Die Bedeutung von CpG-Oligonukleotiden für den myokardialen Reperfusionsschaden

Die angeborene Immunabwehr spielt mit ihren Toll-like Rezeptoren eine wichtige Rolle in der myokardialen Ischämie und Reperfusion (I/R). TLR9 ist der primäre Rezeptor für bakterielle DNA. Sie zeichnet sich durch spezielle Sequenzen aus, sog. CpG-Motive, die zu 98% unmethyliert vorliegen. Verschiedenste Studien konnten zeigen, dass eine repetitive Schädigung (Präkonditionierung) vor myokardialer Ischämie kardioprotektive Wirkungen besitzt. In der vorgestellten Studie sollte die Bedeutung von CpG-Oligonukleotiden im Ischämie-Reperfusionsversuch eruiert und zudem die diagnostische Relevanz des kardialen Troponin T (cTNT) untersucht werden. Darüber hinaus sollten die funktionellen und hämodynamischen Aspekte einer CpG-Vorbehandlung sowie molekulare Mechanismen eingehend untersucht werden. Während der Initaloperation (Instrumentierung) wurde die linke anteriore deszendierende Koronararterie (LAD) bei C57BL/6 Mäusen mit einem Okkluder versehen, um nach abgeschlossener Wundheilung („closed-chest“-Modell) eine 60-minütige Ischämie hervorrufen zu können. 16 Stunden vor Infarkt erfolgte die jeweilige Vorbehandlung mit einem der drei synthetischen Äquivalente der bakteriellen DNA, sog. CpG-ODNs, oder PBS (Kontrolle). Anhand der Reperfusionszeiten können drei Teilstudien unterschieden werden. Nach 3 h Reperfusion wurden die Expressionen proinflammatorischer Zytokine im Herzen bestimmt. Nach 24 h Reperfusion die Herzen histologisch untersucht, kardiale Plasmatroponin T-Werte ermittelt und erste funktionelle und hämodynamische Parameter evaluiert. Einer 2-wöchigen Reperfusionsphase schloss sich erneut die histologische Herzfärbung und planimetrische Vermessung der Infarktgrößen an sowie die Darstellung der myokardialen Funktion und Hämodynamtik mit Hilfe der Echokardigraphie und Herzkatheterisierung. Mäuse, welche eine 5 nmolare 1668-ODN-Vorbehandlung erhielten, hatten kleinere Infarkte und geringere cTNT-Konzentrationen nach I/R als die Kontrolltiere mit einer äquivalenten PBS-Gabe. Andere 1668-Stimulationskonzentrationen (1, 10 und 20 nmol) hatten keinen nachweisbaren präkonditionierenden Effekt, vielmehr führte die erhöhte ODN-Gabe zum Anstieg der Mortalitätsraten auf bis zu 80%. Tiere mit einer 1612- bzw. H154- Vorbehandlung zeigten dagegen keine signifikant reduzierten Infarktgrößen im Vergleich zur Kontrollgruppe. Damit ist die Aktivierung von TLR9 durch CpD-ODNs maßgeblich für das Ausmaß der myokardialen Schädigung mitverantwortlich. Außerdem war nach einer 1668-ODN-Stimulation tendenziell eine Reduktion der inflammatorischen Antwort auf I/R in Form eines verringerten Zytokinanstiegs nachweisbar. Zusätzlich führte sie zu tendenziell verbesserten diastolischen Blutdruckwerten sowie Ejektionsfraktionen. Hier bedarf es jedoch weiterer Untersuchungen. Die vorgestellten Ergebnisse demonstrieren erstmals, dass CpG-Oligonukleotide Mediatoren des Ischämie-Reperfusionsschadens sind. Eine TLR9-vermittelte CpGPräkonditionierung führte konzentrations- und sequenzabhängig zur Reduktion des Myokardinfarktes sowie zur Verringerung der kardialen Troponin T-Plasmakonzentration. Demnach besitzt CpG-ODNs ein therapeutisches Potential nicht nur bei malignen Krankheiten und Infektionserkrankungen, sondern auch bei der Behandlung von Myokardinfarkt und sepsisähnlichen Erkrankungen.The importance of CpG oligonucleotides for the myocardial reperfusion injury Toll-like receptors are important regulators of the innate immunity, e.g. TLR9 is responsible for the detection of bacterial DNA. Bacterial DNA is characterized by special sequences, so called CpG-motives, of which 98% are unmethylated. Interestingly, TLRs also play a role in myocardial ischemia and reperfusion (I/R) injury. Several studies have shown pretreatment (preconditioning) of animals with lipopolysaccharide (LPS), a TLR4 ligand, reduces a cardiac injury caused by I/R up to 16 h after LPS challenge. This study examined whether TLR9 stimulation may also function as preconditioning signal cardiac I/R injury. Therefore CpG oligonucleotides were applied as stimuli pior to ischemia/reperfusion injury of the murine heart. Ischemia was induced in a “closed chest model”. In an initial operation an occluder was implanted around left anterior descending coronary artery (LAD) of the heart of C57BL/6 mice. After wound healing mice underwent 60 minutes of ischemia by pulling at the occluder without opening the thorax of the mouse. 16 hours before infarction they were pre-treated with one of three different synthetic equivalents of bacterial DNA, referred to as CpG-ODNs, or PBS (control). On the basis of reperfusion periods three different protocols can be distinguished. After 3 h of reperfusion the expression of cardiac cytokines was measured. After 24 h the myocardial infarct size was assessed by triphenyltetrazolium chloride (TTC) staining, cardiac troponin T concentration was evaluated as marker of infarct size and hemodynamic parameters were recorded. A period of two weeks of reperfusion was again followed by TTC staining and evaluation of infarct size by a planimetry software. In the same group echocardiography and intracardiac catheter measurements were applied to examine cardiac function and hemodynamics. Animals which had been pre-treated with 5 nmol of CpG-ODN 1668, developed significantly smaller infarct sizes and lower cTNT-concentrations after I/R than the control group. Pre-treatment with other concentrations of 1668 (1, 10 and 20 nmol) did not reduce infarct size. In contrast higher concentrations resulted in an increased mortality up to 80%. Mice pre-treated with 1612- or H154-ODN (sequences without or less CpG-motives) did not show significant reduction of infarct area compared to controls emphasizing that the activation of TLR9 plays a decisive role for the extent of myocardial damage. In addition, the 1668-ODN group tended to show a reduced inflammatory response after I/R as indicated by a decreased cytokine rise. Furthermore, a tendency toward lower diastolic blood pressure and higher ejection fractions could be demonstrated in this group. These data demonstrate for the first time that CpG oligonucleotides play a role in the ischemia-reperfusion injury. TLR9-mediated CpG-preconditioning results in a reduction of myocardial infarction in a dose- and sequence-dependent manner and causes a decrease of cardial troponin T concentrations. In conclusion, CpG-ODNs may offer a therapeutic potential not only in respect to cancer and infections, but also for the treatment of myocardial infarction

Clinical retention force development of double crowns

This study deals with the development of the retentive forces of double crowns intraorally measured. Twenty-five combined fixed-removable prostheses with a total of 84 double crowns were included in the study. The intraoral measurement was performed at 72 defined measuring points directly adjacent to the double crowns of the dentures. The measurement was performed 4-6weeks (baseline), 6months (recall 1), and 18months (recall 2) after the insertion of the restoration. A specifically designed measuring device was used. The median values for the single measuring points reached 4.705N at the baseline, 5.190N after 6months, and 3.740N after 18months. The measured values were analyzed according to differences between the median retention forces at the three defined points in time. The statistical analysis of the median values showed no statistical difference for the retention force change after 6months but for the decrease until the second recall (Mann-Whitney test). The retention force per denture was calculated by a summation of the single measuring points. At the baseline, 12.9N was reached. The forces did only decrease slightly and were not statistically significant. The results indicate that retention force values of double crowns, measured intraorally at the patient, do not relevantly change clinically within the first 1.5years. Within the limitations of this study, it can be stated that wear does not influence the retentive forces of double crowns within the first 18months. After this period the retention force should be still sufficient for denture retentio

Clinical implementation of axial angulation of incisors in the course of routine fixed appliance treatment - a retrospective cohort study

PURPOSE To identify clinically relevant factors for changes in axial angulation of incisors during routine fixed appliance orthodontic treatment. METHODS A total of 106 patients (grades 1-2 of IOTN, 64 females, 42 males; mean age: 15.5 years) from a private practice and treated with metal or ceramic brackets were included in this retrospective cohort study. The axial angulation of the upper and lower incisors was measured on lateral cephalograms before insertion of the first rectangular 0.016 × 0.022-in NiTi archwire (T0) and at the end of treatment about 8 weeks after insertion of the working 0.019 × 0.025-in stainless steel archwire (T1). Treatment-related changes according to bracket type, initial situation, premolar extraction, angle class, and skeletal vertical configuration were analyzed. RESULTS Although statistically significant treatment-related changes were seen for both the upper incisors (+ 1.3°) and the lower incisors (- 5.2°), only in ten patients (9.4%) was the prescribed torque value of 17° for the upper incisors and in no patient for the lower incisors achieved. A negative association between the induced change of axial angulation of incisors and the initial values was detected for the upper incisors as well as for the lower incisors. A comparison of the angle classes revealed significant differences in incisor changes. At the end of therapy, only a slight change for the upper central incisors in patients in angle class I cases and a significantly greater change in patients with angle class II/2 was observed. Cases with premolar extraction ended with lower axial angulation of the incisor than cases without extraction. The individual analysis of possible influencing factors also revealed an association with the vertical skeletal configuration. CONCLUSIONS For the first time, the presented data show clinically relevant influencing factors for incisor axial angulation changes of the upper and lower incisors in relation to the torque value of the applied brackets in the course of routine clinical practice. For the orthodontist, it remains mandatory to decide whether a customized system must be individualized in order to achieve individual therapy goals

Administration of a VEGFR‑2-specific MRI contrast agent to assess orthodontic tooth movement

Purpose It is thought that orthodontic forces initially reduce periodontal blood flow during orthodontic tooth movement (OTM) via tissue compression with cells responding to concomitant oxygen deprivation with expression of vascular endothelial growth factor (VEGF) triggering angiogenesis via binding to its receptor VEGFR‑2. To test this hypothesis, we performed a pilot study to establish a protocol for molecular magnetic resonance imaging (MRI) of rat jaws administering a VEGFR-2-specific contrast agent. Methods Mesial OTM of a first upper left rat molar was initiated in one male Fischer 344 rat 4 days prior to MRI by insertion of an elastic band between the first and second upper molars with the contralateral side left untreated (internal control). T1-weighted MRI sequences including dynamic contrast-enhanced MRI (DCE-MRI) were recorded before and after administration of a molecular VEGFR‑2 MRI marker with a 7 T MRI dedicated for small animal use. Results After injection of anti-VEGFR2-albumin-gadolinium-DTPA, volume enhancement on T1-weighted images was increased at the OTM side distally of the moved first upper molar (M1) compared to the control side, whereas the T1 relaxation time was reduced on the OTM side. DCE-MRI resulted in an increased area under the curve (AUC), whereas time-to-peak (TTP) and washout rate were reduced during OTM distally of the moved M1 compared to the contralateral side. Conclusions OTM resulted in uptake of the VEGFR-2-specific MRI contrast agent in tension areas of the periodontal ligament. The imaging protocol presented here is useful for the assessment of VEGFR‑2 expression in tension areas of the periodontal ligament in vivo

Imputation of Orofacial Clefting Data Identifies Novel Risk Loci and Sheds Light on the Genetic Background of Cleft Lip ± Cleft Palate and Cleft Palate Only.

Abstract Nonsyndromic cleft lip with or without cleft palate (nsCL/P) is among the most common human birth defects with multifactorial etiology. Here, we present results from a genome-wide imputation study of nsCL/P in which, after adding replication cohort data, four novel risk loci for nsCL/P are identified (at chromosomal regions 2p21, 14q22, 15q24 and 19p13). On a systematic level, we show that the association signalswithin this high-density datasetare enriched in functionally-relevant genomic regions that are active in both human neural crest cells (hNCC) and mouse embryonic craniofacial tissue. This enrichment is also detectable in hNCC regions primed for later activity. Using GCTA analyses, we suggest that 30% of the estimated variance in risk for nsCL/P in the European population can be attributed to common variants, with 25.5% contributed to by the 24 risk loci known to date. For each of these, we identify credible SNPs using a Bayesian refinementapproach, with two loci harbouring only one probable causal variant. Finally, we demonstrate that there is no polygenic component of nsCL/P detectable that is shared with nonsyndromic cleft palate only (nsCPO). Our data suggest that, while common variants are strongly contributing to risk for nsCL/P, they do not seem to be involved in nsCPO which might be more often caused by rare deleterious variants. Our study generates novel insights into both nsCL/P and nsCPO etiology and provides a systematic framework for research into craniofacial development and malformation

Extending the allelic spectrum at noncoding risk loci of orofacial clefting

Genome-wide association studies (GWAS) have generated unprecedented insights into the genetic etiology of orofacial clefting (OFC). The moderate effect sizes of associated noncoding risk variants and limited access to disease-relevant tissue represent considerable challenges for biological interpretation of genetic findings. As rare variants with stronger effect sizes are likely to also contribute to OFC, an alternative approach to delineate pathogenic mechanisms is to identify private mutations and/or an increased burden of rare variants in associated regions. This report describes a framework for targeted resequencing at selected noncoding risk loci contributing to nonsyndromic cleft lip with/without cleft palate (nsCL/P), the most frequent OFC subtype. Based on GWAS data, we selected three risk loci and identified candidate regulatory regions (CRRs) through the integration of credible SNP information, epigenetic data from relevant cells/tissues, and conservation scores. The CRRs (total 57 kb) were resequenced in a multiethnic study population (1061 patients; 1591 controls), using single-molecule molecular inversion probe technology. Combining evidence from in silico variant annotation, pedigree- and burden analyses, we identified 16 likely deleterious rare variants that represent new candidates for functional studies in nsCL/P. Our framework is scalable and represents a promising approach to the investigation of additional congenital malformations with multifactorial etiology

Effectiveness of early orthopaedic treatment with headgear: a systematic review and meta-analysis

BACKGROUND Although the headgear appliance has been used extensively to correct anteroposterior discrepancies, its treatment effects have not yet been adequately assessed in an evidence-based manner. OBJECTIVE Aim of this systematic review was to assess the therapeutic and adverse effects of early headgear treatment from controlled clinical trials on human patients in an evidence-based manner. SEARCH METHODS An unrestricted electronic search of six databases from inception to December 2015. SELECTION CRITERIA Randomized and prospective non-randomized controlled trials assessing the effects of headgear treatment on human patients. DATA COLLECTION AND ANALYSIS After duplicate study selection, data extraction, and risk of bias assessment according to the Cochrane guidelines, random-effects meta-analyses of mean differences (MDs) and relative risks (RRs), including their 95% confidence intervals (CIs) were performed, followed by subgroup and sensitivity analyses. RESULTS A total of 18 unique studies with a total of 930 (56% male/44% female) patients were included. Headgear treatment was associated with a posterior translation of the anterior maxilla border in the short term, as seen by the mean annualized change in the SNA angle (MD = -1.63°/year; 95% CI = -2.20 to -1.06°/year; high quality evidence) compared to untreated patients. This effect was independent of the rotation of the palatal plane and the inclination of the upper incisors, while a proportional relationship with the initial discrepancy in SNA was seen. The clinical significance of this improvement diminished in the long term, although only limited evidence existed. Additionally, early headgear treatment might decrease the risk of dental trauma during the following years (RR = 0.34; 95% CI = 0.14 to 0.80; moderate quality evidence). Low quality evidence on the effect of headgear on the rotation of the palatal plane, the nasolabial angle, the occlusal outcome, and signs of temporomandibular disorders precluded robust assessments, due to risk of bias, inconsistency, imprecision, and small-study effects. CONCLUSIONS Based on existing trials, headgear is a viable treatment option to modify sagittal growth of the maxilla in the short term in Class II patients with maxillary prognathism. REGISTRATION PROSPERO (CRD42015029837). FUNDING None

Nickel ion concentrations in the saliva of patients treated with self-ligating fixed appliances: a prospective cohort study

OBJECTIVE Orthodontic appliances are considered to be highly biocompatible although adverse effects attributed to the release of nickel ions (Ni(2+)) have been documented. Self-ligating brackets have grown in popularity for economic reasons and supposed friction reduction. The aim of the present prospective cohort study was therefore to determine salivary Ni(2+) concentrations in patients undergoing orthodontic treatment with self-ligating fixed appliances. MATERIALS AND METHODS A group of 30 patients between 10 and 13 years of age were treated with self-ligating brackets (SmartClip™), molar bands, and nickel-titanium (NiTi) archwires. Unstimulated saliva samples were collected after different time points (before treatment, after self-ligating bracket and band placement, before archwire insertion, after archwire insertion, and finally 4 and 8 weeks afterwards) and analyzed with an ICP mass spectrometer followed by generalized estimating equation modelling with α = 5 %. RESULTS The baseline median salivary Ni(2+) concentration was 21.85 µg/l, while the Ni(2+) concentrations at the following visits ranged between 13.73 and 85.34 µg/l. Significant increases in Ni(2+) levels compared to the baseline levels were detected after band/bracket placement [+59.76 µg/l; 95 % confidence interval (CI) 44.88-74.64 µg/l; P < 0.001] and after archwire insertion (+53.55 µg/l; 95 % CI 25.57-81.52 µg/l; P < 0.001). After 4 weeks, Ni(2+) concentrations returned to initial control levels or were lower. CONCLUSION Self-ligating orthodontic appliances may affect salivary Ni(2+) concentrations in vivo over the short term. However, levels resembled those documented in conjunction with conventional bracket use and remained below the daily dietary Ni intake

Changes in retention force with electroplated copings on conical crowns: a comparison of gold and zirconia primary crowns

The aim of this study was to examine the wear behavior of conical crowns with electroplated gold copings that are used to connect implants and teeth to a removable denture. Gold alloy and zirconium dioxide ceramic crowns were compared

Wear of double crown systems: electroplated vs. casted female part

The wear of telescopic crowns is a common problem often reducing the patient's satisfaction with the denture and resulting in a renewal of the denture. The study aims to compare the wear behavior of conical crowns using electroplated copings (group E ) with standard telescopic crowns with cast female parts (group C)