Types Of Granulocytes Research Articles

Origins and functions of eosinophils in two non-mucosal tissues.

Eosinophils are a type of granulocyte named after the presence of their eosin-stained granules. Traditionally, eosinophils have been best known to play prominent roles in anti-parasitic responses and mediating allergic reactions. Knowledge of their behaviour has expanded with time, and they are now recognized to play integral parts in the homeostasis of gastrointestinal, respiratory, skeletal muscle, adipose, and connective tissue systems. As such, they are implicated in a myriad of pathologies, and have been the target of several medical therapies. This review focuses on the lifespan of eosinophils, from their origins in the bone marrow, to their tissue-resident role. In particular, we wish to highlight the functions of eosinophils in non-mucosal tissues with skeletal muscle and the adipose tissues as examples, and to discuss the current understanding of their participation in diseased states in these tissues.

Density Gradient Centrifugation-Independent Purification of Human Basophils.

Basophils represent the rarest type of granulocyte in human peripheral blood. Thus, researching basophils has historically been challenging and has often been reliant on enrichment protocols using density gradient centrifugation. This article describes a novel, fast, and cost-effective method to purify highly viable human basophils from peripheral blood through negative immunomagnetic selection, foregoing the density centrifugation step in the Basic Protocol. The technique is easy to use and consistently produces purities >96%. Furthermore, the Support Protocols describe procedures to determine basophil yield, purity, and viability, and how to investigate functional activity of the purified basophils through flow cytometry and visualize the basophils through microscopy. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Gradient centrifugation-independent basophil isolation Support Protocol 1: Flow cytometry staining to assess basophil yield, purity, and viability Support Protocol 2: Giemsa staining Support Protocol 3: Calcium flux analysis Support Protocol 4: Basophil activation test.

Teleost fish IgM+ plasma-like cells possess IgM-secreting, phagocytic, and antigen-presenting capacities.

Plasma cells are terminally differentiated antibody-secreting B lymphocytes that contribute to humoral immunity by producing large numbers of antibodies. Increasing evidence suggests that teleost fish B cells share certain characteristics with mammalian B1 B cells, including antibody-secreting, phagocytic, and antigen-presenting capacities. However, the difference between mature B cells and plasma cells remains unclear. In this study, we found that, based on their light-scattering characteristics, tilapia anterior kidney (AK) leukocytes can be categorized into two IgM+ B-cell subsets: the lymphoid (L) gate and granulocyte–monocyte/macrophage (G-M) subsets. G-M gate cells are more numerous than L-gate cells and have higher mean fluorescence, but lower forward scatter and side scatter. We analyzed the morphological and ultrastructural features of sorted IgM+ cells and found that L-gate IgM+ cells have a high nucleus–cytoplasm ratio and lymphocyte-like morphology, whereas G-M gate IgM+ cells have a small nucleus, more abundant endoplasmic reticulum, and a larger number of mitochondria, and have a plasma cell-like or macrophage-like morphology. To further characterize the cell types, we examined the specific patterns of expression of B-cell- and T-cell-related genes. We found that B-cell-specific genes were expressed by both L-gate and G-M gate IgM+ cells, and that G-M gate IgM+ cells secreted extremely high levels of IgM. However, T-cell-related genes were highly expressed only in L-gate IgM– cells. These results suggest that G-M gate IgM+ cells are similar to plasma-like cells, with high antibody-secreting capacity. Given that G-M gate cells include the granulocyte, monocyte, and macrophage cell types, but not B cells, monocyte/macrophage markers were used to investigate the cell types further. A macrophage receptor with a collagenous structure was frequently observed, and macrophage-expressed gene-1 was highly expressed, in the G-M gate IgM+ cells. Phagocytic capacity, as determined by ingestion of beads or bacteria, was significantly higher in G-M gate IgM+ cells than in L-gate IgM+ cells, as was antigen-processing capacity. Our findings show that tilapia AK leukocytes can be divided into two IgM+ B-cell subsets and that G-M gate IgM+ cells resemble plasma-like cells, having high antibody-secreting, phagocytic, and antigen-presenting capacities. Thus, this study increases our understanding of the functions of teleost fish plasma-like cells.

The genetics of chronic myeloid leukaemia and recent advancements in therapy

Each year, approximately 750 people (commonly >65 years) are diagnosed with leukaemia in the United Kingdom (UK). Of every 100 cases of leukaemia, 15 are diagnosed with Chronic Myeloid Leukaemia (CML), also referred to as chronic myelogenous leukaemia. CML is a form of cancer affecting immature white blood cells (i.e. leukocytes). Individuals with CML present with a raised white blood cell count. CML is characterised by the unregulated growth of myeloid cells arising from a transformed haematopoietic stem cell (HSC). Leukocytes serve vital roles in the innate immune system. For example, neutrophils (a type of granulocyte) are highly mobile and first responders in inflammation. Neutrophils are activated for phagocytosis or the release of cytokines, and recent studies have shown their role in neutrophil extracellular traps. This article outlines the pathophysiological mechanisms and genetic basis underlying the development of CML. It also highlights recent advancements in targeted therapies to combat disease severity and progression.

Microscopic anatomy of gonadal area in the deep-sea clam Calyptogena pacifica (Bivalvia: Vesicomyidae) with emphasis on somatic cells

Somatic cells in the gonadal area of male and female deep-sea clams, Calyptogena pacifica, were examined using light and transmission electron microscopy. Acini both at the pre-spawning stage and at the stage of active spermatogenesis were observed to be simultaneously present in sections through a male gonad. Oocytes of various degrees of maturity were simultaneously present in female acini. No storage tissue and cells similar to adipogranular cells or vesicular connective tissue of other mollusks were found in the gonadal area of C. pacifica. Instead, in both males and females, numerous hemocytes surround the acini. Among hemocytes, two types of granulocytes and erythrocytes were identified. Bundles of muscle cells were also found in the interacinar space. Male intraacinar accessory cells were rather large, glycogen-rich, with lipid inclusions, and phagosomes contained spermatogenic cells. Female accessory cells had well-developed endoplasmic reticulum, but they did not form any follicles around oocytes, being in their basal part, closer to basal lamina. Such a specific pattern of gonad organization can presumably be explained by both symbiosis with sulfide-oxidizing bacteria and phylogenetic aspects that should be further studied. Some evidence for continuous gametogenesis are discussed.

Diagnostic approach to eosinophilia in children

Hematopoiesis is the process which maintains lifelong production of haemopoietic (blood) cells from bone marrow. Blood consists of three types of cells (Erythrocyte – Leukocytes - palates), and plasma. Leukocytes are divided into 2 types of granulocytes (including neutrophils, eosinophils and basophils); and agranulocytes (including lymphocytes and monocytes). Eosinophil attack and kill parasites and cancer cells, and help with allergic responses, so their role is to protect the body from bacteria and parasites. They are found in the peripheral blood and also tissue, so they are closer to where the site of infection may occur. The eosinophil is a much-overlooked cell, more usually considered as a curious form of phagocytic leucocyte with functions similar to its more numerous cousins, the neutrophil. However, a dramatic blood and tissue eosinophilia is an important part of adaptive immunity to parasitic helminthic infections, whilst more detrimentally, the eosinophil is thought to make a major contribution to the inflammation underlying the pathogenesis of disorders such as asthma, allergic rhinitis and atopic dermatitis .[1]. Eosinophilia is a common finding in clinical practice and presented in a broad spectrum of diseases, some of them hematologic disorders and others non – hematologic (infection disease – allergic disease – medication reaction, and autoimmune disease). Key words: eosinophilia, children, diagnostic approach

Shotgun analysis to identify differences in protein expression between granulocytes and hyalinocytes of the European flat oyster Ostrea edulis

Recovery of wild populations of the European flat oyster Ostrea edulis is important for ecosystem health and conservation of this species, because native oyster populations have dramatically declined or disappeared in most European waters. Diseases have contributed to oyster decline and are important constrains for oyster recovery. Understanding oyster immune system should contribute to design effective strategies to fight oyster diseases. Haemocytes play a pivotal role in mollusc immune responses protecting from infection. Two main types of haemocytes, granulocytes and hyalinocytes, are distinguished in O. edulis. A study aiming to explore differential functions between both haemocyte types and, thus, to enrich the knowledge of Ostrea edulis immune system, was performed by comparing the proteome of the two haemolymph cell types, using a shotgun approach through liquid chromatography (LC) coupled to mass spectrometry (MS). Cells from oyster haemolymph were differentially separated by Percoll density gradient centrifugation. Shotgun LC-MS/MS performance allowed the identification of 145 proteins in hyalinocytes and 138 in the proteome of granulocytes. After a comparative analysis, 55 proteins with main roles in defence were identified, from which 28 were representative of granulocytes and 27 of hyalinocytes, plus 11 proteins shared by both cell types. Different proteins involved in signal transduction, apoptosis, oxidative response, processes related with the cytoskeleton and structure, recognition and wound healing were identified as representatives of each haemocyte type. Important signalling pathways in the immune response such as MAPK, Ras and NF-κβ seemed to be more relevant for granulocytes, while the Wnt signalling pathway, particularly relevant for wound healing, more relevant in hyalinocytes. The differences in proteins involved in recognition and in cytoskeleton and structure suggest differential specialisation in processes of phagocytosis and internalisation of pathogens between haemocyte types. Apoptosis seemed more active in granulocytes. The differences in proteins involved in oxidative response also suggest different redox processes in each cell type.

Distinct Outcome Based on Fusion Transcript in t(16;21) Rearranged Pediatric AML, a Collaborative Retrospective Analysis By the I-BFM SG

Despite the use of of intensive chemotherapy, and allogeneic stem cell transplant (HSCT) in selected cases, children with acute myeloid leukemia (AML) have 5-year overall survival (OS) rates of ~65%. Apart from early clinical response, cytogenetic and molecular aberrations are the most reliable prognostic factors for survival. Identifying prognostic subgroups to stratify patients for treatment in future trials is clinically important. The study of rare AML subtypes can be facilitated by international collaborations. AML with t(16;21) rearrangements includes 2 different translocations: t(16;21)(p11;q22) resulting in the FUS-ERG fusion, and t(16;21)(q24;q22) resulting in the RUNX1-CBFA2T3 fusion.To study outcomes and associated prognostic factors, clinical data from children with t(16;21) AML were collected from 14 study groups participating in the international Berlin-Frankfurt-Munster (I-BFM) network. Patients (age 0-18 years) diagnosed from January 1, 1995, to January 1, 2016, were included. The AML-BFM study group provided data from 1326 unselected patients diagnosed between 1997 and 2013 as a reference cohort. All karyotypes were centrally reviewed by 2 independent cytogeneticists according to the International System for Human Cytogenetic Nomenclature (ISCN 2005). Patients with inconclusive karyotypes were screened by reverse-transcription PCR. Chi-squared and Fisher exact tests were used to compare clinical characteristics. OS and event free survival (EFS) analysis was performed by the Kaplan-Meier method. Cumulative incidence of relapse (CIR) was calculated according to Kalbfleisch and Prentice. The Cox proportional hazards model was used for multivariate analysis, considering as covariables age, white blood cell (WBC) count at diagnosis, cytogenetic risk group and allogeneic hematopoietic stem cell transplantation (HSCT) as time-dependent variable.There were 54 patients with t(16;21): 32 with FUS-ERG and 22 with RUNX1-CBFA2T3 . There were no significant differences in sex and age between patients in the FUS-ERG- or RUNX1-CBFA2T3- groups and the reference cohort. Median WBC at diagnosis was significantly lower for patients with RUNX1-CBFA2T3 (12.5×109/L, range 0.01-185×109/L) than for the reference cohort (19.4×109/L, range 0.01-1900×109/L, P=0.030). Patients with FUS-ERG had no predominant French-American-British (FAB) type, whereas 76.4% of those with RUNX1-CBFA2T3 had a granulocytic FAB type (M1, M2) compared with 42.1% in the reference cohort (P=0.004).The 4-year EFS, OS, and CIR for the reference cohort were 51% [standard error (SE)=1%], 68% (SE=1%), and 32% (SE=1%), respectively. Median follow up for survivors in the t(16;21) cohort was 1.6 years for those with FUS-ERG and 5.0 years for RUNX1-CBFA2T3 . Patients with FUS-ERG had a 4-year EFS of 7% (SE=5%, P <0.0001), 4-year OS of 21% (SE=8%, P <0.0001), and CIR of 74% (SE=8%, P <0.0001). Patients with RUNX1-CBFA2T3 had a 4-year EFS of 77% (SE=9%, P=0.06), OS of 81% (SE=8%, P=0.34) and a CIR of 0%. When patients in the reference cohort were classified in SR patients comprising (inv(16)/t(8;21), and HR patients comprising other cytogenetic subtypes excluding t(16;21), EFS rates were 74% (SE=3%) and 45% (SE=2%), respectively (Fig. 1). Hence, patients with RUNX1-CBFA2T3 had a similar outcomes as SR patients (Figure 1). In multivariate analysis, presence of FUS-ERG and RUNX1-CBFA2T3 were independent risk factors for EFS, with hazard ratios of 1.93 (P <0.0001) and 0.325 (P=0.025), respectively. HSCT was performed in 14 (45.2%) patients with FUS-ERG in first complete remission (CR1). The 4-year EFS for transplanted patients was 15% (SE=15%) compared with 0% (SE=13%) for patients receiving chemotherapy only (p=NS).We identified 2 clinically relevant, distinct subtypes of pediatric AML patients with different t(16;21) rearrangements: FUS-ERG had poor outcomes, whereas RUNX1-CBFA2T3 had favorable outcomes. Patients with RUNX1-CBFA2T3 -rearranged AML might benefit from inclusion in the SR group and receiving less intensive treatment, whereas those with FUS-ERG rearranged AML should be stratified in the HR group. In this study, only patients with FUS-ERG receiving HSCT in CR1 did not relapse. Most current pediatric AML trials consider children with a 5-year OS below 25%-30% for HSCT in CR1. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Лейкоцитарный состав крови ужа обыкновенного Natrix natrix (Serpentes: Colubridae) Мордовского государственного заповедника (Россия)

The state of the grass snake (Natrix natrix) population in the Mordovian State Nature Reserve was assessed using the hematological approach. The leukocyte blood count was estimated together with calculation of the associated leukocyte indices (neutrophil:lymphocyte (N:L), eosinophil:lymphocyte (E:L), heterophil:eosinophil (H/E) and heterophil:lymphocyte (H:L) ratios). The surface area of leukocytes (heterophils, basophils, eosinophils, azurophils, monocytes, and lymphocytes) was measured. The leukocyte blood composition of Natrix natrix was characterized by the predominance of agranulocytes, and the granulocyte fraction was 31– 37% (u = 1.99, p = 0.04). The population pattern of the leukocyte blood composition of Natrix natrix was as follows: heterophils – 8.07±0.6%, basophils – 12.33±0.95%, eosinophils – 8.33±0.65%, azurophils – 5.25±0.53%, monocytes – 9.77±0.42%, and lymphocytes – 56.22±1.7%. According to the averaged area, the leukocytes were arranged in the following order: eosinophils – monocytes – azurophils, basophils – heterophils – lymphocytes. The diameter of the largest cells (eosinophils) was (17.5±2.42) μm. The smallest lymphocytes had diameters within (7.22±1.21) μm. The absence of any differences in the quantitative content of all types of granulocytes and agranulocytes in the blood of the grass snake from different parts of the reserve indicated a comparable level of impact and the identity of the physiological mechanisms of adaptation that occur in the body of animals in protected habitat conditions. Most of the leukocyte integral indices revealed the same type of variability in the parameters of the white blood cell system of the Natrix natrix in three areas of the reserve, with the exception of an increased value of the heterophiles/lymphocytes ratio in the sample from the village Pushta. The increase in this indicator made it possible to draw a conclusion about the stress effect on the individuals of this sample. The principal component method was used to differentiate the samples of the grass snake with a more complete quantitative description of the leukocyte composition of the objects of study; the results obtained are presented in a visual, integrated and generalized form. The use of the principal component method made it possible to combine samples of Natrix natrix with similar indicators of leukocyte composition, as well as to identify a population of Natrix natrix that differs in its indicators in the urbanized territory of the Samara region. The blood pattern and the dynamics of blood leukocyte indices reflected the active response of the Natrix natrix body to the complex of environmental factors of the habitat on the territory of the Mordovian State Nature Reserve.

Elevated sputum IL-36 levels are associated with neutrophil-related inflammation in COPD patients.

Interleukin (IL)-36, including IL-36α, IL-36β, and IL-36γ in the IL-1 family, are agonists of their receptors. IL-36 expression is associated with inflammation, including lung infection in humans. However, there is no information on its role in the inflammation of different types of chronic obstructive pulmonary disease (COPD). This study focused on the sputum IL-36α, IL-36β, and IL-36γ levels in stable COPD patients and their relationship with lung function and other cytokines in different inflammatory types of COPD patients. Sputum specimens were collected from 73 stable COPD patients and 20 age- and gender-matched healthy controls. The levels of sputum IL-36α, IL-36β, and IL-36γ and other cytokines were quantified and sputum cells were characterized. The potential relationship between the levels of sputum IL-36α, IL-36β, or IL-36γ and lung functional measures, inflammatory cells, and cytokines was analyzed. In comparison with the healthy controls, sputum IL-36α and IL-36γ levels significantly increased in COPD (106.8pg/mL vs. 76.9pg/mL P=.001, 397.9pg/mL vs. 359.5pg/mL P=.006). The sputum IL-36α and IL-36γ levels were significantly higher in the neutrophilic and mixed granulocytic types than that in the eosinophilic and paucigranulocytic types of COPD patients. The sputum IL-36α levels were positively correlated with sputum IL-36γ levels and the numbers of sputum neutrophils, and the sputum IL-36γ levels were positively correlated with the numbers of sputum lymphocytes in COPD patients. Elevated levels of sputum IL-36α and IL-36γ were detected in COPD patients and may provide insights into the inflammatory pathways in neutrophilic COPD.

Basophils Orchestrating Eosinophils' Chemotaxis and Function in Allergic Inflammation.

Eosinophils are well known to contribute significantly to Th2 immunity, such as allergic inflammations. Although basophils have often not been considered in the pathogenicity of allergic dermatitis and asthma, their role in Th2 immunity has become apparent in recent years. Eosinophils and basophils are present at sites of allergic inflammations. It is therefore reasonable to speculate that these two types of granulocytes interact in vivo. In various experimental allergy models, basophils and eosinophils appear to be closely linked by directly or indirectly influencing each other since they are responsive to similar cytokines and chemokines. Indeed, basophils are shown to be the gatekeepers that are capable of regulating eosinophil entry into inflammatory tissue sites through activation-induced interactions with endothelium. However, the direct evidence that eosinophils and basophils interact is still rarely described. Nevertheless, new findings on the regulation and function of eosinophils and basophils biology reported in the last 25 years have shed some light on their potential interaction. This review will focus on the current knowledge that basophils may regulate the biology of eosinophil in atopic dermatitis and allergic asthma.

Potentially modifiable predictors of cell collection efficiencies and product characteristics of allogeneic hematopoietic progenitor cell collections.

Hematopoietic progenitor cell (HPC) and immune effector cell (IEC) therapies often require high doses of mononuclear cells (MNCs), whether CD34+ cells, lymphocytes, or monocytes. Cells for IEC can be sourced from HPC products. We thus examined potentially modifiable variables affecting collection efficiencies (CEs) of MNC subsets in HPC collection and also of the typically undesired cell types of platelets, granulocytes, and red cells, which hinder downstream processing. Finally, we sought to confirm previously indeterminate studies of the effect of an adjusted collect flow rate (CFR) on CD34+ CE. We performed univariate and multivariate regression analyses of all 135 National Marrow Donor Program (NMDP) HPC collections in 2019 and compared these fixed CFR procedures to previous NMDP collections using adjusted CFRs. Target cell CEs decreased with increasing peripheral blood (PB) concentration and were associated with different cell type locations within the MNC layer. CEs of undesired cell types varied with standard procedural parameters (inlet flow rate, whole blood processed, etc.). Interestingly, some CEs increased with preapheresis hematocrit. Finally, adjusting the CFR by PB MNC count improved MNC CE but not CD34+ CE. Correlation of target cell CEs with their PB concentration and different cell type locations by depth within the MNC layer indicates the importance of investigating the compensatory fine-tuning of procedure variables to improve CE. Correlation of CEs with PB hematocrit, and CFR adjustment by a modified PB MNC and/or PB CD34 algorithm should be further explored. Adjusting standard procedural parameters may reduce product contamination.

Eosinophils and childhood asthma.

Eosinophils are a type of granulocyte with eosinophilic granules in the cytoplasm that play an important role in allergic and parasitic diseases. Eosinophils are important in the pathogenesis of asthma, and many studies have examined the relationship between them. In allergic eosinophilic asthma, eosinophils act not only as important effector cells but also as antigen-presenting cells in allergic inflammatory reactions. In nonallergic eosinophilic asthma, type 2 innate lymphoid cells in the airways play an important role in eosinophil activation. Direct methods, including bronchial biopsy, bronchoalveolar lavage, and the induced sputum test, are used to evaluate eosinophilic inflammatory reactions in patients with asthma, however, because of difficulty with their implementation, they are sometimes replaced by measurements of blood eosinophils, fraction of exhaled nitric oxide, and serum periostin level. However, these tests are less accurate than direct methods. For the treatment of patients with severe eosinophilic asthma, anti-interleukin-5 preparations such as mepolizumab, reslizumab, and benralizumab have recently been introduced and broadened the scope of asthma treatment. Although eosinophils are already known to play an important role in asthma, we expect that further studies will reveal more details of their action.

Developmental pathways of myeloid-derived suppressor cells in neoplasia

Immunotherapy has become a major weapon against the war on cancer. This has culminated from decades of seminal work that led to the discovery of innovative approaches to drive adaptive immunity. Notably, was the discovery of immune checkpoint inhibitory receptors on T cells, and the subsequent development of monoclonal antibodies that target those receptors, known as immune checkpoint inhibitors (ICIs). Blocking those receptors using ICIs leads to sustained effector function, which has translated to enhanced antitumor responses across multiple human cancer types. However, these treatments are effective in subsets of patients, implicating significant barriers limiting therapeutic potential. While numerous mechanisms may hinder immunotherapy potency, one prominent mechanism is the production of myeloid-derived suppressor cells (MDSCs). MDSCs comprise monocytic and granulocytic cell types and mediate pro-tumorigenic and immune suppressive activities. Here, we summarize several pathways by which MDSCs arise in cancer, providing a conceptual framework for identifying unique combination therapeutic interventions.

The Expression and Role of Multiple Forms of Granulocyte Colony-Stimulating Factor in Medaka Fish

Diversity in the repertoires of granulocytes, such as neutrophils, basophils, and eosinophils, has been known among more than seventy thousand vertebrate species. Granulocyte colony-stimulating factor (G-CSF; csf3) affects the differentiation, survival, and proliferation of neutrophils. However, the function of G-CSF in many animal species whose repertoire of granulocytes differs from that of mammals is not clear. We, therefore, attempt to investigate G-CSF function in adult d-rR strain of Medaka fish (Oryzias latipes). Of the three types of granulocytes, Medaka fish possess only neutrophils. First of all, we assessed the distribution of neutrophils by detecting myeloperoxidase (MPO) -positive cells in the various organs, including the kidney, spleen, heart, liver, and intestine. Among those tissues, the cell proportion of neutrophils was the highest in the kidney (approx. 15%). We then identified the genome sequences of Medaka G-CSFa1x1 and G-CSFa1x2, as orthologs of the mammalian genes encoding G-CSF. They are transcriptional variants and located on chromosome 19. Phylogenetic analysis and locus analysis revealed that these sequences are classified as G-CSFa of teleost fish. The synteny and functional domains of the Medaka G-CSF receptor (G-CSFR; csf3r) is also conserved with those of other species of G-CSFR. They are different from typical mammalian G-CSFs because they have N-linked glycans. We compared the expression of Olcsf3a1 and Olcsf3r in each tissue and found higher expression in the spleen, kidney, and bone of the Medaka fish. The Olcsf3a1x2 expressed much higher than that of Olcsf3a1x1 in the kidney and spleen. In the Medaka exposed to breeding water containing lipopolysaccharide (LPS; Escherichia coli 055: B5), the number of peripheral neutrophils at 3 hours significantly elevated; while that of splenic neutrophils at 3 hours reduced, suggesting that splenic neutrophils were released to the circulation. The expression of Olcsf3a1 mRNA increased at 12 hours after LPS exposure in the bone The number of neutrophils in the kidney increased at 24 hours after LPS exposure, demonstrating that Olcsf3a1 stimulated myelopoiesis shortly after splenic neutrophil release. To elucidate the role of OlG-CSFa1 in a steady-state, we generated the Olcsf3a1 deficient Medaka (G-CSFa1-deficient) by CRISPR/Cas9 genome editing using short guide RNA for the shared sequence of both G-CSFa1x1 and G-CSFa1x2. As we expected, the heterozygous G-CSFa1-deficient Medaka exhibited severe neutropenia. The number of peripheral neutrophils significantly decreased to 1.80×109 cells/L, whereas that of wild-type Medaka was 14.2×109 cells/L. Remarkably, the number of peripheral erythrocytes displayed 2.91×1012 cells/L in the G-CSFa1-deficient Medaka, whereas that of the wild-type Medaka was 5.85×1012 cells/L. We, therefore, tested the gene expressions of hematopoietic growth factors and found that the relative gene expression of erythropoietin decreased in the kidney of G-CSFa1-deficient Medaka. The results show that G-CSF-deficient Medaka should be a valuable model to examine specific functions of G-CSF in producing granulocytes and its synergistic effect on erythropoiesis. Disclosures No relevant conflicts of interest to declare.

Eosinophils and Neutrophils-Molecular Differences Revealed by Spontaneous Raman, CARS and Fluorescence Microscopy.

Leukocytes are a part of the immune system that plays an important role in the host’s defense against viral, bacterial, and fungal infections. Among the human leukocytes, two granulocytes, neutrophils (Ne) and eosinophils (EOS) play an important role in the innate immune system. For that purpose, eosinophils and neutrophils contain specific granules containing protoporphyrin-type proteins such as eosinophil peroxidase (EPO) and myeloperoxidase (MPO), respectively, which contribute directly to their anti-infection activity. Since both proteins are structurally and functionally different, they could potentially be a marker of both cells’ types. To prove this hypothesis, UV−Vis absorption spectroscopy and Raman imaging were applied to analyze EPO and MPO and their content in leukocytes isolated from the whole blood. Moreover, leukocytes can contain lipidic structures, called lipid bodies (LBs), which are linked to the regulation of immune responses and are considered to be a marker of cell inflammation. In this work, we showed how to determine the number of LBs in two types of granulocytes, EOS and Ne, using fluorescence and coherent anti-Stokes Raman scattering (CARS) microscopy. Spectroscopic differences of EPO and MPO can be used to identify these cells in blood samples, while the detection of LBs can indicate the cell inflammation process.

Enterospora nucleophila (Microsporidia) in Gilthead Sea Bream (Sparus aurata): Pathological Effects and Cellular Immune Response in Natural Infections.

Enterospora nucleophila is a microsporidian responsible for an emaciative disease in gilthead sea bream (Sparus aurata). Its intranuclear development and the lack of in vitro and in vivo models hinder its research. This study investigated the associated lesions, its detection by quantitative polymerase chain reaction, and the cellular immune response of naturally infected fish. The intensity of infection in the intestine was correlated with stunted growth and reduced body condition. At the beginning of the outbreaks, infection prevalence was highest in intestine and stomach, and in subsequent months, the prevalence decreased in the intestine and increased in hematopoietic organs and stomach. In heavy infections, the intestine had histologic lesions of enterocyte hypercellularity and proliferation of rodlet cells. Infected enterocytes had E. nucleophila spores in the cytoplasm, and a pyknotic nucleus, karyorhexis or karyolysis. Lymphocytes were present at the base of the mucosa, and eosinophilic granule cells were located between the enterocytes. In intestinal submucosa, macrophage aggregates containing spores were surrounded by lymphocytes and granulocytes, with submucosal infiltration of granulocytes. Macrophage aggregates appeared to develop into granulomata with necrotic areas containing parasite remnants. Immunohistochemistry revealed mast cells as the main type of granulocyte involved. Abundant IgM+ and IgT+ cells were identified by in situ hybridization in the submucosa when intracytoplasmic stages were present. This study describes the lesions of E. nucleophila in gilthead sea bream, an important aquaculture species.

Công thức bạch cầu của máu ngoại vi cùng các cơ quan tạo máu ở loài cá lóc đồng Channa striata (Bloch, 1793) và lươn Monopterus albus (Zuiew, 1793) thu từ Vườn Quốc gia Cát Tiên (Việt Nam)

The composition of leukocytes of peripheral blood of the snakehead Channa striata and rice eel Monopterus albus and the hematopoietic organs of the snakehead was investigated. It was established that the leukocytes of C. striata and M. albus were heterogeneous in terms of morphofunctional characteristics and different by cell structures: lymphocytes, monocytes, neutro-, eosinophils and blast cells. The compositions of blood leukocytes and hematopoietic organs were dominated by lymphocytes, following by blast forms of cells and a small percentage of granulocytes and monocytes/macrophages. Two types of granulocytes were revealed in the striped snakehead: neutrophils and eosinophils, while in rice eel - only neutrophils.

Kinetics of adult hematopoietic stem cell differentiation in vivo.

Adult hematopoiesis has been studied in terms of progenitor differentiation potentials, whereas its kinetics in vivo is poorly understood. We combined inducible lineage tracing of endogenous adult hematopoietic stem cells (HSCs) with flow cytometry and single-cell RNA sequencing to characterize early steps of hematopoietic differentiation in the steady-state. Labeled cells, comprising primarily long-term HSCs and some short-term HSCs, produced megakaryocytic lineage progeny within 1 wk in a process that required only two to three cell divisions. Erythroid and myeloid progeny emerged simultaneously by 2 wk and included a progenitor population with expression features of both lineages. Myeloid progenitors at this stage showed diversification into granulocytic, monocytic, and dendritic cell types, and rare intermediate cell states could be detected. In contrast, lymphoid differentiation was virtually absent within the first 3 wk of tracing. These results show that continuous differentiation of HSCs rapidly produces major hematopoietic lineages and cell types and reveal fundamental kinetic differences between megakaryocytic, erythroid, myeloid, and lymphoid differentiation.

Pike intestinal reaction to Acanthocephalus lucii (Acanthocephala): immunohistochemical and ultrastructural surveys

BackgroundThe Northern pike, Esox lucius, is a large, long-lived, top-predator fish species and occupies a broad range of aquatic environments. This species is on its way to becoming an important model organism and has the potential to contribute new knowledge and a better understanding of ecology and evolutionary biology. Very few studies have been done on the intestinal pathology of pike infected with helminths. The present study details the first Italian record of adult Acanthocephalus lucii reported in the intestine of E. lucius.ResultsA total of 22 pike from Lake Piediluco (Central Italy) were examined, of which 16 (72.7%) were infected with A. lucii. The most affected areas of gastrointestinal tract were the medium and distal intestine. The intensity of infection ranged from 1 to 18 parasites per host. Acanthocephalus lucii penetrated mucosal and submucosal layers which had a high number of mast cells (MCs) with an intense degranulation. The cellular elements involved in the immune response within the intestine of pike were assessed by ultrastructural techniques and immunohistochemistry using antibodies against met-enkephalin, immunoglobulin E (IgE)-like receptor (FCεRIγ), histamine, interleukin-6, interleukin-1β, substance P, lysozyme, serotonin, inducible-nitric oxide synthase (i-NOS), tumor necrosis factor-α (TNF-α) and the antimicrobial peptide piscidin 3 (P3). In intestines of the pike, several MCs were immunopositive to 9 out of the 11 aforementioned antibodies and infected fish had a higher number of positive MCs when compared to uninfected fish.ConclusionsPike intestinal tissue response to A. lucii was documented. Numerous MCs were seen throughout the mucosa and submucosal layers. In infected and uninfected intestines of pike, MCs were the dominant immune cell type encountered; they are the most common granulocyte type involved in several fish-helminth systems. Immunopositivity of MCs to 9 out of 11 antibodies is of great interest and these cells could play an important key role in the host response to an enteric helminth. This is the first report of A. lucii in an Italian population of E. lucius and the first account on positivity of MCs to piscidin 3 and histamine in a non-perciform fish.