Microbicidal Activity Of Neutrophils Research Articles

Neutrophil phenotype, effector functions and microbicidal activity in SARS-CoV-2-associated ARDS patients.

Critically ill patients admitted to the intensive care unit (ICU) for SARS-CoV-2-induced acute respiratory distress syndrome (ARDS) are at increased risk of bacterial and fungal secondary pulmonary infections due to acquired immune dysfunction. Given that the activity of neutrophils has not been described in these patients, we aimed to investigate the function of neutrophils at ICU admission and on Day 7 (D7) post admission. Neutrophil maturation and several functional indicators were investigated. We detected a significant decrease in reactive oxygen species production at D7, but we did not observe any other significant alterations in neutrophil function. Furthermore, bronchoalveolar lavage obtained from patients displayed no inhibitory effect on the function of neutrophils from healthy donors. These findings indicate that patients admitted to the ICU for SARS-CoV-2-induced ARDS do not acquire neutrophil dysfunction within the first week of their stay, which suggests that nosocomial infections among these patients are not due to acquired neutrophil dysfunctions.

Synthetic thymic hexapeptide in the correction of alterations of antibacterial immune defense and normalization of the profile of proinflammatory cytokines in immunocompromized Children with local unlimited acute peritonitis

The study of dysregulatory disorders of the immune system underlying the immunopathogenesis of severe purulent-inflammatory diseases (PIDs) is important for development of new therapeutic tactics of restoring antibacterial defense. Acute peritonitis (AP) is a severe PID of the abdominal cavity, the course of which are dependent on the treatment, cytokine balance and adequate functioning of the immunity. Objective: to evaluate the modulating effects on the immune system and the levels of proinflammatory cytokines of the synthetic thymic hexapeptide, active substance of Imunofan, included in the complex postoperative treatment (CPOT) of immunocompromised children with local AP. Clinical and immunological examination of 20 immunocompromised children aged 5–12 years with local AP was carried out before (study group 1, SG1) and after (study group 1a, SG1a) CPOT including synthetic thymic hexapeptide (Arginyl-alpha-AspartylLysyl-Valyl-Tyrosyl-Arginine, НР), alongside 20 conditionally healthy children (comparison group, SG). The content of T and B lymphocytes, natural killer cells (NK), levels of serum pro-inflammatory cytokines IL-1β, IL-6, TNFα, IL-8, IL-18, phagocytic and microbicidal activity of neutrophils (NG) were assessed. In SG1, before treatment, a decrease in the number of T lymphocytes, T helpers, CTL-lymphocytes, NK and an increase in the level of B lymphocytes was revealed. Defects in the effector functions of NG were determined: impaired bacterial antigen killing and decreased NADPH oxidase activity. It was established that in case of AP in immunocompromised children, the cytokine profile is characterized by overproduction of studied proinflammatory and neutrophil-associated cytokines. After complex treatment including immunomodulatory therapy, there was a restoration of the content of T lymphocytes, T helper cells, TCTL lymphocytes, an increase in the number of NK and decrease in the level of B lymphocytes. In addition, regression of the levels of inflammatory, including neutrophil-associated, cytokines and emergence of effector functions of NG due to restoration of NADPH oxidases activity, was noted. Thus, the restoration of immunological parameters in AP leads to earlier regression of the purulent-inflammatory process in the abdominal cavity and to the absence of postoperative complications. The clinical and immunological effects of the immunomodulatory therapy program with inclusion of the drug based on HP determines the feasibility of its use in the postoperative period in immunocompromised children with local AP.

Dynamics of the innate immune response profile in patients with coronary heart disease at different terms after coronary artery stenting

Atherosclerosis is accompanied by damage to the vascular endothelium of arteries followed by development of inflammatory response and formation of atherosclerotic plaques. Innate immunity is an important component of this response being the earliest non-specific key mechanism. Our objective was to perform a comprehensive assessment of the cellular link of innate immunity, and to compare the results obtained at various terms after coronary stenting.The study involved 50 patients with coronary atherosclerosis (Group 1), who had clinical indications for stenting of coronary arteries, and 20 volunteers (Group 2), who have no signs of coronary artery disease. The study of immune parameters was carried out before surgery, at 4-5, 9-10 and 28-30 days after operation (during early postoperative period), as well as 6 and 12 months after stenting, i.e. over the late post-surgical period. Phenotyping of peripheral blood monocytes and lymphocytes was performed by flow cytometry using monoclonal antibodies (Beckman Coulter, USA). Intracellular content of Granzyme B was carried out with an FC500 flow laser cytofluorimeter. Metabolic activity of neutrophils was assessed by the NBT test. Alpha defensin was determined in blood plasma by ELISA technique (Hycult Biotech, USA). Statistical analysis was performed using the Statistica 12.0 program (StatSoft, USA). Statistical significance was considered significant at p ≤ 0.05.The numbers of natural killer cells and their activity, as well as those of monocytes, were increased in patients with coronary atherosclerosis. We have also shown a suppression of antigen presentation processes, an imbalance in microbicidal activity of neutrophils, with predominant secretion of antimicrobial peptides. Over the early post-surgical period, significant changes included only decreased content of intracellular Granzyme B on days 4-5, and expression of TLR4 and HLA-DR on days 4-5 and 9-10. During the late period, the patients with coronary artery disease exhibited a significant decrease in the content of some lymphocyte subsets: CD3+CD16+, CD16+Gr+ as well as amounts of monocytes: CD14+CD282+, CD14+CD284+, CD14+CD289+, along with HBT-test activity and α-defensin contents, and increased numbers of HLA-DR-expressing monocytes.There are changes in cellular component of innate immunity, indicating persistent inflammation in patients with coronary heart disease. The dynamics of revealed changes following coronary artery stenting may reflect a lability of assessed indicators mostly over the late postoperative period, thus serving a basis for predicting the outcome of coronary stenting.

Phytochemical characterization and immunomodulatory effects of aqueous and ethanolic extracts and essential oil of Moroccan Laurus nobilis L. (Lauraceae) on human neutrophils

Introduction: The leaves of Moroccan bay laurel (Laurus nobilis L.) have been used in several forms of extracts to cure rheumatic pain due to their anti-inflammatory properties. Our work aimed to evaluate the effects of aqueous and ethanolic extracts, as well as the essential oil (EO) from laurel, on the microbicidal activity of human neutrophils when compared to the effect of eucalyptol. Methods: The extracts (ethanolic and aqueous) were subject to phytochemical profiling and high-performance liquid chromatography (HPLC) analyses. The EO obtained by hydrodistillation from laurel was analyzed by gas chromatography-mass spectrometry (GC-MS). The immunomodulatory effects on neutrophil microbicidal activity of the extracts, EO, and eugenol were carried out by 3-(4,5-diméthylthiazol-2-yl)-2,5-diphényltétrazolium (MTT) assay. Results: The phytochemical analysis of the extracts revealed the presence of flavonoids, coumarins, phenols, flavone aglycones, and tannins. HPLC analysis showed the presence of numerous phenolic molecules such as syringic acid, ferulic acid, gallic acid, caffeine, and quercetin. The chemical composition of EO revealed that the major components were eucalyptol (44.14%), α-terpinyl acetate (11.11%), and β-phellandrene (6.74%). Aqueous and ethanolic extracts and EO revealed a significant and dose-dependent ability to inhibit neutrophils microbicidal activity with maximal inhibition at 200 µg/mL concentration with 30.42%, 24.7%, and 38.13%, respectively (P<0.001). Conclusion: The obtained results revealed the immunomodulatory properties of laurel as a potential natural anti-inflammatory agent that would also allow the development of new anti-inflammatory drugs.

Glycine induces enhancement of bactericidal activity of neutrophils

Severe bacterial infections are frequently accompanied by depressed neutrophil functions. Thus, agents that increase the microbicidal activity of neutrophils could add to a direct antimicrobial therapy. Lysophosphatidylcholine augments neutrophil bactericidal activity via the glycine (Gly)/glycine receptor (GlyR) α2/TRPM2/p38 mitogen-activated protein kinase (MAPK) pathway. However, the direct effect of glycine on neutrophil bactericidal activity was not reported. In this study, the effect of glycine on neutrophil bactericidal activity was examined. Glycine augmented bactericidal activity of human neutrophils (EC50 = 238 μM) in a strychnine (a GlyR antagonist)-sensitive manner. Glycine augmented bacterial clearance in mice, which was also blocked by strychnine (0.4 mg/kg, s.c.). Glycine enhanced NADPH oxidase-mediated reactive oxygen species (ROS) production and TRPM2-mediated [Ca2+]i increase in neutrophils that had taken up E. coli. Glycine augmented Lucifer yellow uptake (fluid-phase pinocytosis) and azurophil granule-phagosome fusion in neutrophils that had taken up E. coli in an SB203580 (a p38 MAPK inhibitor)-sensitive manner. These findings indicate that glycine augments neutrophil microbicidal activity by enhancing azurophil granule-phagosome fusion via the GlyRα2/ROS/calcium/p38 MAPK pathway. We suggest that glycine could be a useful agent for increasing neutrophil bacterial clearance.

Features of the immune status in chronic hepatitis C in patients with lymphopriliferative diseases

Tumor transformation of immunocompetent cells in patients with chronic lymphoproliferative diseases (CLPD) leads to the formation of secondary immune deficiency, which increases the prevalence of infectious complications. Whether the presence of hepatitis C virus (HCV) and the formation of chronic hepatitis (HCV) further weaken the immunoreactivity of the patient, remains poorly understood. The purpose of this work was to reveal the patterns of HCG formation in patients with CLPD with a detailed analysis of immune disorders associated with the chronicity of viral hepatitis C. Another aim of the study was to disclose the patterns of HCG formation in patients with CLP with a detailed analysis of immune disorders associated with the chronicity of viral hepatitis C. The comparative assessment of immunological and biochemical parameters was carried out in 110 patients with HCV (85 – chronic lymphocytic leukemia, 25 – non- Hodgkin lymphoma), with the presence of hepatitis C virus markers (n=20) and 90 patients negative for HCV. To identify intergroup differences, the method of nonparametric statistics was used: the Mann-Whitney criterion. Results. It was found that the group of HCV-infected patients is represented by younger persons compared to uninfected patients (median age – 44 years vs. 60 years, p=0.004). Analyzing the state of the immune system, we have revealed that regardless of the presence of chronic hepatitis C, all patients had a defect in both cellular and humoral immunity. The degree of immune deficiency did not have a direct dependence on viral liver damage. Moreover, in the presence of HCV infection, there was an increase in the microbicidal activity of neutrophils, as well as an increase in the expression of CD95+ on lymphoid cells. Cytokine levels during HCV contamination fluctuated in different directions. There was an increase in the level of ALT in the group of patients with HCV. Findings. The formation of immune deficiency in patients with CLPD is largely associated with the presence of oncopathology and practically does not depend on the presence of chronic hepatitis C.

Функциональная и корреляционная зависимость между показателями метаболической активности нейтрофилов и α-дефензинов у животных различных возрастных групп в динамике острой соматической боли

В последние годы все чаще встречаются штаммы микроорганизмов, устойчивые к антибиотикам. Необходим поиск новых средств, обеспечивающих надежный антибактериальный эффект. Актуальным является изучение антимикробных полипептидов. Возникает вопрос, α-дефензины активируются только при микробной атаке или существуют и иные триггеры их вовлечения в иммунологический процесс. Если исходить из того, что любая рана всегда болезненна, но не любая острая боль связана с внешним повреждением тканей, встает вопрос о возможности вовлечения нейтрофилов и α-дефензинов в долорогенный процесс как таковой. Цель работы - выявление кореляционной связи и особенностей вовлечения нейтрофилов и α-дефензинов в острый долорогенный процесс на этапах онтогенеза. Методика. Объект исследования - белые беспородные крысы 3 возрастных групп: месячные (30-35-дневные, n=36), взрослые половозрелые (2-3 месячные, n=36), старые (старше 1 года, n=36).). Каждая группа делилась на контрольную и экспериментальную подгруппы. В экспериментальных подгруппах моделировали острую соматическую боль методом электростимуляции. После болевого воздействия брали материал для исследования через 2, 30-60, 120-180 мин. Содержание α-дефензинов в сыворотке крови определяли методом иммуноферментного анализа. Для оценки микробицидной активности нейтрофилов использовали спонтанный и стимулированный НСТ - тест. Для оценки корреляционной связи между изучаемыми показателями применяли метод ранговой корреляции Спирмена. Результаты. Острая краткосрочная соматическая боль активирует кислородзависимые механизмы микробицидности нейтрофилов и приводит к увеличению уровня α-дефензинов в периферической крови. В онтогенезе реакция имеет свои особенности, но ее стандартный и непродолжительный характер очевидны. Установлена прямая корреляционная зависимость между функциональной активностью нейтрофилов и уровнем α-дефензинов в периферической крови у 30-35-дневных и 2-3-месячных животных. Заключение. Полученные данные имеют значение при разработке препаратов на основе α-дефензинов и их применении пациентами различного возраста при наличии в анамнезе острого болевого синдрома. Microorganism strains resistant to antibiotics have become more common. This fact prompts searching for new means that would provide a reliable antibacterial effect. Studying antimicrobial polypeptides is relevant. A question arises whether α-defensins are activated only during a microbial attack or there are other triggers for their involvement in the immunological process. Since any wound is always painful but acute pain is not always associated with an external injury, the question is whether neutrophils and α-defensins can be involved in the dolorogenic process as such. The aim of the study was to determine correlation and features of neutrophil and α-defensin involvement in an acute dolorogenic process at stages of ontogenesis. Methods. The study was performed on white rats divided into three age groups (30-35-day-old, 2-3-month-old, and older than one year). Each group was divided into control and experimental subgroups. In experimental subgroups, acute somatic pain was modeled by electrostimulation. Material was collected at 2, 30-60, and 120-180 min after electrostimulation. Serum content of α-defensins was measured by enzyme immunoassay. Neutrophil microbicidal activity was evaluated using the spontaneous and stimulated NBT tests. Values were compared using the non-parametric Mann-Whitney U-test, and correlations were analyzed using the Spearman rank correlation test. Results. Acute short-term somatic pain activated oxygen-dependent mechanisms of neutrophil microbicidity and induced increased levels of α-defensins in peripheral blood. This reaction has specific features in ontogenesis but it is obviously standard and short-term. The functional activity of neutrophils was directly correlated with the level of α-defensins in peripheral blood of 30-35-day-old and 2-3-month-old animals. Conclusion. At stages of ontogenesis, oxygen-dependent neutrophil microbicidity and peripheral blood content of α-defensins increase in response to acute, short-term somatic pain. The type of response is determined by age-related features. The study results are important for development of α-defensin-based drugs and prescribing them to patients of different age with a history of acute pain.

The effect of acute somatic pain on the killing activity of neutrophils in newborn rats

The immune system is subject to all sorts of influences. Pain is one of them, accompanying an organism’s existence. It is essential to be aware of and account for age-related characteristics of the innate immunity in order to adequately assess their dynamics in ontogenesis. The literature is scarce on the changes to the killing activity of neutrophils occurring in newborns in response to acute pain. The aim of this study was to detect potential changes to the phagocytic activity of neutrophils in response to an algogenic stimulus in newborn rats. The experiments were carried out in 3-5-day-old rats. Two groups were formed: the control group and the main group, in which acute pain was modelled. Blood samples were collected 2, 30–60 and 120–180 minutes after exposure to the algogenic stimulus. The microbicidal activity of neutrophils was measured using a spectrophotometric modification of the spontaneous/stimulated nitroblue tetrazolium (NBT) reduction test. The results were compared using the Mann-Whitney U test. In the first hour following pain modeling, the stimulated NBT reduction test demonstrated an increase in the measured parameters from 71.5 to 87.4 a.u. (р < 0.001); the spontaneous NBT reduction test showed an increase from 50.7 to 58.6 a.u. (p < 0.01) 30 to 60 min after exposure. The most pronounced change of the microbicidal activity coefficient was observed 2 min after pain modeling, increasing from 1.40 to 1.72 a.u (р < 0.001). By the end of the experiment, the measured parameters approximated their initial values. During the analysis, we accounted for the fact that the neutrophil response to the algogenic stimulus was unfolding in the setting of microbial colonization occurring in newborns.

The Role of Opioid Receptors in Immune System Function.

Research on the effects of opioids on immune responses was stimulated in the 1980s by the intersection of use of intravenous heroin and HIV infection, to determine if opioids were enhancing HIV progression. The majority of experiments administering opioid alkaloids (morphine and heroin) in vivo, or adding these drugs to cell cultures in vitro, showed that they were immunosuppressive. Immunosuppression was reported as down-regulation: of Natural Killer cell activity; of responses of T and B cells to mitogens; of antibody formation in vivo and in vitro; of depression of phagocytic and microbicidal activity of neutrophils and macrophages; of cytokine and chemokine production by macrophages, microglia, and astrocytes; by sensitization to various infections using animal models; and by enhanced replication of HIV in vitro. The specificity of the receptor involved in the immunosuppression was shown to be the mu opioid receptor (MOR) by using pharmacological antagonists and mice genetically deficient in MOR. Beginning with a paper published in 2005, evidence was presented that morphine is immune-stimulating via binding to MD2, a molecule associated with Toll-like Receptor 4 (TLR4), the receptor for bacterial lipopolysaccharide (LPS). This concept was pursued to implicate inflammation as a mechanism for the psychoactive effects of the opioid. This review considers the validity of this hypothesis and concludes that it is hard to sustain. The experiments demonstrating immunosuppression were carried out in vivo in rodent strains with normal levels of TLR4, or involved use of cells taken from animals that were wild-type for expression of TLR4. Since engagement of TLR4 is universally accepted to result in immune activation by up-regulation of NF-κB, if morphine were binding to TLR4, it would be predicted that opioids would have been found to be pro-inflammatory, which they were not. Further, morphine is immunosuppressive in mice with a defective TLR4 receptor. Morphine and morphine withdrawal have been shown to permit leakage of Gram-negative bacteria and LPS from the intestinal lumen. LPS is the major ligand for TLR4. It is proposed that an occult variable in experiments where morphine is being proposed to activate TLR4 is actually underlying sepsis induced by the opioid.

IPLA2 Activation Mediates Granular Exocytosis and Corrects Microbicidal Defects in ROS-Deficient and CGD Human Neutrophils.

The ubiquitous calcium-independent phospholipase A2 enzyme (iPLA2) is inhibited by calmodulin binding and known to be responsible for phospholipid remodeling housekeeping functions including granule exocytosis-associated membrane fusion in normal human neutrophils. We evaluate in human neutrophils the iPLA2 secretagogue effects using normal neutrophils, where reactive oxygen species (ROS) generation has been blocked by diphenyleneiodonium, as well as in neutrophils from chronic granulomatous disease (CGD) patients. Neutrophils were pretreated with W7, a calmodulin inhibitor known to activate iPLA2 and exocytosis of granules, and vesicles as well as intra- and extra-microbicidal activity against Staphylococcus aureus and Aspergillus fumigatus were evaluated. W7 increases exocytosis of primary, secondary, and tertiary granules and vesicles and improves neutrophil microbicidal activity against S. aureus and A. fumigatus. In neutrophils, calmodulin-mediated iPLA2 inhibition controls granule and vesicle exocytosis in the phagosome and in the extracellular microenvironment. Relieving iPLA2 inhibition results in increased exocytosis of primary, secondary, and tertiary granules and secretory vesicles with correction of defective intracellular and extracellular microbicidal activity. In CGD patients presenting ROS defective production, this increase in the non-oxidative killing pathway partially corrects their microbicidal defects.

Functional features of neutrophils in subclinical hypothyroidism compared to euthyroid status

Neutrophil or polymorphonuclear leukocytes are critical to host defense. They act by migrating (chemotaxis) to site of inflammation or infection, phagocytoze the microorganisms to form phagosome. Reactive Oxygen species and hydrolytic enzymes are released (respiratory burst activity) into this phagosome to kill ingested microorganisms (microbicidal activity). Thyroid hormones are essential for proper differentiation, growth and metabolism of an organism. Increasing evidence indicates that thyroid hormones also play an active role in immunity. Subclinical hypothyroidism is associated with normal thyroid hormone levels with increased thyroid stimulating hormone level. While studies indicate elevated TSH to have some effects on immune functions, there is very little evidence regarding its effect on functional features of neutrophils. In this cross-sectional comparative study, we evaluated the phagocytic, chemotactic, candidacidal, and respiratory burst activity of neutrophils in subclinical hypothyroid and euthyroid subjects. Phagocytosis and metabolic function of the neutrophils was assessed using Nitro-blue tetrazolium test. Chemotaxis, directional movement of the PMNs was measured using the N-Formyl methionyl-leucyl-phenylalanine as chemoattractant. Phagocytic activity was evaluated by exposing leukocytes to candida suspension and counting the number of candida ingested and killed. Microbicidal activity of neutrophils was assessed by the ability of leukocytes to kill Candida albicans in both subclinical hypothyroid and euthyroid subjects. Our study demonstrated that chemotactic and mean phagocytic activity was significantly less in those with subclinical hypothyroidism as compared to euthyroid control group (p value = 0.005 and 0.045 respectively). Respiratory burst activities of both unstimulated & stimulated neutrophils were relatively reduced in subclinical hypothyroidism (p value of 0.08 and 0.07). Candidicidal activity was similar in both groups. We conclude that subclinical hypothyroid individuals having excess of thyroid stimulating hormones are associated altered functional characteristics of neutrophils.

L-Arginine and tetrahydrobiopterin supported nitric oxide production is crucial for the microbicidal activity of neutrophils

Recent report from this lab has shown role of Rac2 in the translocation of inducible nitric oxide synthase (iNOS) to the phagosomal compartment of polymorphonuclear leukocytes (PMNs) following phagocytosis of beads. This study was undertaken to further assess the status and role of tetrahydrobiopterin (BH4), a redox-sensitive cofactor, L-arginine, and the substrate of nitric oxide synthase (NOS) in sustained nitric oxide (˙NO) production in killing of phagocytosed microbes (Escherichia coli) by human PMNs. Time-dependent study revealed consistent NO and reactive oxygen species (ROS) production in the PMNs following phagocytosis of beads. In addition, levels of L-arginine and BH4 were maintained or increased simultaneously to support the enzymatic activity of NOS in the bead activated PMNs. Moreover, translocation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) subunits along with iNOS was reconfirmed in the isolated phagosomes. We demonstrate that increase in the level of NO was supported by L-arginine and BH4 to kill E. coli, by using PMNs from NOS2−/− mice, human PMNs treated with biopterin inhibitor, N-acetyl serotonin (NAS), or by suspending human PMNs in L-arginine deficient medium. Altogether, this study demonstrates that following phagocytosis, sustained. NO production in the PMNs was well-maintained by redox sensitive cofactor, BH4 and substrate, and L-arginine to enable microbial killing. Further results suggest NO production in the human PMNs, along with ROS and myeloperoxidase (MPO) is important to execute antimicrobial activity.

Impact of dichloroethane on the microbicidal activity of neutrophils and mononuclear phagocytes

Aim. To study the damaging effect of dichloroethane on the functional state of neutrophils and peritoneal macrophages. 
 Methods. Dichloroethane was administered to animals intragastrically daily in olive oil at a dose of 0.84 mg/kg of body weight for 60 days so that the total dose was 0.1 of 50% lethal dose. The number of leukocytes, neutrophils and lymphocytes in peripheral blood, intensity of oxygen-dependant metabolism (induced test with nitro blue tetrazolium), antimicrobial activity in the conditions of functioning and blockade (by sodium azide), oxygen-dependent factors of microbicidity, content of myeloperoxidase and cationic proteins in neutrophils and peritoneal macrophages were measured. The results were recorded the next day after introduction of the toxicant. Fungicidal activity was measured by the number of colony-forming units of C. albicans, growing on day 3 at culture medium. 
 Results. Intoxication with dichloroethane for 60 days leads to the formation of leukopenia, mainly due to the decreased number of neutrophils while reducing the number of lymphocytes. This is accompanied by inhibition of oxygen-dependant killing of neutrophils as a result of suppression of peroxidase-dependant mechanisms of microbicidity (the formation of oxygen active forms decreases). Besides, decrease of activity of oxygen-independant mechanisms of killing develops, which correlates with a reduction of cationic proteins level. Suppression of oxidative and non-oxidative mechanisms of microbicidity of peritoneal macrophages. This is accompanied by a decrease of oxygen-dependant metabolism intensity, myeloperoxidase activity and cationic protein level in these cells. 
 Conclusion. Intoxication with dichloroethane for 60 days in a total dose of 0.1 of 50% lethal dose has a profound damaging effect on the cells of phagocytic link of nonspecific resistance: formation of leukopenia, suppression of oxidative metabolism and microbicidal activity of neutrophils and mononuclear phagocytes occur.

Evaluation of peripheral neutrophil functions in aggressive periodontitis patients and their family members in Indian population: An assessment of neutrophil chemotaxis, phagocytosis, and microbicidal activity.

Background:Association of neutrophil function abnormalities with localized aggressive periodontitis (LAP) has been reported in Indian population. There are no published studies on the familial aggregation of aggressive periodontitis (AP) and neutrophil function abnormalities associated with it in Indian population. The present study aimed to assess neutrophil chemotaxis, phagocytosis, and microbicidal activity in AP patients and their family members of Indian origin, who may or may not be suffering from AP.Materials and Methods:Eighteen families with a total of 51 individuals (18 probands, 33 family members) were included. Neutrophil chemotaxis was evaluated against an alkali-soluble casein solution using Wilkinson's method. Phagocytosis and microbicidal activity assay were performed using Candida albicans as an indicator organism.Statistical Analysis Used:The magnitude of association between the presence of defective neutrophil function and LAP or GAP was calculated using odds ratio and relative risk. Total incidence of AP, and in particular, LAP in the families attributable to the presence of defective neutrophil function was calculated by attributable risk.Results:The association between depressed neutrophil chemotaxis and presence of AP and LAP or GAP in all the family members (n = 51) was found to be significant (P < 0.05) while that for phagocytic and microbicidal activity were observed to be nonsignificant.Conclusion:The results of the present study suggest high incidence of AP (LAP and GAP) within families was associated with depressed neutrophil chemotaxis. High prevalence of depressed neutrophil chemotaxis in the family members (61%) of LAP probands exhibiting depressed chemotaxis suggests that the observed abnormalities in neutrophil functions may also be inherited by the family members.

Microbicidal activity of neutrophils is inhibited by isolates from recurrent vaginal candidiasis (RVVC) caused by Candida albicans through fungal thioredoxin reductase

Vulvovaginal candidiasis (VVC) is characterized by an infection of the vulva and vagina, mainly caused by Candida albicans, a commensal microorganism that inhabits the vaginal, digestive, and respiratory mucosae. Vulvovaginal candidiasis affects approximately 75% of women, and 5% develop the recurrent form (RVVC). The aim of the present study was to evaluate whether neutrophils microbicidal response is triggered when activated with RVVC isolates caused by C. albicans. Our results showed that RVVC isolates induced neutrophil migration but significantly decrease the microbicidal activity of neutrophils, compared with VVC and ASS isolates. The microbicidal activity of neutrophils is highly dependent on the production of reactive oxygen species/reactive nitrogen species (ROS/RNS). However, this isolate induced detoxification of ROS/RNS produced by neutrophils, reflected by the high level of thiol groups and by the oxygen consumption. Therefore, RVVC isolates induced biochemical changes in the inflammatory response triggered by neutrophils, and these effects were mainly related to the detoxification of ROS/RNS through the thioredoxin reductase (TR), a key antioxidant enzyme in fungi. This might be one of the resistance mechanisms triggered by RVVC caused by C. albicans.

Functional status of neutrophils at 2,4-dichlorophenoxyacetic acid amine salt intoxication

Aim. To study the influence of 2,4-dichlorophenoxyacetic acid amine salt subacute intoxication on the functional status of neutrophils. Methods. 2,4-dichlorophenoxyacetic acid amine salt (42 mg/kg) was administered intragastrically during 28 days. The number of leukocytes, neutrophils and lymphocytes in peripheral blood, intensity of oxygen-dependent metabolism (spontaneous and induced tetrazolium test), absorbency, microbicidal activity of neutrophils in conditions of oxygen-dependent microbicidal factors functioning and sodium azide mediated blockade, the level of myeloperoxidase and cation proteins in neutrophils were registered at days 7th and 14th after the toxicant administration discontinuation. Results. Subacute 2,4-dichlorophenoxyacetic acid amine salt intoxication resulted in leucopenia due to neutropenia, accompanied by inhibition of oxygen-dependent neutrophils killing as the result of peroxidase-independent microbicidal mechanisms suppression (reduced synthesis of oxygen active forms). A reduced activity of oxygen-independent killing mechanisms correlating with a decrease of cation proteins levels was also registered. On the 14 day, leucopenia was replaced by leukocytosis due to elevated numbers of neutrophils and lymphocytes in the peripheral blood. At the same time, the activity of both oxidative peroxidase-dependent and oxygen-independent microbicidal mechanisms in neutrophils continued to decrease, accompanied by reduction of mieloperoxidase activity, neutrophil absorbency and cation proteins levels. Conclusion. Subacute intoxication with 2,4-dichlorophenoxyacetic acid amine salt leaded to quantitative and functional disorders in neutrophils that persisted by the 14 day of follow-up, indicating a need for further study of of 2,4-dichlorophenoxyacetic acid amine salt immunotoxic action to find the possible opportunities for its pharmacological correction.

Global Transcriptome Analysis of Staphylococcus aureus Biofilms in Response to Innate Immune Cells

The potent phagocytic and microbicidal activities of neutrophils and macrophages are among the first lines of defense against bacterial infections. Yet Staphylococcus aureus is often resistant to innate immune defense mechanisms, especially when organized as a biofilm. To investigate how S. aureus biofilms respond to macrophages and neutrophils, gene expression patterns were profiled using Affymetrix microarrays. The addition of macrophages to S. aureus static biofilms led to a global suppression of the biofilm transcriptome with a wide variety of genes downregulated. Notably, genes involved in metabolism, cell wall synthesis/structure, and transcription/translation/replication were among the most highly downregulated, which was most dramatic at 1 h compared to 24 h following macrophage addition to biofilms. Unexpectedly, few genes were enhanced in biofilms after macrophage challenge. Unlike coculture with macrophages, coculture of S. aureus static biofilms with neutrophils did not greatly influence the biofilm transcriptome. Collectively, these experiments demonstrate that S. aureus biofilms differentially modify their gene expression patterns depending on the leukocyte subset encountered.

Assessment of peripheral neutrophil functions in patients with localized aggressive periodontitis in the Indian population

Background:Localized aggressive periodontitis (LAP) patients exhibit abnormal neutrophil functions to a variety of environmental and host stimuli. The aim of the present study was to evaluate neutrophils chemotaxis, phagocytosis, microbicidal activity and superoxide generation in LAP patients of Indian origin.Materials and Methods:Eleven LAP patients and nine healthy subjects were included in the study. Neutrophil chemotaxis was evaluated against an alkali-soluble casein solution using Wilkinson's method. Phagocytosis and microbicidal activity assay were performed using Candida albicans as an indicator organism. Nitrobluetetrazolium (NBT) test was used to assess superoxide generation by neutrophils using E. coli endotoxin.Results:The chemotactic activity and phagocytic and microbicidal activity were observed to be significantly reduced (P < 0.01) in LAP neutrophils. On the contrary, superoxide generation was observed to be significantly increased (P < 0.01) in LAP neutrophils compared with healthy individuals.Conclusion:The results of the present study suggest that neutrophil functions, namely chemotaxis, phagocytosis and microbicidal activity, are deficient LAP patients. However, superoxide generation was significantly increased when stimulated by endotoxins, which may explain the tissue damage seen in LAP. These abnormal neutrophil functions may predispose to increased susceptibility for LAP. Further large-scale studies are required in the Indian population to ascertain the cause-and-effect relationship of defective host factors and aggressive periodontitis and to develop treatment strategies for more predictable periodontal treatment outcome.

Antioxidant effects of quercetin and naringenin are associated with impaired neutrophil microbicidal activity.

Naringenin and quercetin are considered antioxidant compounds with promising activity against oxidative damage in human cells. However, no reports have described their effects on reactive oxygen species (ROS) production by phagocytes during microbicidal activity. Thus, the present study evaluated the effects of naringenin and quercetin on ROS production, specifically hypochlorous acid (HOCl), and their involvement in the microbicidal activity of neutrophils. Naringenin and quercetin inhibited HOCl production through different systems, but this inhibition was more pronounced for quercetin, even in the cell-free systems. With regard to the microbicidal activity of neutrophils, both naringenin and quercetin completely inhibited the killing of Staphylococcus aureus. Altogether, these data indicate that the decrease in the oxidant activity of neutrophils induced by these compounds directly impaired the microbicidal activity of neutrophils. Naringenin and quercetin exerted their effects by controlling the effector mechanisms of ROS production, with both positive and negative effects of these antioxidant agents in oxidative stress conditions and on ROS in the microbicidal activity of phagocytes. The present results challenge the traditional view of antioxidants as improvers of pathological conditions.

Functional status of neutrophils during carbon tetrachloride intoxication in rats

Aim. To study the damaging effect of acute carbon tetrachloride intoxication on the functional status of neutrophils in an experiment. Methods. The experiments were performed on 90 non-inbred white rats weighing 200-220 g; two groups were identified - experimental group and control group (45 animals in each). Carbon tetrachloride was administered intragastrically to the experimental group (1.25 ml/kg 50% solution in olive oil) for the duration of four days. Determined were the number of leukocytes, neutrophils and lymphocytes in the peripheral blood, the intensity of the oxygen-dependent metabolism (through spontaneous and induced nitro blue tetrazolium test), the absorption and antimicrobial activity, the content of myeloperoxidase and cationic proteins in neutrophils. The results were recorded on the 7th, 14th and 28th day from the end of the toxicant administration. Results. On the 7th day in rats of the experimental group leukopenia was registered in the form of reduction in the number of lymphocytes, the inactivation index decreased, which was confirmed by reduction of the number of myeloperoxidase-positive cells and of the mean cytochemical coefficient; noted was a decrease in the absorptive capacity of neutrophils. On the 14th day leukopenia persisted, which was now in the form of an even decrease in the number of neutrophils and lymphocytes; with the continued decrease in the phagocytic index an increase in the number of cells involved in phagocytosis was registered. By the 28th day the number of lymphocytes increased in the presence of neutropenia; the decrease in the number of myeloperoxidase-positive cells and of the mean cytochemical coefficient in the myeloperoxidase system, reduction of the indices of the induced test with nitro blue tetrazolium and of the proportion of cells positive for cationic proteins persisted. Conclusion. Acute intoxication with carbon tetrachloride leads to profound dysfunction of neutrophils (leukopenia, decreased absorption and microbicidal activity of neutrophils, reduced myeloperoxidase activity and the content of cationic proteins), which is not eliminated by the 28th day of observation.