and analysis of new indicators that could be used in the diagnosis, follow-up, and prognosis of patients with purulent neck infections. Potentially, such a marker could be the delta neutrophil index (DNI), which is increasingly entering clinical practice as a prognostic indicator in critically ill patients with life-threatening illnesses, including sepsis and systemic inflammatory response syndrome (SIRS). In the world literature, there are no data that have been studied in patients with purulent diseases of the neck of non-odontogenic origin, which is the aim of this original article. Materials and methods This retrospective study included 40 men with an average age of 46 (18-87) years with non-odontogenic abscesses and phlegmons of the neck who were hospitalized and operated on. In all of them, the concentration of leukocytes, eosinophils (Eo), neutrophils, and mature polymorphonuclear neutrophil leukocytes (PMN) was examined on an automatic 5-Diff hematology analyzer, ADVIA 2120i (SiemensMedical Solutions USA, Malvern, PA). Thus, it was calculated according to the formula for calculating DNI (DNI% = (Neu%+Eo%) - PMN%). Retrospectively, 30 healthy men with an average age of 42 (18-81) years were used as a control group, in which the same indicators were examined during a preventive examination. Results and discussion Comparing the mean values of WBC, Neu, and DNI between the studied patients with non-odontogenic purulent neck infections (n=40) and the healthy male controls (n=30) it was found that all three indicators of inflammation are significantly higher in the ill men, and these differences are statistically significant (p<0.05): 10.19 ±2.68x103/L versus 7.37 ±1.93x103/L for leukocytes, 7.68 ±2, 76x103/L versus 4.13 ±1.48x103/L for neutrophils, and 1.11±0.83% versus -1.07±1.22% for DNI. Therefore, the high mean numbers of measured WBC and Neu in men with non-odontogenic purulent neck infections were associated with an increase in their mean calculated DNI. This gives us reason to think that while WBC and Neualone are not sufficient for definitive diagnosis, treatment follow-up, and prediction of disease outcome, in combination with DNI they become reliable indicators in purulent neck infections. Conclusions The DNI correlates well with other well-known and established indicators of inflammation, such as the concentration of leukocytes and neutrophils in the peripheral blood of patients. Its calculation is fast as an implementation procedure and is economically beneficial. Its independent use in the diagnosis and treatment of these diseases is about to be investigated and analyzed.

Життєздатність та види загибелі лейкоцитів крові щурів з синдромом полікістозних яєчників за умов переривчастого холодового впливу

The aim of our work is to assess the viability and determine the types of death of blood leukocytes in rats with polycystic ovary syndrome (PCOS) under conditions of intermittent cold exposure. The study was performed on 40 immature female rats of the WAG population, aged 27 days, divided into 5 groups of 8 animals each. Group 1 included intact rats. In group 2, rats were subcutaneously injected with 0.2 ml of purified and sterilized olive oil daily for 25 days. In group 3, rats were exposed to periodic cold exposure daily for 25 days. In group 4, dehydroepiandrosterone-induced PCOS was modeled in rats. In group 5, rats were injected with dehydroepiandrosterone against the background of periodic cold exposure. Leukocyte viability and death pathways were determined using a FACS Canto II flow cytometer (Becton Dickinson, USA). Rats with dehydroepiandrosteroneinduced PCOS showed a decrease in the percentage of viable leukocytes and an increase in the percentage of leukocytes in the early stage of apoptosis, while the number of leukocytes in the late stage of apoptosis and necrotic leukocytes did not change. In rats, on the background of the introduction of dehydroepiandrosterone under conditions of intermittent cold exposure, the percentage of viable leukocytes, as well as leukocytes at different stages of apoptosis and necrosis, corresponded to the physiological norm. Thus, the authors revealed a decrease in the viability of neutrophilic leukocytes in the blood of rats with dehydroepiandrosterone-induced PCOS due to the activation of apoptosis processes, as evidenced by an increase in the percentage of leukocytes at the initial stages of apoptosis. Intermittent cold exposure (4 h daily at +4°C for 25 days) against the background of dehydroepiandrosterone administration to rats had an anti-apoptotic effect, normalizing the viability of neutrophilic blood leukocytes.

Воздействие бовгиалуронидазы азоксимера на бактериальные биопленки в эякуляте пациентов с хроническим простатитом.

Introduction. The use of molecular biological methods in the analysis of bacterial diversity (metataxonomics) of prostate secretion and ejaculate to assess the male microbiota made it possible to identify the bacterial diversity of the microbiota in chronic prostatitis. At the same time, many strains of uropathogens identified in chronic prostatitis show the ability to form biofilms, while there are no generally accepted methods for treating infections caused by bacterial biofilms. Purpose of the study. To study the effect of bovhyaluronidase azoximer in chronic prostatitis, which may be due to the enzymatic action of hyaluronidase on the matrix of bacterial biofilms, which increases the bioavailability of antibiotics, leading to the transition of bacteria into a planktonic state. Material and methods. An electron microscopic study of the ejaculate of 42 patients with chronic prostatitis was performed, of which 32 randomly selected patients received combined antibiotic treatment (4-6 weeks) together with bovhyaluronidase azoxymer (12 weeks) (group I), 10 patients received only antibiotic therapy according to clinical guidelines (group II). On ultrathin sections, the number of bacterial microcolonies and neutrophilic leukocytes per 100 spermatozoa was counted. Results. In the semen of 30 patients, bacterial biofilms with a characteristic morphology were detected: the presence of heterogeneous microorganisms in one microcolony and the presence of a fibrous matrix in which bacteria are immersed. After therapy, in group I, a decrease in the number of bacterial microcolonies, the disappearance of the extracellular matrix, and isolated single bacteria (planktonic state) were detected, in some cases, damage to the bacterial cell wall was detected. In group II quantitative changes in the content of microcolonies and neutrophilic leukocytes were also revealed, however, the morphology of microcolonies in group II did not change. Conclusions. electron microscopy in the study of the ejaculate of patients with chronic prostatitis allows us to reveal the features of the structure of bacterial biofilms. In combined antibiotic therapy together with bovhyaluronidase azoxymer, the biofilm matrix changes and the bacteria transform into a planktonic form.

IRBESARTAN REDUCES LIVER DAMAGE INDUCED BY LIPOPOLYSACCHARIDE VIA INHIBITION OF TOTAL OXIDANT STATUS, INTERLEUKIN-1B AND CASPASE-3 LEVELS

Objective&#x0D; In septic conditions, hyperinflammatory response&#x0D; and hepatotoxicity are caused by oxidative stress,&#x0D; inflammation, and apoptosis. Irbesartan (IB), an&#x0D; adrenergic receptor blocker, has anti-inflammatory and&#x0D; antioxidant properties. This study aimed to investigate&#x0D; the protective effect of IB on lipopolysaccharide (LPS)-&#x0D; induced acute hepatotoxicity.&#x0D; Material and Method&#x0D; A total of eight rats were used in three groups; a control&#x0D; group; LPS group [5 mg/kg, intraperitoneally (IP)];&#x0D; and LPS + IB group [5 mg/kg LPS (IP) + 50 mg/kg IB&#x0D; (orally)]. After sacrification, tissues from the liver and&#x0D; blood were obtained for immunohistochemical and&#x0D; biochemical evaluations, such as interleukin-1 beta&#x0D; (IL-1β), caspase-3 (Cas-3) alanine aminotransferase&#x0D; (ALT), aspartate aminotransferase (AST), oxidative&#x0D; stress index (OSI), total oxidant status (TOS), and&#x0D; total antioxidant status (TAS).&#x0D; Results&#x0D; Compared with the control group, increased AST&#x0D; and ALT levels in the blood, biochemically increased&#x0D; TOS and OSI and decreased TAS levels in the&#x0D; tissue, immunohistochemically increased IL-1β, Cas-&#x0D; 3, detected. Also, in liver tissue, histopathologically&#x0D; hyperemia, hemorrhage, vacuolization, and&#x0D; significant neutrophilia infiltration were found in the&#x0D; LPS group. IB administration significantly reversed&#x0D; all these parameters. TAS levels were increased&#x0D; by IB administration, whereas TOS and OSI levels&#x0D; were decreased (p = 0.001). IB also decreased&#x0D; AST and ALT values (p = 0.001). In the IB group,&#x0D; Cas-3 and IL-1β levels were significantly decreased&#x0D; by IB administration (p = 0.001). In addition, the&#x0D; IB ameliorated histopathological findings showed&#x0D; enhanced hyperaemia, haemorrhages, vacuolisation&#x0D; and significant neutrophilic leukocyte infiltration&#x0D; (p = 0.001). IB treatment attenuated LPS-induced&#x0D; hepatotoxicity by its antioxidant, anti-inflammatory and&#x0D; antiapoptotic properties.&#x0D; Conclusion&#x0D; Attenuating liver injury and restoring liver function lowers&#x0D; morbidity and mortality rates in patients with sepsis.&#x0D; IB protects liver tissue from hepatotoxicity caused by&#x0D; LPS thanks to its antioxidant, anti-inflammatory, and&#x0D; anti-apoptotic properties. Further investigation of the&#x0D; liver’s role in sepsis may lead to the development of&#x0D; new therapeutic targets and strategies. IB may be&#x0D; an alternative therapeutic agent for the prevention of&#x0D; acute hepatotoxicity during sepsis.

Association of urine Interleukin-8 (IL-8) with renal impairment in lupus patients

Background: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with clinical manifestations that vary between individuals with progressive and irreversible SLE organ damage. Recently, it has become known that Interleukin-8 (IL-8) levels in the blood rise in SLE patients compared to healthy people. The increase of IL-8 causes kidney inflammation by promoting the release of the Neutrophil Extracellular Traps (NET) complex, which supports further renal damage. This study aims to evaluate the association of urine interleukin-8 with renal impairment in SLE patients. Methods: A total of 45 lupus participants—22 with and 23 without renal abnormalities—had their urine collected. The interleukin-8 concentration was measured using an enzyme-linked immunosorbent assay (ELISA)—renal impairment criteria based on renal biopsy or ACR 1997 criteria. Mann-Whitney and Spearman correlation tests were employed in the statistical analysis. Data were analyzed using SPSS version 25.0 for Windows. Results: There was no significant difference in IL-8 urine levels between the SLE group with renal impairment (111.27±59.03 pg/ml) and the SLE group without renal impairment (125.76±66.62 pg/ml) was not significant (p=0.67). Urine IL-8 also did not significantly correlate with blood neutrophil count, leukocyte count and leukocyturia (p&gt;0.05). Conclusion: In lupus patients, urine Interleukin-8 levels are not associated with renal impairment.

Increased genital mucosal cytokines in Canadian women associate with higher antigen-presenting cells, inflammatory metabolites, epithelial barrier disruption, and the depletion of L. crispatus

BackgroundCervicovaginal inflammation has been linked to negative reproductive health outcomes including the acquisition of HIV, other sexually transmitted infections, and cervical carcinogenesis. While changes to the vaginal microbiome have been linked to genital inflammation, the molecular relationships between the functional components of the microbiome with cervical immunology in the reproductive tract are understudied, limiting our understanding of mucosal biology that may be important for reproductive health.ResultsIn this study, we used a multi’-omics approach to profile cervicovaginal samples collected from 43 Canadian women to characterize host, immune, functional microbiome, and metabolome features of cervicovaginal inflammation. We demonstrate that inflammation is associated with lower amounts of L. crispatus and higher levels of cervical antigen-presenting cells (APCs). Proteomic analysis showed an upregulation of pathways related to neutrophil degranulation, complement, and leukocyte migration, with lower levels of cornified envelope and cell-cell adherens junctions. Functional microbiome analysis showed reductions in carbohydrate metabolism and lactic acid, with increases in xanthine and other metabolites. Bayesian network analysis linked L. crispatus with glycolytic and nucleotide metabolism, succinate and xanthine, and epithelial proteins SCEL and IVL as major molecular features associated with pro-inflammatory cytokines and increased APCs.ConclusionsThis study identified key molecular and immunological relationships with cervicovaginal inflammation, including higher APCs, bacterial metabolism, and proteome alterations that underlie inflammation. As APCs are involved in HIV transmission, parturition, and cervical cancer progression, further studies are needed to explore the interactions between these cells, bacterial metabolism, mucosal immunity, and their relationship to reproductive health.BGqtALm3-LNazbdZNFi1WRVideo

Impact of Plasmodium falciparum infection on DNA methylation of circulating immune cells.

The regulation of immune cell responses to infection is a complex process that involves various molecular mechanisms, including epigenetic regulation. DNA methylation has been shown to play central roles in regulating gene expression and modulating cell response during infection. However, the nature and extent to which DNA methylation is involved in the host immune response in human malaria remains largely unknown. Here, we present a longitudinal study investigating the temporal dynamics of genome-wide in vivo DNA methylation profiles using 189 MethylationEPIC 850K profiles from 66 children in Burkina Faso, West Africa, sampled three times: before infection, during symptomatic parasitemia, and after malaria treatment. The results revealed major changes in the DNA methylation profiles of children in response to both Plasmodium falciparum infection and malaria treatment, with widespread hypomethylation of CpGs upon infection (82% of 6.8K differentially methylated regions). We document a remarkable reversal of CpG methylation profiles upon treatment to pre-infection states. These changes implicate divergence in core immune processes, including the regulation of lymphocyte, neutrophil, and myeloid leukocyte function. Integrative DNA methylation-mRNA analysis of a top differentially methylated region overlapping the pro-inflammatory gene TNF implicates DNA methylation of TNF cis regulatory elements in the molecular mechanisms of TNF regulation in human malaria. Our results highlight a central role of epigenetic regulation in mounting the host immune response to P. falciparum infection and in response to malaria treatment.

RESPONSE OF PARODONTAL TISSUES TO THE USE OF METAL-CERAMIC DENTURES WITH DIFFERENT TOPOGRAPHY OF THE GINGIVE EDGE OF CROWNS

Clinical, radiological and cytoenzymochemical studies of the results of using 243 metal-ceramic crowns during the year in 90 patients with healthy periodontium were carried out. The edges of the crowns were located in an equal number of teeth subgingivally and supragingivally. The activity of myeloperoxidase, alkaline phosphatase and cationic proteins in peripheral blood leukocytes was studied. It has been established that the subgingival location of the edge of the crowns causes the activation of the lysosomal apparatus of neutrophilic leukocytes and leads to the development of an inflammatory process in the marginal part of the gum. With the supragingival location of the edge of the crowns, there were no manifestations of the inflammatory process in the gingival margin. Changes in the biological activity of neutrophilic leukocytes precede the clinical manifestations of inflammation. Of the cytochemical parameters used in the study, the highest sensitivity was obtained when assessing the amount of cationic proteins in leukocytes.

Level of geneexpression of pro- and anti-inflammatory cytokines in the blood ofcows with chronic endometritis

The level of expression of genes of proinflammatory (IL-1α and IL-1β) and antiinflammatory (IL-10) cytokines in the blood of cows was studied on 20 animals, divided according to the principle of analogues into two groups: the first group (n=10) - clinically healthy cows, the second one (n=10) - diagnosed with chronic endometritis. Diagnosis of chronic endometritis was carried out on the basis of the results of transrectal examination and echographic data obtained using a portable ultrasound scanner. The experiment included cows 60 or more days after calving. It has been found that the level of expression of IL-1α, IL-1β in cows with chronic endometritis is by 1.92 and 4.61 times higher, respectively, than in clinically healthy animals, and the anti-inflammatory cytokine IL-10, on the contrary, is by 118.6 times lower. The dominance of proinflammatory cytokine gene expression over anti-inflammatory ones indicates the development of an inflammatory process in the uterus, which is confirmed by the results of histological studies of endometrial biopsy material. In clinically healthy animals, the integumentary epithelium is lined with cells of a prismatic or cubic shape, the endometrium is slightly infiltrated with neutrophils, lymphocytes and histiocytes. The uterine glands are presented in a significant amount, the glandular cells are integral, prismatic in shape. In the cows with chronic endometritis, desquamation of the integumentary epithelium was diagnosed, the cells of which in most cases are in a state of dystrophy and necrobiosis. The lumen of the uterine glands is narrowed, glandular cells are rejected into the lumen of the glands, which indicates a decrease in the functional activity of the organ. The endometrium looked edematous, abundantly infiltrated, predominantly with neutrophilic leukocytes and, to a lesser extent, with lymphoid cells, indicating the presence of inflammation in the uterine mucosa.