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Fabry disease (FD) is a rare X-linked lysosomal storage disorder caused by a deficiency in α-galactosidase A leading to the accumulation of globotriaosylceramide (Gb3) and subsequent increase in globotriaosylsphingosine (lyso-Gb3) in different cells and organs, including the gastrointestinal (GI) tract. GI symptoms represent some of the earliest manifestations of FD and significantly impact quality of life. The origin of these symptoms is complex, and the exact mechanisms remain poorly understood. Here, we sought to determine whether lyso-Gb3 contributes to the pathophysiology of GI symptoms associated with FD by examining its effects on mouse colonic ion transport and motility ex vivo using Ussing chambers and organ baths, respectively. Lyso-Gb3 significantly increased colonic baseline short-circuit current (Isc). This increase in Isc was insensitive to inhibition of the cystic fibrosis transmembrane conductance regulator and Na-K-Cl cotransporter 1, suggesting that the increase in Isc is Cl− ion independent. This response was also insensitive to inhibition by the neurotoxin, tetrodotoxin. In addition, pretreatment with lyso-Gb3 did not significantly influence subsequent responses to either veratridine or capsaicin implying that the response to lyso-Gb3 does not involve the enteric nervous system. In terms of colonic motility, lyso-Gb3 did not significantly influence colonic tone, spontaneous contractility, or cholinergic-induced contractions. These data suggest that lyso-Gb3 significantly influences ion transport in mouse colon, but that accumulation of Gb3 may be a prerequisite for the more pronounced disturbances in GI physiology characteristic of FD. NEW & NOTEWORTHY: Fabry disease-associated lyso-Gb3 significantly influences mouse colonic ion transport in a Cl− ion-independent manner. [ABSTRACT FROM AUTHOR]

The gustatory system is responsible for detecting and evaluating the palatability of the various chemicals present in food and beverages. Taste bud cells, located primarily on the tongue, communicate with the gustatory sensory neurons by means of neurochemical signals, transmitting taste information to the brain. It has also been found that the endocannabinoid system (ECS) may modulate food intake and palatability, and that taste bud cells express cannabinoid receptors. The purpose of this study was to investigate the expression of cannabinoid and cannabinoid-related receptors in the gustatory cells of the papillae vallatae and foliatae of ten piglets. Specific antibodies against the cannabinoid receptors (CB1R and CB2R), G protein-coupled receptor 55 (GPR55), transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1) were applied on cryosections of lingual tissue; the lingual tissue was also processed using Western blot analysis. Cannabinoid and cannabinoid-related receptors were found to be expressed in the taste bud cells and the surrounding epithelial cells. The extra-papillary epithelium also showed strong immunolabeling for these receptors. The results showed that these receptors were present in both the taste bud cells and the extra-gustatory epithelial cells, indicating their potential role in taste perception and chemesthesis. These findings contributed to understanding the complex interactions between cannabinoids and the gustatory system, highlighting the role of the ECS within taste perception and its potential use in animal production in order to enhance food intake. [ABSTRACT FROM AUTHOR]

Simple Summary: The endocannabinoid system (ECS) plays a crucial role in various processes in animals, including pain, inflammation, and immune function. In this study, the presence and distribution of specific ECS receptors (CB1R, CB2R, and GPR55) in the joints of middle-aged dogs was investigated. By analysing the synovial tissues from the hip and the stifle joints, it was found that both CB2R and GPR55 were more prominently expressed by the synoviocytes as compared to CB1R. In addition, immune cells, such as macrophages and neutrophils, also exhibited some of these receptors. This intriguing finding suggested that the receptors in the ECS, particularly CB2R and GPR55, could be promising targets for therapeutic interventions, such as using Cannabis sativa extract, to address arthropathies in dogs. The endocannabinoid system (ECS) has emerged as a potential therapeutic target in veterinary medicine due to its involvement in a wide range of physiological processes including pain, inflammation, immune function, and neurological function. Modulation of the ECS receptors has been shown to have anti-inflammatory, analgesic, and immunomodulatory effects in various animal models of disease, including dogs with osteoarthritis. The goal of this study was to identify and compare the cellular expression and distribution of cannabinoid receptor type 1 (CB1R) and type 2 (CB2R) and the cannabinoid-related G protein-coupled receptor 55 (GPR55) on the synovial cells of hip and stifle joints of seven dogs of different breeds without overt signs of osteoarthritis (OA). The synovial membranes of seven hips and seven stifle joints were harvested post mortem. The expression of the CB1R, CB2R, and GPR55 present in the synovial tissues was investigated using qualitative and quantitative immunofluorescence and Western blot (Wb) analysis. Synoviocytes of the stifle and hip joints expressed CB1R, CB2R, and GPR55 immunoreactivity (IR); no significant differences were observed for each different joint. Cannabinoid receptor 2- and GPR55-IR were also expressed by macrophages, neutrophils, and vascular cells. The ECS receptors were widely expressed by the synovial elements of dogs without overt signs of OA. It suggests that the ECS could be a target for the therapeutic use of Cannabis sativa extract in canine arthropathies. [ABSTRACT FROM AUTHOR]

To improve pathogenetic studies in cancer development and reliable preclinical testing of anti-cancer treatments, three-dimensional (3D) cultures, including spheroids, have been widely recognized as more physiologically relevant in vitro models of in vivo tumor behavior. Currently, the generation of uniformly sized spheroids is still challenging: different 3D cell culture methods produce heterogeneous populations in dimensions and morphology, that may strongly influence readouts reliability correlated to tumor growth rate or antitumor natural killer (NK) cell-mediated cytotoxicity. In this context, an increasing consensus claims the integration of microfluidic technologies within 3D cell culture, as the physical characterization of tumor spheroids is unavoidably demanded to standardize protocols and assays for in vitro testing. In this paper, we employed a flow-based method specifically conceived to measure weight, size and focused onto mass density values of tumor spheroids. These measurements are combined with confocal and digital imaging of such samples. We tested the spheroids of four colorectal cancer (CRC) cell lines that exhibit statistically relevant differences in their physical characteristics, even though starting from the same cell seeding density. These variations are seemingly cell line-dependent and associated with the number of growing cells and the degree of spheroid compaction as well, supported by different adenosine-triphosphate contents. We also showed that this technology can estimate the NK cell killing efficacy by measuring the weight loss and diameter shrinkage of tumor spheroids, alongside with the commonly used cell viability in vitro test. As the activity of NK cells relies on their infiltration rate, the in vitro sensitivity of CRC spheroids proved to be exposure time- and cell line-dependent with direct correlation to the cell viability reduction. All these functional aspects can be measured by the system and are documented by digital image analysis. In conclusion, this flow-based method potentially paves the way towards standardization of 3D cell cultures and its early adoption in cancer research to test antitumor immune response and set up new immunotherapy strategies. [ABSTRACT FROM AUTHOR]

Gathering precise information on mass density, size and weight of cells or cell aggregates, is crucial for applications in many biomedical fields with a specific focus on cancer research. Although few technical solutions have been presented for single-cell analysis, literature does not cover this aspect for 3D models such as spheroids. Since the research interest on such samples is notably rising, here we describe a flow-apparatus, and the associated physical method and operative protocol for the accurate measurements of mass density, size and weight. The technique is based on the detection of the terminal velocity of a free-falling sample into a specifically conceived analysis flow-channel. Moreover, in order to demonstrate the accuracy and precision of the presented flow-device, analyses were initially carried out on standardized polystyrene beads. Finally, to display the application of the proposed system for biological samples, mass density, size and weight of live SW620 tumor spheroids were analyzed. The combined measurements of such parameters can represent a step toward a deeper understanding of 3D culture models. [ABSTRACT FROM AUTHOR]

Background: Fabry disease (FD) is a hereditary X‐linked metabolic storage disorder characterized by deficient or absent lysosomal α‐galactosidase A (α‐Gal A) activity. This deficiency causes progressive accumulation of glycosphingolipids, primarily globotriaosylceramide (Gb3), in nearly all organ systems. Gastrointestinal (GI) symptoms can be very debilitating and are among the most frequent and earliest of the disease. As the pathophysiology of these symptoms is poorly understood, we carried out a morphological and molecular characterization of the GI tract in α‐Gal A knockout mice colon in order to reveal the underlying mechanisms. Methods: Here, we performed the first morphological and biomolecular characterization of the colon wall structure in the GI tract of the α‐Gal A knock‐out mouse (α‐Gal A −/0), a murine model of FD. Key Results: Our data show a greater thickness of the gastrointestinal wall in α‐Gal A (−/0) mice due to enlarged myenteric plexus' ganglia. This change is paralleled by a marked Gb3 accumulation in the gastrointestinal wall and a decreased and scattered pattern of mucosal nerve fibers. Conclusions and Inferences: The observed alterations are likely to be a leading cause of gut motor dysfunctions experienced by FD patients and imply that the α‐Gal A (−/0) male mouse represents a reliable model for translational studies on enteropathic pain and GI symptoms in FD. The mouse model of Fabry disease shows gastrointestinal histopathological, morphological, and molecular alterations. A marked globotriaosylceramide accumulation in the gastrointestinal wall and a decreased and scattered pattern of mucosal nerve fibers are showed. [ABSTRACT FROM AUTHOR]