Henk S. Brand

Interleukin-6 production by human liver (myo)fibroblasts in culture. Evidence for a regulatory role of LPS, IL-lβ and TNFα

Abstract Background/Aims: Interleukin-6 is a major trigger forthe synthesis of acute phase proteins by liver parenchymal cells. Acute phase proteins may contribute to the regulation of liver fibrosis by inhibition of proteases (e.g. collagenase) and by binding of cytokines. Since liver (myo)fibroblasts play an important role in the production of extracellular matrix in fibrotic livers, a study was undertaken into whether these cells are able to synthesize interleukin-6, which would give them the opportunity to contribute to regulation of synthesis of acute phase proteins by neighbouring parenchymal cells. Methods: In the present study we investigated interleukin-6production by two cell types obtained from human liver tissue: human fat-storing cells obtained from 5–15% Percoll fractions, which transformed in culture into myofibroblasts co-expressing α -smooth muscle actin and vimentin (VA cells) and fibroblasts obtained from 30–40% Percoll fractions which express vimentin only (V vells). Interleukin-6 production was measured in culture media of these cells using an enzyme-linked immunosorbent assay after incubation with lipopolysaccharide, and mediators like interleukin-1 β , tumor necrosis factor- a , transforming growth factor- β and interferon-gamma, known to be present in elevated concentrations in fibrotic livers. Results: Unstimulated human liver (myo)fibroblasts produced considerable amounts of interleukin-6 (287 ng/mg cellular protein (VA cells), and 54 ng/mg cellular protein (V cells), within 48 h). Biological activity of these high concentrations of interleukin-6 measured in the enzyme-linked immuno-sorbent assay was confirmed in the B9-bioassay for interleukin-6 and by stimulation of α 2-macroglobulin production in rat liver parenchymal cell cultures. Lipopolysaccharide, interleukin-1 β and tumor necrosis factor- α were potent stimulators of basal interleukin-6 production by VA and V cells. 1 μ g/ml lipopolysaccharide enhanced basal interleukin-6 production 3-fold within 48 h. 100 U/ml interleukin-1 β and 1000 U/ml tumor necrosis factor- α each stimulated basal interleukin-6 production by VA cells 2–5 fold, whereas V cells were stimulated 10–25 fold. These effects were specific since the stimulation by lipopolysaccharide was completely inhibited by polymyxin B and the enhancing effects of interleukin-1 β and tumor necrosis factor- α were neutralized by specific antibodies. Transforming growth factor- β and interferon gamma did not influence interleukin-6 synthesis by either cell type in culture. Conclusions: These results indicate that transformed fat-storing cells (VA cells) and fibroblasts (V cells) may function as a local source of interleukin-6 in the human liver. Since interleukin-6 plays a key role in the regulation of the production of acute phase proteins by liver parenchymal cells, we hypothesize that human liver (myo)fibroblasts may stimulate local production of acute phase proteins in the fibrotic liver, thus contributing to local regulation of inflammatory and fibrogenic reactions.

Transforming growth factor-β-induced collagen synthesis by human liver myofibroblasts is inhibited by α2-macroglobulin

Abstract Background: Transforming growth factor-β (TGFβ) plays a central role in the stimulation of matrix production during liver fibrosis. The action of TGFβ in different systems has been shown to be influenced by α 2 -macroglubulin (α 2 M), a serum protein with strong protease-scavenging and cytokine-binding properties. Aims: In the present study, α 2 M derived from normal human plasma has been tested for its ability to modulate the TGFβ-induced collagen production by human liver fat-storing cells (FSC), which had transformed into α-smooth muscle actin-expressing myofibroblasts in culture. Methods: α 2 M has been tested after activation with methylamine (α 2 M-Me), an in vitro equivalent of protease activated α 2 M. The binding of 125 I-TGFβ1 to activated forms of α 2 M was demonstrated by rate electrophoresis. Collagen synthesis was examined in human liver myofibroblast cultures obtained from three different human livers by incorporation of 3 H-proline into TCA-precipitable, specific collagenase degradable proteins. Uptake of α 2 M was studied by means of immunofluorescence. Results: TGFβ (1 ng/ml) significantly stimulated collagen synthesis of controls in the absence of TGFβ. α 2 M-Me reduced this TGFβ-induced collagen synthesis dose-dependently, reaching significant inhibition from 10 μg/ml α 2 M-Me onward. Upon addition of 100 μg/ml α 2 M-Me the effect of TGFβ was reduced by 60% to 128±31% (mean±SD) of control values in the absence of TGFβ. Human liver myofibroblasts endocytosed α 2 M-Me added to the cultures as detected by immunofluorescence. Accordingly, reduction of TGFβ-activity by α 2 M-Me may be explained by receptor-mediated clearance of α 2 M-TGFβ complexes by the cells. Conclusions: TGFβ-induced collagen formation by human liver myofibroblasts obtained from three different livers is reduced in vitro by activated α 2 M. From these results, we hypothesize that α 2 M may have an antifibrogenic effect in vivo by interference with TGFβ-induced matrix synthesis during liver fibrosis.

The management of xerostomia in patients on haemodialysis: comparison of artificial saliva and chewing gum

Many patients on haemodialysis (HD) therapy suffer from a dry mouth and xerostomia. This can be relieved by mechanical and gustatory stimulation or palliative care. The aim of this crossover study was to investigate the effect and preferences of a sugar-free chewing gum (Freedent WhiteTM) and a xanthan gum-based artificial saliva (XialineTM) in the management of xerostomia in chronic HD patients. Sixty-five HD patients participated in a 6-week crossover trial. The artificial saliva was rated significantly lower than the chewing gum for effectiveness, taste and a global assessment. No preference differences were found for gender and age, although older subjects rated the artificial saliva with a higher mark. Thirty-nine subjects (60%) preferred chewing gum, 15% (n = 10) preferred the artificial saliva. Therefore, both chewing gum and artificial saliva could play an important role in the palliative care of xerostomia in HD patients.

Interindividual variation, correlations, and sex‐related differences in the salivary biochemistry of young healthy adults

A cross-sectional observational study was conducted to evaluate interindividual biochemical variation in unstimulated whole saliva in a population of 268 systemically healthy young students, 18-30 yr of age, with no apparent caries lesions or periodontal disease. Salivary flow rate, protein content, pH, buffering capacity, mucins MUC5B and MUC7, albumin, secretory IgA, cystatin S, lactoferrin, chitinase, amylase, lysozyme, and proteases were measured using ELISAs and enzymatic activity assays. Significant differences were found between male and female subjects. Salivary pH, buffering capacity, protein content, MUC5B, secretory IgA, and chitinase activity were all lower in female subjects compared with male subjects, whereas MUC7 and lysozyme activity were higher in female subjects. There was no significant difference between sexes in salivary flow rate, albumin, cystatin S, amylase, and protease activity. Principal component analysis (PCA) and spectral clustering (SC) were used to assess intervariable relationships within the data set and to identify subgroups. Spectral clustering identified two clusters of participants, which were subsequently described. This study provides a comprehensive overview of the distribution and inter-relations of a set of important salivary biochemical variables in a systemically healthy young adult population, free of apparent caries lesions and periodontal disease. It highlights significant gender differences in salivary biochemistry.

On the ecosystemic network of saliva in healthy young adults

A dysbiotic state is believed to be a key factor in the onset of oral disease. Although oral diseases have been studied for decades, our understanding of oral health, the boundaries of a healthy oral ecosystem and ecological shift toward dysbiosis is still limited. Here, we present the ecobiological heterogeneity of the salivary ecosystem and relations between the salivary microbiome, salivary metabolome and host-related biochemical salivary parameters in 268 healthy adults after overnight fasting. Gender-specific differences in the microbiome and metabolome were observed and were associated with salivary pH and dietary protein intake. Our analysis grouped the individuals into five microbiome and four metabolome-based clusters that significantly related to biochemical parameters of saliva. Low salivary pH and high lysozyme activity were associated with high proportions of streptococcal phylotypes and increased membrane-lipid degradation products. Samples with high salivary pH displayed increased chitinase activity, higher abundance of Veillonella and Prevotella species and higher levels of amino acid fermentation products, suggesting proteolytic adaptation. An over-specialization toward either a proteolytic or a saccharolytic ecotype may indicate a shift toward a dysbiotic state. Their prognostic value and the degree to which these ecotypes are related to increased disease risk remains to be determined.

MUC5B levels in submandibular gland saliva of patients treated with radiotherapy for head-and-neck cancer: A pilot study

BackgroundThe salivary mucin MUC5B, present in (sero)mucous secretions including submandibular gland (SMG) saliva, plays an important role in the lubrication of the oral mucosa and is thought to be related to the feeling of dry mouth. We investigated if MUC5B levels in SMG saliva could distinguish between the presence or absence of severe dry mouth complaints 12u2009months after radiotherapy (RT) for head-and-neck cancer (HNC).FindingsTwenty-nine HNC patients with a residual stimulated SMG secretion rate of ≥0.2u2009ml/10u2009min at 12u2009months after RT were analyzed. MUC5B (in U; normalized to 1) and total protein levels (mg/ml) were measured in SMG saliva at baseline and 12u2009months after RT using ELISA and BCA protein assay, respectively. Overall, median MUC5B levels decreased after RT from 0.12 to 0.03 U (pu2009=u20090.47). Patients were dichotomized into none/mild xerostomia (nu2009=u200912) and severe xerostomia (nu2009=u200917) based on a questionnaire completed at 12u2009months. SMG and whole saliva flow rates decreased after RT but were comparable in both groups. The median MUC5B level was higher in patients with no or mild xerostomia compared to patients with severe xerostomia (0.14 vs 0.01 U, pu2009=u20090.22). Half of the patients with severe xerostomia had no detectable MUC5B at 12u2009months after RT. No differences in total protein levels were observed.ConclusionsQualitative saliva parameters like MUC5B need further investigation in RT-induced xerostomia. This pilot study showed a trend towards lower MUC5B levels in the SMG saliva of patients with severe xerostomia 12u2009months after RT for HNC.

Saliva and wound healing.

Oral wounds heal faster and with less scar formation than skin wounds. One of the key factors involved is saliva, which promotes wound healing in several ways. Saliva creates a humid environment, thus improving the survival and functioning of inflammatory cells that are crucial for wound healing. In addition, saliva contains several proteins which play a role in the different stages of wound healing. Saliva contains substantial amounts of tissue factor, which dramatically accelerates blood clotting. Subsequently, epidermal growth factor in saliva promotes the proliferation of epithelial cells. Secretory leucocyte protease inhibitor inhibits the tissue-degrading activity of enzymes like elastase and trypsin. Absence of this protease inhibitor delays oral wound healing. Salivary histatins in vitro promote wound closure by enhancing cell spreading and cell migration, but do not stimulate cell proliferation. A synthetic cyclic variant of histatin exhibits a 1,000-fold higher activity than linear histatin, which makes this cyclic variant a promising agent for the development of a new wound healing medication. Conclusively, recognition of the many roles salivary proteins play in wound healing makes saliva a promising source for the development of new drugs involved in tissue regeneration.

The effect of physical exercise on salivary secretion of MUC5B, amylase and lysozyme.

OBJECTIVESnSaliva secretion is regulated by the autonomic nervous system. Parasympathic stimuli increase the secretion of water and mucin MUC5B, whereas sympathetic stimuli such as physical exercise increase the secretion of amylase and other proteins. In the present study we investigated the effect of physical exercise, as a sympathetic stimulus, on salivary flow rate and output of MUC5B, amylase, lysozyme and total protein.nnnDESIGNnUnstimulated whole saliva was collected before exercise (1), after 10 min exercise with moderate intensity by running with a heart rate around 130 beats per minute (2), followed by 10 min exercise with high intensity by running to exhaustion (3) and after 30 min recovery (4). Salivary flow rate, protein and MUC5B concentration, and amylase and lysozyme activity were determined. Saliva protein composition was analysed using SDS-PAGE and immunoblotting.nnnRESULTSnSalivary flow rate, protein and lysozyme secretion increased after exercise with moderate intensity and increased further after exercise with high intensity (p<0.01). Amylase and MUC5B increased after exercise with moderate intensity (p<0.0001), but did not differ significantly between moderate and high exercise intensity. SDS-PAGE analysis and immunoblotting showed that, especially after exercise with high intensity, the concentrations of several other salivary proteins, including MUC7, albumin, and extra-parotid glycoprotein, also increased.nnnCONCLUSIONSnExercise may not only lead to the anticipated increase in amylase and protein secretion, but also to an increase in salivary flow rate and MUC5B secretion.

Interference of electronic apex locators with implantable cardioverter defibrillators

INTRODUCTIONnThe purpose of this in vitro study was to evaluate the potential electromagnetic interference of electronic apex locators (EALs) on implantable cardioverter defibrillators (ICDs).nnnMETHODSnFour different EALs were tested for their ability to interfere with the correct function of 3 different ICDs. Each ICD was placed in a plastic container with 1.5 L physiological saline, and the EAL unit was placed at a distance of 2.5 cm from the ICD. The file electrode and lip clip were placed directly against the ICD. The EAL was turned on for 30 seconds while continuously showing the APEX mark. As a negative control, the ICD was tested without EAL for 30 seconds. An electrosurgical unit served as a positive control. During each test, the ICD output was monitored continuously by real-time telemetry, and after completion of the experiment, intracardiac electrocardiograms were printed. The tests were repeated 3 times for each device. The electrocardiograms were examined for interference on ICD ventricular activity.nnnRESULTSnAll EALs tested and the negative control failed to produce electromagnetic interference in each of the ICDs tested. The electrosurgical unit induced interference in the ICDs, which were detected as episodes of ventricular tachycardia and led to the initiation of electrical shocks in all ICDs.nnnCONCLUSIONSnThe 4 EALs tested did not interfere with the correct functioning of ICDs in vitro.

In vivo amino acid fluxes in regenerating liver after two-thirds hepatectomy in the rat

BACKGROUND/AIMSnRecent reports in the literature suggest that liver cell swelling following amino acid influx exerts anabolic and anti-catabolic effects. We have tested the possibility that rapid liver growth after partial hepatectomy is promoted by an increased amino acid-influx and that this is associated with an increased hepatic water content and a decreased rate of proteolysis.nnnMETHODSnTwo-thirds hepatectomy was performed in rats. Plasma liver flow and amino acid fluxes were measured after 24 or 48 h.nnnRESULTSnPlasma liver flow was increased 24 and 48 h after partial hepatectomy or sham-operation in pair-fed animals. At these time points, in both groups there was a specific two- to threefold increased net hepatic uptake of the amino acids alanine and glycine, both being transported by the sodium-coupled amino acid transport system A/ASC. No changes in uptake of system N transported amino acids were observed. Both in partially hepatectomized and sham-operated pair-fed animals, the hepatic uptake of alanine and glycine was accompanied by a minor increase in tissue water (from 68 to 70%). Proteolysis, measured by leucine efflux, was only reduced in regenerating livers.nnnCONCLUSIONSnWe conclude that cell swelling is not an important factor in the stimulation of net protein synthesis during liver regeneration.