The integrity of esophageal epithelial cells in patients with gastroesophageal reflux disease (GERD) or GERD-like symptoms is the first mechanism of protection to decrease the sensitivity to gastric reflux and heartburn symptoms. We investigated the protective effects of Poliprotect® (PPRO), a CE-marked medical device, on esophageal epithelial integrity using in vitro and ex vivo models. In vitro, the protective effects of PPRO were tested on Caco-2 cells. PPRO demonstrated safety and protection against oxidative damage induced by hydrogen peroxide. It also preserved epithelial integrity by maintaining transepithelial electrical resistance (TEER) against damage from calcium removal or bile acid exposure (taurodeoxycholic acid, TDCA). Ex vivo, esophageal biopsies from patients subjected to endoscopy were mounted in Ussing chambers and exposed to damaging agents (HCl or HCl + TDCA). Untreated biopsies (control) showed significant loss of epithelial resistance (up to −33%). In contrast, low concentrations of PPRO (50–100 µg/mL) provided strong protection against these damages (p < 0.001), even after 60 min of washing. Histological analysis confirmed the barrier-enhancing effect of PPRO. Overall, PPRO effectively protected the esophageal epithelium from damage in both models, suggesting its potential role in alleviating GERD or GERD-like symptoms by strengthening mucosal barriers and reducing epithelial sensitivity to reflux.

Background/Objectives: Unhealthy nutrition and lifestyles contribute to the development of chronic diseases such as cardiovascular disease, type 2 diabetes, and colorectal cancer. The Western diet can impair gastrointestinal motility and function. The underlying mechanisms that lead to changes in the mucus barrier and mucin profiles in response to these dietary patterns are still being studied. In mice, dietary fiber intake can improve the intestinal mucosal barrier function, enhance the differentiation process of goblet cells, and increase acidic mucin production. Our study aimed to investigate the effects of a high-fat diet (HFD) on colonic mucin expression and to assess whether chickpea accessions, known for their nutritional benefits, can mitigate these adverse effects. Methods: We investigated the effects of an HFD and an HFD associated with two accessions of chickpeas (HFD + MG_13; HFD + PI358934) on the mucin expression in murine colons of mice by conventional histochemistry and lectin-histochemistry, combined with chemical treatment and enzymatic digestion and immunohistochemistry. We evaluated possible alterations of Muc2, the main mucin secreted by the mucous cells of the colon. Results: HFD significantly reduced the expression of the mucin Muc2 and altered its composition in the colon. Compared to the CTRL group, distal and proximal measurements for HFD + PI, respectively, showed reductions of 78% and 36%; for the distal colon, a reduction of 34% was also observed for both the HFD and HFD + MG_13 diets. Changes in mucin glycosylation, including sialylation and sulfation, as well as residues such as N-acetylglucosamine, GalNAc, Mannose, and Galactose, were observed, suggesting a beneficial influence of chickpeas on mucosal integrity. In HFD + MG_13 these effects were reduced and resulted similar to the control. Conclusions: HFD reduces Muc2 expression in the colon and alters mucin composition: chickpea accessions, particularly MG_13, partially restore Muc2 levels and mucin oligosaccharide profiles, suggesting protective effects on the intestinal mucosal barrier.

Mucomics is the study of mucus and its biochemical properties. This discipline has gained increasing attention due to the critical roles mucus plays in protection, adhesion, and communication across species. Ethical restrictions on vertebrate research have driven the interest in invertebrate models such as mollusks. Mollusks produce large amounts of mucus which has several functions, including immune defense, digestion, and environmental adaptation. Mollusks—terrestrial, freshwater, or marine—are valuable models for investigating mucus composition and its responses to environmental stressors, including heavy metal contamination. Histochemical and glycomic techniques have revealed variations in mucin glycosylation patterns that influence mucus functionality, such as its viscoelastic and adhesive properties. Bivalves and gastropods, widely used as bioindicators and generally not subject to regulatory constraints in experimental use, accumulate pollutants in their mucus, reflecting environmental health. Investigative techniques such as lectin histochemistry, proteomic, and glycomic analyses provide insights into the impact of contaminants on mucus composition. Further research on molluscan mucins can enhance understanding of their physiological roles, environmental interactions, and potential biomedical applications. By integrating molecular and histochemical approaches, mucomic studies offer a comprehensive perspective on mucus function, advancing both ecological monitoring and biotechnological applications.

The effects of treatments of pool frog embryos of Pelophylax lessonae with engineered zerovalent nanoparticles of Fe Co, and Ni at increasing concentrations (½ LC50, LC50, and 2 × LC50) were studied at macroscopical and microscopical levels, focusing on the epidermis that is often subject to physiological changes in response to environmental factors. Total body length and eye diameter were statistically higher in the control groups. A significantly higher percentage of embryos in the controls reached later Gosner developmental stages (19-23) than the treatments. Malformations such as ventral blister, microcephalia, bent body axis, and microphthalmia were significantly more frequent in the Co and Ni treatments than in the controls. In treated epidermis, ciliated, goblet, and small secretory cells resulted in a significantly larger volume than the same cell types in control embryos. At the ultrastructural level, ciliated cells showed stuck cilia and mitochondrial swelling, as well as cytoplasmic inclusions. Small secretory cells exhibited a significantly higher number of secreting vacuoles, suggesting an increased secretion. In conclusion, nanoparticles affect the development of frog embryos in multiple ways, even if the mechanisms are still mostly unknown.

Microplastics (MPs) pose an increasing and significant threat to marine biodiversity and there is a current need to determine the effects of exposure on benthic sessile invertebrates. This study examines the filtration capacity and retention of MP particles, as well as their physiological impacts in the marine sponge Spongia officinalis, a bioindicator species. The findings revealed a very high filtration capacity for MPs within the size range of 1-5 μm, along with a rapid turnover rate, as a large portion of particles were expelled within 48 h of exposure. Histological analyses detected MP particles within the cellular structures of the analyzed tissues, indicating that MPs of this size can penetrate cellular barriers. In terms of physiological effects, detoxification activity was activated during the depuration phase, and lipid peroxidation was observed during both the exposure and depuration phases. Overall, this study provides critical insights into the filtration and retention capacity, intercellular integration of MP particles, and the physiological effects of MP exposure in S. officinalis, providing a baseline for future research.

Coral reefs are typically widespread in shallow waters from tropical seas, while in the Mediterranean Sea they currently have a very reduced extension. Only recently, a true coral reef has been identified on mesophotic bottoms along the coasts of southeastern Italy. The reef was mainly built by the non-symbiotic Caryophylliidae Phyllangia americana mouchezii and Polycyathus muellerae. Investigating the reproductive cycle of a scleractinian is a useful tool for assessment and conservation purposes, especially in the case of engineering species that play a key role in biogenic habitats. In the Mediterranean Sea, reproductive biology has been studied only in a minority of the 33 species of scleractinians recorded so far. This contribution represents the first attempt of describing the sexual reproductive cycle of P. a. mouchezii and P. muellerae, taking into account its possible relationship with budding and water temperature. In general, the study revealed that both species are oviparous and hermaphroditic. They showed an irregularly cyclical trend of gametogenesis, with a greater energy investment during summer. There was a substantial stasis of oogenesis during winter and the concentration of spermatogenesis in spring/summer. P. muellerae showed a slight advance in gametogenesis, with peak production one month before that of P. a. mouchezii. Budding did not appear to interfere with sexual reproduction, nor did an anomalous peak in water temperature recorded during one of the monthly samplings. Finally, the study allowed to intercept gametes’ spawning in some colonies of the target species reared in tanks under controlled conditions.

Lanthanum (La), a rare earth element, has unique properties that make it valuable in various industries. Extraction activities pose environmental and health risks due to pollution, habitat disruption, and human exposure to hazardous chemicals. Animal models are needed to evaluate La effects on biological systems. We focused on the Manila clam, Ruditapes philippinarum, a common bivalve introduced in the Mediterranean Sea for commercial purposes. This study evaluates La-induced alterations in the Manila clam using an integrated method. Specimens were exposed to varying concentrations of lanthanum chloride, and subsequent changes in mucus secretion and microbial communities were observed. Inductively coupled plasma mass spectrometry analyses supported the bioaccumulation data. Histochemical and lectin-histochemical analyses revealed modifications in glycosaminoglycan composition and secretion patterns, indicating the clam's defensive response to La exposure. Bacterial counts in intervalvar water increased with higher La concentrations. These findings highlight the Manila clam's potential use as a bioindicator for La pollution and underline the importance of responsible management in La extraction to minimize environmental impact.

Aquaporins (AQPs) are important for water transport in the gastrointestinal tract. Changes in their expression and/or localization could cause in disorders and be used as therapeutic targets. Aquaporin-4 (AQP4) is expressed predominantly on the basolateral membrane of the parietal cells in the corpus of the murine gastric glands. Although the secretion of gastric juice is not affected in AQP4-deficient knockout, we evaluated by light microscopy whether the lack of AQP4 affects the glycopatterns of secreting gastric cells. Wild type (WT) and AQP4-deficient knockout mice (KO) were fed a standard diet ad libitum before sacrifice. Segments of stomach corpus were collected, fixed in buffered formalin, and embedded in paraffin wax. Sections, 5-mu m thick, were analyzed by histochemical methods (Periodic acid-Schiff, Alcian Blue pH 2.5), and binding of lectins specific to GalNAc (SBA, DBA), Gal (PNA) GlcNAc (WGA, GSAII) mannose and/or glucose (ConA), and fucose (UEA-I, AAA, LTA). Immunohistochemical methods such as anti-Muc6 for neck cells and anti- beta- H+/K+-ATPase for parietal cells were also performed. Compared to WT mice, in the mucous cells of KO lower amounts of glycans with galactosyl/galactosaminylated, glycosyl/glycosaminylated, and fucosylated residues were observed; lower fucosylation resulted also in the parietal cells. The observed differences of KO in respect to WT could lead to severer pathological conditions.Research Highlights Glycopatterns in gastric glands were compared between wild type (WT) and AQP4-deficient knockout (KO) mice by histochemical and lectin-binding methods. In the mucous cells of KO lower amounts of glycans with galactosyl/galactosaminylated, glycosyl/glycosaminylated and fucosylated residues were observed. In the parietal cells lower fucosylation also resulted. AQP4-deficiency affects glycosylation and could result in altered functionality and pathological conditions. Altered glycosylation patterns in both mucous and parietal gastric cells were evidenced by histochemistry, lectin-histochemistry, and immunohistochemistry in AQP4-deficient KO mice, with possible physio-pathological consequences. image

Antipathella subpinnata is a black coral species with Atlantic-Mediterranean distribution, capable of forming dense forests at mesophotic depths, thereby providing essential ecological goods and services. Limited data are available on its reproductive biology and ecology. Understanding the reproductive patterns of this species is pivotal for effective conservation strategies. This study proposes a histological protocol for the treatment of A. subpinnata samples previously preserved in ethanol, aiming to restore the internal morphology of the polyps and consequently improve the detection of internal structures and the investigation of the reproductive traits of each polyp. The success of this protocol in A. subpinnata suggests its potential applicability to other black corals, providing a valuable tool for ecological and biological research.

Aluminum (Al) is used in everyday life and present in food drugs, packaging, industry, and agriculture. Although it is the most common metal in the Earth crust, a correlation has been demonstrated between its presence and various pathologies, even serious ones, especially of a neurological type. However, there is a histological gap regarding the role Al can have in contact with the covering and secreting epithelia. The alterations of the ventral and dorsal foot mucocytes and their secretions of the snail Eobania vermiculata caused by Al were investigated in situ by histochemical and lectin-histochemical techniques. Administration to different experimental groups took place for 3 and 9 days with 50 and 200 mu M of AlCl3. Several types of mucocytes were detected with a prevalent secretion of acid glycans in the foot of E. vermiculata. Sulfated glycans prevail in the dorsal region, with one type showing only fucosylated residues and another also having galactosaminylated and glycosaminylated residues. Carboxylated glycans prevail in the ventral region, with presence of galactosaminylated, glycosaminylated, and fucosylated residuals in both cells. Snails treated presented a general decrease of mucin amount in the secreting cells and affected the mucus composition. These changes could alter the rheological and functional properties of the mucus with possible implications for the health of the treated animals. Research Highlights Snails were fed with Al-contaminated lettuce at different concentrations. In the foot mucocytes produced mucus with prevailing acidic glycans. In the treated resulted a reduction in the amount of mucus and an alteration of glycan composition.

We analysed the embryonic epidermis of the pool frog, Pelophylax lessonae, by light and electron microscopy techniques, and characterized preliminarily its mucous secretions with conventional histochemistry (Toluidine Blue, Periodic Acid-Schiff, Alcian blue pH 2.5 and Alcian blue pH 1.0). At Gosner’s developmental stage 21, the epidermis consisted of a bilayered epithelium with five cell types. Basal cells (BCs) constituted the basal layer, presented a large nucleus and were sometimes observed in mitosis. Ciliated cells (CCs) had a ciliated apical membrane, with carboxylated glycans in the sub-apical portion. TEM analysis revealed small vesicles just below the surface, a central nucleus, and evident yolk plates. Goblet cells (GCs) and small secretory cells (SSCs) showed secreting vesicles and vacuoles, respectively. In the mucus secretion of GCs sialosulphomucins appeared, while in that of SSCs sulphation was not observed. Under TEM, GCs had irregular shaped nuclei and a less electron-dense cytoplasm with respect to SSCs. Ionocytes (INs) presented apical microvilli and a high electron-dense cytoplasm with vacuoles. SEM analysis revealed that GCs outnumbered the other cell types and that SSCs and INs were usually located together near to GCs. The relatively simple structure of the epidermis and its direct interfacing with the environment renders it a good model for the study of structure and development of mucociliary epithelia, as well as of its alterations in the presence of toxicants.

An in situ analysis of mucin secretions along the annual cycle was performed on the striped Venus clam, Chamelea gallina. Individuals of both sexes from an exploited stock of Margherita di Savoia (Southern Adriatic Sea, Central Mediterranean Sea) were made in June 2020 and 2021, and in January 2021 and 2022, representative of summer and winter seasons, respectively. Tissues from the foot and the gills were analyzed for histochemical and lectin-histochemical analyses. Staining with Periodic Acid-Schiff, Alcian Blue pH 2.5 and High-Iron-Diamine indicated that mucins from both the foot and the gills were acidic, mostly sulfated. Lectin-binding analyses with PNA, SBA, WGA, LTA, UEA-I, AAA, SNA, MAA-II and ConA indicated the presence of N-acetyl-glycosaminylated, mannosylated and fucosylated residuals in the saccharidic chains. In the gills, the amount of acidic and glycosaminylated residuals was higher in summer in both sexes, whereas fucosylation was similar along the sampling period and mannosylation was never observed. In the foot, both surface epithelial and subepidermal secreting cells increased sulfation in winter in males, but not in females. Glycosaminylation and fucosylation were observed only in the surface cells of males. Mannosylated residuals were observed in all the foot cell types in both sexes. It is hypothesized that the observed qualitative and quantitative variations in mucin secretion is linked to the reproductive cycle.

The striped venus clam Chamelea gallina is an important shellfish resource in coastal area of Mediterranean Basin, playing a fundamental role in the ecosystems. In the last two decades there has been a reduction in the stocks of this bivalve with several mortality events along Adriatic Sea due to pollutants. Aiming to evaluate the effects on clams of the anthropogenic increase of some metals in the sea, we focused on aluminium. In particular, we tested whether Al could affect mucus secretion, since mucins are involved in protection from pathogens and toxicants and transport of alimentary particles and pseudofeces. Aluminium was administered in the form of aluminium chloride at concentrations of 25, 50 and 200 μM. Histochemical and lectin-histochemical - analyses were used to evaluate in situ qualitative and quantitative variation of the muciparous secretions produced by both the gills and foot epithelia. Treatments resulted in quali-quantitative alterations in the secretions of both gills and foot epithelia, in particular reduction of glycosylation resulting in a general decrease of acidic residuals and of mannosylated, glycosaminylated and fucosylated chains. This can result in a decrease of acidity and viscosity of mucus and thus it could affect its functionality and the survival of the animals.

Angelini, Ines; Centrone, Mariangela; D'Agostino, Mariagrazia; Semeraro, Daniela; Mastrodonato, Maria; Caponio, Giusy Rita; Di Mise, Annarita; Ranieri, Marianna; Valenti, Giovanna; Tamma, Grazia Author Information

The glycopatterns of the glycans secreted by the mucosa of stomach and duodenal ampulla of the striped dolphin, Stenella coeruleoalba were studied by histochemical (Periodic acid-Schiff, Alcian Blue pH 2.5, High Iron Diamine) and lectin-binding (SBA, DBA, PNA, WGA, MAA-II, SNA, ConA, UEA-I, AAA, LTA) techniques. The stomach can be divided into four compartments: main stomach, two connecting chambers and pylorus. The pylorus is followed by the duodenal ampulla. Mucins are secreted by surface cells and intramucosal glands specific for each compartment. In the main stomach glands, neck cells were weakly sulphated, with prevailing glycosaminylated, glycosylated/mannosylated, and fucosylated residuals. Parietal and chief cells in general were scarcely reactive. In the connecting chambers glands, there were high levels of sulphation, glycosaminylation, glycosylation/mannosylation, and fucosylation, the latter with more complex patterns than those observed in the main stomach glands. In the pyloric glands sulphated, glycosaminylated and fucosylated residuals decreased, whereas the opposite was observed for galactosyl/galactosaminylated residuals. Glycosylation patterns in the glands of the duodenal ampulla differed from those of the pyloric ones, with similar levels of sulphation, lower levels of galactosyl/galactosaminylation and glycosaminylation, and higher level of fucosylation. The results are compared with those available in literature.

Aluminum is the most abundant metal in the Earth's crust and is widely used in everyday life. In sea waters, the presence of dissolved Al is naturally limited to a few micrograms per litre, however anthropogenic activities tend to alter its concentration. Toxic effects caused by this metal have already been demonstrated on several taxa, as well as a direct correlation with various diseases, including neurodegenerative ones. Since bivalves proved good bioindicators for environmental and possibly human health, we tested by histochemical methods the effects of Aluminum on the muciparous tissues of the striped venus clam, Chamelea gallina. Aluminum chloride at concentrations of 25, 50, and 200 µM, respectively were administered to experimental groups of twenty clams housed under controlled conditions. Image analysis of tissues stained by conventional and lectin-histochemistry indicated both qualitative and quantitative alterations of the mucous secretion produced by the gills and feet epithelia of the clams. The acidity and viscosity of the mucus itself is altered, with possible repercussions on the health and survival of animals.

This contribution supplies the first description of the reproductive cycle of Phyllangia americana mouchezii and Polycyathus muellerae (Cnidaria, Anthozoa), the main builders of the Mediterranean coral reef recently described for the first time along the Apulian coasts, focusing on both the sexual and asexual reproductive activity and trying to assess the relationship between gametogenesis and seawater temperature. Sample collection was performed monthly, from December 2020 to January 2022, at depths between 35 and 45 m, and at the same time seawater temperature was measured at 1 m and 40 m of depth. At the laboratory, samples were subjected to histological procedures aimed at highlighting the presence of reproductive elements in the polyp tissues. A multiple regression analysis was then performed to test the relationship between gametogenesis in the two species, depth, and surface and deep-water temperature. On the whole, both species resulted oviparous and gonochoric, with a sex ratio between male and female colonies of 1:2.3 in P. a. mouchezii and 1:1.6 in P. muellerae. However, hermaphrodite colonies were sometimes found during the investigation, accounting for about 7% of the total reproductive colonies in the former species and 11% in the latter. In P. a. mouchezii about 32% of the colonies resulted in gametogenesis, in P. muellerae about 37%. Both species showed an irregularly cyclical trend of gametogenesis, with a greater energy investment during Summer. In general, the oogenesis was less intense during Winter and the spermatogenesis was concentrated in Spring/Summer. Statistical analysis revealed that in both species surface water temperature was a predictor for spermatogenesis, while oogenesis seemed to be less linked to water temperature fluctuations. No significant regression was observed for the other variables considered. The data provided may in the future contribute to the conservation of the Mediterranean coral reef, a peculiar and still poorly known habitat recently proposed as belonging to a real mesophotic system present along the Apulian coasts.

The localization of intestinal glycoconjugates of the blunthead pufferfish Sphoeroides pachygaster and the grey triggerfish Balistes capriscus from the north-western Ionian Sea was analysed by histochemical methods (PAS, AB pH 2.5, HID) and lectin binding experiments (WGA, LFA, SBA, sialidase-SBA, PNA, sialidase-PNA, ConA, AAA, UEA-I, LTA) to assess how evolutionary loss of a functional stomach in S. pachygaster affects intestinal secretions relative to the B. capriscus, which retains the plesiomorphic gastric condition. Sphoeroides pachygaster had a lower content of acid mucins but more complex sialylation patterns than B. capriscus. GalNAc and GlcNAc residuals were present in both, but GalNAc residuals in S. pachygaster were subterminal to sialic acid. Balistes capriscus lacked galactosylated residuals and its enterocytes had a glycocalyx that differed in composition between the small intestine and the rectum and was missing from S. pachygaster. Functional and ecological implications of these findings are discussed.