Free Granules Research Articles

Utilization of rice husk substituting fossil fuel for pelletization process of goethite iron ore

The study investigated the pelletization of goethite iron ore using rice husk as a renewable carbon and energy substitute for coke breeze, which is a traditional fuel used as pellet feed in integrated steel plants. Cold Crushing Strength and porosity of the indurated pellets with rice husk addition were assessed to determine the utilization potential in the blast furnace iron making process. The X-ray diffraction studies showed the presence of iron rich magnetite and wustite, along with hematite and other oxide slag phases, which may impart high strength to the indurated pellets. FESEM studies showed the absence of free granules, which in turn confirmed the occurrence of particle-particle fusion. 3D skeleton image analyses of the indurated pellets in an X-ray micro-CT show uniform formation of round pores with good pore compactness and narrow pore size distribution inside the pellets, which are favorable properties to achieve high strength and porosity of the indurated pellets.

Pollen morphology in the genus Dyckia (Bromeliaceae): endemic species from southern Brazil comprising the proposed infrageneric grouping “D. selloa complex”

Abstract Dyckia, belonging to the Bromeliaceae, consists of species that share several morphological similarities. Recently, a proposal has grouped 13 species of the genus into a “D. selloa complex” based on their limited geographical distribution and seed characteristics. However, further investigation is required to gain a better understanding of this grouping. This study aimed to analyze the pollen grain morphology of the grouped species. The analysis was conducted using scanning electron microscopy (SEM), as well as light microscopy equipped with bright field and fluorescence optics. Various structures were measured. The pollen grains are heteropolar, oblate, and range in size from small to medium. They possess a simple sulcus type and exhibit a sexine (composed of tectum and infratectum columellate) and a nexine 1. Different patterns of ornamentation were identified, including reticulate, microreticulate, reticulate with free granules, reticulate with a partially discontinuous reticulum, and reticulate with a discontinuous reticulum. The aperture margin of some species differs from the described characteristics for the genus (Puya type) and is referred to here as the “Other” type. The results align with existing data for the genus Dyckia and contribute to understanding the pollen morphology within the genus and the studied group. The data obtained in this study, along with the observed variations, could facilitate a better understanding of the “D. selloa complex”.

The Dynamic Accumulation Rules of Chemical Components during the Medicine Formation Period of Angelica sinensis and Chemometric Classifying Analysis for Different Bolting Times Using ATR-FTIR

The dried roots of the perennial herb Angelica sinensis (Oliv.) Diels (AS) are commonly used as medicinal and edible resources. In commercial planting, early bolting and flowering (EB) of ca. 60% in the medicine formation period reduces root yield and quality, becoming a significant bottleneck in agricultural production. In the cultivation process, summer bolting (SB) occurs from June to July, and autumn bolting (AB) occurs in September. The AB root is often mistaken for the AS root due to its similar morphological characteristics. Few studies have involved whether the root of AB could be used as herbal medicine. This study explored and compared the accumulation dynamics of primary and secondary metabolites in AS and EB roots during the vegetative growth stage (from May to September) by light microscopy, ultraviolet spectrometry, and HPLC methods. Under a microscope, the amount of free starch granules and oil chamber in the AS root increased. On the contrary, they decreased further from EB-Jul to EB-Sep. By comparison, the wall of the xylem vessel was slightly thickened and stacked, and the cell walls of parenchyma and root cortex tissue were thickened in the EB root. Early underground bolting reduces soluble sugar, soluble protein, free amino acids, total C element, total N element, ferulic acid, and ligustilide accumulation, accompanied by the lignification of the root during the vegetative growth stage. Furthermore, a total of 55 root samples from different bolting types of AS root (29 samples), SB root (14 samples), and AB root (12 samples) were collected from Gansu Province during the harvesting period (October). The later the bolting occurred, the less difference there was between unbolted and bolted roots in terms of morphological appearance and efficacy components. Fourier transform infrared spectroscopy with the attenuated total reflection mode (ATR-FTIR) provides a “holistic” spectroscopic fingerprinting of all compositions in the tested sample. The ATR-FTIR spectrum of the AB root was similar to that of the AS root. However, the number and location of absorption peaks in the spectra of SB were different, and only one strong absorption peak at 1021 cm−1 was regarded as the characteristic peak of C-O stretching vibration in lignin. The ATR-FTIR spectra can be effectively differentiated based on their various characteristics using orthogonal partial least squares discrimination analysis (OPLS-DA). Results were assessed using multiple statistical techniques, including Spearman’s correlation, principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and OPLS-DA. Among these methods, the ATR-FTIR data demonstrated the most effective outcomes in differentiating between viable and non-viable roots for their application in herbal medicine. Essential substances are ferulic acid and flavonoid, which are much more abundant in the AB root. It provides a material basis for the pharmacological action of the AB roots and a theoretical basis for improving their availability.

Catfish Epidermal Club Cell Morphologic and Immunologic Attributes: Heterogeneous S-100 Immunoreactivity and Possible Neuroendocrine Function.

The fish epidermis (EP) contains several cell types, and it has several functional roles, though the secretory role prevails. The alarm or epidermal club cells (ECCs) represent a voluminous immune cell secreting an alarm substance. Our work targeted the histochemical and immunologic attributes of the ECCs in catfish. Matrix metalloproteinase (MMP-9) immunostaining revealed strong immunoreactive signals in the cytoplasm of all ECCs, while S-100 protein immunoreactivity selectively marked the ECCs. Individual ECCs exhibit intense S-100 immunoreactivity compared to neighboring ECCs. The difference in the intensity of the S-100 immunostaining was associated with the difference in the ECC size. This was confirmed by the semithin results where several developmental stages of ECCs could be distinguished. Some eosinophilic granular cells with their characteristic metachromatic granules were recorded juxtaposing the ECCs. Moreover, some free electron-dense granules could be demonstrated in the intercellular spaces surrounding the ECCs. Collectively, these findings suggest several functional profiles for the catfish ECCs depending on their developmental stage. Most importantly, S-100 immunoreactivity in the ECCs suggests its neuroendocrine function. Moreover, the difference in the intensity and polar distribution of S-100 staining is a sign of its role in the differentiation of the ECCs.

Effects of pH during dry-heat preparation on the physicochemical and emulsifying properties of rice starch and whey protein isolate mixtures

Dry-heat and pH-shifting are simple and effective methods for the modification of starch and protein. To improve the ability of native rice starch (RS) to form stable emulsions, whey protein isolate (WPI) was incorporated into RS samples (RS/WPI mixtures) under different pH conditions (pH 3.0, 5.0, 6.5, 9.0, and 11.0, and pH-shifting) using a dry-heat treatment (130°C, 6 h). The physicochemical properties and emulsifying abilities of the RS/WPI samples with and without dry heating were investigated. Both particle aggregation and contact angle of the dry-heated samples increased with increasing pH. The particle size of D (4, 3) and the contact angle for the sample prepared at pH 11.0 were 34.78 μm and 95.06°, respectively. Pickering emulsions stabilized by the dry-heated RS/WPI samples prepared at pH 6.5, 9.0 and 11.0 showed lower amounts of creaming. Micrographs showed that the RS/WPI-stabilized Pickering emulsions were packed with numerous starch granules adsorbed spherical droplets and a lot of free starch granules and aggregates filling in the droplet gaps. The results suggest that a combination of pH-shifting and dry heating is an effective strategy in improving the emulsifying ability of RS/WPI mixtures.

Bronchiectasis in severe asthma is associated with eosinophilic airway inflammation and activation.

Bronchiectasis is a common comorbidity in severe asthma; causative pathogenic mechanisms are not fully understood but may differ from other causes of bronchiectasis. The role of eosinophilic airway inflammation, a classic feature of asthma predominantly driven by IL-5 and IL-13, in bronchiectasis is unclear, but association with disruption of the airway epithelium through eosinophil degranulation and increased mucus production is plausible. We sought to describe the prevalence of bronchiectasis in an unselected population of patients with severe asthma, and the association with the airway eosinophilic inflammation and activation. All patients with severe asthma according to European Respiratory Society/American Thoracic Society criteria (high-dose inhaled corticosteroids/oral corticosteroids), attending 4 respiratory clinics over a 1-year period, were included. All patients underwent high-resolution computed tomography and induced sputum was collected and analyzed for a cell differential count, free eosinophilic granules, and airway messenger RNA expression of T2 inflammatory pathways. Bronchiectasis was present in 31% (34 of 108) of patients with severe asthma, and half (52%) of these patients had airway eosinophilia whereas only 16% of patients without bronchiectasis had airway eosinophilia. Patients with bronchiectasis had a significantly higher sputum eosinophil count (5.3 vs 0.8; P= .001) as well as more extensive eosinophil degranulation, compared with those without bronchiectasis (13% vs 2%; P= .05), suggesting a higher degree of eosinophil activation. Pairwise analyses identified significantly higher messenger RNA expression of Charcot-Leyden crystal galectin in patients with bronchiectasis (P= .02). Bronchiectasis in severe asthma was associated with eosinophilic airway inflammation and eosinophilic degranulation as well as messenger RNA expression of Charcot-Leyden crystal galectin.

Autoantibody-mediated Macrophage Dysfunction in Patients with Severe Asthma with Airway Infections.

Rationale: Localized autoimmune responses have been reported in patients with severe eosinophilic asthma, characterized by eosinophil degranulation and airway infections. Objective: To determine the presence of autoantibodies against macrophage scavenger receptors within the airways and their effects on macrophage function and susceptibility to infection. Methods: Anti-EPX (eosinophil peroxidase), anti-MARCO (macrophage receptor with collagenous structure) IgG titers, and T1 and T2 (type 1/2) cytokines were measured in 221 sputa from 143 well-characterized patients with severe asthma. Peripheral monocytes and MDMs (monocyte-derived macrophages) isolated from healthy control subjects were treated with immunoprecipitated immunoglobulins from sputa with high anti-MARCO titers or nonspecific IgG to assess uptake of Streptococcus pneumoniae or response to the bacterial product LPS. Measurements and Main Results: Anti-MARCO IgG was detected in 36% of patients, with significantly higher titers (up to 1:16) in patients with mixed granulocytic sputa, indicative of airway infections. Multivariate regression analysis confirmed increased frequency of degranulation (free eosinophil granules), increased blood eosinophils (indicative of high T2 burden), increased sputum total cell count, peripheral blood leukocytes (indicative of infection), and lymphopenia were associated with increased anti-MARCO IgG titers; IL-15 (odds ratio [OR], 1.79; confidence interval [CI], 1.19-2.70), IL-13 (OR, 1.06; CI, 1.02-1.12), and IL-12p70 (OR, 3.34; CI, 1.32-8.40) were the associated cytokines. Patients with anti-MARCO antibodies had higher chances of subsequent infective versus eosinophilic exacerbations (P = 0.01). MDMs treated with immunoprecipitated immunoglobulins (anti-MARCO+ sputa) had reduced bacterial uptake by 39% ± 15% and significantly reduced release of IL-10 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (P < 0.05) in response to an LPS stimulus. Conclusions: Autoantibodies against macrophage scavenger receptors in eosinophilic asthma airways may impede effective host defenses and lead to recurrent infective bronchitis.

Proposal for Structured Histopathology of Nasal Secretions for Endotyping Chronic Rhinosinusitis: An Exploratory Study

Background: The EPOS guidelines promote cellular analysis as a primary goal in endotyping chronic rhinosinusitis (CRS). Current analysis is mainly based on biopsy or operative tissue collection, whereas the use of sinonasal secretions for inflammatory endotyping is not advocated in clinical practice. Early endotyping is crucial though, especially regarding the increasing evidence of patient-tailored therapy. We aimed to investigate the diagnostic value and reproducibility of sinonasal secretions sampling. Methods: First, preoperative secretion analysis of 53 Caucasian CRS patients was compared to subsequent operative tissue analysis. Second, secretion analysis at two different time points was compared for 10 postoperative Caucasian CRS patients with type 2 (T2) inflammation and 10 control participants. Secretions were collected by both endoscopic aspiration and nasal blown secretions in all participants. Results: The sensitivity to detect T2 inflammation was higher in nasal aspiration samples (85%) compared to nasal blow secretions (32%). A specificity of 100% for both techniques was obtained. A 90% reproducibility for T2 eosinophil detection was found by sampling at different time points regardless of the technique. Of the T2 patients, 60% showed no T2 inflammatory pattern more than one year after endoscopic sinus surgery. Conclusions: Nasal secretion sampling, especially aspiration of nasal secretions, is useful in the detection of T2 inflammation in CRS pathology. We proposed a structured histopathology analysis to be useful in daily clinical practice, which includes Congo red staining sensitive for eosinophilic cells and free eosinophil granules. Analysis of nasal secretions enables endotyping in an early stage, allowing more directed therapy.

In Vivo ETosis of Human Eosinophils: The Ultrastructural Signature Captured by TEM in Eosinophilic Diseases.

Eosinophilic diseases, also termed eosinophil-associated diseases (EADs), are characterized by eosinophil-rich inflammatory infiltrates and extensive eosinophil degranulation with clinically relevant organ pathology. Recent evidence shows that eosinophil cytolytic degranulation, that is, the release of intact, membrane-delimited granules that arises from the eosinophil cytolysis, occurs mainly through ETosis, meaning death with a cytolytic profile and extrusion of nucleus-originated DNA extracellular traps (ETs). The ultrastructural features of eosinophil ETosis (EETosis) have been studied mostly in vitro after stimulation, but are still poorly understood in vivo. Here, we investigated in detail, by transmission electron microscopy (TEM), the ultrastructure of EETosis in selected human EADs affecting several tissues and organ systems. Biopsies of patients diagnosed with eosinophilic chronic rhinosinusitis/ECRS (frontal sinus), ulcerative colitis/UC (intestine), and hypereosinophilic syndrome/HES (skin) were processed for conventional TEM. First, we found that a large proportion of tissue-infiltrated eosinophils in all diseases (~45-65% of all eosinophils) were undergoing cytolysis with release of free extracellular granules (FEGs). Second, we compared the morphology of tissue inflammatory eosinophils with that shown by in vitro ETosis-stimulated eosinophils. By applying single-cell imaging analysis, we sought typical early and late EETosis events: chromatin decondensation; nuclear delobulation and rounding; expanded nuclear area; nuclear envelope alterations and disruption; and extracellular decondensed chromatin spread as ETs. We detected that 53% (ECRS), 37% (UC), and 82% (HES) of all tissue cytolytic eosinophils had ultrastructural features of ETosis in different degrees. Eosinophils in early ETosis significantly increased their nuclear area compared to non-cytolytic eosinophils due to excessive chromatin decondensation and expansion observed before nuclear envelope disruption. ETosis led not only to the deposition of intact granules, but also to the release of eosinophil sombrero vesicles (EoSVs) and Charcot-Leyden crystals (CLCs). Free intact EoSVs and CLCs were associated with FEGs and extracellular DNA nets. Interestingly, not all cytolytic eosinophils in the same microenvironment exhibited ultrastructure of ETosis, thus indicating that different populations of eosinophils might be selectively activated into this pathway. Altogether, our findings captured an ultrastructural signature of EETosis in vivo in prototypic EADs highlighting the importance of this event as a form of eosinophil degranulation and release of inflammatory markers (EoSVs and CLCs).

Impact of former smoking exposure on airway eosinophilic activation and autoimmunity in patients with severe asthma.

Severe eosinophilic asthma is characterised by frequent exacerbations and a relative insensitivity to steroids. Experimentally, smoking may induce eosinophilic airway inflammation, but the impact in patients with severe asthma is not clear. To investigate the association between smoking exposure in patients with severe asthma, and eosinophilic inflammation and activation, as well as airway autoimmunity and steroid responsiveness. Patients with severe asthma according to European Respiratory Society/American Thoracic Society criteria were assessed with sputum samples, analysed by cell differential count, and for the presence of free eosinophil granules (FEGs), autoantibodies against eosinophil peroxidase (EPX) and macrophage receptor with collagenous structure (MARCO). A subgroup of patients with eosinophilic airway inflammation was re-assessed after a 2-week course of prednisolone. 132 severe asthmatics were included in the study. 39 (29.5%) patients had ≥10 pack-years of smoking history: 36 (27.3%) were former smokers and three (2.3%) current smokers; and 93 (70.5%) had <10 pack-years exposure. Eosinophilic airway inflammation was more prevalent among patients with ≥10 pack-years (66.7%), compared to patients with <10 pack-years (38.7%, p=0.03), as was the level of FEGs (p=0.001) and both anti-EPX and anti-MARCO (p<0.05 and p<0.0001, respectively). Omitting current smokers did not affect these associations. Furthermore, prednisolone reduced, but did not normalise, sputum eosinophils in patients with a ≥10 pack-year smoking history. In patients with severe asthma, a former smoking history is associated with eosinophilic airway inflammation and activation and relative insensitivity to steroids, as well as airway autoimmunity.

Preparation of Veterinary Premix with Dual Active Ingredients in Granular Form

The aim of the study was to investigate the preparation of granular veterinary premix containing two different antibiotics by fluid bed granulation process. The particle size and density of the active ingredients were investigated for the proper selection of the filler material. The used antibiotics were tylosin tartarate and colistin sulfate, while isomalt sugar alcohol and cellulose materials were selected as filler and binder, respectively. The colistin sulfate was fed together with the binder solution because of its low density, fine particle size and relatively low (1.2%) concentration in the product. The type and concentration of the binder in its solution, the feeding rate and the total amount of added binder solution were optimized for obtaining dust free granules with desirable abrasion resistance and good flowability. The active ingredient content for both antibiotics was preserved even at the elevated temperature applied during the fluid granulation process.

Underestimation of airway luminal eosinophilia by quantitative sputum cytometry

RationaleOn Wright-stained sputum cytospins, eosinophil differential of ≥ 1.2% is considered abnormal, and ≥ 2.3% identifies an eosinophilic endotype. We hypothesized that failure to consider free eosinophil granules (FEG), and the re-emergence (unmasking) of eosinophilia due to various reasons underestimate the prevalence of the eosinophilic endotype.MethodsThis is a retrospective analysis of our Institutional Review Board-approved clinical sputum database. Of the 24,176 examinations of sputa from patients with various airway diseases, 17,693 were viable cell counts from 9570 patients (6604 on a single occasion, 2967 from multiple occasions). The prevalence of intact eosinophil % at 1.2 and 2.3% thresholds was first examined. Then, additional evidence of eosinophilia was assessed by semi-quantitative enumeration of FEGs. In those patients whose sputa were examined on multiple occasions (at the time of an exacerbation or after corticosteroid dose was reduced), re-emergence (unmasking) of eosinophilia was assessed.ResultsUsing the threshold of eosinophilia ≥ 1.2%, 6289/17693 (35.6%) of sputa were classified as eosinophilic. This increased to 7850/17693 (44.4%) when the presence of FEGs was considered. Using the threshold of eosinophilia ≥ 2.3%, 4647/17693 (26.3%) of sputa were classified as eosinophilic. This increased to 5435/17693 (30.7%) when the presence of FEG were considered. Extrapolating from the prevalence of re-emergence observed in the 2967 patients who had sputa examined on multiple occasions to the whole sample, we estimated that eosinophilia at 1.2% threshold would be observed in at least 60% of the samples, and a clinically relevant eosinophilia at 2.3% threshold would be observed in at least 48.5% of the samples.ConclusionsUsing a large sputum cytometry clinical database (17,693 viable cell counts), we demonstrate that a single time point intact cell count underestimates the prevalence of eosinophilia in a variety of airway diseases. The prevalence of eosinophilia increases from 35.6 to 60% (40% underestimation) at the 1.2% threshold, and from 26.3 to 48.5% (45% underestimation) at the 2.3% clinically relevant threshold, when free granules and a second examination are considered. This has important implications to identify the eosinophilic and Th2 high endotype both for clinical trials of anti-eosinophil therapies, and to select patients who may respond well to glucocorticosteroids and anti-IL5 therapies.

Immunohistochemical and ultrastructural evidence for the pathogenesis of white matter degeneration in patients with panencephalopathic-type Creutzfeldt-Jakob disease: Inducible nitric oxide synthase overexpression in bizarre astrocytes.

Excessive production of nitric oxide (NO) due to the overinduction of inducible nitric oxide synthase (iNOS) has a severe cytotoxic effect, which may relate to the pathogenesis of neurodegenerative disorders. In this study, we report the novel finding that iNOS is overinduced in a large number of bizarre astrocytes in the white matter of patients with panencephalopathic (PE)-type Creutzfeldt-Jakob disease (CJD). This study was carried out on brain tissue from seven patients with PE-type CJD. As controls, 12 normal individuals and nine patients with cerebral infarction were examined. We identified a large number of bizarre astrocytes in the degenerative cerebral white matter in PE-type CJD. Using immunohistochemistry, only bizarre astrocytes in PE-type CJD showed strong immunoreactivity for both iNOS and superoxide dismutase 1 (SOD1). Ultrastructural examination demonstrated that these bizarre astrocytes contained many free polyribosome-like granules. No significant iNOS immunoreactivity was observed in either the astrocytes of patients with cerebral infarcts or in the normal controls. This study suggests that the iNOS-overexpressing astrocytes, especially iNOS-overexpressing bizarre astrocytes, could play an important role in the development of white matter lesions in PE-type CJD. Our data also suggest that the bizarre astrocytes could be protecting themselves from the cytotoxicity of NO by producing SOD1. These immunohistochemical findings are supported by the ultrastructural observation of numerous polyribosome granules restricted to the cytoplasm of these bizarre astrocytes.

The improvement effects of Yiqi Huashi Tongluo Formula on oxidative stress and renal fibrosis of experimental CRF rats

Effects of Yiqi Huashi Tongluo Formula on oxidative stress and renal fibrosis of residual kidney were investigated in five/sixth nephrectomy rats. The rat model of chronic renal failure after nephrectomy was established by Platt method. Two weeks after the operation, the rats were randomly divided into model group, Yiqi Huashi Tongluo Formula (YHT) group, benazepril online (BH) group and sham group, with 8 rats in each group. Treatment was initiated once a day for 12 weeks after successful modeling. Animals were treated once a day with intragastric administration for 12 weeks. (Aqueous solution of free decoction granules in YHT group was 0.276 g/100 g·d. BH group benazepril hydrochloride tablet aqueous solution 0.09 mg/100 g·d gavage; sham group and model group were gavage with 1 ml/100 g normal saline). Urine was collected with a metabolic cage at the end of the 12th week, and urine protein content was detected for 24 hours. The rats were then anesthetized to extract blood from the abdominal aorta and the kidneys. The pathological changes of left kidney were observed by HE staining and Masson staining. The activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in kidney homogenate were determined by colorimetry. Western blot assay was used to detect the expressions of nuclear factor-erythroid 2-related factor 2 (Nrf2), kelch-like ech-associated protein-1 (Keap1), NADPH oxidase 4 (Nox4), transforming growth factor-binding 1(TGF-β1), type I collagen (Collagen1) and Nrf2 in the nucleus in renal tissue. Compared with sham group, model group rats had severe glomerular injury and obvious fibrosis. Levels of Scr, BUN, MDA and 24-hour urine protein excretion, protein expressions of Keap1, Nox4, TGF-β1 and Collagen1 were significantly increased (P＜0.01), while SOD activity and Nrf2 expression were significantly decreased (P＜0.01).Compared with the model group, the degree of glomerular lesion was reduced and fibrosis was less after YHT or BH intervention, and the levels of Scr, BUN, MDA, 24-hour urine protein excretion, protein expressions of Keap1, Nox4, TGF-β1 and Collagen1 were significantly decreased (P＜0.01), while SOD activity and Nrf2 expression were significantly increased (P＜0.01). Through affecting the Nrf2/Keap1 signaling pathway and down-regulating the expression of TGF-β1 protein, Yiqi Huashi Tongluo Formula improved the oxidative stress damage and fibrosis degree of residual kidney in the model rats with renal failure.

Hypereosinophilic syndrome with abundant Charcot-Leyden crystals in spleen and lymph nodes

Hypereosinophilic syndrome, which is characterized by eosinophilia in the peripheral blood, often causes various organ disorders. Charcot-Leyden crystals are recognized features of various diseases, such as parasite infection and asthma, and are known to be classic hallmarks of eosinophilic inflammation. Our recent study revealed the mechanism of Charcot-Leyden crystal formation (i.e., galectin-10 crystallization), namely the involvement of eosinophil extracellular trap cell death, a nonapoptotic cell death. Here we report an autopsy case of a 57-year-old man who had died of hypereosinophilic syndrome. We found numerous eosinophil extracellular trap cell death-associated Charcot-Leyden crystals in the spleen and lymph nodes. Observation of abdominal lymph nodes by electron microscopy revealed eosinophil extracellular traps and free extracellular granules, which are characteristic of typical eosinophil extracellular trap cell death. In this case, we observed various sizes of Charcot-Leyden crystals that were stained with anti-galectin-10 immunofluorescent staining. Further studies are required to understand the pathophysiological roles of Charcot-Leyden crystals and these may lead to the development of novel therapeutic modalities for severe eosinophilic inflammation.

Corticosteroid Withdrawal-Induced Loss of Control in Mild to Moderate Asthma Is Independent of Classic Granulocyte Activation

Loss of asthma control and asthma exacerbations are associated with increased sputum eosinophil counts. However, whether eosinophils, or the also present neutrophils, actively contribute to the accompanying inflammation has not been extensively investigated. Twenty-three patients with mild to moderate asthma were included in a standardized prospective inhaled corticosteroid (ICS) withdrawal study; 22 of the patients experienced loss of asthma control. The study assessed various immune, inflammatory, and oxidative stress parameters, as well as markers of eosinophil and neutrophil activity, in exhaled breath condensate, plasma, and sputum collected at three phases (baseline, during loss of control, and following recovery). Loss of asthma control was characterized by increased sputum eosinophils, whereas no differences were detected between the three phases for most inflammatory and oxidative stress responses. There were also no differences detected for markers of activated eosinophils (eosinophil cationic protein and bromotyrosine) and neutrophils (myeloperoxidase and chlorotyrosine). However, free eosinophilic granules and citrullinated histone H3, suggestive of eosinophil cytolysis and potentially eosinophil extracellular trap formation, were enhanced. Baseline blood eosinophils and changes in asymmetric dimethylarginine (an inhibitor of nitric oxide synthase) in plasma were found to correlate with the decrease in FEV1 percent predicted upon ICS withdrawal (both, rs= 0.46; P= .03). The clinical effect in mild to moderate asthma upon interruption of ICS therapy is not related to the classic inflammatory activation of eosinophils and neutrophils. It may, however, reflect another pathway underlying the onset of loss of disease control and asthma exacerbations. The Netherlands Trial Register; No.: NTR3316; URL: trialregister.nl/trial/3172.

Mast cell involvement in human cervical ripening

ObjectiveCervical ripening resembles an inflammatory process in many aspects, involving invasion of inflammatory cells, collagen breakdown and remodelling of the extracellular matrix. Mast cells produce a variety of inflammatory agents and are attributed a functional role in cervical ripening. The aim of this study was to examine if cervical mast cells are increased in number and stimulated during pregnancy. Study designCervical biopsies were obtained with a biopsy needle prior to surgical termination of pregnancy in the first trimester, surgery for first-trimester miscarriage, elective caesarean section, and benign gynaecological surgery in non-pregnant women. After fixation, semithin sections were prepared and stained with toluidine blue. The number of mast cells was counted under a light microscope and their secretory activity was scored (0.5–4) according to specified criteria and further visualised with electron microscopy. For pairwise comparison between groups Fisher’s nonparametric permutation test was used. ResultsThe number of mast cells was increased from 3.4 ± 1.65 mast cells per 10 visual fields in non-pregnant women to 7.70 ± 0.35 per 10 visual fields in first trimester control women (p < 0.05). The highest number of mast cells was observed at term with 10.8 ± 2.1 per 10 visual fields, a number that was significantly higher than in first trimester control women (p < 0.05). At term mast cell activity scores were 3.39 ± 0.37 compared with 2.69 ± 0.27 in control first trimester women and 2.21 ± 0.86 in women with missed miscarriage (p < 0.05). The percentage of mast cells with activity score 4 was significantly higher at term compared with in the first trimester. Free mast cell granules were predominantly observed in areas with disorganized collagen fibres. ConclusionThe findings confirm that an increased influx of mast cells to the cervix occurs during pregnancy. The stimulated mast cell secretory activity in conditions associated with cervical tissue remodelling, such as term pregnancy and symptomatic miscarriage, provides further evidence that mast cells play a physiological role in cervical ripening.

Eosinophils in anti-neutrophil cytoplasmic antibody associated vasculitis

BackgroundAnti-neutrophil cytoplasmic antibodies associated vasculitides (AAV) are characterized by autoimmune small vessel inflammation. Eosinophils are multifunctional cells with both pro-inflammatory and immunoregulatory properties. Tissue activated eosinophils secrete cyto- and chemokines and form extracellular traps (EETs), they release free granules and produce reactive oxygen species. The role of eosinophils is well established in eosinophilic granulomatosis with polyangiitis (EGPA) but very little is known about their role in granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA).MethodsThe expression of surface markers CD11c, CD11b, CD16, CD35, CD62L, CD64, CD88, Siglec-8 and CD193 and reactive oxygen species production by peripheral blood eosinophils were studied using flow cytometry. Fluorescence microscopy was used to visualize the release of eosinophil extracellular DNA traps (EETs). 98 GPA and MPA patients and 121 healthy controls were included in the study.ResultsBoth GPA and MPA patients had decreased frequency of eosinophils in peripheral blood compared with healthy controls (p < 0.0001), which could not solely be explained by corticosteroid treatment. The patient’s eosinophils showed increased surface expression of the Fc receptors CD16 (p < 0.0001) and CD64 (p = 0.0035) as well as CCR3 (CD193) (p = 0.0022). Decreased expression was found of the complement receptors CD35 (p = 0.0022), CD88 (p < 0,0001) as well as CD11c (p < 0,0001), CD11b (p = 0.0061) and Siglec-8 (p = 0,0015). Moreover, GPA and MPA eosinophils, showed decreased capacity to produce ROS (p < 0.0001). ANCA stimulation of eosinophils from GPA and MPA patients after C5a priming enhanced EETosis (p = 0,0088).ConclusionsThe percentage of eosinophils were decreased in peripheral blood in GPA and MPA patients and showed altered surface marker expression and function. The enhanced EETosis after ANCA stimulation, suggests that eosinophil can contribute to the autoantibody driven inflammatory process.

Design and evaluation of rhubarb total free anthraquinones oral colon-specific drug delivery granules to improve the purgative effect

Rhubarb is commonly used as a cathartic in Asian countries. However, researchers have devotedextensive concerns to the quality control and safety of rhubarb and traditional Chinese preparations composed of rhubarb due to the instable purgative effect and potential nephrotoxicity of anthraquinones. In this study, we aimed to prepare rhubarb total free anthraquinones (RTFA) oral colon-specific drug delivery granules (RTFA-OCDD-GN) to delivery anthraquinones to colon to produce purgative effect. RTFA-OCDD-GN were prepared using chitosan and Eudragit S100 through a double-layer coating process and the formulation was optimized. Continuous release studies were performed in a simulated gastric fluid (pH 1.2), followed by a small-intestinal fluid (pH 6.8) and a colonic fluid (pH 7.4, containing rat cecal contents). The purgative effect test was performed in rats. The dissolution profile of RTFA-OCDD-GN showed that the accumulative dissolution rate of RTFA was about 83.0% in the simulated colonic fluid containing rat cecal contents and only about 9.0% in the simulated gastrointestinal fluids. And the RTFA-OCDD-GN could produce the comparative purgative activity as rhubarb, suggesting it could deliver the free AQs to the colon. The RTFA-OCDD-GN was a useful media to enhance the purgative activity of free anthraquinones after administered orally.

Charcot-Leyden crystal formation is closely associated with eosinophil extracellular trap cell death

AbstractProtein crystallization in human tissue rarely occurs. Charcot-Leyden crystals (CLCs) were described in various eosinophilic diseases >150 years ago, but our understanding of CLC formation still remains limited. In this study, we demonstrate that CLCs observed in varied inflamed human tissues are closely associated with eosinophil cell-free granules and nuclear envelope/plasma membrane disintegration with release of filamentous chromatin (extracellular traps), typical morphologies of a regulated pathway of extracellular trap cell death (ETosis). During the process of eosinophil ETosis, eccentrically localized cytoplasmic and perinuclear CLC protein (galectin-10) is homogeneously redistributed in the cytoplasm. Rapid (1-2 minutes) formation of intracytoplasmic CLCs was observed using time-lapse imaging. Plasma membrane rupture enabled the release of both intracellularly formed CLCs and soluble galectin-10 that further contributed to formation of CLCs extracellularly, in parallel with the expulsion of free intact granules and extracellular traps. CLC formation and galectin-10 release were dependent on nicotinamide adenine dinucleotide phosphate oxidase activation. To our knowledge, this is the first demonstration of natural formation of CLCs in association with an active physiological process (ie, ETosis). These results indicate that dynamic changes in intracellular localization and release of galectin-10 contribute to CLC formation in vivo and suggest that CLC/galectin-10 might serve as an indicator of ETosis.