MRGPRX2 and Immediate Drug Hypersensitivity: Insights From Cultured Human Mast Cells.

BACKGROUND AND OBJECTIVES: Mast cell (MC) degranulation via activation of the Mas-related G protein-coupled receptor X2 (MRGPRX2) plays a key role in immediate drug hypersensitivity (IDH). However, data in humans are limited to observations in specific cell lines. Objective: To study the usefulness of silencing MRGPRX2 in human MCs with the aim of further unveiling the MRGPRX2 pathway in IDH. METHODS: MCs were cultured from CD34+ progenitor cells obtained from peripheral blood (PBCMCs) and incubated with substance P (as a positive control), rocuronium, moxifloxacin, morphine, or amoxicillin. Immunophenotyping of the cells included flow cytometry and microscopy analyses of the expression of CD117, CD203c, and MRGPRX2. Intracellular calcium was measured using Fluo-4. Degranulation was analyzed by quantifying CD63 expression. For MRGPRX2 silencing, MCs were electroporated with Dicer small interference RNAs. RESULTS: Incubation of MCs with substance P, morphine, and moxifloxacin increased intracellular calcium levels and triggered MC degranulation, which, for the drugs, is almost completely abolished by selective MRGPRX2 silencing. Despite an increase in intracellular calcium in MRGPRX2+ cells, incubation with nontoxic concentrations of rocuronium does not result in degranulation of PBCMCs. Amoxicillin has no effect on PBCMCs. CONCLUSION: The use of MRGPRX2 silencing in human MCs can provide important insights into the role of MRGPRX2 in the pathogenesis of IDH. As induction of calcium signals does not necessarily translate into a secretory response, measurement of the degranulation reaction seems more meaningful in the context of drug testing.

Altered embryotrophic capacities of the bovine oviduct under elevated free fatty acid conditions: an in vitro embryo--oviduct co-culture model.

Maternal metabolic stress conditions are of growing importance in both human and dairy cattle settings as they can have significant repercussions on fertility. Upregulated lipolysis is a common trait associated with metabolic disorders and results in systemically elevated concentrations of non-esterified fatty acids (NEFAs). The effects of high NEFA concentrations on the follicular environment, oocyte and embryo development is well documented. However, knowledge on the effects of NEFAs within the oviduct, representing the initial embryonic growth environment, is currently lacking. Therefore, the experiments outlined here were designed to obtain fundamental insights into both the direct and indirect interactions between NEFAs, bovine oviductal cells and developing zygotes. Hence, zygotes were co-cultured with NEFA-pre-exposed bovine oviductal cells or subjected to simultaneous NEFA exposure during the co-culture period. The outcome parameters assessed were embryo development with cleavage (48h post insemination (pi)), morula (120-126h pi) and blastocyst (192h pi) rates, as well as morula intracellular lipid content and blastocyst quality using Bodipy and differential staining respectively. Our data suggest a direct embryotoxicity of NEFAs as well as impaired embryo development through a reduced oviductal ability to support and protect early embryo development.

The effect of elevated non-esterified fatty acid concentrations on bovine spermatozoa and on oocyte in vitro fertilisation.

Elevated non-esterified fatty acid (NEFA) concentrations, present in follicular and oviductal fluid, have been postulated as a causative link between metabolic disorders and subfertility. High NEFA conditions can directly disrupt oocyte maturation and developmental capacity after fertilisation. However, their influence on sperm function and the fertilisation process is not known. This study investigated the fertilisation process under high NEFA conditions. To differentiate between effects on both spermatozoa and oocytes or on spermatozoa only, different experiments were conducted. In the first experiment both gametes were simultaneously incubated during IVF under different conditions: (1) NEFA-free, solvent-free control conditions, (2) solvent control, (3) physiological concentrations of oleic (OA), palmitic (PA) and stearic (SA) acids or (4) pathophysiological concentrations of OA, PA and SA. In the second experiment spermatozoa were incubated (4h) under the same treatment conditions prior to routine IVF. Gamete co-incubation resulted in reduced fertilisation and cleavage rates and increased prevalence of polyspermy. In the second experiment embryo developmental capacity and quality were not affected, although sperm motility and plasma membrane integrity were decreased. In conclusion, lipolytic conditions affected the fertilisation process mainly through an effect on the oocyte. Spermatozoa were still able to fertilise even though these conditions reduced sperm function.

FABP4 blocker attenuates colonic hypomotility and modulates white adipose tissue-derived hormone levels in mouse models mimicking constipation-predominant IBS.

BACKGROUND: The role of fatty acid binding protein 4 (FABP4) in lower gastrointestinal (GI) motility is unknown. We aimed to verify the effect of inhibition of FABP4 on GI transit in vivo, and to determine the expression of FABP4 in mouse and human tissues. METHODS: Fatty acid binding protein 4 inhibitor, BMS309403, was administered acutely or chronically for 6 and 13 consecutive days and its effect on GI transit was assessed in physiological conditions and in loperamide-induced constipation. Intracellular recordings were made to examine the effects of BMS309403 on colonic excitatory and inhibitory junction potentials. Abdominal pain was evaluated using behavioral pain response. Localization and expression of selected adipokines were determined in the mouse colon and serum using immunohistochemistry and Enzyme-Linked ImmunoSorbent Assay respectively. mRNA expression of FABP4 and selected adipokines in colonic and serum samples from irritable bowel syndrome (IBS) patients and control group were assessed. KEY RESULTS: Acute injection of BMS309403 significantly increased GI motility and reversed inhibitory effect of loperamide. BMS309403 did not change colonic membrane potentials. Chronic treatment with BMS309403 increased the number of pain-induced behaviors. In the mouse serum, level of resistin was significantly decreased after acute administration; no changes in adiponectin level were detected. In the human serum, level of adiponectin and resistin, but not of FABP4, were significantly elevated in patients with constipation-IBS (IBS-C). FABP4 mRNA expression was significantly downregulated in the human colon in IBS-C. CONCLUSIONS AND INFERENCES: Fatty acid binding protein 4 may be involved in IBS pathogenesis and become a novel target in the treatment of constipation-related diseases.

Antibody-Induced Internalization of the Human Respiratory Syncytial Virus Fusion Protein.

Respiratory syncytial virus (RSV) infections remain a major cause of respiratory disease and hospitalizations among infants. Infection recurs frequently and establishes a weak and short-lived immunity. To date, RSV immunoprophylaxis and vaccine research is mainly focused on the RSV fusion (F) protein, but a vaccine remains elusive. The RSV F protein is a highly conserved surface glycoprotein and is the main target of neutralizing antibodies induced by natural infection. Here, we analyzed an internalization process of antigen-antibody complexes after binding of RSV-specific antibodies to RSV antigens expressed on the surface of infected cells. The RSV F protein and attachment (G) protein were found to be internalized in both infected and transfected cells after the addition of either RSV-specific polyclonal antibodies (PAbs) or RSV glycoprotein-specific monoclonal antibodies (MAbs), as determined by indirect immunofluorescence staining and flow-cytometric analysis. Internalization experiments with different cell lines, well-differentiated primary bronchial epithelial cells (WD-PBECs), and RSV isolates suggest that antibody internalization can be considered a general feature of RSV. More specifically for RSV F, the mechanism of internalization was shown to be clathrin dependent. All RSV F-targeted MAbs tested, regardless of their epitopes, induced internalization of RSV F. No differences could be observed between the different MAbs, indicating that RSV F internalization was epitope independent. Since this process can be either antiviral, by affecting virus assembly and production, or beneficial for the virus, by limiting the efficacy of antibodies and effector mechanism, further research is required to determine the extent to which this occurs in vivo and how this might impact RSV replication.IMPORTANCE Current research into the development of new immunoprophylaxis and vaccines is mainly focused on the RSV F protein since, among others, RSV F-specific antibodies are able to protect infants from severe disease, if administered prophylactically. However, antibody responses established after natural RSV infections are poorly protective against reinfection, and high levels of antibodies do not always correlate with protection. Therefore, RSV might be capable of interfering, at least partially, with antibody-induced neutralization. In this study, a process through which surface-expressed RSV F proteins are internalized after interaction with RSV-specific antibodies is described. One the one hand, this antigen-antibody complex internalization could result in an antiviral effect, since it may interfere with virus particle formation and virus production. On the other hand, this mechanism may also reduce the efficacy of antibody-mediated effector mechanisms toward infected cells.

Elevated non-esterified fatty acid concentrations hamper bovine oviductal epithelial cell physiology in three different in vitro culture systems.

Elevated non-esterified fatty acids (NEFAs) have been recognized as an important link between lipolytic metabolic conditions and impaired fertility in high-yielding dairy cows. However, NEFA effects on the oviductal micro-environment currently remain unknown. We hypothesize that elevated NEFAs may contribute to the complex pathology of subfertility by exerting a negative effect on bovine oviductal epithelial cell (BOEC) physiology. Therefore, the objectives of this study were to elucidate direct NEFA effects on BOEC physiology in three different in vitro cell culture systems. Bovine oviductal epithelial cells (four replicates) were mechanically isolated, pooled, and cultured as conventional monolayers, as explants, and in a polarized cell culture system with Dulbecco's modified Eagle's medium/F12-based culture medium. Bovine oviductal epithelial cells were exposed to an NEFA mixture of oleic, stearic, and palmitic acids for 24 hours at both physiological and pathologic concentrations. A control (0 µM NEFA) and a solvent control (0 µM NEFA + 0.45% ethanol) group were implemented. Bovine oviductal epithelial cells physiology was assessed by means of cell number and viability, a sperm binding assay, transepithelial electric resistance (TER), and a wound-healing assay. Bovine oviductal epithelial cell morphology was assessed by scanning electron microscopy on cell polarity, presence of microvilli and cilia, and monolayer integrity. Bovine oviductal epithelial cell number was negatively affected by increasing NEFAs, however, cell viability was not. Sperm binding affinity significantly decreased with increasing NEFAs and tended (P = 0.051) to be more affected by the direction of NEFA exposure in the polarized cell culture system. The absolute TER increase after NEFA exposure in the control (110 ± 11 Ω.cm(2)) was significantly higher than that in all the other treatments and was also different depending on the exposure side. Bidirectional exposed monolayers were even associated with a significant TER reduction (-15 ± 10 Ω.cm(2); P < 0.05). Cell proliferation capacity showed a decreased cell migration with increasing NEFA concentrations but was irrespective of the exposure side. Bovine oviductal epithelial cell morphology was not affected. In conclusion, in an in vitro setting, NEFAs exert a negative effect on BOEC physiology but not morphology. Ultimately, these physiological alterations in its microenvironment may result in suboptimal development of the pre-implantation embryo and a reduced reproductive outcome in dairy cattle.

Fibrillin-1 impairment enhances blood-brain barrier permeability and xanthoma formation in brains of apolipoprotein E-deficient mice.

We recently reported that apolipoprotein E (ApoE)-deficient mice with a mutation in the fibrillin-1 gene (ApoE(-/-)Fbn1(C1039G+/-)) develop accelerated atherosclerosis with enhanced inflammation, atherosclerotic plaque rupture, myocardial infarction and sudden death. In the brain, fibrillin-1 functions as an attachment protein in the basement membrane, providing structural support to the blood-brain barrier (BBB). Here, we investigated whether fibrillin-1 impairment affects the permeability of the BBB proper and the blood-cerebrospinal fluid barrier (BCSFB), and whether this leads to the accelerated accumulation of lipids (xanthomas) in the brain. ApoE(-/-) (n=61) and ApoE(-/-)Fbn1(C1039G+/-) (n=73) mice were fed a Western-type diet (WD). After 14 weeks WD, a significantly higher permeability of the BBB was observed in ApoE(-/-)Fbn1(C1039G+/-) mice compared to age-matched ApoE(-/-) mice. This was accompanied by leukocyte infiltration, enhanced expression of pro-inflammatory cytokines, matrix metalloproteinases and transforming growth factor-ß, and by decreased expression of tight junction proteins claudin-5 and occludin. After 20 weeks WD, 83% of ApoE(-/-)Fbn1(C1039G+/-) mice showed xanthomas in the brain, compared to 23% of their ApoE(-/-) littermates. Xanthomas were mainly located in fibrillin-1-rich regions, such as the choroid plexus and the neocortex. Our findings demonstrate that dysfunctional fibrillin-1 impairs BBB/BCSFB integrity, facilitating peripheral leukocyte infiltration, which further degrades the BBB/BCSFB. As a consequence, lipoproteins can enter the brain, resulting in accelerated formation of xanthomas.

Interaction between differential gene expression profile and phenotype in bovine blastocysts originating from oocytes exposed to elevated non-esterified fatty acid concentrations.

Maternal metabolic disorders linked to lipolysis are major risk factors for reproductive failure. A notable feature of such disorders is increased non-esterified fatty acid (NEFA) concentrations in the blood, which are reflected in the ovarian follicular fluid. Elevated NEFA concentrations impact on the maturing oocyte and even alter subsequent embryo physiology. The aetiological mechanisms have not been fully elucidated. Therefore, in the present study, bovine in vitro maturing cumulus-oocyte complexes were exposed (24 h) to three different maturation treatments containing: (1) physiological (72 µM) NEFA concentrations (=control); (2) elevated (75 µM) stearic acid (SA) concentrations (=HIGH SA); and (3) elevated (425 µM) NEFA concentrations (=HIGH COMBI). Zygotes were fertilised and cultured following standard procedures. Transcriptomic analyses in resulting Day 7.5 blastocysts revealed that the major pathways affected are related to lipid and carbohydrate metabolism in HIGH COMBI embryos and to lipid metabolism and cell death in HIGH SA embryos. Furthermore, lower glutathione content and a reduced number of lipid droplets per cell were observed in HIGH SA-exposed oocytes and resulting morulae, respectively, compared with their HIGH COMBI-exposed counterparts. Vitrified embryos originating from HIGH SA-exposed oocytes tended to exhibit lower survival rates compared with controls. These data suggest possible mechanisms explaining why females across species suffering lipolytic disorders experience difficulties in conceiving.

Therapeutic potential of VEGF and VEGF-derived peptide in peripheral neuropathies.

Besides its prominent role in angiogenesis, the vascular endothelial growth factor (VEGF) also exerts important protective effects on neurons. In particular, mice expressing reduced levels of VEGF suffer from late-onset motor neuron degeneration, whereas VEGF delivery significantly delays motor neuron death in ALS mouse models, at least partly through neuroprotective effects. Additionally, VEGF protects dorsal root ganglion (DRG) neurons against paclitaxel-induced neurotoxicity. Here, we demonstrate that VEGF also protects DRG neurons against hyperglycemia-induced neuronal stress as a model of diabetes-induced peripheral neuropathy. Specifically, VEGF decreased expression of the stress-related gene activating transcription factor 3 (ATF3) in DRG neurons isolated from streptozotocin-induced diabetic mice (ex vivo) and in isolated DRG neurons exposed to high glucose concentrations (in vitro). In vivo, local VEGF application also protected against paclitaxel- and diabetes-induced neuropathies without causing side effects. A small synthetic VEGF mimicking pentadecapeptide (QK) exerted similar effects on DRG cultures: the peptide reduced ATF3 expression in vitro and ex vivo in paclitaxel- and hyperglycemia-induced models of neuropathy to a similar extent as the full-length recombinant VEGF protein. By using transgenic mice selectively overexpressing the VEGF receptor 2 in postnatal neurons, these neuroprotective effects were shown to be mediated through VEGF receptor 2. Overall, these results underscore the potential of VEGF and VEGF-derived peptides for the treatment of peripheral neuropathies.

Selective visualisation of neuroepithelial bodies in vibratome slices of living lung by 4-Di-2-ASP in various animal species.

Pulmonary neuroepithelial bodies (NEBs) are extensively innervated organoid groups of neuroendocrine cells that lie in the epithelium of intrapulmonary airways. Our present understanding of the morphology of NEBs is comprehensive, but direct physiological studies have so far been challenging because the extremely diffuse distribution of NEBs makes them inaccessible in vivo and because a reliable in vitro model is lacking. Our aim has been to optimise an in vitro method based on vibratome slices of living lungs, a model that includes NEBs, the surrounding tissues and at least part of their complex innervation. This in vitro model offers satisfactory access to pulmonary NEBs, provided that they can be differentiated from other tissue elements. The model was first optimised for living rat lung slices. Neutral red staining, reported to stain rabbit NEBs, proved unsuccessful in rat slices. On the other hand, the styryl pyridinium dye, 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (4-Di-2-ASP), showed brightly fluorescent cell groups, reminiscent of NEBs, in the airway epithelium of living lung slices from rat. In addition, nerve fibres innervating the NEBs were labelled. The reliable and specific labelling of pulmonary NEBs by 4-Di-2-ASP was corroborated by immunostaining for protein gene-product 9.5. Live cell imaging and propidium iodide staining further established the acceptable viability of 4-Di-2-ASP-labelled NEB cells in lung slices, even over long periods. Importantly, the in vitro model and 4-Di-2-ASP staining procedure for pulmonary NEBs appeared to be equally reproducible in mouse, hamster and rabbit lungs. Diverse immunocytochemical procedures could be applied to the lung slices providing an opportunity to combine physiological and functional morphological studies. Such an integrated approach offers additional possibilities for elucidating the function(s) of pulmonary NEBs in health and disease.