Different Forms of TFF3 in the Human Endocervix, including a Complex with IgG Fc Binding Protein (FCGBP), and Further Aspects of the Cervico-Vaginal Innate Immune Barrier.

TFF3 is a typical secretory poplypeptide of mucous epithelia belonging to the trefoil factor family (TFF) of lectins. In the intestine, respiratory tract, and saliva, TFF3 mainly exists as a high-molecular-mass complex with IgG Fc binding protein (FCGBP), which is indicative of a role in mucosal innate immunity. For the first time, we identified different forms of TFF3 in the endocervix, i.e., monomeric and homodimeric TFF3, as well as a high-molecular-mass TFF3-FCGBP complex; the latter also exists in a hardly soluble form. Immunohistochemistry co-localized TFF3 and FCGBP. Expression analyses of endocervical and post-menopausal vaginal specimens revealed a lack of mucin and TFF3 transcripts in the vaginal specimens. In contrast, genes encoding other typical components of the innate immune defense were expressed in both the endocervix and vagina. Of note, FCGBP is possibly fucosylated. Endocervical specimens from transgender individuals after hormonal therapy showed diminished expression, particularly of FCGBP. Furthermore, mucus swabs from the endocervix and vagina were analyzed concerning TFF3, FCGBP, and lysozyme. It was the aim of this study to illuminate several aspects of the cervico-vaginal innate immune barrier, which is clinically relevant as bacterial and viral infections are also linked to infertility, pre-term birth and cervical cancer.

Expression Profiling along the Murine Intestine: Different Mucosal Protection Systems and Alterations in Tff1-Deficient Animals.

Tff1 is a typical gastric peptide secreted together with the mucin, Muc5ac. Tff1-deficient (Tff1KO) mice are well known for their prominent gastric phenotype and represent a recognized model for antral tumorigenesis. Notably, intestinal abnormalities have also been reported in the past in these animals. Here, we have compared the expression of selected genes in Tff1KO mice and their corresponding wild-type littermates (RT-PCR analyses), focusing on different mucosal protection systems along the murine intestine. As hallmarks, genes were identified with maximum expression in the proximal colon and/or the duodenum: Agr2, Muc6/A4gnt/Tff2, Tff1, Fut2, Gkn2, Gkn3, Duox2/Lpo, Nox1. This is indicative of different protection systems such as Tff2/Muc6, Tff1-Fcgbp, gastrokines, fucosylation, and reactive oxygen species (ROS) in the proximal colon and/or duodenum. Few significant transcriptional changes were observed in the intestine of Tff1KO mice when compared with wild-type littermates, Clca1 (Gob5), Gkn1, Gkn2, Nox1, Tff2. We also analyzed the expression of Tff1, Tff2, and Tff3 in the pancreas, liver, and lung of Tff1KO and wild-type animals, indicating a cross-regulation of Tff gene expression. Furthermore, on the protein level, heteromeric Tff1-Fcgbp and various monomeric Tff1 forms were identified in the duodenum and a high-molecular-mass Tff2/Muc6 complex was identified in the proximal colon (FPLC, proteomics).

The Forms of the Lectin Tff2 Differ in the Murine Stomach and Pancreas: Indications for Different Molecular Functions.

The lectin TFF2 belongs to the trefoil factor family (TFF). This polypeptide is typically co-secreted with the mucin MUC6 from gastric mucous neck cells, antral gland cells, and duodenal Brunner glands. Here, TFF2 fulfills a protective function by forming a high-molecular-mass complex with the MUC6, physically stabilizing the mucus barrier. In pigs and mice, and slightly in humans, TFF2 is also synthesized in the pancreas. Here, we investigated the murine stomach, pancreas, and duodenum by fast protein liquid chromatography (FPLC) and proteomics and identified different forms of Tff2. In both the stomach and duodenum, the predominant form is a high-molecular-mass complex with Muc6, whereas, in the pancreas, only low-molecular-mass monomeric Tff2 was detectable. We also investigated the expression of Tff2 and other selected genes in the stomach, pancreas, and the proximal, medial, and distal duodenum (RT-PCR analysis). The absence of the Tff2/Muc6 complex in the pancreas is due to a lack of Muc6. Based on its known motogenic, anti-apoptotic, and anti-inflammatory effects, we propose a protective receptor-mediated function of monomeric Tff2 for the pancreatic ductal epithelium. This view is supported by a report that a loss of Tff2 promotes the formation of pancreatic intraductal mucinous neoplasms.

Na,K-ATPase α1 and ß-subunits show distinct localizations in the nervous tissue of the large milkweed bug.

The Na,K-ATPase (NKA) is an essential ion transporter and signaling molecule in all animal tissues and believed to consist at least one α and one ß-subunit to form a functional enzyme. In the large milkweed bug, Oncopeltus fasciatus, adaptation to dietary cardiac glycosides (CGs), which can fatally block the NKA, has resulted in gene duplications leading to four α1-subunits. These differ in sensitivity to CGs, but resistance trades off against ion pumping activity, thus influencing the α1-subunits' suitability for specific tissues. Besides, O. fasciatus possesses four different ß-subunits that can alter the NKA's kinetics and should play an essential role in the formation of cellular junctions.Proteomic analyses revealed the distribution and composition of α1/ß-complexes in the nervous tissue of O. fasciatus. The highly CG-resistant, but less active α1B and the highly active, but less resistant α1C predominated in the nervous tissue and co-occurred with ß2 and ß3, partly forming larger complexes than just heterodimers. Immunohistochemical analyses provided a fine scale resolution of the subunits' distribution in different morphological structures of the nervous tissue. This may suggest that α1 as well as ß-subunits occur in isolation without the other subunit, which contradicts the present understanding that the two types of subunits have to associate to form functional complexes. An isolated occurrence was especially prominent for ß3 and ßx, the enigmatic fourth and N-terminally largely truncated ß-subunit. We hypothesize that dimerization of these ß-subunits plays a role in cell-cell contacts.

Different Molecular Forms of TFF3 in the Human Respiratory Tract: Heterodimerization with IgG Fc Binding Protein (FCGBP) and Proteolytic Cleavage in Bronchial Secretions.

The polypeptide TFF3 belongs to the trefoil factor family (TFF) of lectins. TFF3 is typically secreted from mucous epithelia together with mucins. Both intestinal and salivary TFF3 mainly exist as disulfide-linked heterodimers with IgG Fc binding protein (FCGBP). Here, we investigated bronchial tissue specimens, bronchial secretions, and bronchoalveolar lavage (BAL) fluid from patients with a chronic obstructive pulmonary disease (COPD) background by fast protein liquid chromatography and proteomics. For the first time, we identified different molecular forms of TFF3 in the lung. The high-molecular mass form represents TFF3-FCGBP oligomers, whereas the low-molecular mass forms are homodimeric and monomeric TFF3 with possibly anti-apoptotic activities. In addition, disulfide-linked TFF3 heterodimers with an Mr of about 60k and 30k were detected in both bronchial secretions and BAL fluid. In these liquids, TFF3 is partly N-terminally truncated probably by neutrophil elastase cleavage. TFF3-FCGBP is likely involved in the mucosal innate immune defense against microbial infections. We discuss a hypothetical model how TFF3 might control FCGBP oligomerization. Furthermore, we did not find indications for interactions of TFF3-FCGBP with DMBT1gp340 or the mucin MUC5AC, glycoproteins involved in mucosal innate immunity. Surprisingly, bronchial MUC5AC appeared to be degraded when compared with gastric MUC5AC.

Receptor-independent modulation of cAMP-dependent protein kinase and protein phosphatase signaling in cardiac myocytes by oxidizing agents.

The contraction and relaxation of the heart is controlled by stimulation of the ß1-adrenoreceptor (AR) signaling cascade, which leads to activation of cAMP-dependent protein kinase (PKA) and subsequent cardiac protein phosphorylation. Phosphorylation is counteracted by the main cardiac protein phosphatases, PP2A and PP1. Both kinase and phosphatases are sensitive to intramolecular disulfide formation in their catalytic subunits that inhibits their activity. Additionally, intermolecular disulfide formation between PKA type I regulatory subunits (PKA-RI) has been described to enhance PKA's affinity for protein kinase A anchoring proteins, which alters its subcellular distribution. Nitroxyl donors have been shown to affect contractility and relaxation, but the mechanistic basis for this effect is unclear. The present study investigates the impact of several nitroxyl donors and the thiol-oxidizing agent diamide on cardiac myocyte protein phosphorylation and oxidation. Although all tested compounds equally induced intermolecular disulfide formation in PKA-RI, only 1-nitrosocyclohexalycetate (NCA) and diamide induced reproducible protein phosphorylation. Phosphorylation occurred independently of ß1-AR activation, but was abolished after pharmacological PKA inhibition and thus potentially attributable to increased PKA activity. NCA treatment of cardiac myocytes induced translocation of PKA and phosphatases to the myofilament compartment as shown by fractionation, immunofluorescence, and proximity ligation assays. Assessment of kinase and phosphatase activity within the myofilament fraction of cardiac myocytes after exposure to NCA revealed activation of PKA and inhibition of phosphatase activity thus explaining the increase in phosphorylation. The data suggest that the NCA-mediated effect on cardiac myocyte protein phosphorylation orchestrates alterations in the kinase/phosphatase balance.

Lactate metabolism in strictly anaerobic microorganisms with a soluble NAD+ -dependent l-lactate dehydrogenase.

Lactate is a universal metabolite and energy source, yet the mode of lactate metabolism in many strictly anaerobic microorganisms is still enigmatic. This sparked us to investigate the biochemistry and bioenergetics of lactate metabolism in the model acetogenic bacterium Moorella thermoacetica. Growth and metabolism were dependent on CO2 and the chemiosmotic gradient. We discovered a l-lactate:NAD+ oxidoreductase (LDH) in cell-free extracts, exhibiting an average specific activity of 362.8 ± 22.9 mU mg-1 . The enzyme was reversible, most active at 65°C and pH 9, with Km values of 23.1 ± 3.7 mM for l-lactate and 273.3 ± 39.1 µM for NAD+ . In-gel activity assays and mass spectrometric proteomics revealed that the ldh gene encoded the characterized LDH. Transcriptomic and genomic analyses showed that ldh expression was induced by lactate and there was a single nucleotide polymorphism near the predicted NAD+ binding site. Genes encoding central redox and energy metabolism complexes, such as, the energetic coupling site Ech2, menaquinone, and the electron bifurcating EtfABCX and MTHFR were also upregulated in cells grown on lactate. These findings ultimately lead to a redox-balanced metabolic model that shows how growth on lactate can proceed in a microorganism that only has a conventional NAD+ -reducing LDH.

Hypoxia-Responsive Class III Peroxidases in Maize Roots: Soluble and Membrane-Bound Isoenzymes.

Flooding induces low-oxygen environments (hypoxia or anoxia) that lead to energy disruption and an imbalance of reactive oxygen species (ROS) production and scavenging enzymes in plants. The influence of hypoxia on roots of hydroponically grown maize (Zea mays L.) plants was investigated. Gene expression (RNA Seq and RT-qPCR) and proteome (LC-MS/MS and 2D-PAGE) analyses were used to determine the alterations in soluble and membrane-bound class III peroxidases under hypoxia. Gel-free peroxidase analyses of plasma membrane-bound proteins showed an increased abundance of ZmPrx03, ZmPrx24, ZmPrx81, and ZmPr85 in stressed samples. Furthermore, RT-qPCR analyses of the corresponding peroxidase genes revealed an increased expression. These peroxidases could be separated with 2D-PAGE and identified by mass spectrometry. An increased abundance of ZmPrx03 and ZmPrx85 was determined. Further peroxidases were identified in detergent-insoluble membranes. Co-regulation with a respiratory burst oxidase homolog (Rboh) and key enzymes of the phenylpropanoid pathway indicates a function of the peroxidases in membrane protection, aerenchyma formation, and cell wall remodeling under hypoxia. This hypothesis was supported by the following: (i) an elevated level of hydrogen peroxide and aerenchyma formation; (ii) an increased guaiacol peroxidase activity in membrane fractions of stressed samples, whereas a decrease was observed in soluble fractions; and (iii) alterations in lignified cells, cellulose, and suberin in root cross-sections.

The Tumor Suppressor TFF1 Occurs in Different Forms and Interacts with Multiple Partners in the Human Gastric Mucus Barrier: Indications for Diverse Protective Functions.

TFF1 is a protective peptide of the Trefoil Factor Family (TFF), which is co-secreted with the mucin MUC5AC, gastrokine 2 (GKN2), and IgG Fc binding protein (FCGBP) from gastric surface mucous cells. Tff1-deficient mice obligatorily develop antropyloric adenoma and about 30% progress to carcinomas, indicating that Tff1 is a tumor suppressor. As a hallmark, TFF1 contains seven cysteine residues with three disulfide bonds stabilizing the conserved TFF domain. Here, we systematically investigated the molecular forms of TFF1 in the human gastric mucosa. TFF1 mainly occurs in an unusual monomeric form, but also as a homodimer. Furthermore, minor amounts of TFF1 form heterodimers with GKN2, FCGBP, and an unknown partner protein, respectively. TFF1 also binds to the mucin MUC6 in vitro, as shown by overlay assays with synthetic 125I-labeled TFF1 homodimer. The dominant presence of a monomeric form with a free thiol group at Cys-58 is in agreement with previous studies in Xenopus laevis and mouse. Cys-58 is likely highly reactive due to flanking acid residues (PPEEEC58EF) and might act as a scavenger for extracellular reactive oxygen/nitrogen species protecting the gastric mucosa from damage by oxidative stress, e.g., H2O2 generated by dual oxidase (DUOX).

Changes in proinflammatory gene expression in human whole blood after contact with UV-conditioned implant surfaces.

OBJECTIVES: The aim of this in vitro study was to assess changes in the gene expression of proinflammatory cytokines in human whole blood after contact with titanium implant surfaces conditioned by UV light. To this end, expression levels of proinflammatory cytokines were analyzed in vitro in human whole blood. MATERIALS AND METHODS: Dental implants made of grade 4 titanium were conditioned by UV light in a UV device and submerged in human whole blood. Unconditioned implants served as controls, and blood samples without implants served as the negative control group. Sampling was performed at 1, 8, and 24 h. Changes in the expression levels of interleukin-1ß (IL1B) and tumor necrosis factor alpha (TNF) were assessed using RT-qPCR at the mRNA level. RESULTS: The gene expression of IL1B was significantly suppressed in the test group over the observation period compared to the control group during the 1-8 h after having contact between the implant surface and the blood. The gene expression of TNF was not significantly altered by UV conditioning after 1 and 8 h of observation, but both cytokine expression levels were increased significantly after 24 h. CONCLUSIONS: Differences in the gene expression of proinflammatory cytokines after insertion of UV-conditioned titanium implants can be assessed using a human whole blood test. UV-conditioned implant surfaces apparently suppress the release of IL1B in vitro. CLINICAL RELEVANCE: The results of our publication demonstrate that modulation of the early inflammatory response in human whole blood is possible by surface treatment with UV light. In particular, the suppression of IL1B expression, especially after the initial contact of blood cells, may be beneficial in the osseointegration of titanium implants by positively influence the balance between rejection and acceptance of an implant.

Different Forms of TFF3 in the Human Saliva: Heterodimerization with IgG Fc Binding Protein (FCGBP).

The peptide TFF3 is a member of a family of secretory lectins, and is typically synthesized by mucous epithelia together with mucins. It is mainly released from intestinal goblet cells as a high-molecular mass heterodimer with IgG Fc binding protein (FCGBP). Herein, we investigated human saliva by fast protein liquid chromatography (FPLC) and proteomics and identified high- and low-molecular-mass forms of TFF3. Whereas the high-molecular-mass forms represent a heterodimer with FCGBP, the low-molecular-mass forms represent homodimeric TFF3 forms. Proteomic analysis also revealed a C-terminally truncated form of TFF3. We hypothesize that salivary TFF3-FCGBP might play a role in the innate immune defense of the oral cavity and that TFF3 might also bind to microbial glycans. The known interaction of TFF3 with the agglutinin DMBT-1, a typical constituent of human saliva, further supports this protective role.

Commercial Porcine Gastric Mucin Preparations, also Used as Artificial Saliva, are a Rich Source for the Lectin TFF2: In Vitro Binding Studies.

Mucous gels (mucus) cover internal body surfaces. The secretory mucins MUC5AC and MUC6 and the protective peptide TFF2 are characteristic constituents of gastric mucus; TFF2 is co-secreted with MUC6. Herein, we investigated two commercial mucin preparations by FPLC and proteomics, because they are model systems for studying the rheology of gastric mucins. One preparation is also used as a saliva substitute, for example, after radiation therapy. We show that both preparations contain TFF2 (≈0.6 to 1.1 %, w/w). The majority of TFF2 is strongly bound noncovalently to mucin in a manner that is resistant to boiling in SDS. First overlay assays with 125 I-labeled porcine TFF2 revealed that mucin binding is modulated by Ca2+ and can be blocked by the lectin GSA-II and the antibody HIK1083, both recognizing the peripheral GlcNAcα1→4Galß1→R moiety of MUC6. TFF2 binding was also inhibited in the presence of Me-ß-Gal but less so by the α anomer. TFF2 may play a role in the oligomerization and secretion of MUC6, the rheology of gastric mucus, and the adherence of gastric microbiota. TFF2 in artificial saliva may be of benefit. TFF2 might also interact with the sugar moiety of various receptors.

COSMC knockdown mediated aberrant O-glycosylation promotes oncogenic properties in pancreatic cancer.

BACKGROUND: Human pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies in the world and despite great efforts in research types of treatment remain limited. A frequently detected alteration in PDACs is a truncated O-linked N-acetylgalactosamine (GalNAc) glycosylation with expression of the Tn antigen. Changes in O-glycosylation affect posttranslationally modified O-GalNAc proteins resulting in profound cellular alterations. Tn antigen is a tumor associated glycan detected in 75-90 % of PDACs and up to 67 % in its precursor lesions. Since the role of Tn antigen expression in PDAC is insufficiently understood we analyzed the impact of COSMC mediated Tn antigen expression in two human PDAC cell lines on cellular oncogenic properties. METHODS: Forced expression of Tn antigen on O-glycosylated proteins in pancreatic cancer cells was induced by lentiviral-mediated knockdown of the COSMC chaperone, which prevented O-glycan elongation beyond the initial GalNAcα1- residue on O-linked glycoproteins. Altered O-GalNAc glycosylation was analyzed in human pancreatic cancer cell lines Panc-1 and L3.6pl using Western and Far-Western blot as well as immunocytochemical techniques. To assess the biological implications of COSMC function on oncogenic properties, cell viability assays, scratch assays combined with live cell imaging, migration and apoptosis assays were performed. Lectin based glycoprotein enrichment with subsequent mass spectrometric analysis identified new cancer O-GalNAc modified proteins. Expression of Tn antigen bearing Nucleolin in patient derived PDAC tumor specimens was evaluated and correlated with clinicopathological data. RESULTS: Tn antigen expression was induced on various O-GalNAc glycoproteins in COSMC deficient cell lines compared to the control. Proliferation was reduced (p < 0.001) in COSMC knockdown cells, whereas migration was increased (p < 0.001) and apoptosis was decreased (p = 0.03), highlighting the importance of Tn antigen expression on metastatic and anti-apoptotic behavior of PDAC derived cells. Nucleolin was identified as O-GalNAc modified protein in COSMC deficient PDAC cell lines. Interestingly, immunohistochemical staining and co-localization studies of patient derived PDACs revealed poor survival for patients with strong co-localization of Tn antigen and Nucleolin (p = 0.037). CONCLUSION: This study substantiates the influence of altered O-glycan (Tn/STn) expression on oncogenic properties in pancreatic cancer and identifies O-GalNAc modified Nucleolin as novel prognostic marker.

The autism susceptibility kinase, TAOK2, phosphorylates eEF2 and modulates translation.

Genes implicated in translation control have been associated with autism spectrum disorders (ASDs). However, some important genetic causes of autism, including the 16p11.2 microdeletion, bear no obvious connection to translation. Here, we use proteomics, genetics, and translation assays in cultured cells and mouse brain to reveal altered translation mediated by loss of the kinase TAOK2 in 16p11.2 deletion models. We show that TAOK2 associates with the translational machinery and functions as a translational brake by phosphorylating eukaryotic elongation factor 2 (eEF2). Previously, all signal-mediated regulation of translation elongation via eEF2 phosphorylation was believed to be mediated by a single kinase, eEF2K. However, we show that TAOK2 can directly phosphorylate eEF2 on the same regulatory site, but functions independently of eEF2K signaling. Collectively, our results reveal an eEF2K-independent signaling pathway for control of translation elongation and suggest altered translation as a molecular component in the etiology of some forms of ASD.

Impact of vertical loading on the implant-bone interface.

OBJECTIVES: The main aim of this study was to evaluate the impact of vertical loading occurring during removal of cemented restorations on the implant-bone interface. METHODS: Thirty-six titanium implants (Camlog 4.3 × 9 mm) were placed 1 mm supraosseous in the frontal skull of four minipigs. After a 13 week healing period the implants were exposed and the implant stability was measured. Three implants per minipig were vertically loaded using 20 or 100 impulses, respectively with an 18 Ns impulse imitating a crown removal. Three implants were left unloaded as control. The animals were sacrificed after 13 or 18 weeks. The harvested specimens were analyzed using scanning electron microscopy (SEM), light and fluorescence microscopy. RESULTS: No post operative complications or deaths of the minipigs occurred. All implants osseointegrated. The average bone-implant contact area (BIC) was 78 ± 5.1%. No statistically significant difference could be found when comparing the BIC areas of the control and the experimental groups between the sacrificed animals at 13 weeks and 18 weeks (P > 0.05). Therefore, the results of each subgroup were pooled. No significant differences regarding the BIC area could be detected between the control and the experimental groups (P > 0.05). Except one failing implant no cracks due to vertical loading could be evaluated in the SEM. Fluorescence microscopy revealed a significantly higher bone remodeling activity in the vertically loaded groups. CONCLUSIONS: Removal of cemented implant restorations seems not to have an impact on the mechanical implant stability, but seems to increase bone remodeling activity.

Analysis of the intraimplant microflora of two-piece dental implants.

OBJECTIVE: Information about the spectrum of microorganisms in the intraimplant cavities of two-piece dental implants is scarce. The purpose of this study was to assess the intraimplant microflora of two-piece dental implants by conventional biochemical testing, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and 16 s rDNA gene sequencing. MATERIALS AND METHODS: Ten patients (six men and four women; average age = 66.7 years; age range = 58-78 years) received 35 two-piece titanium implants carrying ball attachments. Biofilm sampling was performed with sterile microbrushes, and nonadherent microbial samples were obtained by injection and reuptake of predefined volumes of NaCl solution. The samples were cultured and analyzed by conventional biochemical testing, MALDI-TOF MS, and 16 s rDNA gene sequencing. RESULTS: Of the 103 species detected, 27 and 33 were identified only in the biofilm and nonadherent microbial samples, respectively. Forty-three species were identified in both types of samples. CONCLUSIONS: Two-piece dental implants harbored a broad spectrum of gram-positive and gram-negative aerobes and anaerobes, especially rods and cocci. CLINICAL RELEVANCE: These findings confirm bacterial translocation from the oral cavity to intraimplant cavities. Microbiological methods as used in this study are necessary to reveal the complete vital microflora of intraimplant cavities.

Influence of cement film thickness on the retention of implant-retained crowns.

PURPOSE: The main goal of this study was to establish a new, high precision procedure to evaluate the influence of cement film thickness on the retention of cemented implant-retained crowns. MATERIALS AND METHODS: Ninety-six tapered titanium abutments (6° taper, 4.3 mm diameter, Camlog) were shortened to 4 mm. Computer-aided design was used to design the crowns, and selective laser sintering, using a cobalt-chromium alloy, was used to produce the crowns. This method used a focused high-energy laser beam to fuse a localized region of metal powder to build up the crowns gradually. Before cementing, preset cement film thicknesses of 15, 50, 80, or 110 µm were established. Glass ionomer, polycarboxylate, or resin cements were used for cementation. After 3 days storage in demineralized water, the retention of the crowns was measured in tension using a universal testing machine. RESULTS: The cement film thicknesses could be achieved with a high level of precision. Interactions between the factors cement and cement film thickness could be found (p ≤ 0.001). For all cements, crown retention decreased significantly between a cement film thickness of 15 and 50 µm (p ≤ 0.001). At 15 µm cement film thickness, the resin cement was the most retentive cement, followed by the polycarboxylate and then the glass ionomer cement (p ≤ 0.05). CONCLUSIONS: The results suggest that cement film thickness has an influence on the retentive strength of cemented implant-retained crowns.

Surface contamination of dental implants assessed by gene expression analysis in a whole-blood in vitro assay: a preliminary study.

AIM: We aimed at evaluating pyrogen contamination of dental implants made of titanium and zirconia by using gene expression analysis in a whole-blood in vitro assay. MATERIAL AND METHODS: Titanium and zirconia implants (five each) were incubated in human whole blood. Samples were assayed for gene expression levels of toll-like receptor 4 (TLR4), TLR9, interleukin (IL)-1ß, nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-kB), tumour necrosis factor (TNF)-α, and Fas-associated protein with death domain (FADD) as indicators of surface contamination resulting in lipopolysaccharides (LPS)-stimulated TLR- or TNF-mediated immune responses. Gene expression was assayed using real-time quantitative polymerase chain reaction (RT-qPCR). Non-stimulated blood from the same donor served as a negative control, and blood stimulated with LPS served as a positive control. After dry-heat treatment with dry heat, all implants were re-analysed as described above. RESULTS: Both implant systems contained surface contaminants evoking a pro-inflammatory response similar to that induced by LPS. After dry-heat treatment, gene expression was significantly decreased to levels similar to those of negative control samples. CONCLUSIONS: The results demonstrated LPS-like surface-bound contaminants in both tested implant systems. Depyrogenation with dry heat seems to be an effective means of reducing such contamination in dental implants.

In vitro influence of ultrasonic stress, removal force preload and thermocycling on the retrievability of implant-retained crowns.

OBJECTIVES: The main goals of this in vitro study were to evaluate the influence of thermocycling, ultrasonic stress and the removal force preload on the retrievability of cemented implant crowns using a clinical removal device (Coronaflex) and evaluating the tensile strength using a universal testing machine (UTM). METHODS: Thirty-six crowns were cast from a Co-Cr alloy for 36 tapered titanium abutments (5° taper, 4.3 mm diameter, 6 mm height, Camlog, Germany). The crowns were cemented with a glass-ionomer (Ketac Cem) or a polycarboxylate (Durelon) cement, followed by 3 days of storage in ionized water without thermocycling or 150 days of storage with 37,500 thermal cycles between 5°C and 55°C. Before removal, the crowns were subjected to ultrasonic stress for 0, 5 or 10 min with a contact pressure of either 50 or 500 g. The Coronaflex was used with a removal force preload of 50 or 400 cN, respectively, applied on the point of loading. Scanning electronic microscopy (SEM) was used to evaluate the impact of the removal on the abutment screws. RESULTS: Crowns cemented with the glass-ionomer cement were significantly easier to remove with the Coronaflex or the UTM than crowns cemented with the polycarboxylate cement (P≤0.05). Ultrasonic stress showed no significant impact on the retrievability regardless of the contact pressure or duration applied (P>0.05). No significant differences could be found for both cements when removed with the Coronaflex or the UTM (P>0.05) after thermocycling was applied. A removal force preload of 400 cN resulted in significantly reduced removal attempts in comparison with 50 cN for both cements (P≤0.05). CONCLUSIONS: Ultrasound and thermal cycling did not result in reduced cement strength, but to retrieve the crowns, the full impact of a removal instrument has to be applied. Ketac Cem can be used as a "semipermanent" solution, whereas Durelon might serve for permanent cementation. None of the abutment screws showed signs of wear caused by the removal process.

Assessment of lipopolysaccharide microleakage at conical implant-abutment connections.

OBJECTIVE: The aim of this in vitro study was to assess lipopolysaccharide microleakage at conical implant-abutment connections of two-piece dental implants in terms of the expression levels of genes involved in lipopolysaccharide-mediated proinflammatory cytokine production. MATERIALS AND METHODS: Two implant systems with conical implant-abutment connections were inoculated with lipopolysaccharide and submerged in human whole blood. Positive-control blood samples (without implants) were stimulated with 4 µg/ml, 2 µg/ml, 200 ng/ml, and 20 ng/ml lipopolysaccharide. Sampling was performed after 1, 8, and 24 h of incubation. Changes of gene expression levels of Toll-like receptor 9, tumor necrosis factor-α, nuclear factor kappa light chain enhancer of activated B cells, interleukin-1ß, and interferon-γ were assessed by real-time quantitative PCR. In addition, protein expression levels of interleukin-6, tumor necrosis factor-α, interleukin-1ß, and interferon-γ were determined by immunoassay. RESULTS: Changes in cytokine expression at the genomic and proteomic levels indicated lipopolysaccharide leakage at the interfaces of both tested implant systems, although some implants showed no sign of microleakage. Any tested concentration of lipopolysaccharide stimulated similar gene expression. CONCLUSIONS: Conical implant-abutment connections of two-piece dental implants do not prevent microleakage on a molecular level. Changes in lipopolysaccharide-induced proinflammatory cytokine gene expression facilitate the detection of lipopolysaccharide microleakage at implant-abutment interfaces. CLINICAL RELEVANCE: Small amounts of lipopolysaccharide released from intra-implant cavities can stimulate a detectable immunological response in human whole blood and may induce alveolar bone resorption via the osteoclast-activating pathway.