Polymorphonuclear Neutrophil Leukocytes Research Articles

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.

The role of the host-Neutrophil biology.

Neutrophilic polymorphonuclear leukocytes (neutrophils) are myeloid cells packed with lysosomal granules (hence also called granulocytes) that contain a formidable antimicrobial arsenal. They are terminally differentiated cells that play a critical role in acute and chronic inflammation, as well as in the resolution of inflammation and wound healing. Neutrophils express a dense array of surface receptors for multiple ligands, ranging from integrins to support their egress from bone marrow into the circulation and from the circulation into tissues, to cytokine/chemokine receptors that drive their navigation to the site of infection or tissue damage and also prime them for a second stimulus, to pattern recognition receptors and immunoglobulin receptors to facilitate the destruction and removal of infective agents or debridement of damaged tissues. When afferent neutrophil signals are proportionate and coordinated they will phagocytose opsonized and unopsonized bacteria, activating the nicotinamide adenine dinucleotide phosphate oxidase (respiratory burst) to generate reactive oxygen species, which augment the proteolytic destruction of microbes secured within the phagosome. A highly orchestrated process of apoptosis follows, forming membrane-bound substructures that are removed by macrophages. Neutrophils are capable of various other forms of programmed cell death, such as NETosis and pyroptotic cell death, as well as nonprogrammed cell death by necrosis. In recent years, research has revealed that neutrophils are capable of far more subtle cell-cell interactions than previously thought possible. This includes synthesis of various inflammatory mediators and also myeloid cell training within bone marrow, where epigenetic and metabolic signals associated with returning neutrophils that undergo reverse egress from tissues into the vasculature and back to bone marrow program a hyperreactive subset of neutrophils during myelopoiesis that are capable of hypersensitive reactions to microbial aggressors. These characteristics are evident in various neutrophil subsets/subpopulations, creating broad heterogeneity in the behavior and biological repertoire of these seemingly schizophrenic immune cells. Moreover, neutrophils are critical effector cells of adaptive and innate immunity, binding to opsonized bacteria and destroying them by extracellular and intracellular methods. The former creates substantial collateral host tissue damage, as they are less specific than T-cytotoxic cell-killing mechanisms, and in conditions such as peri-implantitis, where plasma cells and neutrophils dominate the immune infiltrate, bone and tissue destruction are rapid and appear relentless. Finally, the role of neutrophils as conduits for periodontal-systemic disease connections and for oxidative damage to act as a causal link between the two has only recently been realized. In this chapter, we attempt to expand on these issues, emphasizing the contributions of European scientists throughout a detailed appraisal of the benefits and side effects of neutrophilic inflammation and immune function.

Oral Epithelial Cell Culture Model for Studying the Pathogenesis of Chronic Inflammatory Disease.

The interactions between bacteria, epithelium, and neutrophilic polymorphonuclear leukocytes (neutrophils) are the key to the initiation and progression of many chronic inflammatory-immune diseases. In addition, all can be influenced by external factors, such as micronutrients, thereby providing potentially novel approaches to therapy. This chapter will therefore provide detailed methods for core techniques involved in studying cellular and molecular epithelial responses to a bacterial challenge in relation to chronic inflammatory disease pathogenesis and therapy.

Distinct behavior of bovine-associated staphylococci species in their ability to resist phagocytosis and trigger respiratory burst activity by blood and milk polymorphonuclear leukocytes in dairy cows

Mastitis affects a high proportion of dairy cows and is still one of the greatest challenges faced by the dairy industry. Staphylococcal bacteria remain the most important cause of mastitis worldwide. We investigated how distinct staphylococcal species evade some critical host defense mechanisms, which may dictate the establishment, severity, and persistence of infection and the outcome of possible therapeutic and prevention interventions. Thus, the present study investigated variations among distinct bovine-associated staphylococci in their capability to resist phagocytosis and to trigger respiratory burst activity of blood and milk polymorphonuclear neutrophil leukocytes (PMNL) in dairy cows. To do so, PMNL of 6 primiparous and 6 multiparous dairy cows were used. A collection of 38 non-aureus staphylococci (NAS) and 12 Staphylococcus aureus were included. The phagocytosis and intracellular reactive oxygen species (ROS) production by blood and milk PMNL were analyzed by flow cytometry. Phagocytosis, by both blood and milk PMNL, did not differ between S. aureus and NAS as a group, although within-NAS species differences were observed. Staphylococcus chromogenes (a so-called milk-adapted NAS species) better resisted phagocytosis by blood PMNL than the so-called environmental (i.e., Staphylococcus fleurettii) and opportunistic (i.e., Staphylococcus haemolyticus) NAS species. Otherwise, S. haemolyticus was better phagocytosed by blood PMNL than S. aureus, S. fleurettii, and S. chromogenes. No influence of the origin of the isolates within the staphylococci species in the resistance to phagocytosis by blood and milk PMNL was found. Overall, both S. aureus and NAS did not inhibit intracellular ROS production in blood and milk PMNL. Non-aureus staphylococci induced fewer ROS by milk PMNL than S. aureus, which was not true for blood PMNL, although species-specific differences in the intensity of ROS production were observed. Staphylococcus chromogenes induced more blood PMNL ROS than S. fleurettii and S. haemolyticus, and as much as S. aureus. Conversely, S. chromogenes induced fewer milk PMNL ROS than S. aureus. The origin of the isolates within the staphylococci species did not affect the ROS production by blood and milk PMNL. In conclusion, our study showed differences in staphylococci species in evading phagocytosis and triggering ROS production, which may explain the ability of some staphylococci species (i.e., S. aureus and S. chromogenes) to cause persistent infection and induce inflammation.

Clinical endometritis with Trueperella pyogenes reduces reproductive performance and milk production in dairy cows.

The aim of this study was to identify the impact of Trueperella pyogenes in cows with clinical endometritis (CE) on reproductive performance and milk production in affected cows. In total, 230 lactating Holstein dairy cows from six commercial dairy herds were sampled once between 28 and 33days post-partum. Cows included in the present study did not receive antibiotic or anti-inflammatory treatments prior to the experimental period. Clinical endometritis (CE) was characterized as cow with vaginal mucus score=3 (>50% of purulent vaginal discharge) and >18% polymorphonuclear neutrophilic leukocyte (PMNL). The body condition scores (BCS) and milk production were evaluated at the time of enrolment. The identification of isolated bacteria was carried out through the analysis of MALDI-TOF MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry). According to uterine health, three groups of dairy cows were formed: healthy control cows without T.pyogenes (n=147), CE cows with T.pyogenes (n=22) and CE cows without T.pyogenes (n=61). CE cows with T.pyogenes had lower BCS, milk production and conception at first AI (p<.01) than CE cows without T.pyogenes and control cows. Furthermore, CE cows with T.pyogenes had higher (p<.01) service per pregnancy and had greater (p<.01) days to get pregnant than CE cows without T.pyogenes and control cows. This study demonstrates that CE cows with T.pyogenes had impaired reproductive performance and milk production when compared to cows without CE and CE cows without T.pyogenes. This information can contribute to a strategic treatment in cows affected by clinical endometritis, favouring the rational use of antibiotics on dairy farms.

LOX-1 Regulates Neutrophil Apoptosis and Fungal Load in A. Fumigatus Keratitis

ABSTRACT Purpose To determine whether LOX-1 regulates neutrophil apoptosis and fungal load in A. fumigatus keratitis. Methods Fas, FasL, CASP3, CASP8, CASP9 and BCL2 were tested in normal and infected corneas of C57BL/6 mice. Mice corneas were infected with A. fumigatus with or without pretreatment of LOX-1 neutralizing antibody or inhibitor (Poly I). Clinical score was recored and HE staining was tested. Fungal load in mice corneas was observed by plate counting. Poly morphonuclear neutrophilic leukocytes (PMNs) were stimulated with 75% ethanol-killed A. fumigatus with or without pretreatment of LOX-1 neutralizing antibody or Poly I. PCR, western blot and immunostaining tested expression of Fas, FasL, CASP3, CASP8, CASP9, BCL2 and cleaved caspase-3. PMNs infiltration and TUNEL-positive cells were assessed by immunofluorescent staining. Flow cytometry assay tested the percentage of apoptosis neutrophils. Results Fas, Fas ligand, caspase-8, caspase-9 and caspase-3 mRNA levels were significantly higher in C57BL/6 mice corneas infected with A. fumigatus than normal corneas. Poly I treatment alleviated the severity and decreased clinical score at 3, 5 and 7 days post infecrion (p.i.). HE staining showed less infiltration in corneal tissue after LOX-1 inhibition. Plate counting experiment showed that number of viable fungus in corneas of Poly I treated group was significantly less than control group. LOX-1 neutralizing antibody or Poly I treatment significantly decreased neutrophil infiltration, the quantity of TUNEL-positive cells, the expression of Fas, Fas ligand, caspase-8, caspase-9, caspase-3, cleaved caspase-3 and the percentage of apoptosis neutrophils compared with control corneas. LOX-1 neutralizing antibody treatment significantly decreased Fas, FasL, CASP3, CASP8, CASP9 and cleaved caspase-3 expression in neutrophils. Conclusion LOX-1 inhibition decrease neutrophil apoptosis and fungal load in A. fumigatus keratitis.

Galectin-3 plays an important pro-inflammatory role in A. fumigatus keratitis by recruiting neutrophils and activating p38 in neutrophils

PurposeTo determine the role of galectin-3 (Gal-3) in cornea infected by Aspergillus fumigatus (A. fumigatus). MethodsGal-3 was tested in normal and infected corneas of C57BL/6 mice. Mice corneas were pretreated with or without rmGal-3 or Gal-3 siRNA and infected with A. fumigatus. Recombinant mouse (rm) Gal-3 stimulated polymorphonuclear neutrophilic leukocytes (PMNs). PMNs were stimulated with 75% ethanol-killed A. fumigatus with or without pretreatment of Gal-3 siRNA. Disease severity was documented by clinical score and photographs with a slit lamp. PCR, Western blot, and ELISA tested expression of Gal-3, interleukin (IL)-1β, IL-6, macrophage inflammatory protein 2 (MIP-2) and p-p38. PMNs infiltration was assessed by flow cytometry and myeloperoxidase (MPO) assay. ResultsGal-3 expression was significantly elevated by A. fumigatus in mice corneas. rmGal-3 treatment increased clinical scores, PMNs infiltration, and cytokines expression, which were decreased by Gal-3 siRNA treatment. In PMNs, Gal-3 expression was also significantly increased by A. fumigatus. The rmGal-3 treatment upregulated proinflammatory cytokines secretion and p-p38 expression, which was significantly inhibited by Gal-3 siRNA. ConclusionThese data proved that A. fumigatus increased Gal-3 expression and elevated disease clinical scores, PMNs infiltration and cytokines expression through Gal-3. In PMNs, A. fumigatus upregulated IL-1β and IL-6 secretion through the Gal-3 / p38 pathway.

IL-36α Exerts Proinflammatory Effects in Aspergillus fumigatus Keratitis of Mice Through the Pathway of IL-36α/IL-36R/NF-κB.

PurposeTo explore the role of IL-36α in corneas infected by Aspergillus fumigatus.MethodsThe experimental group was comprised of 15 corneas with fungal keratitis, and 15 healthy donor corneas were included in the control group. IL-36α was detected in normal and infected corneas of humans and C57BL/6 mice. Mice corneas were infected with A. fumigatus with or without pretreatment of recombinant mouse (rm) IL-36α and IL-36α neutralizing antibody (Ab). Primary macrophages were stimulated with 75% ethanol-killed A. fumigatus with or without pretreatment of rmIL-36α. The severity of the disease was documented by clinical score and photographs with a slit lamp. PCR, western blot, and immunostaining were used to determine the expression of IL-36α, IL-1β, IL-6, and TNF-α. Polymorphonuclear neutrophilic leukocyte infiltration was assessed by myeloperoxidase (MPO) assay and flow cytometry. Macrophage infiltration was tested by immunofluorescent staining and flow cytometry.ResultsIL-36α mRNA and protein were significantly elevated in human and mice corneas after infection. The rmIL-36α treatment of C57BL/6 mice increased clinical score, MPO levels, macrophage infiltration, and expression of the proinflammatory cytokines IL-1β, IL-6, and TNF-α compared with the infected controls, which showed a decrease due to IL-36α Ab treatment. In primary macrophages, IL-36α expression was also significantly increased by A. fumigatus. The rmIL-36α treatment upregulated IL-1β, IL-6, and phosphorylated nuclear factor (NF)-κB expression, which was significantly inhibited by rmIL-36Ra.ConclusionsIL-36α act as a proinflammatory cytokine in A. fumigatus keratitis by promoting the infiltration of neutrophils and macrophages and increasing the secretion of IL-1β, IL-6, and TNF-α, in addition to regulating expression of phosphorylated NF-κB.

Prevalence of subclinical uterine pathologies diagnosed by biopsy and cytological and bacteriological findings in cyclic bitches.

The study was performed on 45 bitches in different cycle phases that were divided into the following groups: anoestrus (I, n=15), heat (pro-oestrus (n=7) or estrous (n=8) (II, n=15) and metestrus (III, n=15). Moreover, all experimental dogs were grouped according to their age: younger than 5 years (Y, n=35) and older than 5 years (O, n=10). The endometrial status was evaluated using cytological, bacteriological and biopsy samples obtained after ovariohysterectomy. The main uterine pathology diagnosed by biopsy was endometritis, since 40%-66% of bitches, independent of the experimental group, developed this condition. No significant differences were found among the cycle phase groups (p>0.05). By contrast, significant differences were found in the age groups; the prevalence of this pathology was higher in older bitches (p=0.0019). The general prevalence of cystic endometrial hyperplasia (CEH) and a normal endometrium (NE) was lower (6.7-26.7% vs 26.7-53.3%) in all groups, and no statistically significant differences were found between certain groups (p>0.05). The percentage of polymorphonuclear neutrophilic leukocytes (PMNs) in endometrial cytology was generally low (⟨ 2%) and did not differ significantly among the experimental groups (p=0.142). In general, a low degree of correlation was found between the diagnostic results by endometrial cytology and biopsy (Kappa Coefficient= 0.046). Positive bacteriological findings were found in approximately 50% of the bitches, independent of the cycle phase and health status of the endometrium. No correlation was found between the bacteriological and histopathological findings (p=0.883). In conclusion, uterine cytology is not a reliable diagnostic method to detect the subclinical inflammatory and degenerative uterine pathologies in cyclic bitches.

High-mobility group box1 as an amplifier of immune response and target for treatment in Aspergillus fumigatus keratitis.

To determine the roles of high-mobility group box1 (HMGB1) in pro-inflammation, host immune response and its potential target for treatment in Aspergillus fumigatus (A.fumigatus) keratitis. Expression of HMGB1 was tested in C57BL/6 normal and infected corneas. Dual immunostaining tested co-expression of HMGB1 with TLR4 or LOX-1. C57BL/6 mice were pretreated with Box A or PBS and then infected. Clinical scores, polymerase chain reaction, ELISA, and MPO assay were used to assess the disease response. Flow cytometry were used to test the effect of Box A on reactive oxygen species (ROS) expression after A.fumigatus stimulation in polymorphonuclear neutrophilic leukocytes (PMN). C57BL/6 peritoneal macrophages were pretreated with Box B before A.fumigatus stimulation, and MIP-2, IL-1β, TNF-α, HMGB1 and LOX-1 were measured. Macrophages were pretreated with Box B or Box B combined with Poly(I) (an inhibitor of LOX-1) before stimulating with A.fumigatus, and MIP-2, IL-1β, TNF-α, LOX-1, p38-MAPK, p-p38-MAPK were measured. HMGB1 levels were elevated in C57BL/6 mice after infection. HMGB1 co-expressed with TLR4, and LOX-1 in infiltrated cells. Box A vs PBS treated C57BL/6 mice had lower clinical scores and down-regulated corneal HMGB1, MIP-2, IL-1β expression and neutrophil influx. Box B treatment amplified expression of MIP-2, IL-1β, TNF-α, HMGB1 and LOX-1 that induced by A.fumigatus in macrophage. Compared to the treatment of Box B only, the protein expression of IL-1β, TNF-α showed inhibition of Box B combined with Poly(I), which also reduced the A.fumigatus-evoked protein level of LOX-1 and phosphorylation level of p38-MAPK. The production of A.fumigatus-stimulated ROS was significantly declined after Box A pretreatment in PMN. Blocking HMGB1 reduces the disease response in C57BL/6 mice. HMGB1 can amplify the host immune response through p38-MAPK, and is a target for treatment of A.fumigatus keratitis.

The Role of Autophagy in the Innate Immune Response to Fungal Keratitis Caused by Aspergillus fumigatus Infection.

PurposeTo determine the role of autophagy in the innate immune response to fungal keratitis (FK) caused by Aspergillus fumigatus infection.MethodsCorneal samples obtained from patients and mice with FK were visualized via transmission electron microscopy (TEM). Autophagy-related proteins LC3B-II, Beclin-1, LAMP-1, and p62 in A. fumigatus-infected corneas of C57BL/6 mice were tested by Western blot. After treatment with autophagy inhibitors 3-methyladenine (3-MA), chloroquine (CQ), or inducer rapamycin, autophagy-related proteins were detected by Western blot. Corneas were photographed with slit lamp microscopy and pathological changes were observed by hematoxylin and eosin staining. Polymorphonuclear neutrophilic leukocytes (PMNs) were assessed by immunofluorescent staining and observed under TEM. The levels of CXCL-1, IL-1β, HMGB1, IL-18, TNF-α, and IL-10 were tested by reverse transcription polymerase chain reaction and Western blot. The quantification of fungal loads was detected and photographed.ResultsThe accumulation of autophagosomes in corneas of patients and mice with FK was observed with TEM. The expression of LC3B-II, Beclin-1, and LAMP-1 was elevated in corneas after fungal infection, whereas p62 was reduced. Treatment with 3-MA or CQ upregulated clinical scores, pathological changes, and the expression of CXCL-1, IL-1β, HMGB1, IL-18, and TNF-α except IL-10. The morphology of PMNs was changed and PMN recruitment was increased in mice corneas treated with 3-MA or CQ, whereas rapamycin reduced the inflammatory response to keratitis. These results were statistically significant.Conclusions A. fumigatus infection increases the expression of autophagy in corneas. Autophagy plays an anti-inflammatory role in the innate immune response to A. fumigatus keratitis.

The role of netrin-1 in the mouse cornea during Aspergillus fumigatus infection

PurposeTo explore the effects of netrin-1 on inflammation in Aspergillus fumigatus-infected mouse corneas and on proliferation and migration in human corneal epithelial cells (HCECs). MethodsNetrin-1 and the receptor A2BAR were detected in normal and infected corneas from C57BL/6 mice and RAW 264.7 cells. The mice were injected subconjunctivally with recombinant netrin-1. The severity of the disease was determined by clinical scores, photography with a slit lamp, RT-PCR, western blotting, myeloperoxidase (MPO) assays and immunofluorescence staining of polymorphonuclear neutrophilic leukocytes (PMNs). The effects of netrin-1 on RAW 264.7 cells in vitro were determined by RT-PCR. The role of A2BAR was demonstrated in vivo by detecting the expression of IL-1β, TNF-α, and IL-10 in corneas pretreated subconjunctivally with an A2BAR antagonist (PSB1115). RAW 264.7 cells were stimulated with Aspergillus fumigatus (A. fumigates) and netrin-1 with or without PSB1115 pretreatment. A cell counting kit-8 (CCK-8) assay was used to evaluate cell proliferation ability, and cell migration ability was determined by cell scratch experiments with HCECs. ResultsNetrin-1 expression decreased slightly after A. fumigatus infection and then increased to its peak. A2BAR expression increased at 1 day post infection (p.i.), with a subsequent decline. Compared to the PBS control, exogenous netrin-1 attenuated the inflammatory response, PMN infiltration, and expression of the proinflammatory factors IL-1β and TNF-α, while IL-10 expression was up-regulated. In RAW 264.7 cells, recombinant netrin-1 obviously inhibited the mRNA expression of IL-1β and TNF-α and promoted the mRNA expression of the anti-inflammatory cytokine IL-10. Pretreatment with PSB1115 resulted in disease aggravation and higher levels of the proinflammatory factors IL-1β and TNF-α both in vivo and in vitro. And the effect of netrin-1 on inflammatory factors was abolished by PSB1115. Moreover, compared to the control treatment, exogenous netrin-1 significantly facilitated the proliferation and migration of HCECs. ConclusionsNetrin-1 attenuates inflammation in C57BL/6 mice infected with A. fumigatus, and it may play this role via the receptor A2BAR. Additionally, netrin-1 can promote the proliferation and migration of HCECs.

POSTNATAL INFECTION AND IMMUNE SYSTEM IN CHILDREN OF THE FIRST YEAR OF LIFE

Objective. The aim of the work is to investigate the relationship between the indices of the cellular and humoral components of immunity, the absolute and relative quantity of immunocompetent blood cells in children infected postnatal.Material and methods. 20 children aged 3 months to one year were divided into two groups: 1) control (n = 7); 2) in utero infected (n = 13). The study of blood counts and immune homeostasis was carried out from a single portion of heparinized venous blood taken from the ulnar vein of children with the permission of mothers. The determination was made by conventional methods [8-10]. Statistical processing of the results of the studies was carried out using standard methods for the program Statistica-6.0. To determine the statistical significance, the t-test of the Student was used.Results. The conducted studies aimed at establishing the absolute and relative number of immunocompetent cells and immunohematological indexes and coefficients at the age of 3 months to one year, with postnatal infection (Table 1), showed that these children were reduced by the absolute number of erythrocytes (by 22 , 4%), leukocytes (41.5%), the relative amount of polymorphonuclear neutrophilic leukocytes (by 80.0%), eosinophils (57.0%), absolute (58.1%) and relative quantities (13 , 0%) of lymphocytes, the relative amount of monocytes ( by 66.1%), while the number of segmented neutrophils increased by 43.8%, and the ESR by 13.6%. Such changes in the absolute number of immunocompetent cells affect the characteristics of the immune-haematological indices and coefficients. Thus, in children aged 3 months to 1 year with PNI, the leukocyte index increases by 2.7 times, the neutrophilic leukocyte coefficient increases by 54.9%, the leukocyte shift index by 55.3%, the index of immune reactivity - by 45.8%. Against this background, the neutrophil shift index is reduced by 2.6 times, the allergy index by 40.0% and the index of unspecific resistance by 31.5%. Versatile changes in the absolute and relative quantity of immunocompetent cells, immunohematological indices and coefficients indicate the presence of intoxication in the body of children from 3 months to 1 year with PNI. So, the leukocyte index of intoxication in these children increases by 64.3%, and the leukocyte index of intoxication by Ya.Ya. Calf-Caliph in 2,1 times. Thus, in children aged 3 months to 1 year, patients with PNI, there are a variety of significant changes in the absolute and relative quantity of immunocompetent cells and immunohumatological indices and coefficients.Conclusion. Postanital infection in children from 3 months to one year is formed and runs against the backdrop of lower indicators of factors and mechanisms of non-specific anti-infection protection, as well as against the background of acquired immunodeficiency state.

Adenovirus infection in adults: reactive response of polymorphonuclear neutrophilic leukocytes study

The role of adenovirus infection in the SARS is significant but every year this group of infections makes up to 30 % of general infectious morbidity. Nowadays features of nonspecific immune defence in patients with adenovirus infection are insufficiently studied. Particularly acute AVI problem appears to clinicians and scientists as a contagious disease, which is rapidly spreading in organized collectives, among military personnel, students. Purpose. To study the reactive response of polymorphonuclear granulocytes in the peripheral blood of adult patients with adenovirus infection. Materials and methods. 37 volunteers with SARS signs (Unified Protocol Criteria) were involved in this “case – control” type study. Absolute and relative quantities of the main immune cells populations in the peripheral blood were analyzed. Immunohematological indicators which characterize reactive response of the main non-specific immune cells were calculated. Clinical diagnosis was confirmed by the serological method of complement fixation test in paired sera and using adenoviruses common soluble complement-fixing antigen. Results. Neutrophil granulocytes reactivity increased by 83.92 % indicating these cells activation by cytokine system. The inflammatory process is accompanied by an increase in leukocytes absolute count – 19.76 %, band neutrophil leukocytes - in 2.18 times; lymphocytes – 30.30 % and monocytes – 48.15 %. Conclusions. Aforementioned means that there are different types of immune response to adenovirus infection antigens. Patients immune reactivity decreasing by 14.86 % means that specific immune response is formed later. Method of neutrophils reactive response evaluation can be used by practicing doctors for the early prediction of possible anti-infectious protection system alteration.

Nicorandil reduces burn wound progression by enhancing skin blood flow

We assessed whether nicorandil, an adenosine triphosphate-sensitive K + channel opener, reduces burn wound progression in a rat comb burn model. A total of 24 rats were used. Following thermal injury, one dose of nicorandil (10 or 30 mg/kg) was administered intragastrically twice daily for 3 days. At days 1 and 3 after injury, skin was harvested for histopathological examination and protein isolation. Rats treated with the 10-mg/kg and 30-mg/kg doses of nicorandil exhibited significantly increased tissue survival in the zone of stasis at days 1 and 3 after injury. The 10-mg/kg and 30-mg/kg nicorandil doses also significantly increased skin perfusion in the zone of stasis at days 1 and 3 after injury. At 30 mg/kg, nicorandil significantly reduced hypoxia-inducible factor-1α expression in the zone of stasis at day 1 after injury and reduced inflammatory responses in the zone of stasis. The latter effect included decreased polymorphonuclear neutrophil leukocyte infiltration and interleukin-1β release at day 1 after injury. At 30 mg/kg, nicorandil also significantly reduced expression of nuclear factor-κB p65, akey transcriptional factor in the regulation of inflammatory mediators, in the zone of stasis at day 1 after injury. Our study demonstrates that a 30 mg/kg dosing schedule of nicorandil increases tissue survival in the zone of stasis by attenuating ischemia-reperfusion injury. This effect is mediated by the enhancement of skin blood flow and reduction in the inflammatory response. Therefore, our findings suggest that nicorandil has potential clinical applications for patients with burns.

Intracellular reactive oxygen species production and phagocytosis of Staphylococcus aureus by milk neutrophils as tool to diagnose mastitis and identify susceptible dairy cows

ABSTRACT: The immune response capacity of the mammary gland plays a major role to determine if mastitis will or not be established. Thus, we hypothesize that a better understanding of polymorphonuclear neutrophil leukocyte (PMN) function will elucidate mechanisms that will improve our knowledge of how we could avoid an inflammatory process by increasing the immune capacity of the cow, and even further, to search for a tool to diagnose mastitis or a possible way to select and identify non-susceptible animals. The present study utilized 112 quarters from 28 Holstein dairy cows that were divided into quarters milk samples with somatic cell count (SCC) &lt;2×105 cells mL-1 (n=72) and SCC &gt;2×105 cells mL-1 (n=40). The percentages of milk PMNs and the levels of intracellular reactive oxygen species (ROS) and the phagocytosis of Staphylococcus aureus by milk neutrophils were evaluated by flow cytometry. Our results showed a higher percentage of neutrophils in quarter milk samples with high SCC (P=0.0003), and this group also had a significantly higher percentage of neutrophils that produced ROS (P=0.008). On the other hand, the phagocytosis intensity of S. aureus by milk neutrophils was higher in quarters with low SCC (P=0.003), suggesting a better mammary gland immunity against invading pathogens. Analyzing the results of the predictive values of the measured PMN functions, they cannot be used isolated as a good diagnosis test since none of them had a satisfactory sensitivity and specificity values, which was also confirmed by the Youden index values being far from one. In conclusion, the assessment of milk bovine neutrophil functions could improve our understanding of the cellular basis of mastitis. Although, the intracellular ROS production and S. aureus phagocytosis by milk neutrophil did not have high predictive values to detect intramammary infections, our results strengthen the idea that that poor bovine mammary gland neutrophil phagocytic ability may be associated with high SCC, and might be considered to identify susceptible dairy cows to mastitis.

Aspergillus fumigatus Increased PAR-2 Expression and Elevated Proinflammatory Cytokines Expression Through the Pathway of PAR-2/ERK1/2 in Cornea.

To determine the role of protease-activated receptor-2 (PAR-2) in cornea infected by Aspergillus fumigatus. PAR-2 was tested in normal and infected corneas of C57BL/6 mice. Mice corneas were infected with A. fumigatus with or without pretreatment of PAR-2 antagonist (FSLLRY-NH2). Polymorphonuclear neutrophilic leukocytes (PMNs) were stimulated with 75% ethanol-killed A. fumigatus with or without pretreatment of FSLLRY-NH2. Disease severity was documented by clinical score and photographs with a slit lamp. PCR, Western blot, and ELISA tested expression of PAR-2, IL-1β, TNF-α, IFN-γ, MIP-2, and p-ERK1/2. PMN infiltration was assessed by myeloperoxidase assay and immunofluorescent staining. PAR-2 expression was significantly elevated by A. fumigatus, whereas the upregulation was significantly inhibited by FSLLRY-NH2 in mice corneas. FSLLRY-NH2 decreased disease response, PMN infiltration, and proinflammatory cytokine expression compared with infected control. In PMNs, PAR-2 expression was also significantly increased by A. fumigatus, which was significantly inhibited by FSLLRY-NH2. FSLLRY-NH2 significantly inhibited proinflammatory cytokine protein expression, as compared with that in infected control cells, which may be modified by p-ERK1/2. These data provide evidence that A. fumigatus increased PAR-2 expression and elevated disease, PMN infiltration, and proinflammatory cytokine expression through PAR-2, which may be modified by p-ERK1/2.

Acute febrile neutrophilic dermatosis in a patient with Crohn’s disease: case report and review of the literature

Crohn's disease is a chronic inflammatory bowel disease. The disease is characterized by acute exacerbations with diarrhea, abdominal pain, fever, anorexia, intestinal bleeding, and weight loss. Immune-mediated diseases that are frequently associated with Crohn's disease include arthritis, ankylosing spondylitis, sacroiliitis, episcleritis, uveitis, and skin lesions, such as erythema nodosum and pyoderma gangrenosum. The authors present the case of a 22-year-old female patient that was admitted to their hospital due to diarrhea, fever, arthralgias, and diffuse erythematous papules and plaques with vesicles and pustules affecting the patient's face, lips, arms, trunk, and legs. Six months prior to onset, the patient was diagnosed with terminal ileitis and Crohn's disease of the sigmoid colon. Treatment with mesalazine and budesonide had been introduced. In the diagnostic procedure, a skin biopsy was taken from the patient. Histology confirmed dense infiltration of neutrophilic polymorphonuclear leukocytes, or Sweet's syndrome. This condition is a rare manifestation of chronic inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. This syndrome is thought to be a hypersensitivity reaction and may be associated with various inflammatory, infectious, or neoplastic diseases. The patient was treated with high doses of methylprednisolone (60 mg IV), which resulted in rapid improvement of Crohn's disease and skin lesions.

Estrogen-dependent efficacy of limb ischemic preconditioning in female rats.

Our aim was to examine the effects of ischemic preconditioning (IPC) on the local periosteal and systemic inflammatory consequences of hindlimb ischemia-reperfusion (IR) in Sprague-Dawley rats with chronic estrogen deficiency (13 weeks after ovariectomy, OVX) in the presence and absence of chronic 17beta-estradiol supplementation (E2, 20 µg kg-1 , 5 days/week for 5 weeks); sham-operated (non-OVX) animals served as controls. As assessed by intravital fluorescence microscopy, rolling and the firm adhesion of polymorphonuclear neutrophil leukocytes (PMNs) gave similar results in the Sham + IR and OVX + IR groups in the tibial periosteal microcirculation during the 3-h reperfusion period after a 60-min tourniquet ischemia. Postischemic increases in periosteal PMN adhesion and PMN-derived adhesion molecule CD11b expressions, however, were significantly reduced by IPC (two cycles of 10'/10') in Sham animals, but not in OVX animals; neither plasma free radical levels (as measured by chemiluminescence), nor TNF-alpha release was affected by IPC. E2 supplementation in OVX animals restored the IPC-related microcirculatory integrity and PMN-derived CD11b levels, and TNF-alpha and free radical levels were reduced by IPC only with E2. An enhanced estrogen receptor beta expression could also be demonstrated after E2 in the periosteum. Overall, the beneficial periosteal microcirculatory effects of limb IPC are lost in chronic estrogen deficiency, but they can be restored by E2 supplementation. This suggests that the presence of endogenous estrogen is a necessary facilitating factor of the anti-inflammatory protection provided by limb IPC in females. The IPC-independent effects of E2 on inflammatory reactions should also be taken into account in this model. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:97-105, 2018.

Detection of PR-39, a porcine host defence peptide, in different cell sub-linages in pigs infected with Actinobacillus pleuropneumoniae.

Innate immunity is critically important for the outcome of infection in many diseases. It was previously shown that cathelicidin PR-39, an important porcine multifunctional host defence peptide, is elevated in bronchoalveolar lavage fluid and respiratory tract tissue after experimental infection with Actinobacillus pleuropneumoniae (A.pp.). To date, neutrophil polymorphonuclear leukocytes (PMNs) are thought to be the only source of PR-39. The aim of this study was to further characterize PR-39⁺ cells and selected immune cell populations in lung tissue during the peracute (7-10 hours), acute (2 days), reconvalescent (7 days) and chronic (21 days) stages of experimental infection with A.pp. serotype 2. In total, six mock-infected control pigs and 12 infected pigs were examined. Using immunofluorescence double-labeling, antibodies against PR-39 were combined with antibodies against CD3 (T-cells), CD79 (B-cells), Iba1 (activated macrophages), TTF-1 (lung epithelial cells expressing surfactant proteins), macrophage/L1 protein and myeloperoxidase (MPO, cells of the myeloid linage). In the peracute and acute phases of infection, total PR-39⁺ cells and myeloid linage cells increased, whereas CD3⁺ cells and TTF-1⁺ cells decreased. Double labeling revealed that most Macrophage/L1 protein+ cells and to a lesser extent MPO⁺ cells co-expressed PR-39. In addition, few bronchial epithelial cells and type 2 alveolar epithelial cells (both identified with TTF-1) produced PR-39. Occasionally, CD3⁺ T cells expressing PR-39 were seen in infected animals. Taken together, this study identifies cell types, other than PMNs, in lungs of A.pp.-infected pigs that are capable of producing PR-39. In addition, these findings provide further insights into the dynamics of different immune cell populations during A.pp.-infection.