Granulocyte Function Research Articles

Mechanisms of Beta-Blocker Induced Psoriasis, and Psoriasis De Novo at the Cellular Level.

Beta-blockers are a commonly prescribed medication, but the increase in use goes hand in hand with increasing side effects; one of particular interest lately has been its dermatological reactions. Although rare, beta-blockers can exacerbate pre-existing psoriasis and also cause de novo psoriasis in patients naïve to the disease. The mechanism by which this occurs is still unclear, although numerous articles have been published throughout the years as to how this unusual effect takes place. The most common mechanism suggests that beta-blockers cause intracellular changes in calcium, affecting both keratinocyte proliferation and granulocyte function via decreased cyclic adenosine monophosphate (cAMP) levels. Several inflammatory mediators are known to play a role, as well as reduced expression and desensitization of the beta-adrenergic receptor itself. We discuss these posed pathways in-depth and how each contributes to the worsening or formation of new psoriasis. With this knowledge, future physicians may be more mindful of this side effect should it occur, and why they occur, to better manage our patients on this widely used medication.

Phagocyte function of peripheral neutrophil granulocytes and monocytes in endometriosis before and after surgery

IntroductionEndometriosis is a chronic systemic disease, which influence negatively the quality of life of affected women and responsible for infertility and chronic pelvic pain. Pathophysiology of the disease is still enigmatic, but insufficient immune surveillance may play a role in it. Peripheral natural immune cell function is rarely examined. The aim of the study was to examine phagocyte function of peripheral neutrophil granulocytes and monocytes, whether this phagocytic activity is affected by the presence or removal of endometriotic lesions in women with endometriosis. Material and methodsTwenty-six preoperative, 13 postoperative samples from women with endometriosis, 23 samples from healthy women, 14 pre- and postoperative samples from the surgical control group were enrolled. Cells were isolated from peripheral blood samples, marked and evaluated for the phagocytosis index with immunofluorescent microscope after phagocyting the zymosane molecules. ResultsPhagocyte function of monocytes and neutrophil granulocytes decreased significantly women with endometriosis before surgery compared to healthy controls. However, 7 days after surgery the postoperative values showed significant improvement compared to the preoperative results of women with endometriosis. This increment reached the values of the healthy women. In the surgical control group no difference was detected between the pre- and postoperative outcomes. DiscussionDecreased phagocyte function of the examined cells, which can be the result of the circulating immunosuppressive factors, may play a role in the deficient clearance of ectopic endometrial tissue. Based on the postoperative results, these immunosuppressive factors may be reduced or eliminated 7 days after surgery in women with endometriosis.

Observation of granulocyte function during ex vivo thrombus formation for patients with ANKRD26-associated thrombocytopenia

ANKRD26-associated thrombocytopenia is a non-syndromic hereditary thrombocytopenia for which there are currently no formal diagnostic criteria. It is known that the probability of myeloid leukemia in patients with pathogenetic variants in the ANKRD26 gene significantly increases, however, studies of the functioning of granulocytes in this pathology have not been conducted. Aims: Analysis of the functioning of granulocytes and platelets during ex vivo thrombosis in patients with ANKRD26-associated thrombocytopenia. The study was approved by the Independent Ethics Committee of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, and Immunology. Two patients and 10 healthy volunteers were included in the study. Intracellular signaling and platelet functional responses were observed by continuous flow cytometry. Ex vivo thrombus formation and granulocyte functioning were observed on a fluorescence microscope in parallel-plane flow chambers containing fibrillar collagen. Upon physiological activation (ADP, collagen) of patients’ platelets in vitro, there were no significant differences between the platelets of patients and healthy donors. However, the observed ex vivo size of platelet aggregates was significantly reduced in comparison with healthy donors and published data on patients with other thrombocytopenias. The observed number and activity (movement velocity) of granulocytes of patients was within normal values. However, significant morphological differences were observed for granulocytes of patients compared with granulocytes of healthy donors: there was an increased spreading of granulocytes, in particular, expressed in a large number of thin pseudopodia, as well as an increased curvature of the motion trajectories of granulocytes. Ex vivo observation of thrombus formation in patients with ANKRD26- associated thrombocytopenia, a significantly reduced thrombus size is observed with normal platelet activity and increased variability in the shape of granulocytes.

Early Post-Stroke Infections Are Associated with an Impaired Function of Neutrophil Granulocytes.

To investigate whether neutrophil granulocytes’ function relates to post-stroke infections and clinical outcome after stroke, we prospectively recruited 95 patients after ischemic stroke and tested them for their microbiocidal neutrophil functions in this exploratory study. Additionally, 24 age-adjusted controls were examined regarding neutrophil function. Phagocytic capacity and the ability of the neutrophil granulocytes to produce reactive oxygen species (ROS) as well as CD11b and CD16 receptor expression profile were measured by flow cytometry at days 1, 3, 7, and 90 after symptom onset. Primary outcome was the development of an infection within the first week after stroke. Results of neutrophil functional measurements were compared between patients with and without infection as well as between all stroke patients and controls. Further risk factors for the development of infections were summarized in an infection-risk score for the purpose of multivariate statistical analysis. The ROS production in neutrophils after stimulation with formyl-methionyl-leucyl-phenylalanine (fMLP) was reduced at baseline in patients with post-stroke infections compared to those without (p = 0.013). This difference proved to be independent from the infection-risk score in the binary logistic regression (p = 0.011). Phagocytosis and oxidative bursts were not significantly reduced in the whole stroke patient group compared to controls. Dysfunction of neutrophil granulocytes seems to play a significant role in the development of post-stroke infections. Further studies are warranted to investigate neutrophil granulocytes´ function as a potential biomarker of post-stroke infections.

Enhanced neutrophil functions during Opisthorchis viverrini infections and correlation with advanced periductal fibrosis

Millions of people are infected with the liver fluke, Opisthorchis viverrini (OV), but only ~25% of those infected develop liver disease and even fewer develop cholangiocarcinoma. The reasons for these differential outcomes following infection are unknown but it has been proposed that differential immune responses to the parasite may play a role. We therefore measured granulocyte (neutrophil) function in OV-infected individuals, with and without advanced periductal fibrosis, to determine if these cells have a “pro-inflammatory” phenotype that may contribute to liver disease post-infection. A case-controlled study (n = 54 in each cohort) from endemic OV-infected areas of northeastern Thailand measured neutrophil functions in whole blood from non-infected (healthy controls) and OV-infected individuals with and without APF. We measured reactive oxygen species production, phagocytosis, receptor expression and apoptosis. Secreted products from OV cultures (obtained after in vitro culture of parasites) stimulated reactive oxygen species production in non-infected healthy controls, but levels were two-fold greater after OV infection (P < 0.0001); neutrophil reactive oxygen species production in individuals with APF was double that observed in those without APF (P < 0.0001). OV-infected neutrophils had elevated CD11b expression and greater phagocytic capacity, which was even three-fold higher in those with advanced periductal fibrosis (P < 0.0001). This “activated” phenotype of circulating neutrophils was further confirmed by the observation that isolated neutrophils had delayed apoptosis ex vivo. We believe this is the first study to show that circulating blood neutrophil function is enhanced following OV infection and is more activated in those with advanced periductal fibrosis. We propose that this activated phenotype could contribute to the pathology of liver disease. These data support the hypothesis of an activated innate inflammatory phenotype following OV infection and provide the first evidence for involvement of neutrophils in disease pathology.

Патогенетическое обоснование применения иммунотерапии в лечении рекуррентных ко-инфекций верхних дыхательных путей у детей с врожденными расщелинами губы и нёба

The synergism of the action of co-infecting microorganisms contributes to their overcoming the epithelial barrier, modification of the function of cells of the immune system (IS) and evading the immune response. In children with congenital cleft lip and palate (CCLP), despite elimination of the anatomical defect at an earlier age, the frequency of infectious and inflammatory diseases of the respiratory tract and oral cavity involving pathogen associations increases as they get older, at the same time defects in the functioning of IS are preserved. Objective. To develop immunotherapy program for children with CCLP, suffering from recurrent co-infections of the upper respiratory tract and oral cavity, based on the study of the dynamics of the microbial landscape transformation, IS dysfunction, clinical manifestations of infectious and inflammatory diseases of the upper respiratory tract and oral cavity in children with CCLP at different ages. Patients and methods. The study included 120 children from 1 to 12 years of age with CCLP at different stages of surgical treatment. The study was carried out: T and B lymphocytes, natural killer cells (FC500 "Beckman Coulter", USA), phagocytic and microbicidal functions of neutrophilic granulocytes (NG), serum IgA, IgM, IgG and sIgA levels in the oral fluid (ELISA), detection of microbial pathogens (microbiological analysis, ELISA) and viral (PCR). Results. Comprehensive examination of children with CCLP at different age periods, including characteristics of the clinical manifestations of infectious and inflammatory diseases, assessment of the microbial landscape of the oral cavity and upper respiratory tract with the identification of co-infection features, clarification of the variants of IS dysfunction, made it possible to reveal children with the most severe clinical manifestations of recurrent co-infections and to optimize treatment. Conclusion. The developed program of combined local interferon and systemic immunotherapy for immunocompromised children with CCLP contributes to the restoration of IS functions, anti-infective resistance and achievement of positive clinical effects in the form of regression of clinical signs of immunocompromise in patients with recurrent co-infections, which allows to obtain a protective effect and provide timely surgical care, to avoid complications in the postoperative period and at the stage of recovery, and by improving anti-infective immunity to enhance the quality of life of children with CCLP. Key words: children, congenital cleft lip and palate, co-infection, systemic immunity, microbiocenosis, immunotherapy

Gene Knockout in Hematopoietic Stem and Progenitor Cells Followed by Granulocytic Differentiation.

In this chapter, we present an optimized CRISPR/Cas9 RNP nucleofection approach for gene knockout (KO) in hematopoietic stem and progenitor cells (HSPCs). With experimentally proved active locus-specific sgRNAs, we routinely reach over 80% gene KO in HSPCs, thus avoiding the need for cell sorting or enrichment of targeted cell population. Additionally, we provide a protocol for in vitro granulocytic differentiation of HSPCs after gene KO and detailed description of granulocyte function tests which can be applied to study the effects of a particular gene KO.

The Influence of Exogenous ATP on Functional Responses of Murine Bone Marrow Granulocytes

Extracellular ATP is a potent inflammatory mediator and regulator of neutrophil activity in an inflammatory site. The role of ATP in the regulation of bone marrow granulocyte functions remains unexplored. The aim of this work was to study effects of ATP on the cytoplasmic Ca2+ concentration ([Ca2+]i), generation of reactive oxygen species (ROS) and adhesion of bone marrow granulocytes. Changes of [Ca2+]i were estimated with Fura-2 AM ratiometric probe. To reveal cells with calcium activity in a population we developed an automated procedure based on the calculation of skewness and kurtosis of the [Ca2+]i value distribution. ROS generation was estimated by luminol-dependent chemiluminescence. Cell adhesion was evaluated by a standard absorbance (492 nm) method. The population of bone marrow granulocytes contained cells that generated calcium spikes in response to 10 μM ATP (35.2 ± 4.0%). The automated scanning allowed us to identify four groups of cells: those responding to ATP (23.4 ± 4.9%) or to the addition of Hanks’ solution (16.9 ± 2.5%) or to both ATP and Hanks’ solution (6.7 ± 1.5%), and cells not responding to either stimulus (53.1 ± 8.2%). ATP increased the adhesion of granulocytes, while apyrase, which hydrolyzes ATP, did not significantly change the adhesion. ATP did not influence the respiratory burst initiated by the bacterial peptide N-formyl-MLF (fMLF). Apyrase significantly enhanced the response to fMLF, and cytochalasin D inhibited this effect. In summary, exogenous ATP enhances adhesion and generation of calcium spikes in the population of bone marrow granulocytes. Endogenous ATP likely reduces respiratory burst through a mechanism involving the cytoskeleton. The studied granulocyte population contains cells with different ATP sensitivity.

Granulocyte concentrates prepared from residual leukocyte units produced by the Reveos automated blood processing system

BackgroundGranulocyte concentrates are mainly derived by apheresis technique from donors stimulated with granulocyte colony-stimulating factor and steroids. The automated blood processing system Reveos, which is now increasingly used across the world, separates whole blood into four components, including a residual leukocyte unit containing granulocytes. The aim of this study was to produce an alternative granulocyte concentrate from leukocyte units produced by the Reveos system, and to assess the function of the granulocytes. MethodsThe number of granulocytes was measured in residual leukocyte units, derived from whole blood donations, with different volumes ranging from 10 to 40 ml. After deciding the optimal volume of the leukocyte unit (30 ml), ten ABO-matched units were pooled to form a granulocyte concentrate. The function of the granulocytes from residual leukocyte units was assessed by analyzing surface markers, phagocytosis of yeast, and production of reactive oxygen species. ResultsResidual leukocyte units with a volume of 30 ml contained a median number of 0,7 × 109 granulocytes, and granulocyte concentrates prepared from ten pooled 30 ml-leukocyte units contained a median number of 6,3 × 109 granulocytes. Granulocytes derived from residual leukocyte units displayed surface markers associated with granulocyte function, and capability to phagocytose yeast and produce reactive oxygen species. ConclusionsGranulocyte concentrates prepared from residual leukocyte units contain in vitro functional granulocytes and may be considered as an alternative product in acute situations before regular granulocyte concentrates from stimulated donors are available.

Defects in Signal Recognition Particle (SRP) Components Reveal an Essential and Non-Redundant Role for Granule Biogenesis and Differentiation of Neutrophil Granulocytes

Neutrophil granulocyte play pivotal roles in inflammatory responses, immune defence, tissue remodeling, and cancer control. Studying rare patients with defects in differentiation and/or function of neutrophil granulocytes highlights genes and pathways orchestrating these important cellular functions. A previously not appreciated role of the signal recognition particle (SRP) has emerged when monoallelic mutations in SRP54 were associated with congenital neutropenia and pancreatic insufficiency. The eukaryotic SRP is composed of six distinct polypeptides (SRP9, SRP14, SRP19, SRP54, SRP68, SRP72) bound to an RNA molecule (the 7SL RNA). SRP and its receptor (SRPRA and SRPRB) cooperatively transport nascent proteins emerging from ribosome to the endoplasmic reticulum (ER) lumen via a protein-conducting channel. We hypothesized that other constituents of the SRP may be involved in the pathophysiology of congenital neutropenia and that disturbances of the SRP may result in defective proteostasis in neutrophil granulocytes. By virtue of our international Care-for-Rare Alliance we identified three kindred with molecularly unsolved congenital neutropenia. Exome-sequencing revealed a de novo monoallelic mutation in SRPRA in one individual in one family and a homozygous splice-site mutation in SRP19 in five individuals from two families. Clinically, all patients with mutations in SRPRA and SRP19 suffered from various degrees of neutropenia and susceptibility to bacterial infections. Pancreatic insufficiency was only seen in patients with mutation in SRPRA, whereas SRP19 deficiency was not associated with any non-hematopoietic symptoms. To functionally validate these novel genetic variants, we set up an in-vitro neutrophil differentiation system allowing us to model the genetic defects in human SRP complex subunits. We introduced patient-specific mutations (SRPRA Q464E and SRP19 c.189+5G&amp;gt;A) into wildtype iPS cells using CRISPR-Cas9 editing. The engineered cells were differentiated into bona fide neutrophil granulocytes. In vitro generated wildtype neutrophil granulocytes had a high degree of resemblance to primary peripheral blood neutrophil granulocytes, as evidenced by phenotypic (light microscopy, cell surface markers, gene expression) and functional (phagocytosis, bacterial killing, rolling and adhesion, chemotaxis) characteristics. In striking contrast to wildtype iPS cells, both SRPRA and SRP19 mutated iPS cells had a significantly reduced capacity to differentiate into neutrophil granulocytes, as shown by colony-forming unit assays and phenotypic analysis. Furthermore, in vitro generated neutrophil granulocytes had higher susceptibility to apoptosis and evidence of increased unfolded protein responses. We went on to characterize the proteome of primary neutrophil granulocytes from patients with SRP-defects by data-independent proteomics technology. Interestingly, neutrophil granulocytes with defects in SRP constituents shared a significantly reduced abundance of the proteins Pentraxin-3 (PTX3) and Glucuronidase Beta (GUSB). These findings were confirmed in both non-hematopoietic HELA cells and promyelocytic HL60 leukemia cells engineered to express the respective SRP mutants. Upon genetic reconstitution, the aberrant protein expression was re-established. In summary, our studies define novel genetic defects causing congenital neutropenia and highlight the relevance of coordinated protein trafficking pathways and homeostasis for physiological differentiation and function of human neutrophil granulocytes. Disclosures No relevant conflicts of interest to declare.

CRISPR/Cas9 Mediated ELANE Knock-out Restores Survival and Granulocytic Differentiation of HL60 Cells Expressing Mutant Neutrophil Elastase: Is Neutrophil Elastase a Dispensible Granulocyte Protease?

Background : Mutations in ELANE are the most common cause of both cyclic and severe congenital neutropenia. ELANE encodes neutrophil elastase (NE), a tissue specific serine protease expressed primarily in neutrophils. Expression of the mutant protein impairs survival and maturation of myeloid precursors in bone marrow. More than 130 different ELANE mutations have been found in patients with cyclic and congenital neutropenia, and genotype-phenotype studies suggest that specific mutations cause more severe disease. (Curr Op Hematol. 2015;22:3-11) Mutant NE is also implicated as the primary cause of ELANE associated neutropenia by studies showing that cell permeable inhibitors of NE correct the defect in cell survival and maturation in cellular models. (Makaryan et al. J Leukoc Biol. 2017;102:1143). Hypothesis: If inhibition of NE can correct the cellular defect, deletion of ELANE will have similar effects and permit studies to see if NE is an essential neutrophil protease. Methods: We used CRISPR/Cas9 mediated gene editing to create a cellular model of congenital neutropenia in the commercially available human promyelocytic cell line, HL60. We used CRISPR/Cas9 editing technology to create engineered HL60 cell lines with knock-in (KI) ELANE P139L, C151Y and G214R single point heterozygous mutations. Using the same technology, we then performed ELANE gene complete knock out (KO) of ELANE in each mutant cell line. All engineered cell lines as well as wild-type HL60 cells were cultured for 5-7 days in complete RPMI supplemented with 2uM all-trans retinoic acid (ATRA) to trigger myeloid differentiation. Survival of these cell lines was investigated using Annexin V-PE staining and flow cytometric analysis. Granulocytic differentiation was evaluated using CD11b surface marker staining and flow cytometry and by performing manual differential cell counts. We measured the unfolded protein response (UPR) by western blotting using UPR specific antibodies, phagocytosis with E. coli particles using fluorescence detection, IL-8 stimulated chemotaxis in trans-well system and PMA activated respiratory burst by flow cytometry. Results: Cells expressing the P139L and C151Y mutant ELANE had increased Annexin V staining more than a 2-fold increase in apoptotic cells at 7 days in culture. Granulocytic differentiation, measured by surface CD11b expression, was significantly impaired. (p&amp;lt;0.0001). Cytospins stained with Diff-Quik showed a typical block of myeloid differentiation and a significant deficiency of mature neutrophils in 7-day cultures. Western blot analysis using antibodies to GRP78/BiP and ATF6 showed a typical UPR signature in both ELANE mutant cell lines compared to wild type. Chemotaxis to the IL8 chemo-attractant was severely impaired in cells expressing mutant NE. Respiratory burst and phagocytosis was also altered in cells expressing mutant NE. ELANE KO corrected all these cellular and functional abnormalities, reverting these functions toward the wild phenotype. Studies are in progress for the third cell line expressing the G214R mutation. Conclusions: CRISPR/Cas9 engineered HL60 cell lines expressing mutant NE are a highly reproducible and reliable cellular model for investigating ELANE associated neutropenia. The presence of mutant NE severely impairs neutrophil functions, and its deletion through KO of ELANE corrects the cellular abnormalities and reverses the induced abnormalities. These results suggest that CRISPR/Cas9 mediated genetic knock down of NE is a novel therapeutic approach for treatment of this disorder. This data also suggests that total absence of NE does not alter basic functions of granulocytes. Disclosures Dale: Sanofi Aventis: Consultancy, Honoraria; Athelas: Equity Ownership; Amgen: Consultancy, Research Funding; Cellerant: Other: Scientific Advisory Board; Hospira: Consultancy; Prolong: Consultancy; Beheringer/Ingelheim: Consultancy; Coherus: Consultancy; x4pharma: Consultancy, Honoraria, Research Funding.

CNS Infections in Immunoincompetent Patients : Neuroradiological and Clinical Features.

In patients with immunodeficiency the pathogen spectrum of central nervous system (CNS) infections is broader and different from that of immunocompetent patients. Numerous opportunistic infections are characterized by a high prevalence of viral, bacterial and parasitic pathogens, and depend on the type of impaired immune defense, for example impaired T‑cell or monocyte function, monoclonal antibody treatment, and impaired granulocyte function. Neuroradiological features as well as laboratory findings are often different and versatile in comparison to immunocompetent individuals and pathognomonic imaging findings do not exist; however, knowledge of possible pathways of pathogens in the CNS and preferred tissue affection may help in narrowing down differential diagnoses. Therefore, knowledge of the type of patient and the performed immunomodulatory therapy is essential for the neuroradiological assessment and the differential diagnostic considerations. Moreover, parenchymal reactions in the sense of an immune reconstitution inflammatory syndrome (IRIS) can occur when immunocompetence is restored. This review focus on the most common pathologies in immunocompromised patients, and an overview of imaging features but also of pathology and clinical aspects is given. The synopsis of anamnestic information, clinical findings and structured analysis of the lesion pattern, its spread and short-term follow-up may increase the correct diagnostic classification; however, the gold standard is still determination of the pathogen in the cerebrospinal fluid (CSF), blood cultures or biopsies.

Buccal space hematoma- A rare presentation in chronic myeloid leukemia

Chronic Myeloid Leukemia (CML) typically seen in older patients, increases with age. Patients often are asymptomatic at the time of diagnosis and the disease is found incidentally on examination of blood. Oral ulceration, petechiae, mucosal bleeding, gingival enlargement, acute periodontitis, noma, trismus and oral infections are the most common oral manifestations. It occurs either due to direct infiltration of leukemic cells (primary) or secondary to underlying thrombocytopenia, neutropenia or impaired granulocyte function. We present a rare case of buccal space hematoma secondary to CML. Keywords: Chronic myeloid leukemia, Buccal space hematoma, Facial swelling, Oral manifestation.

Short communication: Pegbovigrastim treatment in vivo does not affect granulocyte ability to migrate to endometrial cells and kill bacteria in vitro in healthy cows

In periparturient dairy cows, immune suppression, resulting in decreased neutrophil numbers and function, leads to increased susceptibility to postpartum conditions such as mastitis, retained placenta, and metritis. Administration of polyethylene glycol-conjugated bovine granulocyte colony stimulating factor (pegbovigrastim, Imrestor; Elanco Animal Health, Greenfield, IN) 7 d before and within 24 h of calving, effectively improves granulocyte production and function in vivo as well as in milk. A recently developed coculture assay was adapted for use with endometrial epithelial cells to assess the effects of pegbovigrastim application on directed granulocyte migration and bactericidal activity in vitro on a per-cell basis in endometrial cell cultures. Granulocytes from treated and untreated periparturient cows (6 and 5 per group, respectively) were evaluated for their ability to migrate to and kill bacteria after treatment, in context of the infected endometrium. We hypothesized that in addition to increasing the absolute concentration of circulating neutrophil granulocytes, pegbovigrastim treatment in vivo alters the ability of granulocytes to migrate to endometrial cells in vitro. The results clearly show a marked increase in the total concentration of granulocytes and monocytes between the 2 treatment groups as early as 2 d after the first injection, and this increased between the samples taken 2 d after calving. No migratory or killing differences were identified between granulocytes of both groups, suggesting that pegbovigrastim-induced granulocytes were as effective as non-induced cells. This may also be due to the absence of negative energy balance in the study animals and leads us to conclude that the positive effects seen in vivo are most likely based on the larger number of granulocytes present rather than a direct effect of pegbovigrastim treatment on the functionality of cells for the parameters tested in this study.

ГЕМОЦИТИ ІНТАКТНОЇ ТА ІНВАЗОВАНОЇ ТРЕМАТОДАМИ ЖАБУРНИЦІ КИТАЙСЬКОЇ (MOLLUSCA, UNIONIDAE, ANODONTINAE)

The hemolymph of Sinanodonta woodiana (Lea, 1834) consists of plasma and cells of four types: prohemocytes (cambial cells), macronucleocytes, basophilic and eosinophilic granulocytes. The three latter cell types derive from the cambial cells though mitosis. The cellular sizes are: prohemocytes 14.3±0.4, basophilic granulocytes 20.9±0.7, young eosinophilic granulocytes 23.8±0.6, older eosinophilic granulocytes 25.7±0.1, macronucleocytes 26.1±0.3. All cells and their nuclei are roundish. Nuclear chromatin is either fine-grained fairly evenly distributed in the karyoplasm (in basophilic granulocytes), or more or less grouped dark-colored small (2-5-6) chromatin blocks. NC-ratio is maximum in macronucleocytes (0.6±0.01). NC-ratio of prohemocytes is 0.4±0.01, that of basophilic granulocytes is 0.3±0.01. NC-ratio of eosinophilic granulocytes is 0.2±0.01. Eosinophilic cells prevail in hemolymph elements: the young eosinophilic granulocytes make up 25.1±0.4 % and the older eosinophilic cells are up to 27.9±0.6 % of all hemocytes. The major functions of hemolymph cells are transport and protection.Transport is particularly pronounced in basophilic granulocytes in the form of phagocytosis. The protective function of hemocytes of different categories is manifested in a different way. Thus, some of the basophilic granulocytes develop into nephrocytes, which accumulate numerous (11-23) vacuoles of yellow-green-brown color. The vacuoles are subsequently excreted by the mollusk through the kidneys. The protective function of eosinophilic granulocytes is realized as false agglutination (these hemocytes clog into lumps that close wounds), which helps preventing blood loss. Also, eosinophilic granulocytes merge into multinucleated plasmodium, accumulating in large quantities around various foreign bodies (parasites or fragments of destroyed tissues) and encapsulating them to isolate from host tissues. At Northern Black Sea Coast, Chinese pond mussel is a common intermediate host of the trematode Rhipidocotyle companula Dujardin, 1845.The trematode inhabits the mollusk’s gonads. The parasitic sporocysts and cercariae were found in 29 % of examined mollusk specimens. Infestations were weak (up to 10 % of gonads were affected) in 22.4 % of infected mollusks, moderate (10 to 50 % of gonads) in 70 % of contaminated mollusks. Only 7.6 % of infected pond mussels were hyperinfected (100 % of gonads were affected).Weak trematode infestation is accompanied by localized damage. The total number and volume of parasitic focal lesions are generally insignificant. Moderate infection, and especially a hyperinfection cause not only the lesions in the hostal biotope, but also the overall pathological process in mollusk hosts. Simultaneously, the prohemocytes and basophilic granulocytes as well as their nuclei reduce in size. The total number of prohemocytes also declines by 1.7 times. The greatest decrease in the nuclear-cytoplasmic ratio (by a factor of 1.5-2) is noted also for prohemocytes and basophilic granulocytes. The vacuolization of karyoplasm and cytoplasm, the basophilization of cytoplasm, the degenerative changes in hemocyte nuclei (karyopicnosis, karyorexis, karyolysis), and the total number of aging and dying hemocytes are directly related to the level of infection intensity.

Functional characteristics of circulating granulocytes in severe congenital neutropenia caused by ELANE mutations

BackgroundNeutrophils and eosinophils are multifunctional granulocytes derived from common myelocytic-committed progenitor cells. Severe congenital neutropenia 1 (SCN1) caused by ELANE mutations is a rare disease characterized by very low numbers of circulating neutrophils. Little is known about the functional characteristics of the SCN1 granulocytes, except that eosinophilia has been noticed in both bone marrow and peripheral blood. In this study, we profiled the number and function of granulocytes in patients suffering from SCN1.MethodsNine patients diagnosed with SCN1 were enrolled in this study and absolute counts of eosinophils and neutrophils from bone marrow aspirates and peripheral blood samples were analysed. In addition, Ficoll-Paque enriched granulocytes from patients and healthy controls were analysed for specific eosinophil and neutrophil markers using flow cytometry and for NADPH-oxidase activity-profile by chemiluminescence.ResultsOur data demonstrate a skewed granulocyte population in SCN1 patients dominated by eosinophils in both bone marrow and peripheral blood. The latter was detected only by blood smear examination, but not by automated blood analysers. Furthermore, we show that the SCN1 eosinophils exerted normal production of reactive oxygen species generated by the NADPH-oxidase, however the response was profoundly different from that of healthy control neutrophils.ConclusionsSCN1 patients with ELANE mutations suffer from neutropenia yet display eosinophilia in the bone marrow and blood, as revealed by smear examination but not by automatic blood analysers. The SCN1 eosinophils are functionally normal regarding production of reactive oxygen species (ROS). However, the ROS profile produced by eosinophils differs drastically from that of neutrophils isolated from the same blood donor, implying that the eosinophilia in SCN1 cannot compensate for the loss of neutrophils regarding ROS-mediated functions.

Short Communication: Effect of Seminal Plasma on Functions of Monocytes and Granulocytes.

Most HIV-1 transmissions occur at mucosae and involve exposure to semen. Semen contains immunomodulatory factors, which inhibit anti-HIV-1 natural killer cell and T cell responses. We demonstrate high concentrations (1:2 dilution) of seminal plasma (SP) inhibit monocyte phagocytosis and anti-HIV-1 Fc-dependent functions of both neutrophils and monocytes. In addition, slightly lower SP concentrations (1:2-1:10 dilutions) inhibit granulocyte phagocytosis and oxidative burst of both monocytes and granulocytes. These observations may have implications for HIV-1 infectivity after mucosal exposure.

PF456 BETA‐CATENIN/TCF SIGNALING PATHWAY PROMOTES GRANULOCYTIC DIFFERENTIATION OF HEMATOPOIETIC PROGENITORS BY FACILITATING IL‐1 RECEPTOR SIGNALING

Background:The canonical Wnt signaling pathway is an essential regulator of differentiation and cell fate decisions in many types of somatic stem cells. However, in hematopoietic stem and progenitor cells (HSPCs), the role of the canonical Wnt signaling remains unclear. A critical event in Wnt‐target gene activation is the interaction of ß‐catenin with TCF/LEF transcription factors which enables transcription of regulated regions. The majority of previous studies relied on ß‐catenin manipulation. Intriguingly, ß‐catenin has been described to have also important cytoplasmic functions and therefore, it is not clear whether the previously observed phenotypes are really caused by the alteration of expression of Wnt‐target genes.Aims:Our aim was to investigate how specific loss of ß‐catenin interaction with TCF/LEF factors will affect the function of HSPCs in vivo and to decipher molecular mechanism that is responsible for the functional changes.Methods:To investigate the function of canonical Wnt signaling pathway in HSPCs, we disrupted ß‐catenin interaction with TCF/LEF factors by expressing a dominant negative form of human TCF4 (dnTCF4) specifically in hematopoietic tissues. dnTCF4 forms a heterodimers with endogenous TCF factors and as it lacks ß‐catenin binding domain, it prevents the transactivation of Wnt‐target genes. The hematopoietic tissues of transgenic mice were analyzed using flow cytometry, while the function of HSPCs was assessed by bone marrow transplantation assays in vivo and colony formation assay in vitro. NF‐κB activation was assessed by intracellular staining of IκBa and analyzed by flow cytometry in various cell populations. RNA sequencing was performed in sorted short‐term HSC, defined as lin−, c‐kit+, Sca1+, CD48+, CD150−, using Illumina platform.Results:dnTCF4 transgenic mice presented decreased levels of granulocytes in peripheral blood accompanied by accumulation of HSPCs in bone marrow. Consistently, dnTCF4 bone marrow cells displayed granulocytic differentiation block, augmented colony‐forming abilities, and retained immature phenotype longer than wild type control in colony formation assays. The accumulation of HSPCs was also reflected by enhanced engraftment rate of dnTCF4‐expressing bone marrow after transplantation. Transcriptomic analysis revealed that dnTCF4‐expressing HSPCs have reduced expression of genes involved in granulocyte differentiation and function, including Csf3r, Il18r, and Il1r1r. Interestingly, IL‐1ß treatment fail to activate NF‐κB signaling pathway, a well described inducer of myeloid differentiation, in dnTCF4‐expressing HSPCs.Summary/Conclusion:Altogether, our data suggest that the canonical Wnt signaling promotes HSPCs differentiation towards granulocytes by upregulating IL‐1 receptor levels that allow proper NF‐κB activation. Since our murine model does present intact cytoplasmic ß‐catenin activity, our data supports that ß‐catenin controls HSPCs fate and differentiation via transactivation of Wnt‐target genes.

Efficiency of Phytoimmunocorrection in Case of Recurrent Respiratory Infections and in the Case of Their Combined Course in Children Against the Background of Undifferentiated Connective Tissue Dysplasia

The objective: to study the effectiveness of treatment of recurrent respiratory infections, as well as in the case of their combined course in children with undifferentiated connective tissue dysplasia (NDST).Materials and methods. The analysis of the effectiveness of the use of the phytoimmunomodulator Imupret® was carried out in 89 children who were divided into two groups: 1st group – 43 children with recurrent respiratory infections (RRI), 2nd group – 46 children with RRI on the background of NDST. The program for studying the functional state of the system was complex.Results. In the group of children with RRI without NDCT, after the treatment, positive dynamics was revealed, which manifested a significant (p &lt;0.05) increase in the proportion of Lymphocyte tact from 38.7 ± 0.74% to 42.6 ± 0.8%; a significant (p &lt;0.05) increase in the specific weight of CD3+lymphocytes from 55.2 ± 0.84% to 57.4 ± 0.42%; a significant (p &lt;0.05) increase in the specific gravity of CD4+lymphocytes from 37.59 ± 0.85% to 42.9 ± 0.73% and, in fact, «normalization» of IC RBTL (before treatment – 24.8 ± 1.5 u, after treatment – 28.7 ± 1.3 units).Under the influence of the phytoimmunomodulator, the phagocytic and metabolic functions of granulocytes improved significantly (p &lt;0.05) (the NBT test), including significantly (ph &lt;0.05) the phagocytic number increased from 50.6 ± 1.5% to 57.5 ± 1.3%, respectively, and phagocytic index – from 5.1 ± 0.2 to 5.9 ± 0.2.Conclusion. The results of the study indicate the benefits of improving the functional state of granulocytes under the influence of the drug Imupret®. In general, the state of T-, B- and phagocytic chains of immune protection after treatment is characterized by full compliance of immunograms with reference values.

Two paternal mosaicism of mutation in ELANE causing severe congenital neutropenia exhibit normal neutrophil morphology and ROS production

Severe congenital neutropenia caused by ELANE gene mutation is a rare disease. To date, only four families were reported with mosaicism. Here we examined the morphology and function of granulocytes isolated from two patients and their mosaic fathers. Analysis of granulocytes isolated from the fathers revealed no genetic mutations. DNA extracted from fractionated peripheral blood mononuclear cells (PBMCs) and fingernails obtained from both fathers did harbor the mutation, suggesting mosaicism. Granulocytes isolated from the patients displayed significantly weaker ionomycin-induced intracellular reactive oxygen species (ROS) responses than those isolated from the fathers. Both patients showed increased expression of neutrophil elastase, whereas the mosaic fathers showed normal expression. Taken together, the results suggest that granulocytes from these SCN patients are immunocompromised, whereas those from the mosaic fathers are normal. These findings may provide new insight into disease diagnosis, prognosis, therapy and genetic counseling.