Platelets retain function and can be stored following disruption of human leucocyte antigens.

BACKGROUND AND OBJECTIVES: Antibodies to human leucocyte antigen (HLA) Class-I antigens can lead to refractoriness to platelet transfusion. Although this can be overcome by transfusion of HLA-compatible platelets, they are not always available. Disruption of HLA antigens on platelets by acid treatment may be a suitable alternative when no other components are available. The aim of this study was to assess the effect of HLA disruption and subsequent storage of platelet components. MATERIALS AND METHODS: Platelet components were treated with 0.9% saline or citric acid solution (pH 3.0), and then stored until expiry (Day 7). HLA and platelet glycoprotein expression, platelet viability, activation and sialylation were measured by flow cytometry. Release of soluble factors was measured by ELISA and metabolism by biochemistry analyser. Reactivity to patient anti-sera containing anti-HLA antibodies was measured using platelet immunofluorescence tests (PIFTs) and monoclonal antibody immobilization of platelet antigen (MAIPA) assays. Platelet function was measured using aggregometry and thromboelastography (TEG). RESULTS: Acid treatment reduced detection of HLA Class-I on platelets by 75%, with significant reductions in reactivity to patient anti-sera. Acid treatment reduced platelet content and viability, increased platelet activation and accelerated metabolism. Glycan cleavage was increased by acid treatment. Treatment reduced platelet activation following agonist stimulation by ADP and TRAP-6, but platelets remained functional, displaying increased aggregation response and reduced time to clot formation by TEG. CONCLUSION: Although HLA disruption had some detrimental effects, acid-treated platelets remained functional, retaining their capacity to respond to agonists and form clots, and with further development could be used to support refractory patients.

Mechanotransduction-induced interplay between phospholamban and yes-activated protein induces smooth muscle cell hypertrophy.

The gastrointestinal system is a hollow organ affected by fibrostenotic diseases that cause volumetric compromise of the lumen via smooth muscle hypertrophy and fibrosis. Many of the driving mechanisms remain unclear. Yes-associated protein-1 (YAP) is a critical mechanosensory transcriptional regulator that mediates cell hypertrophy in response to elevated extracellular rigidity. In the type 2 inflammatory disorder, eosinophilic esophagitis (EoE), phospholamban (PLN) can induce smooth muscle cell hypertrophy. We used EoE as a disease model for understanding a mechanistic pathway in which PLN and YAP interact in response to rigid extracellular substrate to induce smooth muscle cell hypertrophy. PLN-induced YAP nuclear sequestration in a feed-forward loop caused increased cell size in response to a rigid substrate. This mechanism of rigidity sensing may have previously unappreciated clinical implications for PLN-expressing hollow systems such as the esophagus and heart.

Plasminogen Activator Inhibitor-1 as a Marker of Esophageal Functional Changes in Pediatric Eosinophilic Esophagitis.

BACKGROUND & AIMS: Esophageal remodeling in eosinophilic esophagitis (EoE) can lead to esophageal rigidity with eventual luminal compromise and stenoses. Gauging esophageal functional alterations in EoE is challenging. An epithelial marker of functional remodeling would impact EoE management. METHODS: Esophageal biopsy specimens from children with and without EoE and primary human esophageal epithelial cells were used for PAI-1 immunohistochemistry, and cell proliferation experiments. PAI-1 immunostaining and basal cell hyperplasia were assessed in the context of concurrently obtained esophageal compliance measures on endoscopic functional lumen imaging probe (EndoFLIP). RESULTS: EndoFLIPs were performed in 45 children (32 with and 13 without EoE). Epithelial PAI-1 was increased in patients with active EoE versus inactive or control patients (P < .01). Esophageal compliance was lower in EoE patients versus controls, particularly in the proximal esophagus (P < .001). Proximal compliance was the strongest predictor of EoE (AUROC 0.88, 95% CI 0.77, 0.98) with esophageal compliance of less than 2.6%mL/mmHg demonstrating 82% sensitivity and 84% specificity for EoE. PAI-1 inhibition significantly diminished esophageal epithelial cell proliferation, suggesting PAI-1 could trigger basal cell hyperplasia. A composite mid-esophageal BZH + PAI-1 score was the strongest predictor of altered compliance (P = .02, AUROC 0.89 (95% CI 0.80, 0.99). CONCLUSIONS: PAI-1 is significantly elevated in pediatric EoE and distinguishes altered compliance in children. PAI-1 may be a novel disease marker and therapeutic target.

Development and Application of a Functional Human Esophageal Mucosa Explant Platform to Eosinophilic Esophagitis.

There is an increasing prevalence of esophageal diseases but intact human tissue platforms to study esophageal function, disease mechanisms, and the interactions between cell types in situ are lacking. To address this, we utilized full thickness human donor esophagi to create and validate the ex vivo function of mucosa and smooth muscle (n = 25). Explanted tissue was tested for contractile responses to carbachol and histamine. We then treated ex vivo human esophageal mucosa with a cytokine cocktail to closely mimic the Th2 and inflammatory milieu of eosinophilic esophagitis (EoE) and assessed alterations in smooth muscle and extracellular matrix function and stiffening. We found that full thickness human esophagus as well as the individual layers of circular and longitudinal muscularis propria developed tension in response to carbachol ex vivo and that mucosa demonstrated squamous cell differentiation. Treatment of mucosa with Th2 and fibrotic cytokines recapitulated the majority of the clinical Eosinophilic Esophagitis Diagnostic Profile (EDP) on fluidic transcriptional microarray. Transforming growth factor-beta-1 (TGFß1) increased gene expression of fibronectin, smooth muscle actin, and phospholamban (p < 0.001). The EoE cocktail also increased stiffness and decreased mucosal compliance, akin to the functional alterations in EoE (p = 0.001). This work establishes a new, transcriptionally intact and physiologically functional human platform to model esophageal tissue responses in EoE.

Interleukin 9 Alters Epithelial Barrier and E-cadherin in Eosinophilic Esophagitis.

BACKGROUND: Eosinophilic esophagitis (EoE) is a chronic TH2-assocated inflammatory condition accompanied by substantial impairments in epithelial barrier function and increased numbers of interleukin 9 (IL-9) expressing inflammatory cells. While IL-9 is known to affect barrier function in the intestine, the functional effects of IL-9 on the esophagus are unclear. Herein we aimed to understand the expression of the IL-9 receptor and effects of IL-9 on the epithelium in EoE. METHODS: We used esophageal biopsies from pediatric EoE patients with active and inactive disease to analyze the expression of the IL-9 receptor, the adherens junction protein E-cadherin and the tight junction protein claudin-1. We treated primary human esophageal epithelial cells with IL-9 to understand its effects on E-cadherin expression and function. RESULTS: Active EoE subjects had increased epithelial expression of IL-9 receptor mRNA and protein (P < 0.05) and decreased membrane bound E-cadherin (P < 0.01) and claudin-1 (P < 0.05) expression. IL-9 receptor expression and mislocalized claudin-1 positively correlated and while membrane bound E-cadherin expression negatively correlated with the degree of histologic epithelial remodeling (P < 0.05). IL-9 decreased epithelial resistance in stratified primary human esophageal epithelial cells (P < 0.01) and membrane bound E-cadherin in epithelial cell monolayers (P < 0.01). CONCLUSIONS: These data suggest that IL-9, its receptor, and its effects on E-cadherin may be important mechanisms for epithelial barrier disruption in EoE.

TGF-ß1-induced PAI-1 contributes to a profibrotic network in patients with eosinophilic esophagitis.

BACKGROUND: Eosinophilic esophagitis (EoE) is an allergic disease of increasing worldwide incidence. Complications are due to tissue remodeling and involve TGF-ß1-mediated fibrosis. Plasminogen activator inhibitor 1 (PAI-1/serpinE1) can be induced by TGF-ß1, but its role in EoE is not known. OBJECTIVE: We sought to understand the expression and role of PAI-1 in patients with EoE. METHODS: We used esophageal biopsy specimens and plasma samples from control subjects and patients with EoE, primary human esophageal epithelial cells, and fibroblasts from patients with EoE in immunohistochemistry, quantitative PCR, and immunoassay experiments to understand the induction of PAI-1 by TGF-ß1, the relationship between PAI-1 and esophageal fibrosis, and the role of PAI-1 in fibrotic gene expression. RESULTS: PAI-1 expression was significantly increased in epithelial cells of biopsy specimens from patients with active EoE compared with that seen in biopsy specimens from patients with inactive EoE or control subjects (P < .001). Treatment of primary esophageal epithelial cells with recombinant TGF-ß1 increased PAI-1 transcription, intracellular protein expression, and secretion. Esophageal PAI-1 expression correlated with basal zone hyperplasia, fibrosis, and markers of esophageal remodeling, including vimentin, TGF-ß1, collagen I, fibronectin, and matrix metalloproteases, and plasma PAI-1 levels correlated with plasma TGF-ß1 levels. PAI-1 inhibition significantly decreased baseline and TGF-ß1-induced fibrotic gene expression. CONCLUSIONS: PAI-1 expression is significantly increased in the epithelium in patients with EoE and reflects fibrosis, and its inhibition decreases TGF-ß1-induced gene expression. Epithelial PAI-1 might serve as a marker of EoE severity and form part of a TGF-ß1-induced profibrotic network.

The TGFß1 Promoter SNP C-509T and Food Sensitization Promote Esophageal Remodeling in Pediatric Eosinophilic Esophagitis.

BACKGROUND: Eosinophilic esophagitis (EoE) is a chronic antigen mediated disease associated with substantial esophageal remodeling and fibrosis. The functional TGFß1 promoter SNP C-509 associates with renal fibrosis and asthma. The effect of TGFß1 genotype and EoE severity or potential gene-environment interactions have not been previously reported in EoE. METHODS: Genotype at TGFß1 C-509T and remodeling was analyzed in 144 subjects with EoE. The severity of remodeling and inflammation was analyzed in the context of IgE sensitization to food antigens and C-509T genotype. RESULTS: The TGFß1 promoter C-509 genotypes CC, CT, and TT were 35%, 52%, and 13%, respectively. Sixty-six percent of subjects were sensitized to foods by positive skin prick test (SPT) or serum specific IgE. TT genotype subjects had significantly more TGFß1 (CC subjects = 1300 per mm2; TT = 2250 per mm2) (p<0.05) and tryptase (CC subjects = 145 per mm2: TT = 307 per mm2) (p<0.05) positive cells and higher epithelial remodeling scores (2.4 vs 3.7, p<0.001) than CC subjects. The differences in TGFß1 and tryptase positive cells as well as fibrosis were significantly increased when there was concurrent food sensitization. Food sensitization alone did not associate with any parameters of inflammation or remodeling. CONCLUSIONS: Our data support a gene-environment interaction between food and genotype at C-509 that modulates disease severity in EoE. Since EoE subjects often continue to consume foods to which they are sensitized, these findings may have clinical relevance for disease management.

Extracellular vesicles from a muscle cell line (C2C12) enhance cell survival and neurite outgrowth of a motor neuron cell line (NSC-34).

INTRODUCTION: There is renewed interest in extracellular vesicles over the past decade or 2 after initially being thought of as simple cellular garbage cans to rid cells of unwanted components. Although there has been intense research into the role of extracellular vesicles in the fields of tumour and stem cell biology, the possible role of extracellular vesicles in nerve regeneration is just in its infancy. BACKGROUND: When a peripheral nerve is damaged, the communication between spinal cord motor neurons and their target muscles is disrupted and the result can be the loss of coordinated muscle movement. Despite state-of-the-art surgical procedures only approximately 10% of adults will recover full function after peripheral nerve repair. To improve upon such results will require a better understanding of the basic mechanisms that influence axon outgrowth and the interplay between the parent motor neuron and the distal end organ of muscle. It has previously been shown that extracellular vesicles are immunologically tolerated, display targeting ligands on their surface, and can be delivered in vivo to selected cell populations. All of these characteristics suggest that extracellular vesicles could play a significant role in nerve regeneration. METHODS: We have carried out studies using 2 very well characterized cell lines, the C2C12 muscle cell line and the motor neuron cell line NSC-34 to ask the question: Do extracellular vesicles from muscle influence cell survival and/or neurite outgrowth of motor neurons? CONCLUSION: Our results show striking effects of extracellular vesicles derived from the muscle cell line on the motor neuron cell line in terms of neurite outgrowth and survival.

Characterisation of antisera to recombinant IgA of the common brushtail possum (Trichosurus vulpecula).

One of the limiting factors in understanding immune responses in marsupials is the scarcity of marsupial specific immunological reagents. This paper describes the characterisation of an antiserum raised against a recombinant protein of the constant region of the heavy chain of IgA (C(alpha)) of the common brushtail possum (Trichosurus vulpecula). The availability of a marsupial specific anti-IgA provides a useful tool for the characterisation of mucosal immune responses in possums. Anti-C(alpha) specifically detects IgA in possum serum and secretions using ELISAs, immuno-dot blots and Western blots without any cross-reactivity to IgG. The possum anti-C(alpha) cross-reacts with IgA of koala (Phascolarctos cinereus), tammar wallaby (Macropus eugenii) and eastern grey kangaroo (Macropus giganteus), demonstrating the potential for use in other marsupials.