José Cancela

Islet-cell-to-cell communication as basis for normal insulin secretion.

The emergence of pancreatic islets has necessitated the development of a signalling system for the intra‐ and inter‐islet coordination of β cells. With evolution, this system has evolved into a complex regulatory network of partially cross‐talking pathways, whereby individual cells sense the state of activity of their neighbours and, accordingly, regulate their own level of functioning. A consistent feature of this network in vertebrates is the expression of connexin (Cx)‐36‐made cell‐to‐cell channels, which cluster at gap junction domains of the cell membrane, and which adjacent β cells use to share cytoplasmic ions and small metabolites within individual islets. This chapter reviews what is known about Cx36, and the mechanism whereby this β‐cell connexin significantly regulates insulin secretion. It further outlines other less established functions of the protein and evaluates its potential relevance for the development of novel therapeutic approaches to diabetes.

Beta cells preferentially exchange cationic molecules via connexin 36 gap junction channels

Aims/hypothesisPancreatic beta cells are connected by gap junction channels made of connexin 36 (Cx36), which permit intercellular exchanges of current-carrying ions (ionic coupling) and other molecules (metabolic coupling). Previous studies have suggested that ionic coupling may extend to larger regions of pancreatic islets than metabolic coupling. The aim of the present study was to investigate whether this apparent discrepancy reflects a difference in the sensitivity of the techniques used to evaluate beta cell communication or a specific characteristic of the Cx36 channels themselves.MethodsWe microinjected several gap junction tracers, differing in size and charge, into individual insulin-producing cells and evaluated their intercellular exchange either within intact islets of control, knockout and transgenic mice featuring beta cells with various levels of Cx36, or in cultures of wild-type and Cx36-transfected MIN6 cells.ResultsWe found that (1) Cx36 channels favour the exchange of cations and larger positively charged molecules between beta cells at the expense of anionic molecules; (2) this exchange occurs across sizable portions of pancreatic islets; and (3) during glibenclamide (known as glyburide in the USA and Canada) stimulation beta cell coupling increases to an extent that varies for different gap junction-permeant molecules.Conclusions/interpretationThe data show that beta cells are extensively coupled within pancreatic islets via exchanges of mostly positively charged molecules across Cx36 channels. These exchanges selectively increase during stimulation of insulin secretion. The identification of this permselectivity is expected to facilitate the identification of endogenous permeant molecules and of the mechanism whereby Cx36 signalling significantly contributes to the modulation of insulin secretion.

Effect of Photodynamic Therapy, Diode Laser, and Deep Scaling on Cytokine and Acute-Phase Protein Levels in Gingival Crevicular Fluid of Residual Periodontal Pockets

BACKGROUND There is an ongoing controversy on the benefits of treatment protocols, including dental lasers and photodynamic therapy (PDT). The purpose of this study is to compare the local biologic effects of PDT, diode soft laser (DSL) therapy, and conventional deep scaling and root planing (SRP) in residual pockets. METHODS Thirty-two individuals were included based on a history of previous treatment for periodontitis and the persistence of sites with probing depths >4 mm and bleeding on probing. Residual pockets were debrided with an ultrasonic device and then randomly assigned either to PDT, DSL, or SRP. Gingival crevicular fluid was collected before treatment, after 14 days, and at 2 and 6 months. Levels of 13 cytokines and nine acute-phase proteins were measured using a bead-based multiplexing analysis system. RESULTS Treatment with PDT, DSL, or SRP led to significant changes in several cytokines and acute-phase proteins: Compared with baseline, levels of interleukin-17, basic fibroblast growth factor, granulocyte colony-stimulating factor, granulocyte macrophage colony-stimulating factor, and macrophage inflammatory protein 1-α were lower 14 days and 2 months after treatment. Except for granulocyte colony-stimulating factor, these differences remained significant throughout the observation period. The levels of five acute-phase proteins (α-2 macroglobulin, haptoglobin, serum amyloid P, procalcitonin, and tissue plasminogen activator) were significantly higher at 6 months than at baseline. No significant differences were observed among the three treatment modalities at any time point for any biochemical parameter. CONCLUSIONS Levels of several cytokines and acute-phase proteins significantly changed after treatment regardless of treatment modality. There was no evidence for a specific DSL- or PDT-enhanced expression of inflammatory mediators.

Connexin36 and pancreatic β-cell functions

Abstract Most cell types are functionally coupled by connexin (Cx) channels, i.e. exchange cytoplasmic ions and small metabolites through gap junction domains of their membrane. This form of direct cell-to-cell communication occurs in all existing animals, whatever their position in the phylogenetic scale, and up to humans. Pancreatic β-cells are no exception, and normally cross-talk with their neighbors via channels made of Cx36. These exchanges importantly contribute to coordinate and synchronize the function of individual cells within pancreatic islets, particularly in the context of glucose-induced insulin secretion. Compelling evidence now indicates that Cx36-mediated coupling, and/or the Cx36 protein per se, play significant regulatory roles in various β-cell functions, ranging from the biosynthesis, storage and release of insulin. Recent preliminary data further suggest that the protein may also be implicated in the balance of β-cell growth versus necrosis and apoptosis, and in the regulated expression of specific genes. Here, we review this evidence, discuss the possible involvement of Cx36 in the pathophysiology of diabetes, and evaluate the relevance of this connexin in the therapeutic approaches to the disease.

Subgingival air-polishing with erythritol during periodontal maintenance: randomized clinical trial of twelve months.

Objectives To evaluate repeated subgingival air-polishing in residual pockets with a new erythritol powder containing 0.3% chlorhexidine. Material and Methods Single-centre, examiner masked, randomized clinical trial of 12 months with a two-arm, within-subject parallel design. Fifty patients in periodontal maintenance were monitored in 3-month intervals. At months 0, 3, 6 and 9, all sites presenting with a probing depth (PD) >4 mm were subject to subgingival air-polishing (test side) or ultrasonic debridement (control side). The primary endpoint was presence/absence of PD >4 mm after 12 months. Results Totally 6918 sites were monitored at baseline, 457 of them had a PD >4 mm (range 5–9 mm). The number of pockets >4 mm per subject, PD and bleeding on probing were significantly lower at month 12. Differences between test and control were not significant. There was a significant difference in favour of air-polishing for the perception of pain/discomfort. Differences of frequencies at >1000 and >100,000 cells/ml of six microorganisms between baseline and month 12 were not significant. At month 12, test sites were less frequently positive for Aggregatibacter actinomycetemcomitans at >1000 cells/ml than controls, and counts never exceeded 100,000 cells/ml. Conclusions Repeated subgingival air-polishing reduced the number of pockets >4 mm similar to ultrasonic debridement. It was safe and induced less pain.

The intercellular synchronization of Ca2+ oscillations evaluates Cx36-dependent coupling.

Connexin36 (Cx36) plays an important role in insulin secretion by controlling the intercellular synchronization of Ca2+ transients induced during stimulation. The lack of drugs acting on Cx36 channels is a major limitation in further unraveling the molecular mechanism underlying this effect. To screen for such drugs, we have developed an assay allowing for a semi-automatic, fluorimetric quantification of Ca2+ transients in large populations of MIN6 cells. Here, we show that (1) compared to control cells, MIN6 cells with reduced Cx36 expression or function showed decreased synchrony of glucose-induced Ca2+ oscillations; (2) glibenclamide, a sulphonylurea which promotes Cx36 junctions and coupling, increased the number of synchronous MIN6 cells, whereas quinine, an antimalarial drug which inhibits Cx36-dependent coupling, decreased this proportion; (3) several drugs were identified that altered the intercellular Ca2+ synchronization, cell coupling and distribution of Cx36; (4) some of them also affected insulin content. The data indicate that the intercellular synchronization of Ca2+ oscillations provides a reliable and non-invasive measurement of Cx36-dependent coupling, which is useful to identify novel drugs affecting the function of β-cells, neurons, and neuron-related cells that express Cx36.

Cutting Edge: Lack of Evidence for Connexin-43 Expression in Human Epidermal Langerhans Cells

A provocative study has shown that viral peptides may be transferred in vitro from epithelial cells to APC through connexin-43 gap junction channels. In support of this cross-presentation pathway, the study also reported that human dendritic cells, including Langerhans cells of skin, express connexin-43. In this report we show that if this was the case, the levels of connexin-43 are below those detectable by immunofluorescence, flow cytometry, quantitative PCR of purified CD1a+ cells, and electron microscopy, raising questions about the relevance of the connexin-43-dependent mechanism for Langerhans cells of noninflamed human skin.

Connexin Modulators of Endocrine Function

The emergence of multicellular organisms has necessitated the specialization of short- and long-range chemical signaling systems, including that provided by the endocrine system. Conversely, the existence of an endocrine system conceptually demands a multicellular organism, to which proper signaling usually also imposes a multicellular gland. Accordingly, the secretory cells of all endocrine glands have developed mechanisms for interacting with adjacent and distant cells. With evolution, such mechanisms have diversified and have been progressively integrated in a complex regulatory network, whereby individual endocrine cells sense the state of activity of their neighbors and regulate accordingly their own level of functioning. A consistent feature of this network is the expression of connexin-made channels between the hormone-producing cells of all glands so far investigated in vertebrates. In a few instances, these channels have also been documented between the endocrine cells and nearby target cells. Here, we have reviewed the distribution of connexins in the mammalian endocrine system, and have discussed the recent evidence pointing to the participation of these proteins in the functioning of endocrine cells, and on the action of hormones on specific target cells.

Systemic Biomarkers in 2-Phase Antibiotic Periodontal Treatment A Randomized Clinical Trial

Accumulating evidence suggests that periodontal infections may have an impact on systemic health. In patients with untreated periodontitis, very high values for several inflammatory markers in serum are expressed simultaneously. We investigated to what extent these peak values change after nonsurgical and surgical periodontal treatment, with adjunctive antibiotics administered during the first or the second treatment phase. In a single-center, randomized, placebo-controlled, and double-masked clinical trial, 80 patients with chronic or aggressive periodontitis were randomized into 2 treatment groups: group A, receiving systemic amoxicillin and metronidazole during the first, nonsurgical phase of periodontal therapy (phase 1), and group B, receiving the antibiotics during the second, surgical phase (phase 2). Serum samples were obtained at baseline (BL), 3 mo after phase 1 (M3), and 6 and 12 mo after phase 2 (M6, M12). Samples were evaluated for 15 cytokines and 9 acute-phase proteins using the Bio-Plex bead array multianalyte detection system. For each analyte, peak values were defined as greater than mean +2 SD of measurements found in 40 periodontally healthy persons. Sixty-six patients showed a peak value of at least 1 analyte at BL. At M12, the number of these patients was only 36 (P = 0.0002). This decrease was stronger in group A (BL: 35, M12: 19, P = 0.0009) than in group B (BL: 31, M12: 17, P = 0.14). Twenty patients displayed peak values of at least 4 biomarkers at BL. The nonsurgical therapy delivered in the first phase reduced most of these peaks (group A, BL: 9, M3: 4, P = 0.17; group B, BL: 11, M3: 2, P = 0.01), irrespective of adjunctive antibiotics. The reductions obtained at M3 were maintained until M12 in both groups. Initial, nonsurgical periodontal therapy reduced the incidence of peak levels of inflammatory markers. Antibiotics and further surgical therapy did not enhance the effect (Clinicaltrials.gov NCT02197260).

Gap Junctions and Insulin Secretion

About 30 years ago, pancreatic beta cells were shown to be connected by gap junctions and to exhibit glucose-induced oscillatory electrical activity, two features that were hypothetically linked to insulin secretion. Since then, gap junctions have been shown to be an obligatory feature of beta cells in all species and all physiological conditions studied. They are composed of connexin proteins and allow for beta-cell to beta-cell exchanges of current-carrying ions and other small cytosolic metabolites which synchronize the electrical and metabolic activity of beta cells, and recruit these cells for insulin biosynthesis and release. Together, these effects account for the significant contribution of gap junction-dependent signaling to the control of insulin secretion. More recent data suggest that gap junctions, either via the expression of connexin proteins and/or of the intercellular communications that these proteins permit, also significantly influence beta-cell growth, apoptosis, and the resistance of islets to immune attack. The mechanism(s) whereby gap junction signaling exerts these multiple effects is still obscure. Understanding this mechanism is relevant both to our understanding of the physiology of pancreatic islets and to the pathophysiology of beta-cell dysfunction in both type 1 and type 2 diabetes. Furthermore, appropriate expression of gap junctions may be a prerequisite for the engineering of surrogate insulin-producing cells and their proper three-dimensional packaging, which may be important for using these cells as a cell-based therapy for the treatment of diabetic patients. Here, we review the current status of our knowledge in this field and its exciting perspectives.