Echinoderm larvae as bioindicators for the assessment of marine pollution: Sea urchin and sea cucumber responsiveness and future perspectives

Echinoderms play a crucial role in the functioning of marine ecosystems and due to their extensive distribution, rapid response, and the high sensitivity of their planktonic larvae to a large range of stressors, some species are widely used as biological indicators. In addition to sea urchins, sea cucumbers have recently been implemented in embryotoxicity bioassays showing high potential in ecotoxicological studies. However, the use of this species is still hindered by a lack of knowledge regarding their comparative responsiveness. The present study aimed to investigate the responsiveness of different echinoderm species to environmental pollution in order to develop their integration in batteries of ecotoxicological bioassays. To this end, the embryos of two sea urchins (Paracentrotus lividus and Arbacia lixula) and two sea cucumbers (Holothuria polii and Holothuria tubulosa) were incubated with inorganic and organic toxicants (cadmium, copper, mercury, lead, sodium dodecyl sulphate and 4-n-Nonhyphenol) and elutriates from contaminated marine sediments, chosen as a case study model. The results obtained, expressed through the percentage of abnormal embryos and Integrative Toxicity Indices (ITI), indicated species-specific sensitivities to pollutants, with comparable and correlated responsiveness between sea urchins and sea cucumbers. More specifically, sea cucumber larvae exposed to elutriates appear to be more sensitive than sea urchins, especially when incubated with samples containing trace metals, PCB and TBT. These results indicate that toxic responses in embryos exposed to environmental matrices are probably modulated by interactions between different variables, including additive, synergistic and antagonistic effects. These findings suggest that performing a larval test using different echinoderm classes can integrate the interactive effects of bioavailable fraction of contaminants on various levels, providing sensitive, representative and all year-round batteries of bioassays to apply in ecotoxicological studies

Effects of Vitamin and Amino Acid-Enriched Hyaluronic Acid Gel on the Healing of Oral Mucosa: In Vivo and In Vitro Study

Background and Objectives: Wound healing is a dynamic process that can be compromised in patients with chronic and metabolic conditions or unhealthy lifestyles. Numerous medical substances designed for topical use, charged with compounds that promote the healing process, have been developed to improve wound healing, especially in compromised subjects. The present study aimed to extend our understanding of the in vivo effects of a hyaluronic acid gel charged with amino acids (HAplus gel, Aminogam gel® Errekappa Euroterapici spa, Milan, Italy) and study the in vitro effects of the same gel charged with additional substances in an attempt to optimize its formulation. Materials and Methods: In a randomized controlled split-mouth clinical and histological trial, HAplus gel was tested on the gingival tissue of the lower third molar post-extraction socket. The gingiva was collected at the time of extraction (T0) and ten days after the extraction (T1) to be histologically analyzed. During the second stage of the study, culture media with HAplus gel and vitamin C and E at different concentrations (TEST) were tested on human gingival fibroblasts and compared to the HAplus-enriched medium (HA-Control). Results: Histological and immunohistochemical analysis of collected gingiva showed higher microvascular density and collagen fibers organized in closely packed and well-oriented bundles in sites treated with HAplus gel. In the in vitro study, all TEST groups showed an increased viability from 24 h to 48 h. After 24 h, the viability percentage in all experimental groups was below 100% of the HA-Control, demonstrating a mild toxicity. After 48 h from seeding, the TEST groups’ viability grew significantly compared to HA-Control. Conclusions: These encouraging preliminary results suggest that the use of HAplus gel enriched with vitamins C and E may be beneficial in patients with conditions that impair soft tissue healing

Enteric dysfunctions in experimental Parkinson's disease: alterations of excitatory cholinergic neurotransmission regulating colonic motility in rats

Parkinson's disease (PD) is frequently associated with gastrointestinal symptoms, mostly represented by constipation and defecatory dysfunctions. This study examined the impact of central dopaminergic denervation, induced by injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle, on distal colonic excitatory cholinergic neuromotor activity in rats. Animals were euthanized 4 and 8 weeks after 6-OHDA injection. In vivo colonic transit was evaluated by radiological assay. Electrically and carbachol-induced cholinergic contractions were recorded in vitro from longitudinal and circular muscle colonic preparations, while acetylcholine levels were assayed in their incubation media. Choline acetyltransferase (ChAT), HuC/D (pan-neuronal marker), muscarinic M2 and M3 receptors. As compared with control rats, at week 4 6-OHDA-treated animals displayed the following changes: decreased in vivo colonic transit rate; impaired electrically evoked neurogenic cholinergic contractions; enhanced carbachol-induced contractions; decreased basal and electrically stimulated acetylcholine release from colonic tissues; decreased ChAT immunopositivity in the neuromuscular layer; unchanged density of HuC/D immunoreactive myenteric neurons; increased expression of colonic muscarinic M2 and M3 receptors. The majority of such alterations were detected also at week 8 post-6-OHDA injection. These findings indicate that central nigrostriatal dopaminergic denervation is associated with an impaired excitatory neurotransmission characterized by a loss of myenteric neuronal ChAT positivity and decrease in acetylcholine release, resulting in a dysregulated smooth muscle motor activity, which likely contributes to the concomitant decrease in colonic transit rate

Immunohistochemical evaluation of pro-inflammatory molecules in the soft tissue surrounding switching platform implants

Introduction. Switching platform implants have been proposed to reduce the peri-implant bone loss by shifting the implant abutment interface internally and away from the bone tissue. Aim of the present study was to characterize the inflammatory infiltrate in the soft tissue surrounding switching and matching platform implants and to evaluate the expression of pro-inflammatory molecules involved in the bone loss. Material and methods. A total of 32 implants with diameters of 3.8mm, 4.3mm, 4.8mm and 5.5mm were all restored with abutment of 3.8mm of diameter, thus resulting the following implant-abutment mismatches: 0mm (n=10) (control group), 0.25mm (n=7) (test group1), 0.5mm (n=8) (test group2), and 0.85mm (n=7) (test group3). Four years after loading, peri-implant soft tissue samples were harvested and processed for immunohistochemical analysis. Total amount of lymphocytes T (LyT) -B (LyB) infiltrated, and expression of IL-17 and RANKL were detected. Results. At the harvesting time all sites were clinically healthy. No significant differences were found between groups (p>0.05) in terms of infiltrated T and B cells amount, IL-17 and RANKL expression. In all samples lymphocytes T and B were mainly localized close to the junctional epithelium and sparsely detected in the surrounding connective tissue. The distribution of IL-17 and RANKL staining resulted strictly correlated to the inflammatory infiltrated. When pooled data were analyzed, amount of lymphocytes T and IL-17 were higher than respectively lymphocytes B and RANKL. Amount of LyT and LyB were highly correlated (Pearson’s r>0.7) and IL-17 was moderately correlated (Pearson’s r>0.4, <0.7) to LyT and LyB. Conclusions. In the prolonged exposure of the abutment at the oral cavity the configuration of the implant abutment interface may do not affect the inflammatory cellular and molecular pattern responsible for bone loss

Immunohistochemical and molecular analysis of bone remodelling pattern in alveolar socket

Following tooth extraction, the alveolar bone remodelling process starts. Bundle bone and buccal wall resorption occur early with horizontal and vertical bone crest reduction [1]. The use of bone substitutes has been proposed to limit bone resorption, thus allowing further dental rehabilitation [2]. Aim of this project was to characterize by a molecular and morphological approach the physiological remodelling of post-extractive alveolar socket and to compare it with the bone remodelling occurring after alveolar bone reconstruction with an alloplastic material. Thirty-six patients needing tooth extraction were enrolled and equally divided into three groups: A) baseline, B) spontaneous healing, C) biomaterial. In each group, 2 biopsies per site were harvested during tooth extraction (group A) or 4-6 months after tooth extraction (groups B and C). In group B, patients recovered spontaneously, while in group C the alveolar socket was filled with a magnesium-enriched hydroxyapatite. One biopsy was processed for immunohistochemistry to localise TNF-α, IL-6, RANK, RANKL and OPG. The second biopsy underwent a Real-Time PCR analysis for the same biomarkers in order to evaluate gene expression. In groups B and C, a third biopsy was retrieved and processed for ground section aiming to assess tissue composition. Differences between the three groups were investigated using Kruskal Wallis test (p<0,05) followed by post-hoc tests. All samples showed a normal structure without inflammatory infiltrate. At immunohistochemical analysis, all biomarkers except for OPG had increased. Significant differences were found between the three groups for TNF-α (p< 0,05), IL-6 (p<0,001), RANK (p< 0,01) and RANKL (p<0,001), between groups A and C for IL-6 (p≤ 0,001), RANK (p≤ 0,01), RANKL (p≤ 0,001) and between B and C for IL-6 (p≤ 0,01). Gene expression did not show statistical differences. Crumbles of biomaterial surrounded by regenerated bone were evident. A higher percentage of mineral component was obtained in group B than in C. The biomarkers selected in the current study were involved in the alveolar remodelling and the biomaterial used for socket preservation did not influence the process

Qualitative and quantitative analysis of gingival microvessels by capillaroscopy in healthy subjects

Gingiva is composed by attached gingiva and free gingiva that are separated by free gingival line. Attached gingiva covers the alveolar bone and adheres to the bone and root surface by fibres. Free gingiva ends with the gingival margin and in clinical practice it can be displaced from the tooth surface to locate the prosthetic margin. Capillaroscopy allows to take microphotographs of the microvessels and to observe their abnormalities in autoimmune rheumatic diseases (1). Aim of this study was to analyse microvessels of the attached gingiva, free gingiva line and free gingiva by means of capillaroscopy. In correspondence of upper incisors of 12 young healthy volunteers, after placement of liquid vaseline, microphotographs (x200) were taken at level of the free gingiva and 2-3 mm more apically within the attached gingiva. Capillaries structure and organization were evaluated in the three areas of interest. In 10 randomly selected microphotographs of the attached gingiva, the amount and percentage of microvessels per mm2 were also calculated. For each subject, two analyses were performed at 3 weeks of distance for repeatability assessment. At the observation, in attached gingiva vessels appeared as tortuous capillary loops perpendicular to the epithelial surface. At level of free gingival line vessels get linear and parallel to the arch of gingival margin. In free gingiva capillaries run superficially and parallel to the epithelial surface, toward the margin and fell back with a loop on the tooth side. At the quantitative analysis, the method resulted repeatable (Wilcoxon signed-rank test, p>0.05). A mean of 49.8 (± 9.5) microvessels for mm2 was found. Capillaries represented the 10.3% (±3.5) of the attached gingiva. Capillaroscopy is a non-invasive repeatable method to observe gingival capillaries. This method may be proposed in clinical practice to detect and monitor changes or abnormalities after placement of prosthetic margins

Transcriptional dynamics of induced pluripotent stem cell differentiation into β cells reveals full endodermal commitment and homology with human islets.

Abstract Background aims Induced pluripotent stem cells (iPSCs) have the capacity to generate β cells in vitro, but the differentiation is incomplete and generates a variable percentage of off-target cells. Single-cell RNA sequencing offers the possibility of characterizing the transcriptional dynamics throughout differentiation and determining the identity of the final differentiation product. Methods Single-cell transcriptomics data were obtained from four stages across differentiation of iPSCs into β cells and from human donor islets. Results Clustering analysis revealed that iPSCs undertake a full endoderm commitment, and the obtained endocrine pancreatic cells have high homology with mature islets. The iPSC-derived β cells were devoid of pluripotent residual cells, and the differentiation was pancreas-specific, as it did not generate ectodermal or mesodermal cells. Pseudotime trajectory identified a dichotomic endocrine/non-endocrine cell fate and distinct subgroups in the endocrine branch. Conclusions Future efforts to produce β cells from iPSCs must aim not only to improve the resulting endocrine cell but also to avoid differentiation into non-pancreatic endoderm cells

Uncinate process deviation in patients with odontogenic sinusitis: a computed tomographic evaluation

The uncinate process of the ethmoidis is one of the anatomic boundaries of osti- omeatal complex. Its relationship with the maxillary sinus ostium makes it the key landmark for endoscopic sinus surgery. Many authors denied a direct role of the uncinate process in the development of sinonasal infections (1). Nevertheless, chronic sinonasal diseases are often accompanied by an uncinate process antero-medialization, most notably in presence of an odontogenic etiology. This study aimed to retrospectively analyze uncinate process anatomy on computed tomographic (CT) scans, defining the association between uncinate process inclination and sinonasal health status. Sinonasal CT examinations of 46 individuals were reviewed, comparing patients without clinical and radiographic signs of sinonasal diseases (Group I), and patients diagnosed with odontogenic sinusitis according to the criteria proposed by Felisati et al. (2)(Group II). Uncinate process inclination was calculated by Radiant Dicom Viewer software, as the angle between the straight line connecting the antero- superior and the postero-inferior part of uncinate process, and the axis of symmetry, passing through sphenoidal rostrum and perpendicular to bizygomatic line. For each patient three axial scans (the most cranial, median, the most caudal), in which uncinate process was clearly detectable, were selected and a mean value was computed. Descriptive statistics of uncinate process inclination were calculated separately in the two groups. In Group I the mean angle was13.18° ± 10.33°with confidence limits (CL) (99%) between 6.21° and 20.15°,in Group II the mean angle was 29.89°±9.56° with CL between 24.44° and 35.34°. From these preliminary results, a marked medial devia tion of uncinate process was identified in odontogenic sinusitis compared to healthy sites. Additional assessments are required to confirm the role of this anatomical varia- tion in the pathogenesis of odontogenic sinusitis

Treponema pallidum (syphilis) antigen TpF1 induces angiogenesis through the activation of the IL-8 pathway

Over 10 million people every year become infected by Treponema pallidum and develop syphilis, a disease with broad symptomatology that, due to the difficulty to eradicate the pathogen from the highly vascularized secondary sites of infection, is still treated with injections of penicillin. Unlike most other bacterial pathogens, T. pallidum infection produces indeed a strong angiogenic response whose mechanism of activation, however, remains unknown. Here, we report that one of the major antigen of T. pallidum, the TpF1 protein, has growth factor-like activity on primary cultures of human endothelial cells and activates specific T cells able to promote tissue factor production. The growth factor-like activity is mediated by the secretion of IL-8 but not of VEGF, two known angiogenic factors. The pathogen's factor signals IL-8 secretion through the activation of the CREB/NF-\u3baB signalling pathway. These findings are recapitulated in an animal model, zebrafish, where we observed that TpF1 injection stimulates angiogenesis and IL-8, but not VEGF, secretion. This study suggests that the angiogenic response observed during secondary syphilis is triggered by TpF1 and that pharmacological therapies directed to inhibit IL-8 response in patients should be explored to treat this disease