Melatonin compensates silencing of heat shock protein 70 and suppresses ultraviolet radiation‐induced inflammation in human skin ex vivo and cultured keratinocytes

Melatonin, a lipophilic compound synthesized and released from the pineal gland, effectively acts against ultraviolet radiation (UVR), one of the main inducers of epidermal damage, skin cancer, inflammation, and DNA photo damage. One of the common known stress protein induced by UVR is heat shock protein 70 (Hsp70), highly expressed in human keratinocytes, providing cellular resistance to such stressors. Here, using human full‐thickness skin and normal human epidermal keratinocytes (NHEK), we investigated the interaction of melatonin and Hsp70 toward UVR‐induced inflammatory and apoptotic responses. The following observations were made: (i) UVR upregulated Hsp70 gene expression in human epidermis while melatonin significantly inverted this effect, (ii) similar patterns of regulation were observed within Hsp70 protein level, and (iii) mechanistic studies involving silencing of Hsp70 RNA (Hsp70 siRNA) showed prominent decrease of IκB‐α (an inhibitor of NF‐κB) and enhanced gene expression of pro‐inflammatory cytokines (IL‐1β, IL‐6, Casp‐1) and pro‐apoptotic protein (Casp‐3) in NHEK. Parallel investigation using melatonin (10^?3 m ) significantly inverted these responses regardless depletion of Hsp70 RNA suggesting a compensatory action of this compound in the defense mechanisms. Our findings combined with data reported so far thus enrich existing knowledge about the potent anti‐apoptotic and anti‐inflammatory action of melatonin.     

Associations between peri-implant crevicular fluid volume, concentrations of crevicular inflammatory mediators, and composite IL-1A -889 and IL-1B +3954 genotype. A cross-sectional study on implant recall patients with and without clinical signs of peri-implantitis

OBJECTIVES: To assess possible relationships between peri-implant crevicular fluid (PICF) volumes, biochemical markers of the peri-implant immune response, and periodontitis-associated genotype. MATERIAL AND METHODS: PICF samples from 29 implant maintenance patients, 24 wearing overdentures, five having single crowns and bridgework (11 patients with peri-implantitis and 18 individuals with healthy peri-implant conditions), were analyzed for per site and per crevicular-fluid-volume concentrations of interleukin-1beta, plasminogen activator inhibitor type 2, and prostaglandin E2 by ELISA. Associations between the three substance concentrations and to crevicular fluid flow rate were analyzed by linear regression analysis. The possible differentiating influence of the composite interleukin-1A and -1B genotype on the patients' peri-implant health and biochemical inflammatory status was checked formally with t-test statistics and the Wilcoxon' test. One implant per patient was chosen for analysis. RESULTS: In patients with healthy peri-implant conditions, genotype-positive individuals showed elevated crevicular fluid flow rates and at the same time reduced mediator concentrations. In patients with an implant affected from peri-implantitis, no statistically significant influence of the periodontitis-associated genotype around the fixture can be stated. There was no statistical difference between per site and per crevicular-fluid-volume concentration analyses. All three mediator concentrations were positively related to each other, while there was a strong negative correlation between crevicular fluid volume and plasminogen activator inhibitor 2 or prostaglandin E2. CONCLUSIONS: The Interleukin-1 polymorphism investigated exerted only little influence on the peri-implant crevicular immune response, and this influence appeared to be of limited impact in sites with established peri-implantitis lesions.     

The immunohistochemical distribution of collagens type IV, V, VI and of laminin in the human oral mucosa

The distribution of collagens type V (form AB2) and VI was investigated on cryostat sections of normal human oral mucosa by indirect immunofluorescence. For comparison, antibodies to fragments of type IV collagen and laminin were also used to delineate basement membrane containing structures. All antibodies used were raised against human proteins. Type V collagen appeared as a microfibrillar structure throughout the interstitium, apparently touching but not being present within epithelial or vascular basement membranes. Microfibrils in blood vessel walls were limited to the intimal layer. Pericellular areas were not specifically stained. Type VI collagen appeared as an almost amorphous stromal structure becoming more prominent and more fibrillar in the upper connective tissue papillae. Intense staining was observed in the media of blood vessels and around smooth muscle cells. A possible role of type VI collagen in tissue stabilization may be expected from this ubiquitous and abundant distribution. The findings identify types V and VI collagen as important structures in the oral mucosa and serve as a basis for understanding morbid changes.     

Tribonucleation of bubbles

We report on the nucleation of bubbles on solids that are gently rubbed against each other in a liquid. The phenomenon is found to depend strongly on the material and roughness of the solid surfaces. For a given surface, temperature, and gas content, a trail of growing bubbles is observed if the rubbing force and velocity exceed a certain threshold. Direct observation through a transparent solid shows that each bubble in the trail results from the early coalescence of several microscopic bubbles, themselves detaching from microscopic gas pockets forming between the solids. From a detailed study of the wear tracks, with atomic force and scanning electron microscopy imaging, we conclude that these microscopic gas pockets originate from a local fracturing of the surface asperities, possibly enhanced by chemical reactions at the freshly created surfaces. Our findings will be useful either for preventing undesired bubble formation or, on the contrary, for “writing with bubbles,” i.e., creating controlled patterns of microscopic bubbles.Elementary considerations show that a bubble will spontaneously disappear unless its radius r is larger than a critical value r_c = 2γ/ΔP, where γ is the surface tension of the liquid and ΔP is the difference between the pressure of the bubble contents and the surrounding liquid (1). Only bubbles larger than r_c can persist and grow by gas diffusion and liquid evaporation. The classical kinetic theory of nucleation (2) shows that, for water, the spontaneous formation of critical bubbles requires either superheats of 212 °C or negative pressures (i.e., tensions) of 140 MPa. Recent experiments have come close to the quantitative verification of these predictions (3, 4), but only at the cost of a great deal of sophistication and ingenuity. It must therefore be concluded that a different mechanism is responsible for the exceedingly commonplace occurrence of bubbles.The seed for the currently accepted explanation was planted by Gernez (5) who, in 1867, hypothesized that bubbles start from a preexisting gaseous nucleus lodged in solid impurities or the walls of the container. An explanation for the stability of these heterogeneous nuclei was later supplied by Harvey et al. (6) who pointed out that the curvature induced by contact with a hydrophobic solid surface would be able to stabilize a gas pocket even in an undersaturated liquid. This “crevice model” of bubble nucleation explains a large number of observations and has been applied to the development of so-called enhanced boiling surfaces (7, 8). Gas bubbles can be further stabilized by the formation of organic skins at their surface (9, 10).Despite these advances, the nucleation phenomenon still exhibits obscure facets, one of which—tribonucleation—is studied in this paper. It has been known for at least half a century that, as noticed by Hayward in 1967 (11), “extremely gentle rubbing” of two solid objects inside a liquid under tension, which is otherwise stable against most forms of mechanical action (e.g., knocking on the container wall or stirring), readily induces nucleation. This tribonucleation is often cited as a plausible source of the microbubbles found in the limbs of humans and animals after physical exercise (12, 13). Campbell (14) and Ikels (15) attributed the nucleation observed in these conditions to the pressure drop induced by the viscous flow in the space between two separating solid surfaces. Indeed, in highly viscous liquids, bubble formation compatible with this picture has been reported (16–18). However, this explanation cannot easily account for the nucleation observed in low-viscosity fluids like water and ethanol, because in many cases the theoretical gap between the solids would have to be smaller than the surface roughness. More strikingly, it cannot account for the key observation by Hayward that bubbles do not nucleate in the case of a rolling motion, but only in the case of a sliding motion between the solids (11), although for the same force and velocity the pressure drop is expected to be twice as large for rolling than for sliding (19). Another instance of bubble nucleation upon solid–solid contact in a low-viscosity liquid was reported by Theofanous et al. (20). These authors were able to reliably nucleate single bubbles by gently bringing into contact two stainless-steel wires in Freon superheated by up to 60 °C.In this paper, we present experiments in which we rub a bead against a wafer submerged in a low-viscosity liquid. We vary the rubbing force and velocity, the temperature, and the materials of the solids. Our approach is to combine macroscopic observations, revealing a threshold for the rubbing-induced nucleation, with microscopic observations at the smallest scales of the problem: that of the apparent (Hertz) contact between the solids and that of the roughness tips where the actual contact is realized.     

Relevance of Bilateral Cervical Thymectomy in Patients with Renal Hyperparathyroidism: Analysis of 161 Patients Undergoing Reoperative Parathyroidectomy

Background

The most frequent location of ectopic or supernumerary inferior parathyroid is the thymus. Bilateral cervical thymectomy has therefore been recommended as an essential part of the initial surgery for renal hyperparathyroidism (rHPT) to avoid persistent or recurrent cervical disease. The aim of this study was to evaluate how often reoperation might have been avoidable if an appropriate cervical thymectomy had been performed during initial surgery.

Methods

A prospective database of patients with rHPT was screened for patients on permanent dialysis who underwent reoperative parathyroidectomy (PTX) between 1976 and 2010. Data were retrospectively analyzed for the performance of bilateral cervical thymectomy during previous surgeries and the presence of ectopic and/or supernumerary intrathymic parathyroid glands during reoperative PTX.

Results

Of 161 patients who underwent reoperative PTX, 95 had neck reexploration. Among them were 29 patients with total PTX and autotransplantation, seven with subtotal PTX (3.5 glands resected), and 59 with incomplete PTX during the initial surgery. Bilateral cervical thymectomy during the initial PTX was performed in only 12 of 95 patients (12.6 %). It was revealed to be incomplete in six of them, inheriting an intrathymic parathyroid gland during reoperative interventions. Reoperative PTX revealed intrathymic parathyroid glands in 27 of 95 patients (28.4 %). The intrathymic parathyroid glands were ectopic in 17 (63.0 %) patients and supernumerary in 8 (29.6 %). Both ectopic and supernumerary intrathymic parathyroid glands were found in two patients (7.4 %).

Conclusions

The risk for persistent and recurrent disease based on intrathymic parathyroid glands is a relevant problem during initial surgery for rHPT. Thus, routine bilateral cervical thymectomy that is as complete as possible is essential during the initial PTX for rHPT.     

Induction of dermal-epidermal separation in mice by passive transfer of antibodies specific to type VII collagen

Epidermolysis bullosa acquisita (EBA) is a subepidermal blistering disorder associated with tissue-bound and circulating autoantibodies specific to type VII collagen, a major constituent of the dermal-epidermal junction. Previous attempts to transfer the disease by injection of patient autoantibodies into mice have been unsuccessful. To study the pathogenic relevance of antibodies specific to type VII collagen in vivo, we generated and characterized rabbit antibodies specific to a murine form of this antigen and passively transferred them into adult nude, BALB/c, and C57BL/6 mice. Immune rabbit IgG bound to the lamina densa of murine skin and immunoblotted type VII collagen. Mice injected with purified IgG specific to type VII collagen, in contrast to control mice, developed subepidermal skin blisters, reproducing the human disease at the clinical, histological, electron microscopical, and immunopathological levels. Titers of rabbit IgG in the serum of mice correlated with the extent of the disease. F(ab')(2) fragments of rabbit IgG specific to type VII collagen were not pathogenic. When injected into C5-deficient mice, antibodies specific to type VII collagen failed to induce the disease, whereas C5-sufficient mice were susceptible to blister induction. This animal model for EBA should facilitate further dissection of the pathogenesis of this disease and development of new therapeutic strategies.     

Functional Profile of the Isolated Uremic Nephron: IMPAIRED WATER PERMEABILITY AND ADENYLATE CYCLASE RESPONSIVENESS OF THE CORTICAL COLLECTING TUBULE TO VASOPRESSIN

Resistance of the chronically diseased kidney to vasopressin has been proposed as a possible explanation for the urinary concentrating defect of uremia. The present studies examined the water permeability and adenylate cyclase responsiveness of isolated cortical collecting tubules (CCT) from remnant kidneys of uremic rabbits to vasopressin. In the absence of vasopressin the CCTs of both normal and uremic rabbits were impermeable to water. At the same osmotic gradient, addition of a supramaximal concentration of vasopressin to the peritubular bathing medium led to a significantly lower net water flux per unit length (and per unit luminal surface area) in uremic CCTs than in normal CCTs. Transepithelial osmotic water permeability coefficient, P(f), was 0.0232 +/-0.0043 cm/s in normal CCTs and 0.0059+/-0.001 cm/s in uremic CCTs (P < 0.001). The impaired vasopressin responsiveness of the uremic CCTs was observed whether normal or uremic serum was present in the bath.Basal adenylate cyclase activity per microgram protein was comparable in normal and uremic CCTs. Stimulation by NaF led to equivalent levels of activity in both, whereas vasopressin-stimulated activity was 50% lower in the uremic than in the normal CCTs (P < 0.025).The cyclic AMP analogue, 8-bromo cyclic AMP, produced an increase in the P(f) of normal CCTs closely comparable to that observed with vasopressin. In contrast, the P(f) of uremic CCTs was only minimally increased by this analogue and was not further stimulated by theophylline.These studies demonstrate an impaired responsiveness of the uremic CCT to vasopressin. This functional defect appears to be a result, at least in part, of a blunted responsiveness of adenylate cyclase to vasopressin. The data further suggest that an additional defect in the cellular response to vasopressin may exist, involving a step (or steps) subsequent to the formation of cyclic AMP.A unifying concept of the urinary concentrating defect of uremia is proposed which incorporates a number of hitherto unexplained observations on the concentrating and diluting functions of the diseased kidney.     

Pemphigus foliaceus IgG causes dissociation of desmoglein 1-containing junctions without blocking desmoglein 1 transinteraction

Autoantibodies against the epidermal desmosomal cadherins desmoglein 1 (Dsg1) and Dsg3 have been shown to cause severe to lethal skin blistering clinically defined as pemphigus foliaceus (PF) and pemphigus vulgaris (PV). It is unknown whether antibody-induced dissociation of keratinocytes is caused by direct inhibition of Dsg1 transinteraction or by secondary cellular responses. Here we show in an in vitro system that IgGs purified from PF patient sera caused cellular dissociation of cultured human keratinocytes as well as significant release of Dsg1-coated microbeads attached to Dsg-containing sites on the keratinocyte cellular surface. However, cell dissociation and bead release induced by PF-IgGs was not caused by direct steric hindrance of Dsg1 transinteraction, as demonstrated by single molecule atomic force measurements and by laser trapping of surface-bound Dsg1-coated microbeads. Rather, our experiments strongly indicate that PF-IgG-mediated dissociation events must involve autoantibody-triggered cellular signaling pathways, resulting in destabilization of Dsg1-based adhesive sites and desmosomes.