Quantity and Quality of Basophil RNA Depend on the RNA Extraction Technique.

Basophils have been suggested to express low quantities of RNA, challenging the study of gene expression within these cells. However, the purification technique employed might have an impact on the quantity and quality of RNA purified from basophils. This chapter describes a method which gives an optimal RNA output using a TRIzol-based method in contrast to a commercial kit.

Flow cytometric basophil activation tests: Staining of exteriorized basophil granule matrix by fluorescent avidin versus appearance of CD63.

BACKGROUND: Staining of exteriorized basophil granule matrix by fluorescent avidin might be a reliable technique to monitor basophil degranulation. This study compares the avidin-based technique with the upregulation of CD203c and appearance of CD63 in response to various stimuli. METHODS: Fourteen individuals responsive to anti-IgE, nine healthy controls, and five birch pollen-allergic patients, and five nonresponders were studied. Activation experiments included anti-IgE, fMLP, interleukin-(IL)-3, and birch pollen allergen. Basophil activation/degranulation was analyzed by flow cytometry and microscopy using anti-CD63, anti-CD203c, and avidin. RESULTS: Stimulation with anti-IgE, fMLP, and relevant allergen results in upregulation of CD203c, CD63 appearance, and an increase in avidin binding. In response to anti-IgE and allergen, upregulation of CD203c peaks within 10 min, CD63 and avidin binding reach a plateau after 10-20 min. CD63 staining leads to a bimodal distribution, avidin staining causes a unimodal shift with a less clear discrimination between degranulating and nondegranulating cells. In response to fMLP, upregulation of CD203c and CD63 and avidin binding are maximal after 2.5 min. Following incubation with anti-IgE and fMLP, percentages of CD203c+ cells are higher than those of CD63+ and avidin+ cells, pointing to a dissociation between activation and degranulation. Percentages of CD63+ cells are systemically higher than those of avidin+ cells. Incubated with IL-3 only upregulates CD203c, while no CD63 or avidin binding is observed. CONCLUSIONS: Staining of exteriorized proteoglycans by avidin is a reliable technique to quantify basophil degranulation but offers no added value when compared to traditional assays that use CD63 as a readout.

Anti-degranulation response of herbal formula in RBL-2H3 cells.

Allergic diseases not only bring serious economic burden to the patients, but also consume a lot of substantial resources of social medical systems. Thus, the prevention and treatment of allergic diseases are imperative. In this study, the anti-degranulation activity of herbal formula was evaluated using the rat basophil leukemia cells (RBL-2H3) as in vitro model. The morphological and biophysical properties of RBL-2H3 cells before and after treatment with herbal formula were also determined. Notably, the herbal formula exhibits clearly inhibited degranulation by RBL-2H3 cells in a concentration-dependent manner without cytotoxic effect. Therefore, this herbal formula can be used as an alternative and promising therapeutic agent to ameliorate allergic diseases.

Engineered Nanostructures of Haptens Lead to Unexpected Formation of Membrane Nanotubes Connecting Rat Basophilic Leukemia Cells.

A recent finding reports that co-stimulation of the high-affinity immunoglobulin E (IgE) receptor (FcεRI) and the chemokine receptor 1 (CCR1) triggered formation of membrane nanotubes among bone-marrow-derived mast cells. The co-stimulation was attained using corresponding ligands: IgE binding antigen and macrophage inflammatory protein 1α (MIP1 α), respectively. However, this approach failed to trigger formation of nanotubes among rat basophilic leukemia (RBL) cells due to the lack of CCR1 on the cell surface (Int. Immunol. 2010, 22 (2), 113-128). RBL cells are frequently used as a model for mast cells and are best known for antibody-mediated activation via FcεRI. This work reports the successful formation of membrane nanotubes among RBLs using only one stimulus, a hapten of 2,4-dinitrophenyl (DNP) molecules, which are presented as nanostructures with our designed spatial arrangements. This observation underlines the significance of the local presentation of ligands in the context of impacting the cellular signaling cascades. In the case of RBL, certain DNP nanostructures suppress antigen-induced degranulation and facilitate the rearrangement of the cytoskeleton to form nanotubes. These results demonstrate an important scientific concept; engineered nanostructures enable cellular signaling cascades, where current technologies encounter great difficulties. More importantly, nanotechnology offers a new platform to selectively activate and/or inhibit desired cellular signaling cascades.

Comparison of optical data from flow cytometry and microscopy of leukocytes after exposure to specific reagents.

Flow cytometry and microscopy are equally important in cell analysis. However, few reports have compared the optical data (cell size, internal complexity and fluorescent signal) from flow cytometry and microscopy. In this study, we compared the scattergram from XN-series, a flow cytometry based hematology analyzer with microscopic images of similarly treated leukocytes, and investigated the correlation between the appearance in the scattergram and cell size, internal complexity and fluorescence intensity. Healthy human peripheral blood was analyzed using the XN analyzer. For microscopic comparison, five types of leukocytes (monocytes, lymphocytes, basophils, neutrophils and eosinophils) were isolated from the peripheral blood by centrifugation and magnetic cell sorting, treated with a specific reagent and analyzed using electron microscopy, laser microscopy and confocal laser microscopy. Cell size, residual internal structures and fluorescence intensity correlated with intensity of forward-scattering, side scattering and fluorescent light. In this study, optical data from a clinically used hematology analyzer was clarified using microscopic images.

Chlorogenic acid alters the biological characteristics of basophil granulocytes by affecting the fluidity of the cell membrane and triggering pseudoallergic reactions.

It is not clear whether pseudoallergic reactions are caused by similar mechanisms as type I allergic reactions. 3­Caffeoylquinic acid (chlorogenic acid) is an active ingredient in traditional Chinese medicines used for antibacterial, anti-inflammatory and cholagogic purposes. It is assumed to be the reason for the high allergic reaction rates associated with certain traditional Chinese medicine injection solutions. The aim of the present study was to investigate the possible mechanisms through which chlorogenic acid triggers pseudoallergic reactions. The fluidity of the cell membrane was investigated using fluorescence recovery after photobleaching. Western blot analysis was used to measure the phosphorylation levels of the Spleen tyrosine kinase (Syk) protein and Fluo­3/AM fluorescent probes were used to investigate the influx of calcium ions. In addition, fluorescence microscopy and phalloidin were used to determine F­actin depolymerization levels. The secretion rate of ß­hexosaminidase by RBL­2H3 cells clearly increased following treatment with chlorogenic acid and the levels of cytoskeletal disintegration were also markedly increased. Furthermore, we detected an increase in the intracellular calcium ion concentration along with distinct changes in Syk protein phosphorylation and cellular F­actin. These changes indicated that chlorogenic acid affected the restructuring of the cytoskeleton and played a role in cell degranulation. In conclusion, chlorogenic acid may lead to the aggregation of lipid rafts on the cell membrane surface by altering RBL­2H3 cell membrane fluidity, thus triggering Syk­related signal transduction and inducing a truncated type I like allergic reaction.

Xenotransplanted human prostate carcinoma (DU145) cells develop into carcinomas and cribriform carcinomas: ultrastructural aspects.

Androgen-independent, human prostate carcinoma cells (DU145) develop into solid, carcinomatous xenotransplants on the diaphragm of nu/nu mice. Tumors encompass at least two poorly differentiated cell types: a rapidly dividing, eosinophilic cell comprises the main cell population and a few, but large basophilic cells able to invade the peritoneal stroma, the muscular tissue, lymph vessels. Poor cell contacts, intracytoplasmic lumina, and signet cells are noted. Lysosomal activities are reflected by entoses and programmed cell deaths forming cribriform carcinomas. In large tumors, degraded cells may align with others to facilitate formation of blood supply routes. Malignant cells would spread via ascites and through lymphatics.

Molecular analysis of the membrane insertion domain of proteinase 3, the Wegener's autoantigen, in RBL cells: implication for its pathogenic activity.

PR3, also called myeloblastin, is a neutrophil serine protease that promotes myeloid cell proliferation by cleaving the cyclin-dependent kinase inhibitor p21(cip1/waf1). In addition, it is the target of ANCA in GPA, a necrotizing vasculitis. Anti-PR3 ANCA binding to membrane-expressed PR3 triggers neutrophil activation, potentiating vascular inflammation. This study performed in RBL cells identifies the structural motifs of PR3 membrane anchorage and examines its impact on PR3 proinflammatory and proliferative functions. With the use of MD simulations and mutagenesis, we demonstrate that the mutations of four hydrophobic (F180, F181, L228, F229) or four basic (R193, R194, K195, R227) amino acids abrogated PR3 membrane anchorage. The hydrophobic patch-deficient PR3 mutant (PR34H4A) was still able to cleave the synthetic substrate Boc-Ala-Pro-Val in cell lysates. However, in contrast to WT PR3, PR34H4A was not expressed at the plasma membrane after degranulation and failed to cleave extracellular fibronectin, was not externalized after apoptosis and did not impair macrophage phagocytosis of apoptotic cells, did not promote myeloid cell proliferation and failed to cleave p21/waf1. PR3 membrane insertion appears to be pivotal for its proinflammatory activities, such as extracellular proteolysis and impairment of apoptotic cell clearance, but also for myeloid cell proliferation. Targeting membrane-associated PR3 might constitute a novel, anti-inflammatory therapeutic strategy in inflammatory disease especially in vasculitis, but this approach has to be validated in mature neutrophils.

Juvenile ALS with basophilic inclusions is a FUS proteinopathy with FUS mutations.

BACKGROUND: Juvenile amyotrophic lateral sclerosis (ALS) with basophilic inclusions is a form of ALS characterized by protein deposits in motor neurons that are morphologically and tinctorially distinct from those of classic sporadic ALS. The nosologic position of this type of ALS in the molecular pathologic and genetic classification of ALS is unknown. METHODS: We identified neuropathologically 4 patients with juvenile ALS with basophilic inclusions and tested the hypothesis that specific RNA binding protein pathology may define this type of ALS. Immunohistochemical findings prompted us to sequence the fused in sarcoma (FUS) gene. RESULTS: Motor symptoms began between ages 17 and 22. Disease progression was rapid without dementia. No family history was identified. Basophilic inclusions were strongly positive for FUS protein but negative for TAR DNA binding protein 43 (TDP-43). Granular and compact FUS deposits were identified in glia and neuronal cytoplasm and nuclei. Ultrastructure of aggregates was in keeping with origin from fragmented rough endoplasmic reticulum. Sequencing of all 15 exons of the FUS gene in 3 patients revealed a novel deletion mutation (c.1554_1557delACAG) in 1 individual and the c.1574C>T (P525L) mutation in 2 others. CONCLUSION: Juvenile ALS with basophilic inclusions is a FUS proteinopathy and should be classified as ALS-FUS. The FUS c.1574C>T (P525L) and c.1554_1557delACAG mutations are associated with this distinct phenotype. The molecular genetic relationship with frontotemporal lobar degeneration with FUS pathology remains to be clarified.

Extensive FUS-immunoreactive pathology in juvenile amyotrophic lateral sclerosis with basophilic inclusions.

Juvenile amyotrophic lateral sclerosis (ALS) with basophilic inclusions is a well-recognized entity. However, the molecular underpinnings of this devastating disease are poorly understood. Here, we present genetic and neuropathological characterizations in two young women with fatal rapidly progressive ALS with basophilic inclusions. In one case, a germline mutation (P525L) was detected in the fused in sarcoma/translocated in liposarcoma (FUS/TLS) gene, whereas no mutation was identified in the other case. Postmortem examination in both cases revealed severe loss of spinal motor neurons with remaining neurons showing basophilic inclusions that contain abnormal aggregates of FUS proteins and disorganized intracellular organelles, including mitochondria and endoplasmic reticulum. In both patients, the FUS-positive inclusions were also detected in neurons in layers IV-V of cerebral cortex and several brainstem nuclei. In contrast, spinal motor neurons in patients with late-onset sporadic ALS showed no evidence of abnormal accumulation of FUS protein. These results underscore the importance of FUS mutations and pathology in rapidly progressive juvenile ALS. Furthermore, our study represents the first detailed characterizations of neuropathological findings in rapidly progressive juvenile ALS patients with a mutation in the FUS/TLS gene.

Basophils in the giant papillae of chronic allergic keratoconjunctivitis.

AIMS: The essential role of basophils as an initiator of chronic allergic reaction has been elucidated in mouse models. The aim of this present study was to analyse the in situ immunolocalisation of basophils and other relevant inflammatory cells in chronic allergic keratoconjunctivitis. METHODS: Transmission electron microscopic (TEM) analysis was carried out to examine the existence of basophils in the giant papillae obtained from atopic keratoconjunctivitis (AKC) and vernal keratoconjunctivitis (VKC) patients. Cryostat sections of giant papillae were immunostained with basophil-specific antibody BB-1, and with anti-CD4, anti-CD8, anti-CD20, anti-major basic protein (MBP), anti-IgE and anti-FcepsilonRI-beta antibodies. RESULTS: TEM analysis confirmed the existence of basophils in the giant papillae. Small clusters of basophils were observed in the substantia propria of giant papillae, especially at the vicinity of vascular endothelium and subepithelial regions. BB-1-positive basophil clusters were surrounded by T cells, B cells, IgE-positive cells and MBP-positive eosinophils. No BB-1-positive basophils were observed in the control conjunctivae. CONCLUSION: Basophils may infiltrate from either vascular endothelium into the giant papillae. The existence of basophils at the centre of inflammatory cells suggests the role of basophils as an initiator of chronic allergic conjunctivitis.

La apariencia óptica de las células y los tejidos patológicos desentrañada a través del microscopio electrónico. Bases ultraestructurales de la microscopía óptica / The light microscopic appearance of pathologic cells and tissues revealed through the electron microscope

El microscopio electrónico aporta información detallada sobre la estructura de las células y los tejidos, así como sobre las variaciones funcionales que experimentan en situaciones normales y patológicas. Estos datos tienen una traducción directa en el aspecto de esas mismas células en las secciones histológicas convencionales. En este artículo, se revisan las principales características ópticas del citoplasma, del núcleo y de la matriz extracelular y se correlacionan con los rasgos ultraestructurales subyacentes. Conocer el substrato ultraestructural puede ser de gran utilidad para conseguir que la información que se obtiene con el microscopio óptico sea más cuantiosa y más precisa

The electron microscope provides detailed information on cell and tissue structure, as well as on their functional modifications under physiologic and pathologic conditions. All these findings have a direct translation into how these same cells look under the light microscope. In the present article, the main light microscopic features of the cell cytoplasm, the nucleus, and the extracellular matrix are reviewed, in the context of the underlying ultrastructural changes. Acquaintance with this ultrastructural background may prove extremely helpful to withdraw more abundant and more precise information from conventional light microscopy

Role of human mast cells and basophils in bronchial asthma.

Mast cells and basophils are the only cells expressing the tetrameric (alphabetagamma2) structure of the high affinity receptor for IgE (FcepsilonRI) and synthesizing histamine in humans. Human FcepsilonRI+ cells are conventionally considered primary effector cells of bronchial asthma. There is now compelling evidence that these cells differ immunologically, biochemically, and pharmacologically, which suggests that they might play distinct roles in the appearance and fluctuation of the asthma phenotype. Recent data have revealed the complexity of the involvement of human mast cells and basophils in asthma and have shed light on the control of recruitment and activation of these cells in different lung compartments. Preliminary evidence suggests that these cells might not always be detrimental in asthma but, under some circumstances, they might exert a protective effect by modulating certain aspects of innate and acquired immunity and allergic inflammation.