Design of a migration assay for human gingival fibroblasts on biodegradable magnesium surfaces.

A novel regenerative approach to Guided Bone Regeneration (GBR) in dental surgery is based on the development of biodegradable and volume stable barrier membranes made of metallic magnesium. Currently used volume stable barrier membranes are made of titanium-reinforced PTFE or titanium-reinforced collagen membranes, both, however, are accompanied by a high incidence of wound dehiscence resulting in membrane exposure, which leads to an increased infection risk. An exposed membrane could also occur directly after insertion due to insufficient soft tissue coverage of the membrane. In both cases, fast wound margin regeneration is required. As a first step of soft-tissue regeneration, gingival fibroblasts need to migrate over the barrier membrane and close the dehiscent wound. Based on this aim, this study investigated the migration behaviour of human gingival fibroblasts on a magnesium surface. Major experimental challenges such as formation of hydrogen bubbles due to initial magnesium corrosion and non-transparent material surfaces have been addressed to allow cell adhesion and to follow cell migration. The designed scratch-based cell migration assay involved vital fluorescent cell staining on a pre-corroded magnesium membrane to simulate invivo wound dehiscence. The assay has been used to compare cell migration on pre-corroded magnesium to titanium surfaces and tissue culture plastic as control substrates. First results of this assay showed that human gingival fibroblasts migrate slower on pre-corroded magnesium compared to plastic and titanium. However, the scratch was finally closed on all materials. Compared to titanium surfaces and tissue culture plastic, the surface roughness and the surface free energy (SFE) could not explain slower cell migration on magnesium surfaces. Immunohistological investigations of cellular structure revealed, that magnesium ions increased focal adhesion at concentration of additionally 75 mM MgCl2 in cell culture medium. The use of our designed cell migration assay has shown that ionic medium alterations due to magnesium corrosion has a higher impact on the cell migration rate than surface alterations. STATEMENT OF SIGNIFICANCE: The design of a migration assay on non-transparent magnesium surfaces will add the option to study cell response to surface modifications, coatings and the corrosion process itself under life view conditions.

Genome-wide significant risk factors for Alzheimer's disease: role in progression to dementia due to Alzheimer's disease among subjects with mild cognitive impairment.

Few data are available concerning the role of risk markers for Alzheimer's disease (AD) in progression to AD dementia among subjects with mild cognitive impairment (MCI). We therefore investigated the role of well-known AD-associated single-nucleotide polymorphism (SNP) in the progression from MCI to AD dementia. Four independent MCI data sets were included in the analysis: (a) the German study on Aging, Cognition and Dementia in primary care patients (n=853); (b) the German Dementia Competence Network (n=812); (c) the Fundació ACE from Barcelona, Spain (n=1245); and (d) the MCI data set of the Amsterdam Dementia Cohort (n=306). The effects of single markers and combined polygenic scores were measured using Cox proportional hazards models and meta-analyses. The clusterin (CLU) locus was an independent genetic risk factor for MCI to AD progression (CLU rs9331888: hazard ratio (HR)=1.187 (1.054-1.32); P=0.0035). A polygenic score (PGS1) comprising nine established genome-wide AD risk loci predicted a small effect on the risk of MCI to AD progression in APOE-ɛ4 (apolipoprotein E-ɛ4) carriers (HR=1.746 (1.029-2.965); P=0.038). The novel AD loci reported by the International Genomics of Alzheimer's Project were not implicated in MCI to AD dementia progression. SNP-based polygenic risk scores comprising currently available AD genetic markers did not predict MCI to AD progression. We conclude that SNPs in CLU are potential markers for MCI to AD progression.

Microdeletions including FMR1 in three female patients with intellectual disability - further delineation of the phenotype and expression studies.

Fragile X syndrome (FXS) is one of the most common causes of intellectual disability/developmental delay (ID/DD), especially in males. It is caused most often by CGG trinucleotide repeat expansions, and less frequently by point mutations and partial or full deletions of the FMR1 gene. The wide clinical spectrum of affected females partly depends on their X-inactivation status. Only few female ID/DD patients with microdeletions including FMR1 have been reported. We describe 3 female patients with 3.5-, 4.2- and 9.2-Mb de novo microdeletions in Xq27.3-q28 containing FMR1. X-inactivation was random in all patients, yet they presented with ID/DD as well as speech delay, macrocephaly and other features attributable to FXS. No signs of autism were present. Here, we further delineate the clinical spectrum of female patients with microdeletions. FMR1 expression studies gave no evidence for an absolute threshold below which signs of FXS present. Since FMR1 expression is known to be highly variable between unrelated females, and since FMR1 mRNA levels have been suggested to be more similar among family members, we further explored the possibility of an intrafamilial effect. Interestingly, FMR1 mRNA levels in all 3 patients were significantly lower than in their respective mothers, which was shown to be specific for patients with microdeletions containing FMR1.

Follow-up of loci from the International Genomics of Alzheimer's Disease Project identifies TRIP4 as a novel susceptibility gene.

To follow-up loci discovered by the International Genomics of Alzheimer's Disease Project, we attempted independent replication of 19 single nucleotide polymorphisms (SNPs) in a large Spanish sample (Fundació ACE data set; 1808 patients and 2564 controls). Our results corroborate association with four SNPs located in the genes INPP5D, MEF2C, ZCWPW1 and FERMT2, respectively. Of these, ZCWPW1 was the only SNP to withstand correction for multiple testing (P=0.000655). Furthermore, we identify TRIP4 (rs74615166) as a novel genome-wide significant locus for Alzheimer's disease risk (odds ratio=1.31; confidence interval 95% (1.19-1.44); P=9.74 × 10(-)(9)).

Evidence for a polygenic contribution to androgenetic alopecia.

BACKGROUND: Male pattern baldness (androgenetic alopecia, AGA) is a highly heritable trait and the most common form of hair loss in humans. Eight genome-wide significant risk loci for AGA have been identified. OBJECTIVES: To determine whether a polygenic component contributes to the genetic risk for AGA. METHODS: This study used a German case-control sample for AGA, which comprised 581 severely affected patients and 617 controls, to determine the contribution of polygenic variance to AGA risk. The sample was divided evenly into discovery and test samples. An additive polygenic risk score was calculated from risk alleles with increasingly liberal P-values in the discovery dataset, which was then used to test for the enrichment of AGA risk score alleles in the independent test samples. RESULTS: The polygenic score analysis provided significant evidence for a polygenic contribution to AGA where the amount of variance explained was 1·4-4·5%. CONCLUSION: This study provides evidence for the specific contribution of a polygenic component to the overall heritable risk for AGA. To some degree, the polygenic architecture of AGA might reflect the complexity of the biological pathways involved. Further analyses and strategies that complement conventional genome-wide association studies are needed to identify these factors. These may include pathway-based analyses, the analysis of functional candidate genes and tests for epistatic effects with known loci.

Morphine metabolism in human skin microsomes.

For patients with severe skin wounds, topically applied morphine is an option to induce efficient analgesia due to the presence of opioid receptors in the skin. However, for topical administration it is important to know whether the substance is biotransformed in the skin as this can eventually reduce the concentration of the active agent considerably. We use skin microsomes to elucidate the impact of skin metabolism on the activity of topically applied morphine. We are able to demonstrate that morphine is only glucuronidated in traces, indicating that the biotransformation in the skin can be neglected when morphine is applied topically. Hence, there is no need to take biotransformation into account when setting up the treatment regimen.

Susceptibility variants on chromosome 7p21.1 suggest HDAC9 as a new candidate gene for male-pattern baldness.

BACKGROUND: Male-pattern baldness (androgenetic alopecia, AGA) is the most common form of hair loss among humans. Research has shown that it is caused by genetic factors. Numerous studies have unequivocally identified two major genetic risk loci for AGA: the X-chromosomal AR/EDA2R locus, and the PAX1/FOXA2 locus on chromosome 20. OBJECTIVES: To identify further candidate genes for AGA, and thus gain further insights into this phenotype. METHODS: A German sample of 581 severely affected cases and 617 controls was used to perform a genome-wide association study. The identified associated locus was further analysed by fine-mapping, and then independently replicated in an Australian sample. Expression and pathway analyses were performed to characterize the susceptibility gene identified. RESULTS: The most significant association signal was obtained for rs756853 (P = 1·64 × 10(-7) ), which is located intronically in the histone deacetylase 9 (HDAC9) gene. Fine-mapping and a family-based analysis revealed that rs756853 and the 6-kb distal rs2249817 were the most highly associated single nucleotide polymorphisms. The association finding was replicated in an independent Australian sample, when the analysis was restricted to severely affected cases and unaffected controls (P = 0·026). Analysis of rs2249817 in a combined sample of severely affected German and Australian cases and unaffected controls revealed a strong association signal (P = 9·09 × 10(-8) ). Tissue expression studies demonstrated HDAC9 expression in various tissues, including tissues of relevance to AGA. No strong genotypic effects were observed in genotype-specific expression or splice studies. Pathway analyses supported the hypothesis that HDAC9 plays a functional role in AGA via interaction with the AR gene. CONCLUSIONS: The present study suggests that HDAC9 is the third AGA susceptibility gene.

[What can we learn from patients?]. / Was können wir vom Patienten lernen?

Quality management is only complete and sound when it comprehensively takes all comments from patients or staff into consideration, especially negative comments. In this manner, complaints, surveys and registered risks can be seen in a synoptic way. Every comment must be evaluated in relation to isolated cases as well as in the sense of pattern recognition.

[Effects of systemic or topical administration of corticosteroids and vitamin B in patients with olfactory loss]. / Untersuchung der Wirksamkeit von systemischen bzw. topischen Kortikoiden und Vitamin B bei Riechstörungen.

BACKGROUND: Aim of the present, unblinded, multicentric, open trial was to compare effects of 3 treatments in patients with olfactory dysfunction. METHODS: Treatments included administration of systemic corticosteroids (oral prednisolone), local corticosteroids (mometasone nasal spray), and systemic vitamin B, respectively. A total of 192 patients participated (95 women, 97 men; mean age 56 years). Duration of the smell loss ranged from 1 to 288 months (average 45 months). Olfactory dysfunction was due to infections of the upper respiratory tract (n = 72), sinunasal disease (n = 19), and posttraumatic olfactory loss (n = 10); the largest portion was classified as idiopathic (n = 85); other causes were rare (n = 6). RESULTS: Following systemic administration of corticosteroids improvement of olfactory function was observed (p < 0.001). Similarly, improvement of the sense of smell was found 2 (p = 0.03) and 6 months (p = 0.001) after local administration of mometasone, respectively. In contrast, after administration of vitamin B no significant change of olfactory function was seen after 2 months, while improvement was present after 6 months (p = 0.001). Duration of olfactory dysfunction had no effect on changes of smell function. CONCLUSIONS: These results indicate that improvement of olfactory function is found in olfactory dysfunction of different causes. Adequately controlled, blinded studies are needed to further explore potential effects of the various treatments.

Clinical presentation of qualitative olfactory dysfunction.

Many patients with olfactory dysfunction not only experience quantitative reduction of olfactory function, but also suffer from distorted olfactory sensations. This qualitative dysfunction is referred to as parosmia (also called "troposmia") or phantosmia, with the major difference that distorted olfactory sensations are experienced in the presence or absence of an odor, respectively. Our clinical observations corroborate the literature in terms of a general underestimation of the incidence of olfactory distortions. Based on selected cases we try to show that olfactory distortions exhibit a large variance in their clinical appearance. Further, emphasis is placed on the fact that only a detailed and directed history of the patient can provide cues to the correct diagnosis.

Immunohistochemical distribution of galectin-1, galectin-3, and olfactory marker protein in human olfactory epithelium.

The expression pattern of galectin-1 and galectin-3 in the human olfactory epithelium was investigated in relation to olfactory marker protein (OMP) using confocal laser immunofluorescence in human specimens and postmortem biopsies. OMP expression was found in olfactory receptor neurons (ORNs) in the olfactory mucosa and in fibers of the olfactory nerve crossing the submucous connective tissue. Galectin-1 was expressed in both the connective tissue of the nasal cavity and in the basal layer of the olfactory epithelium. In contrast, galectin-3 expression was limited to cells of the upper one-third of the olfactory epithelium. Expression of galectin-3 occurred in a subset of OMP-positive cells. However, between areas of galectin-1 and galectin-3 expression in the lower and upper portion of the epithelium, OMP-positive ORNs did not stain for both galectins. Considering the potential role of galectin-1 and galectin-3 in cell differentiation and maturation, the differential localization of galectins in the olfactory epithelium appears to be consistent with a significant role of these molecules in the physiological turnover of ORNs.

[Gustatory olfactory function test with the Güttich technique: an evaluation of the clinical value]. / Der gustatorische Riechtest in der Technik nach Güttich: Eine Uberprüfung der klinischen Wertigkeit.

BACKGROUND: Testing of retronasal olfactory sensitivity is routinely performed in many ENT-departments in German speaking countries. One of the most popular tests relates to an anecdotal report by Güttich [1]. In this test, liquids are placed on the patient's tongue; the patient is then asked to describe the aroma. Assuming that anosmic patients should not use other descriptors than "sweet," "sour," "salty," and "bitter", this test design is frequently applied in disability compensation settings. Surprisingly though, so far there are no studies regarding the sensitivity or specificity of this test in terms of the differentiation between hyposmic and anosmic patients. METHODS: The present investigation was performed in 50 anosmics, 42 hyposmics, and 13 normosmics. The diagnosis "hyposmia" or "anosmia" was based on 1. the patients' history, 2. psychophysical tests using an established test of olfactory function, the "Sniffin' Sticks," and 3. the recording of olfactory evoked potentials. RESULTS: We obtained the following major results: 1. In comparison to hyposmis and normosmics, anosmic patients had greater difficulty identifying the aromas. However, correct identification was not always possible for normosmics. 2. Regarding the differentiation between anosmic and hyposmic patients, the test's sensitivity was 86% with a specificity of 62%. This clearly limits routine clinical application of this test. CONCLUSIONS: Retronasal testing appears to be an elegant and simple means in order to screen olfactory function. However, the presently investigated approach is limited with regard to the discrimination between anosmic and hyposmic patients.

The use of antibodies for characterization and quantification of a recombinant protein.

In this study, we characterized proteinase A secreted by recombinant Saccharomyces cerevisiae bearing a multicopy plasmid containing the encoding gene (PEP4). Polyclonal and monoclonal antibodies were raised to study the product heterogeneity. Characterization of proteinase A was performed by immunoelectrophoresis and immunoblotting techniques. None of the monoclonal antibodies raised against proteinase A was found to react with the glycosyl side chains; thus cross-reaction with other glycosylated proteins (e.g. carboxypeptidase Y) was very low. This study allowed us to develop an ELISA method for the quantification of proteinase A in culture supernatants as well as the evaluation of monoclonal antibodies for their use in immunoaffinity chromatography.

[Bone changes in long-term therapy with etretinate (Tigason)]. / Knochenveränderungen unter Langzeittherapie mit Etretinat (Tigason).

We report on a 10-year-old girl with ichthyosis vulgaris, suffering from pains in her left arm and wrist after 1 1/2 years' treatment with etretinate (Tigason) The activity of the alkaline phosphatase was increased. Scintigraphy with technetium showed increased accumulation of activity as well as beginning transformation processes of the left ulna, which recovered after the reduction of the maintenance dose and are no longer detectable after a total of 3 1/2 years' treatment. The potential bone toxicity must be taken into consideration in long-term treatment with retinoids. Its manifestation should be prevented by thorough follow-up.

[Prognostic significance of the QT-interval following myocardial infarction]. / Prognostische Bedeutung des QT-Intervalls nach Herzinfarkt.

The prognostic value of QT prolongation was evaluated in 109 patients with myocardial infarction. Patients who suffered from reinfarction during the first month or developed ventricular fibrillation and sudden death did not have longer QT intervals than patients without these complications. On the other hand, QT prolongation was of prognostic value in the prediction of late reinfarction. The reasons for varying results from different investigators are discussed.

[Psoriasis therapy with the aromatic retinoid Ro 10-9359 (Tigason)]. / Zur Therapie der Psoriasis mit dem aromatischen Retinoid Ro 10-9359 (Tigason).

At three University Departments of Dermatology (Berlin, Leipzig, Erfurt) of the GRD, 63 psoriatics (46 men, 17 women) were treated with aromatic retinoid Ro 10-9359 (Tigason) up to a maximum of 84 weeks. In 34 of these cases, photochemotherapy was additionally applied. This combination resulted in a more rapid healing than Tigason alone. The average initial dose amounted to 1 mg per kg body weight. Only in cases of psoriatic erythroderma, the treatment started with a daily rate of 25 mg Tigason. The maintenance dose came to 50 to 25 mg daily. On account of deterioration of the cutaneous condition and/or heavy itching, the treatment had to be discontinued in 4 cases. Side effects as cheilitis, rhagades, loss of hair, and onycholysis disappeared after reduction of the dose. Our study confirmed the good therapeutical effect of Tigason with the various forms of psoriasis.