Secondary Granules Research Articles

Abstract Tu148: cGAS-STING pathway promotes granulopoiesis and neutrophil differentiation in acute ischemia

Contemporary evidence supports neutrophils' complex roles beyond being the simple and powerful destruction tools in MI. However, our understanding remains limited. We especially lack the knowledge of how cGAS-STING regulates neutrophil functions in acute ischemia. The cGAS-STING pathway senses cytosolic DNA to produce IFN and ISGs and is a pillar of innate immunity. Early evidence suggested neutrophils did not express cGAS. Taking advantage of the improved detecting method, we revisited this topic because this pathway can play an essential role in neutrophil function in MI. Using purified neutrophils from WT and knockout mice (as control), we detected cGAS, STING, and IRF3 in these cells. To determine if cGAS-STING regulates neutrophil function, we first generated MI model in cGAS -/- , STING -/- mice, and their WT littermates and performed flow cytometry and RNA-seq analyses. We uncovered that inhibiting cGAS-STING decreases neutrophil production in bone marrow (BM) after ischemia. Interestingly, cGAS- and STING-deficient neutrophils express significantly higher levels of primary granule molecules such as MPO, Prtn3, Elane, Cts- (s, g, b, l) but much lower levels of the secondary and tertiary granule components , including Mmp9, Mmp8, Olfm4, Cd177, Lyz2, and Lyz1. Neutrophils acquire granules sequentially during differentiation. Therefore, our data suggest inhibiting cGAS-STING leads to a stagnated neutrophil differentiation in the BM and mobilizes CD101 - immature neutrophils into the circulation and MI hearts. Consistently, cGAS- and STING-deficient neutrophils release less MMP8 and MMP9 into the ischemic area. Ischemia does not trigger the degranulation of primary granules, as indicated by the minimal changes of CD63. Our data further suggest that TF C/EBP𝜹 functions downstream of cGAS-STING to promote neutrophil differentiation. Adoptive transfer experiments suggest the neutrophil-specific function of cGAS-STING. In conclusion, we identify cGAS-STING as a novel mechanism promoting emergency granulopoiesis and neutrophil differentiation after MI and uncover its functional implications in ischemia-induced remodeling. Studies using Mrp8-Cre-mediated cGAS-STING deletion in granulocyte/macrophage progenitors are in progress.

Eosinophil peroxidase promotes bronchial epithelial cells to secrete asthma-related factors and induces the early stage of airway remodeling

Asthma is a heterogeneous disease characterized by chronic airway inflammation, reversible airflow limitation, and airway remodeling. Eosinophil peroxidase (EPX) is the most abundant secondary granule protein unique to activated eosinophils. In this study, we aimed to illustrate the effect of EPX on the epithelial-mesenchymal transition (EMT) in BEAS-2B cells. Our research found that both EPX and ADAM33 were negatively correlated with FEV1/FVC and FEV1%pred, and positively correlated with IL-5 levels. Asthma patients had relatively higher levels of ADAM33 and EPX compared to the healthy control group. The expression of TSLP, TGF-β1 and ADAM33 in the EPX intervention group was significantly higher. Moreover, EPX could promote the proliferation, migration and EMT of BEAS-2B cells, and the effect of EPX on various factors was significantly improved by the PI3K inhibitor LY294002. The findings from this study could potentially offer a novel therapeutic target for addressing airway remodeling in bronchial asthma, particularly focusing on EMT.

Microbial diversity in polyextreme salt flats and their potential applications.

Recent geological, hydrochemical, and mineralogical studies performed on hypersaline salt flats have given insights into similar geo-morphologic features on Mars. These salt-encrusted depressions are widely spread across the Earth, where they are characterized by high salt concentrations, intense UV radiation, high evaporation, and low precipitation. Their surfaces are completely dry in summer; intermittent flooding occurs in winter turning them into transitory hypersaline lakes. Thanks to new approaches such as culture-dependent, culture-independent, and metagenomic-based methods, it is important to study microbial life under polyextreme conditions and understand what lives in these dynamic ecosystems and how they function. Regarding these particular features, new halophilic microorganisms have been isolated from some salt flats and identified as excellent producers of primary and secondary metabolites and granules such as halocins, enzymes, carotenoids, polyhydroxyalkanoates, and exopolysaccharides. Additionally, halophilic microorganisms are implemented in heavy metal bioremediation and hypersaline wastewater treatment. As a result, there is a growing interest in the distribution of halophilic microorganisms around the world that can be looked upon as good models to develop sustainable biotechnological processes for all fields. This review provides insights into diversity, ecology, metabolism, and genomics of halophiles in hypersaline salt flats worldwide as well as their potential uses in biotechnology.

STAG2 Somatic Mutations Are Associated with Specific Dysplastic Megakaryocytic and Myeloid Cell Features in Myelodysplastic Syndrome

Introduction: Although myelodysplastic neoplasms/myelodysplastic syndromes (MDS) are driven by genetic mutations, their diagnosis relies on morphologic evaluation of abnormal hematopoiesis in the bone marrow. Only a small number of genetic abnormalities define bone marrow morphologic features in MDS, such as those harboring SF3B1 mutations and deletions of chromosome 5q. We hypothesized that there are additional genetic alterations and dysplastic morphologic features in MDS that have not been well-described in the literature. We assessed genetic morphologic associations between 50 commonly mutated genes and 10 morphologic features in a cohort of MDS bone marrows with a high degree of dysplasia. We validated and extended these findings in an independent cohort of myeloid neoplasms. STAG2 mutations were strongly associated with specific dysplastic megakaryocytic and myeloid cell features in both cohorts. Methods: We reviewed bone marrow core biopsies and aspirates from 272 MDS clinical cases (2013-2014) and selected 89 for further study based on the presence of prominent dysplastic features. Each case was independently evaluated by two pathologists for the following dysplastic features: small hypolobated megakaryocytes, widely separated megakaryocyte nuclei; abnormal myeloid nuclear segmentation, myeloid hypogranulation, Auer rods; ring sideroblasts, erythroid binucleation, irregular erythrocyte nuclei, erythroid nuclear cytoplasmic dyssynchrony, and erythroid karyorrhexis. Targeted next generation sequencing was performed using a 50 gene panel comprising the most common somatic myeloid mutations. For each dysplastic feature, univariate risk analysis was used to identify genetic mutations for use in multivariable analysis. Significant associations were validated through blinded morphologic review in an independent cohort of 155 bone marrow biopsies of myeloid neoplasms, comprised of 83 myelodysplastic syndromes, 33 myelodysplastic/myeloproliferative neoplasms and 39 acute myeloid leukemias, diagnosed between 2014 and 2019 and selected for mutations identified in the initial discovery cohort ( STAG2, RUNX1, SRSF2, ASXL1 and SETBP1) . Results: Pairwise analyses demonstrated broad patterns of genetic associations with myeloid/megakaryocytic dysplasia versus erythroid dysplasia (Panel 1). In multivariable analysis, STAG2 and SRSF2 mutations were significantly associated with megakaryocytes with widely separated nuclei ( STAG2, OR = 25.5, 95% CI [4.17, 493.5], P = 0.003; SRSF2, OR = 10.1, 95% CI [2.07, 75.0], P = 0.008). Mutations in STAG2 and SETBP1 were significantly associated with abnormal myeloid nuclear segmentation ( STAG2, OR = 7.08, 95% CI [1.94, 28.0], P = 0.003; SETBP1, OR = 12.2, 95% CI [1.29, 267.4], P = 0.04). Furthermore, STAG2 mutations were significantly associated with myeloid cell hypogranulation (OR = 12.7, 95% CI [3.10, 86.3], P = 0.002). Because this initial cohort was relatively small, which led to wide confidence intervals suggesting estimation instability, we validated and extended these findings in an independent cohort of myeloid neoplasms. In the validation cohort (enriched for mutations in STAG2, SRSF2, and SETBP1), STAG2 mutations were significantly associated with separated megakaryocyte nuclei (univariate OR = 4.18, 95% CI [1.98, 8.80], P = 0.0002), abnormal myeloid nuclear segmentation (univariate OR = 3.47, 95% CI [1.47, 8.22], P = 0.008), and myeloid cell hypogranulation (multivariable OR = 5.28, 95% CI [2.18, 13.4], P = 0.0003) (Panel 2). In contrast, morphologic associations with SRSF2 and SETBP1 mutations were not validated. Conclusion: STAG2 genetic alterations are strongly associated with the presence of separated megakaryocyte nuclei, abnormal myeloid nuclear segmentation and myeloid cell hypogranulation in MDS. Our data suggest that STAG2 may affect pathways involved in nuclear shape control and secondary granule formation. Panel 1: Pairwise associations between gene mutation and bone marrow morphology in MDS. N, number. Panel 2: Multivariable (black) and univariate (white) analysis in two independent cohorts showing significant gene associations with megakaryocytes with separated nuclei, abnormal myeloid nuclear segmentation, and myeloid cell hypogranulation. CI, confidence interval.

The chemorepellent, SLIT2, bolsters innate immunity against Staphylococcus aureus.

Neutrophils are essential for host defense against Staphylococcus aureus (S. aureus). The neuro-repellent, SLIT2, potently inhibits neutrophil chemotaxis, and might, therefore, be expected to impair antibacterial responses. We report here that, unexpectedly, neutrophils exposed to the N-terminal SLIT2 (N-SLIT2) fragment kill extracellular S. aureus more efficiently. N-SLIT2 amplifies reactive oxygen species production in response to the bacteria by activating p38 mitogen-activated protein kinase that in turn phosphorylates NCF1, an essential subunit of the NADPH oxidase complex. N-SLIT2 also enhances the exocytosis of neutrophil secondary granules. In a murine model of S. aureus skin and soft tissue infection (SSTI), local SLIT2 levels fall initially but increase subsequently, peaking at 3 days after infection. Of note, the neutralization of endogenous SLIT2 worsens SSTI. Temporal fluctuations in local SLIT2 levels may promote neutrophil recruitment and retention at the infection site and hasten bacterial clearance by augmenting neutrophil oxidative burst and degranulation. Collectively, these actions of SLIT2 coordinate innate immune responses to limit susceptibility to S. aureus.

Investigation on the effect of nickel and nickel-chromium alloy pulse current plating on copper substrate

ABSTRACT In the present study, nanocrystalline nickel (Ni) and nickel-chromium (Ni-Cr) alloy coatings were developed on copper (Cu) substrate using pulse current. The pulse current parameters include a cycle time of 240 milliseconds at a duty cycle of 0.5 and 4.17 Hz frequency. The surface morphology, texture, hardness, scratch resistance and porosity were investigated for different coatings developed by pulse plating. Energy Dispersive Spectroscopy (EDS) analysis revealed good quality Ni and Ni-Cr alloy coatings developed on the copper substrate. The peak current of the pulse has been observed to have an impact on the coating weight and thickness. The micrographs of the coatings explored under Field Emission Scanning Electron Microscopy (FESEM), depict uniform coatings consisting of fine primary and coarse secondary granules of Ni and Cr. The hard primary and secondary Ni granules deposited on the surface of Cu increase its hardness, corrosion and wear resistance. Further, it was observed that the Ni-Cr alloy plated samples are harder and less porous in nature compared to Ni plated samples.

Butyrate regulates neutrophil homeostasis and impairs early antimicrobial activity in the lung

Short-chain fatty acids (SCFAs) are metabolites that are produced after microbial fermentation of dietary fiber and impact cell metabolism and anti-inflammatory pathways both locally in the gut and systemically. In preclinical models, administration of SCFAs, such as butyrate, ameliorates a range of inflammatory disease models including allergic airway inflammation, atopic dermatitis, and influenza infection. Here we report the effect of butyrate on a bacteria-induced acute neutrophil-driven immune response in the airways. Butyrate impacted discrete aspects of hematopoiesis in the bone marrow resulting in the accumulation of immature neutrophils. During Pseudomonas aeruginosa infection, butyrate treatment led to the enhanced mobilization of neutrophils to the lungs as a result of increased CXCL2 expression by lung macrophages. Despite this increase in granulocyte numbers and their enhanced phagocytic capacity, neutrophils failed to control early bacterial growth. Butyrate reduced the expression of nicotinamide adenine dinucleotide phosphate, oxidase complex components required for reactive oxygen species production, and reduced secondary granule enzymes, culminating in impaired bactericidal activity. These data reveal that SCFAs tune neutrophil maturation and effector function in the bone marrow under homeostatic conditions, potentially to mitigate against excessive granulocyte-driven immunopathology, but their consequently restricted bactericidal capacity impairs early control of Pseudomonas infection.

Eosinophils in normal and pathological conditions. Structure, mediators, development

Eosinophils are the most important cell type. They have a variety of functions. Eosinophil as a separate cellular element was first described by Paul Ehrlich in 1879. Their number usually ranges from 1–4% of the total number of circulating leukocytes. The presence of large specific (secondary) granules is a characteristic feature that distinguishes eosinophils from other granulocytes. Primary granules, lipid bodies are also determined in the cell. Charcot — Leiden crystals are registered in the cytoplasm and granules. Eosinophils are the effector cells of natural immunity. Eosinophils have an ability to rapidly release a vast number of tissue mediators such as granule proteins, cytokines, neuromediators, enzymes and others. It should be noted that some of them are determined only in these cells. The concentrations of many mediators in eosinophils is much higher than in neutrophils. The development of eosinophils is determined by the interaction of a whole complex of transcription factors and cytokines. It is shown that new transcription factors and other molecules involved in the differentiation of these cells to be determined in the future. A more detailed characterization of eosinophil mediators will also be carried out.

Rac2 regulates immune complex-mediated granule polarization and exocytosis in neutrophils.

A key molecule for neutrophil degranulation is Rac2 guanosine triphosphatase (GTPase). Neutrophils from Rac2 knockout mice (Rac2-/-) exhibit impaired primary granule exocytosis in response to cytochalasin B/f-Met-Leu-Phe, while secondary and tertiary granule release is unaffected. Coronin-1A (Coro1A), a protein involved in actin remodeling, is diminished in Rac2-/- neutrophils. However, primary granule exocytosis from Rac2-/- neutrophils has not been determined using more immunologically relevant stimuli. We sought to determine the role of Rac2 in degranulation and actin cytoskeleton rearrangement in response to immobilized immune complexes (IC) and relate this to intracellular Coro1A localization. We used bone marrow neutrophils (BMN) from WT and Rac2-/- mice stimulated with immobilized immune complexes. Secretion of primary (myeloperoxidase, MPO), secondary (lactoferrin, LTF) and tertiary granule (MMP-2 and MMP-9) products was evaluated. Subcellular colocalization of Coro1A with actin and the primary granule marker CD63 was determined by deconvolution microscopy. We found major differences in MPO, MMP-2, and MMP-9, but not LTF, release, along with diminished filopodia formation, CD63 polarization, and colocalization of Coro1A with CD63 in IC-stimulated Rac2-/- BMNs. Rac2 and Coro1A were found associated with granules in CB/fMLF-activated human neutrophils. This report confirms a role for Rac2 in immunologically relevant stimulation of neutrophil granule exocytosis. Rac2 appears to attach to neutrophil granules, polarize CD63+ granules to the cell surface in a manner dependent on Coro1A, and induce filopodia formation. Our studies provide insight into mechanisms of Rac2-mediated regulation of granule exocytosis.

Multiparametric Profiling of Neutrophil Function via a High-Throughput Flow Cytometry-Based Assay.

Neutrophils are a vital component of the innate immune system and play an essential function in the recognition and clearance of bacterial and fungal pathogens. There is great interest in understanding mechanisms of neutrophil dysfunction in the setting of disease and deciphering potential side effects of immunomodulatory drugs on neutrophil function. We developed a high throughput flow cytometry-based assay for detecting changes to four canonical neutrophil functions following biological or chemical triggers. Our assay detects neutrophil phagocytosis, reactive oxygen species (ROS) generation, ectodomain shedding, and secondary granule release in a single reaction mixture. By selecting fluorescent markers with minimal spectral overlap, we merge four detection assays into one microtiter plate-based assay. We demonstrate the response to the fungal pathogen, Candida albicans and validate the assay's dynamic range using the inflammatory cytokines G-CSF, GM-CSF, TNFα, and IFNγ. All four cytokines increased ectodomain shedding and phagocytosis to a similar degree while GM-CSF and TNFα were more active in degranulation when compared to IFNγ and G-CSF. We further demonstrated the impact of small molecule inhibitors such as kinase inhibition downstream of Dectin-1, a critical lectin receptor responsible for fungal cell wall recognition. Bruton's tyrosine kinase (Btk), Spleen tyrosine kinase (Syk), and Src kinase inhibition suppressed all four measured neutrophil functions but all functions were restored with lipopolysaccharide co-stimulation. This new assay allows for multiple comparisons of effector functions and permits identification of distinct subpopulations of neutrophils with a spectrum of activity. Our assay also offers the potential for studying the intended and off-target effects of immunomodulatory drugs on neutrophil responses.

Hyperactive immature state and differential CXCR2 expression of neutrophils in severe COVID-19.

Neutrophils are vital in defence against pathogens, but excessive neutrophil activity can lead to tissue damage and promote acute respiratory distress syndrome. COVID-19 is associated with systemic expansion of immature neutrophils, but the functional consequences of this shift to immaturity are not understood. We used flow cytometry to investigate activity and phenotypic diversity of circulating neutrophils in acute and convalescent COVID-19 patients. First, we demonstrate hyperactivation of immature CD10- subpopulations in severe disease, with elevated markers of secondary granule release. Partially activated immature neutrophils were detectable 12 wk post-hospitalisation, indicating long term myeloid dysregulation in convalescent COVID-19 patients. Second, we demonstrate that neutrophils from moderately ill patients down-regulate the chemokine receptor CXCR2, whereas neutrophils from severely ill individuals fail to do so, suggesting an altered ability for organ trafficking and a potential mechanism for induction of disease tolerance. CD10- and CXCR2hi neutrophil subpopulations were enriched in severe disease and may represent prognostic biomarkers for the identification of individuals at high risk of progressing to severe COVID-19.

Investigation of immunoglobulin G, lactoferrin and zinc levels in blood sera of calves fed fresh and frozen colostrum

Colostrum has high nutritional values, is more easily digestible than milk, and has a comparatively higher concentration of dry matter, fat and non-fat dry matter, protein and most importantly, immunoglobulin (Ig). The most important task of immunoglobulins is to neutralize pathogens and toxins through neuttalization. Lactoferrin is a protein product of the transferrin gene family with iron binding ability. Lactoferrin serves as a major component of the secondary granules of polymorphonuclear neutrophils and is produced by epithelial cells, including those in the mammary gland. Zinc acts as a cofactor and activator of more than 300 enzymes in different metabolic pathways and is known as a biologically important trace mineral. The aim of this study is to examine whether the immunoglobulin G, lactoferrin and zinc concentrations in colostrum, which is vital for newborn calves, show a decrease tendency by freezing. Fresh colostrum was given to one group (n=12) and frozen (-20 °C) colostrum (n=12) was given to one group and blood samples were collected after 32 hours. According to the results obtained, there was no statistical difference between the groups in the initial measurements of IgG, lactoferrin and zinc values in group 1 (fresh colostrum) and group 2 (frozen colostrum). The differences between the first and second measurements among themselves in both groups were found to be statistically significant. In the second measurements between the two groups, no statistical difference was found between the values of immunoglobulin G (p= 0.996), lactoferrin (p = 0.513), zinc (p = 0.605).

Review: Lactoferrin a natural glycoprotein as antiviral agent

Lactoferrin is a great multifunctional natural glycoprotein, it is distributed widely in various tissues especially in exocrine glands. It is abundant in exocrine fluids such as breast milk and colostrum, in mucosal secretions, and in secondary granules of neutrophils. Lactoferrin have many physiological functions, it enhances and strengths the immune system of the body by sequestering iron and prevent pathogenic bacteria and viruses from using iron for their growth and replication, also it competes bacteria and viruses on the binding receptor sites of host cells. The low death rate of infants and children during COVID-19 pandemic is revealed to the antiviral activity of lactoferrin and other bioactive components. Lactation of infants from breast milk for two years will increase immune response against bacteria and viruses.

A prolonged innate systemic immune response in COVID-19

Despite the introduction of vaccines, COVID-19 still affects millions of people worldwide. A better understanding of pathophysiology and the discovery of novel therapies are needed. One of the cells of interest in COVID-19 is the neutrophil. This cell type is being recruited to a site of inflammation as one of the first immune cells. In this project, we investigated a variety of neutrophils phenotypes during COVID-19 by measuring the expression of markers for migration, maturity, activation, gelatinase granules and secondary granules using flow cytometry. We show that neutrophils during COVID-19 exhibit altered phenotypes compared to healthy individuals. The activation level including NETs production and maturity of neutrophils seem to last longer during COVID-19 than expected for innate immunity. Neutrophils as one of the drivers of severe cases of COVID-19 are considered as potential treatment targets. However, for a successful implementation of treatment, there is a need for a better understanding of neutrophil functions and phenotypes in COVID-19. Our study answers some of those questions.

Myeloid-Specific Pyruvate-Kinase-Type-M2-Deficient Mice Are Resistant to Acute Lung Injury.

Infiltration of polymorphonuclear neutrophils (PMNs) plays a central role in acute lung injury (ALI). The mechanisms governing PMN inflammatory responses, however, remain incompletely understood. Based on our recent study showing a non-metabolic role of pyruvate kinase type M2 (PKM2) in controlling PMN degranulation of secondary and tertiary granules and consequent chemotaxis, here we tested a hypothesis that Pkm2-deficient mice may resist ALI due to impaired PMN inflammatory responses. We found that PMN aerobic glycolysis controlled the degranulation of secondary and tertiary granules induced by fMLP and PMA. Compared to WT PMNs, Pkm2-deficient (Pkm2-/-) PMNs displayed significantly less capacity for fMLP- or PMA-induced degranulation of secondary and tertiary granules, ROS production, and transfilter migration. In line with this, myeloid-specific Pkm2-/- mice exhibited impaired zymosan-induced PMN infiltration in the peritoneal cavity. Employing an LPS-induced ALI mouse model, LPS-treated Pkm2-/- mice displayed significantly less infiltration of inflammatory PMNs in the alveolar space and a strong resistance to LPS-induced ALI. Our results thus reveal that PKM2 is required for PMN inflammatory responses and deletion of PKM2 in PMN leads to an impaired PMN function but protection against LPS-induced ALI.

Lactoferrin Can Attenuate SARS-CoV-2: An Analysis of Evidential Relations

Background: Lactoferrin (Lf ) has been shown to have antiviral action against a variety of animal and human viruses, particularly deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) viruses. This review aims to summarize the pharmacological activities that lead to the influential role of Lf against SARS-CoV-2. Methods: An all-inclusive search of published articles was carried out to focus on publications related to Lf and its biological/pharmacological activities using various literature databases, including the scientific databases Science Direct, Scopus, Web of Science, PubMed, Google Scholar, Google, EMBASE, and Scientific Information (SID). Results: By acting on cell targets, Lf prevents viral attachment, surface accumulation on the host cell, and virus penetration. Lf has shown high antiviral effectiveness across a broad spectrum of viruses, suggesting that it might be used to cure and prevent severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). Lf can also attach to viral particles directly, such as hepatitis C virus (HCV), and steer them away from certain sites. LF has a powerful attraction for iron, with a constant of approximately 1020. Lf capacity to link iron relies on the existence of (minute amounts of ) bicarbonate. The bacteriostatic effect of Lf is due to its capability to come together with free iron, which is one of the ingredients necessary for bacterial development. Lf located in neutrophil secondary granules is essential for host defense. Conclusion: Researchers confirmed that Lf activates natural killer (NK) cells in a study. Lf has been shown in certain studies to prevent patronization in pseudovirus severe acute respiratory syndrome (SARS) cases that leads to attenuation of SARS-CoV-2. Lf may decrease inflammation induced by microbial exposure and directly reduce bacterial growth. It is concluded that Lf possesses antibacterial, immunomodulatory, anticancer, antiviral, cytoprotective, and anti-inflammatory activities, which ultimately act as an antiviral against SARS-CoV-2 via various mechanisms.

Histological observations on the oocyte development in the Picnic seabream, Acanthopagrus berda (Forsskål 1775)

Histological analysis was carried out on ovarian development in the picnic seabream, Acanthopagrus berda (Forsskål 1775) caught in the estuarine waters of Calicut, Kerala, India from January to December 2016. Four developmental phases of oocyte development were identified viz., oogonia proliferation and transformation into the primary oocyte, primary growth phase, secondary growth phase and maturation phase and these are similar to those described for other species. The reproductive development process begins in a very young ovary with the formation of oogonia, the cells originally derived from special primordial germ cells formed early in the embryonic development; their multiplication and differentiation are followed by the release of mature oocytes. Oocyte development stages observed in A. berda were chromatin nucleolus, early perinucleolar, late perinucleolar, lipid vesicle stage I oocyte, lipid vesicle stage II oocyte, primary yolk granule stage, secondary yolk granule stage, tertiary yolk granule stage, hydrated oocyte and atretic oocyte. The oocytes entered the maturation phase when they attained the maximum size of 459.6±71.83 µm. Histological examination of the mature ovaries showed the presence of all the different oocyte stages in the ovary of A. berda with an asynchronous mode of ovarian development shortly before spawning, indicating that the species is a multiple spawner.

C3d Elicits Neutrophil Degranulation and Decreases Endothelial Cell Migration, with Implications for Patients with Alpha-1 Antitrypsin Deficiency.

Alpha-1 antitrypsin (AAT) deficiency (AATD) is characterized by increased risk for emphysema, chronic obstructive pulmonary disease (COPD), vasculitis, and wound-healing impairment. Neutrophils play a central role in the pathogenesis of AATD. Dysregulated complement activation in AATD results in increased plasma levels of C3d. The current study investigated the impact of C3d on circulating neutrophils. Blood was collected from AATD (n = 88) or non-AATD COPD patients (n = 10) and healthy controls (HC) (n = 40). Neutrophils were challenged with C3d, and degranulation was assessed by Western blotting, ELISA, or fluorescence resonance energy transfer (FRET) substrate assays. Ex vivo, C3d levels were increased in plasma (p < 0.0001) and on neutrophil plasma membranes (p = 0.038) in AATD compared to HC. C3d binding to CR3 receptors triggered primary (p = 0.01), secondary (p = 0.004), and tertiary (p = 0.018) granule release and increased CXCL8 secretion (p = 0.02). Ex vivo plasma levels of bactericidal-permeability-increasing-protein (p = 0.02), myeloperoxidase (p < 0.0001), and lactoferrin (p < 0.0001) were significantly increased in AATD patients. In endothelial cell scratch wound assays, C3d significantly decreased cell migration (p < 0.0001), an effect potentiated by neutrophil degranulated proteins (p < 0.0001). In summary, AATD patients had increased C3d in plasma and on neutrophil membranes and, together with neutrophil-released granule enzymes, reduced endothelial cell migration and wound healing, with potential implications for AATD-related vasculitis.

Lactoferrin modified by hypohalous acids: Partial loss in activation of human neutrophils

Previously we have shown that lactoferrin (LTF), a protein of secondary neutrophilic granules, can be efficiently modified by hypohalous acids (HOCl and HOBr), which are produced at high concentrations during inflammation and oxidative/halogenative stress by myeloperoxidase, an enzyme of azurophilic neutrophilic granules. Here we compared the effects of recombinant human lactoferrin (rhLTF) and its halogenated derivatives (rhLTF-Cl and rhLTF-Br) on functional responses of neutrophils. Our results demonstrated that after halogenative modification, rhLTF lost its ability to induce mobilization of intracellular calcium, actin cytoskeleton reorganization, and morphological changes in human neutrophils. Moreover, both forms of the halogenated rhLTF prevented binding of N-acetylglucosamine-specific plant lectin Triticum vulgaris agglutinin (WGA) to neutrophils and, in contrast to native rhLTF, inhibited respiratory burst of neutrophils induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine and by two plant lectins (WGA and PHA-L). However, we observed no differences between the effects of rhLTF, rhLTF-Cl, and rhLTF-Br on respiratory burst of neutrophils induced by phorbol 12-myristate 13-acetate (PMA), digitonin, and number of plant lectins with different glycan-binding specificity. Furthermore, all rhLTF forms interfered with PMA- and ionomycin-induced formation of neutrophil extracellular traps. Thus, halogenative modification of LTF is one of the mechanisms involved in modulating a variety of signaling pathways in neutrophils to control their pro-inflammatory activity.

Analysis of Susceptibility to the Antimicrobial and Anti-Biofilm Activity of Human Milk Lactoferrin in Clinical Strains of Streptococcus agalactiae With Diverse Capsular and Sequence Types.

Group B Streptococcus (GBS) is one of the leading infection-related causes of adverse maternal and neonatal outcomes. This includes chorioamnionitis, which leads to preterm ruptures of membranes and can ultimately result in preterm or stillbirth. Infection can also lead to maternal and neonatal sepsis that may contribute to mortality. Currently, treatment for GBS infection include a bolus of intrapartum antibiotic prophylaxis to mothers testing positive for GBS colonization during late pregnancy. Lactoferrin is an antimicrobial peptide expressed in human breast milk, mucosal epithelia, and secondary granules of neutrophils. We previously demonstrated that lactoferrin possesses antimicrobial and antibiofilm properties against several strains of GBS. This is largely due to the ability of lactoferrin to bind and sequester iron. We expanded upon that study by assessing the effects of purified human breast milk lactoferrin against a panel of phenotypically and genetically diverse isolates of GBS. Of the 25 GBS isolates screened, lactoferrin reduced bacterial growth in 14 and biofilm formation in 21 strains. Stratifying the data, we observed that colonizing strains were more susceptible to the growth inhibition activity of lactoferrin than invasive isolates at lactoferrin concentrations between 250-750 µg/mL. Treatment with 750 µg/mL of lactoferrin resulted in differences in bacterial growth and biofilm formation between discrete sequence types. Differences in bacterial growth were also observed between capsular serotypes 1a and III. Maternally isolated strains were more susceptible to lactoferrin with respect to bacterial growth, but not biofilm formation, compared to neonatal sepsis isolates. Finally, high biofilm forming GBS strains were more impacted by lactoferrin across all isolates tested. Taken together, this study demonstrates that lactoferrin possesses antimicrobial and antibiofilm properties against a wide range of GBS isolates, with maternally isolated colonizing strains being the most susceptible.