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Since the adhesion of bacteria to the tooth surface is a prerequisite for dental plaque and subsequent caries development, a promising caries preventive strategy could be to block the lectin-glycan-mediated adherence of cariogenic bacteria. The aim of the study was to evaluate potential differences in glycan-binding specificities of two Streptococcus mutans strains (DSM 20523 and DSM 6178) and Streptococcus sobrinus (DSM 20381). A competitive enzyme-linked lectin-binding assay was used to identify the binding specificities of isolated bacterial surface lectins. Blotting of the microbial proteins on neoglycoprotein-coated PVP membranes enabled a qualitative protein analysis of all specific bacterial lectins. Different glycan-binding sites could be identified for the S. mutans strains in comparison to S. sobrinus. An earlier reported glycan-binding specificity for terminal galactose residues could be confirmed for the S. mutans strains. For the S. sobrinus strain, more than one glycan-binding specificity could be found (oligomannose and terminal sialyl residues). Each of the tested strains showed more than one surface lectin responsible for the specific lectin-binding with varying molecular weight (S. mutans, 90/155 kDa and S. sobrinus, 35/45 kDa). The established experimental setup could be used as future standard procedure for the identification of bacterial lectin-derived binding specificities. The findings from this study might serve as basis for the design of an individual 'glycan cocktail' for the competitive inhibition of lectin-mediated adhesion of mutans streptococci to oral surfaces.

Acute pancreatitis (AP) is a disease whose pathogenesis remains largely obscure. Genetic research has focussed attention upon the role of the pancreatic protease/protease inhibitor system. The aim of this study was to investigate the prevalence of genetic variants of the trypsin inhibitor, SPINK1, in acute pancreatitis.We genotyped 468 patients with AP and 1117 healthy controls for SPINK1 alterations by single-strand conformation polymorphism analysis and by melting curve analysis using fluorescence resonance energy transfer probes.The c.101AG (p.N34S) variant was detected in 24/936 alleles of patients and in 18/2234 alleles of healthy controls (odds ratio=3.240; 95% confidence interval: 1.766-5.945; P0.001). In the UK patients, the mean age of patients with N34S was 11.9 years younger compared with N34S negative patients (P=0.023), but this was not apparent in the German patients. Allele frequencies for the c.163CT (p.P55S) variant did not differ between patients and controls.The SPINK1 N34S variant is associated with acute pancreatitis. This supports the importance of premature protease activation in the pathogenesis of AP and suggests that mutated SPINK1 may predispose certain individuals to develop this disease.

Background and aims A recent study reported that a nonsynonymous SNP rs2241880 (c.898A>G, p.Thr300Ala) within ATG16L1 confers susceptibility to Crohn's disease (CD). We analyzed ATG16L1 c.898A>G in three independent European inflammatory bowel disease (IBD) cohorts from Germany, Hungary and the Netherlands. Methods In total, we included 910 European IBD patients and compared the ATG16L1 c.898A>G genotype frequency with 707 ethnically matched healthy controls. We included patients from 3 populations originating from Germany (CD n =310; ulcerative colitis [UC] n =179), Hungary (CD n =147; UC n =117), and the Netherlands (CD n =157). Subtyping analysis was performed in respect to CARD15 alterations and clinical characteristics. Results We found a highly significant association of c.898A>G to CD. The association was significant ( p =0.0005) for the total CD cohort but also for the individual populations from Germany ( p =0.02) and Netherlands ( p =0.02) whereas in the Hungarian CD patients a clear trend was observed ( p =0.19; OR 1.227, 95% CI 0.910; 1.654). No association was found between c.898A>G and UC. No statistical interactions were observed between ATG16L1 c.898A>G and C ARD15 variants. Furthermore no association to a CD subphenotype was detected. Conclusions We confirm that ATG16L1 variant c898A>G confers a risk variant for CD but is not associated with a distinct CD phenotype.

Purpose To report the treatment of facial skin defects by cultured epithelial autografts and its clinical outcome. Patients and Methods Between 2002 and 2003, 18 patients with secondary facial skin defects (after tumor excision, trauma, or due to chronic wound healing dysfunction) were successfully treated with autologous cultivated keratinocytes. Overall, 12 patients were included in our study. At the time of this evaluation, the average time lapse after treatment with autologous cultivated keratinocytes was 13.1 months. From 9 of 12 patients a skin biopsy was taken, 12 of 12 patients were neurologically tested, and the results of 12 of 12 patients’ esthetics were evaluated by photography and in written form with a standardized questionnaire. Results Histologically, 9 of 12 patients showed a regular epithelial layer with evidence of basal cells of the basal membrane and conspicuously arranged connective tissue. The neurologic quality of the skin was discreetly reduced in 9 of 12 patients, but this was not experienced by the patient as a limitation. The wound closure was permanent in the case of all 12 patients. Scar tissue was found frequently, when the wound size was greater than 2.5 cm 2 . On the basis of the standardized questionnaire, 12 of 12 patients rated the degrees of their subjective satisfaction. Conclusion From the esthetic, histologic, and neurologic points of view, cultured epithelial autografts are an auspicious alternative to conventional grafting methods for facial skin replacement. Optimizing cell growth in vitro to decrease the cultivation period still remains an essential goal for the future to increase patient acceptance of the procedure as well.

Chronic pancreatitis is a common inflammatory disease of the pancreas. Mutations in the genes encoding cationic trypsinogen (PRSS1) and the pancreatic secretory trypsin inhibitor (SPINK1) are associated with chronic pancreatitis. Because increased proteolytic activity owing to mutated PRSS1 enhances the risk for chronic pancreatitis, mutations in the gene encoding anionic trypsinogen (PRSS2) may also predispose to disease. Here we analyzed PRSS2 in individuals with chronic pancreatitis and controls and found, to our surprise, that a variant of codon 191 (G191R) is overrepresented in control subjects: G191R was present in 220/6,459 (3.4%) controls but in only 32/2,466 (1.3%) affected individuals (odds ratio 0.37; P = 1.1 x 10(-8)). Upon activation by enterokinase or trypsin, purified recombinant G191R protein showed a complete loss of trypsin activity owing to the introduction of a new tryptic cleavage site that renders the enzyme hypersensitive to autocatalytic proteolysis. In conclusion, the G191R variant of PRSS2 mitigates intrapancreatic trypsin activity and thereby protects against chronic pancreatitis. The initial experiments were supported by the DFG (Wi 2036/1-1). This work was supported by the Sonnenfeld-Stiftung, Berlin, Germany (to H.W.), the US National Institutes of Health (NIH) (grant DK058088 to M.S.-T.), INSERM (Institut National de la Santé et de la Recherche Médicale) and the Programme Hospitalier de Recherche Clinique (grant PHRC R 08-04 to C.F.)

Salivary secretory IgA (s-IgA) is considered to act as an important first line of defense mechanism in the oral cavity. It has therefore been suggested that an increased antigenic load would induce an increase in salivary IgA production. This study investigated the pure glandular levels of salivary IgA in parotid and submandibular/sublingual (SM/SL) saliva during plaque accumulation leading to experimental gingivitis. Starting from regular oral hygiene, 14 healthy, nonsmoking men refrained from all oral hygiene measures for 12 days. On days -2, 0, 3, 6, and 12 a plaque index, a bleeding index, and unstimulated and stimulated saliva from the parotid and the SM/SL glands were measured. Salivary IgA was quantified using a sandwich ELISA. All subjects developed gingivitis as measured by a bleeding index. Compared to baseline the salivary flow rate was increased on day 12. Regarding the secretion rate of IgA there was a statistically significant increase in stimulated parotid saliva but not SM/SL saliva compared to baseline after 6 and 12 days without oral hygiene. No significant changes were observed for the concentration of IgA during the trial. Thus, in healthy subjects with regular oral hygiene the development of plaque induced gingivitis is associated with increased salivary gland output and increased total IgA output levels in stimulated parotid saliva but not in SM/SL saliva.

There is evidence that the quantity of antigen load is crucial for the activation of IgA immune responses. In order to investigate the relevance of these findings in aggressive periodontitis, salivary antibody responses were measured during non-surgical and antibiotic treatment. Twenty-one patients with generalized aggressive periodontitis were monitored for total salivary IgA and IgA reactive to Porphyromonas gingivalis in resting and stimulated whole saliva. Non-surgical treatment included full-mouth professional tooth cleaning and subgingival scaling and root planing (SRP) under local anesthesia. Patients were recalled at 3 months and 6 months following systemic antibiotic treatment. Non-parametric statistics showed significant improvements in the clinical parameters in all patients. Between baseline and 4 wk following SRP, median concentrations of total IgA decreased both in resting (-46%) and in stimulated (-33%) saliva. The P. gingivalis-specific IgA activity showed a twofold increase at 4 wk after SRP. In addition to these changes, periodontal treatment of aggressive periodontitis did not appear to affect salivary IgA, and there were no significant correlations of IgA to the clinical parameters. In conclusion, salivary IgA responses during periodontal treatment were not found to have a diagnostic or prognostic significance.

The local salivary immunoglobulin A (IgA) response in patients with aggressive periodontitis to oral microorganisms and its role for the pathogenesis has not been determined. This study investigated the hypothesis that aggressive periodontitis patients have impaired oral secretory immunity. Our test group was made-up of 19 aggressive periodontitis patients and 19 age- and gender-matched periodontally healthy controls. Total IgA, IgA subclass 1, IgA subclass 2 and IgA reactive to Actinobacillus actinomycetemcomitans Y4, Treponema denticola ATCC 35404 and Candida albicans DSM 3454 were determined by enzyme-linked immunosorbent assay in whole unstimulated and stimulated saliva. A statistically significantly lower concentration and secretion rate of total salivary IgA (P < 0.01) and IgA1 (P < 0.001) was found in the aggressive periodontitis group in resting and stimulated saliva. A decrease of IgA2 (P < 0.05) was seen in resting saliva. Although only minor differences were detected in the concentration and secretion of bacteria-reactive IgA in both groups, the proportion of bacteria-reactive IgA from the total IgA was significantly higher (P < 0.01) in the aggressive periodontitis group in all three microorganisms tested. Our results indicate an inhibition of total secretory IgA. In particular an IgA subclass 1-specific decrease in aggressive periodontitis was noted, while the bacteria-reactive humoral immune system in saliva was activated. The role of the decrease of IgA1 immunoglobulins in aggressive periodontitis with respect to susceptibility for periodontal diseases has to be elucidated.

Background: Periodontal bacteria are known to invade the systemic circulation. Chronic low-level bacteremia and a systemic inflammatory response have been suggested as a pathogenetic link between periodontal disease and atherosclerosis. The purpose of this study was to examine the systemic effect of a non-surgical therapy on white blood cell count (WBC count) and differential blood count in smoking and non-smoking generalized aggressive periodontitis (GAP) patients. Methods: 27 adult periodontitis patients (13 smokers and 14 non-smokers) with previously untreated GAP were subjected to 3 sessions of oral hygiene procedure. Afterwards, the patients were treated by scaling and root planing under local anaesthesia. Periodontal examinations were performed after supragingival pretreatment and three months after subgingival therapy. Pocket probing depth (PPD) and relative attachment level (RAL) were measured with Florida probe and disc probe. Accompanying clinical evaluation venous blood samples were taken to analyse the WBC counts and differential blood counts. For statistical analysis non-parametric tests were utilized. Results: No clinical or demographic differences were found between smokers (n=13) and non-smokers (n=14). PPD, bleeding on probing (BoP) and suppuration improved significantly after therapy both in smokers and non-smokers. Following periodontal treatment WBC counts, neutrophil and platelet counts decreased significantly in non-smokers (p≤0.004), while in smokers only platelet counts were significantly reduced (p=0.006). Non-smokers showed a significantly higher reduction of WBC counts (p=0.005) and neutrophils (p=0.001) compared to smokers. Conclusion: The results indicate that a therapeutical intervention may have a systemic effect on the blood count in GAP patients. This effect seems to differ between smokers and non-smokers.

Interactions between bacterial adhesins of lectin type and the oligosaccharide part of immobilised glycoconjugates on the tooth surface are involved in the specific colonisation of teeth. The specificity of the adhesion process is determined by the carbohydrate specificity of the bacterial lectins and the availability of the corresponding glycosylation pattern. On the other hand the same carbohydrate structures can specifically prevent the binding of bacteria by competitively blocking their adhesion, if sufficient amounts of this distinct carbohydrate structure are available in the secretion. Since carbohydrate binding receptors are also involved in the colonisation of tooth surfaces by cariogenic bacteria, it has been suggested that the architecture of the oligosaccharide portion of soluble glycoconjugates in saliva may play an important role as a constitutional host defence factor in the aetiology of dental caries. Characterising the availability of distinct carbohydrate patterns in saliva by using a pattern of well–described lectins in a competitive lectin inhibition assay we show that in children of a population–based sample a high caries susceptibility is associated with a reduced binding inhibition against the lectin peanut agglutinin (PNA). PNA is specific for the presence of terminal galactosyl residues and binds to the same O–glycan fractions as a surface lectin from Streptococcus mutans. The data suggest that a reduced availability of glycosylation patterns of galactosyl residues detected by the lectin PNA may act as an additional host–derived factor for an increased caries susceptibility.

The butyrate derivative isobutyramide (IBT) increases fetal hemoglobin (HbF) in patients with β-hemoglobinopathies, but little is known about its usefulness for prolonged therapeutic use. We treated 8 patients with transfusion-dependent β-thalassemia with 350 mg/kg of body weight per day of oral IBT for 126 to 384 days. During the trial period, the hemoglobin level was maintained between 85 g/L (range 82-87 g/L) (pretransfusion) and 115 g/L (range 110-119 g/L) (post-transfusion) (median, interquartile range), corresponding to 4-week transfusion intervals in all patients during the pretreatment phase. Adverse effects (bitter taste, epigastric discomfort) did not cause discontinuation of IBT. HbF increased in all patients from 3.1% (range 1.9%-4.8%) to 6.0% (range 3.3%-8.7) (P = .0017), while free Hb dropped from 0.48 g/L (range 0.39-0.81 g/L) to 0.19 g/L (range 0.16-0.24 g/L) (P 4.5%), high parental HbF, and increased erythropoietin levels (> 150 IU/L). We conclude that IBT prolongs transfusion intervals and reduces parenteral iron burden in some patients with transfusion-dependent β-thalassemia.

Generalised early-onset periodontitis (GEOP) is characterized by acute inflammatory bursts, resulting in rapid destruction of the periodontal apparatus in young adults. An impaired host defense seems to play an improtant role as etilogical factor of periodontitis, especially in the development of GEOP. As the Gram-negative Porphyromonas gingivalis has been indentified as one of the causative anaerobic bacteria, the humoral immune response to this micro-organism is of particular interest in patients with GEOP. To evaluate the local immune status, we measured total and P. gingivalis- reactive salivary IgA in GEOP patients and in age- and gender-matched periodontally normal controls. We found a significantly lower concentration and secretion rate of total salivary IgA in the GEOP group. Although no differences were detected in the concentration or secretion of P. gingivalis-reactive IgA between groups, the specific fraction of P. gingivalis-reactive IgA of the total IgA was significantly higher in the GEOP group. These findings indicate an inhibition of total secretory IgA in GEOP, while the P. gingivalis-reactive humoral immune system in saliva is, however, activated. P. gingivalis seems to selectively activate IgA lymphocyte clones and induces a switch in the fraction of specific IgA.

Objective In chronic pancreatitis (CP), alterations in several genes have so far been described, but only small cohorts have been extensively investigated for all predisposing genes. Design 660 patients with idiopathic or hereditary CP and up to 1758 controls were enrolled. PRSS1 , SPINK1 and CTRC were analysed by DNA sequencing, and cystic fibrosis transmembrane conductance regulator ( CFTR ) by melting curve analysis. Results Frequencies of CFTR variants p.R75Q, p.I148T, 5T -allele and p.E528E were comparable in patients and controls. We identified 103 CFTR variants, which represents a 2.7-fold risk increase (p CFTR compound heterozygous status with variant classes CF-causing severe and mild represented an OR of 16.1 (p Conclusions Accumulation of CFTR variants in CP is less pronounced than reported previously, with ORs between 2.7 and 4.5. Only CF-causing variants reached statistical significance. Compound and trans-heterozygosity is an overt risk factor for the development of CP, but the number of CFTR compound heterozygotes in particular is rather low. In summary, the study demonstrates the complexity of genetic interactions in CP and a minor influence of CFTR alterations in CP development.

Application of immunoapheresis to eliminate pathogenic autoantibodies targeting the second extracellular loop of the β1-receptor (β1-AABs) is currently investigated in patients with cardiomyopathy. Aptamers (single short DNA or RNA strands) are a new class of molecules that bind to a specific target molecule. This property qualifies aptamers for potential use in the apheresis technique. We recently identified an aptamer that specifically binds to β1-AABs, so in the present study we tested whether this aptamer could be used as a binder to prepare an apheresis column suitable for clearing β1-AABs from rat's blood. An apheresis column was designed containing the β1-AAB-targeting-aptamer coupled to sepharose. As tested in vitro, this column (1) binds β1-AABs highly specifically without marked interference with common IgGs, (2) has a capacity for clearing of approximately 1L of β1-AAB-positive serum and (3) can be completely regenerated for subsequent use. Using the column for extracorporeal apheresis of spontaneously hypertensive rats (SHR) positive for both β1-AABs and muscarinic 2-receptor autoantibodies (M2-AABs), only β1-AABs were removed. In a follow-up of 9 weeks, recurrence of β1-AABs in the blood of SHR could not be detected. For the first time, a newly designed apheresis column with a β1-AAB specific aptamer as a binder was successfully used to eliminate β1-AABs from SHR blood.

We have demonstrated label-free optical detection of viral nucleoprotein binding to a polyvalent anti-influenza aptamer by monitoring the surface-enhanced Raman (SERS) spectra of the aptamer-nucleoprotein complex. The SERS spectra demonstrated that selective binding of the aptamer-nucleoprotein complex could be differentiated from that of the aptamer alone based solely on the direct spectral signature for the aptamer-nucleoprotein complex. Multivariate statistical methods, including principal components analysis, hierarchical clustering, and partial least squares, were used to confirm statistically significant differences between the spectra of the aptamer-nucleoprotein complex and the spectra of the unbound aptamer. Two separate negative controls were used to evaluate the specificity of binding of the viral nucleoproteins to this aptamer. In both cases, no spectral changes were observed that showed protein binding to the control surfaces, indicating a high degree of specificity for the binding of influenza viral nucleoproteins only to the influenza-specific aptamer. Statistical analysis of the spectra supports this interpretation. AFM images demonstrate morphological changes consistent with formation of the influenza aptamer-nucleoprotein complex. These results provide the first evidence for the use of aptamer-modified SERS substrates as diagnostic tools for influenza virus detection in a complex biological matrix.

Background: A prototype system for computer-assisted colposcopic diagnosis (CAD) currently achieves a high level of accuracy of 80% (sensitivity 85%, specificity 75%) for the automatic assessment of colposcopic images. This pilot study investigated whether this type of CAD system is, in principle, capable of influencing the quality of the examiner's assessment. Materials and Methods: In this observer study, 24 digitized colposcopic images from patients attending a dysplasia clinic were assessed by 90 participants. All participants had attended a colposcopy training workshop so that they acquired the same basic information and skills. Results: Wide variation was seen among the non-experts, in contrast to the experts. An overall improvement in diagnostic accuracy was noted when the CAD system was used (non-experts: sensitivity 78%, specificity 70%; experts: sensitivity 74%, specificity 70%). Conclusion: The CAD system may serve as an aid in the further diagnosis of cervical intraepithelial neoplasia, and has the potential to improve the diagnostic process.

Summary Serum level of trace elements can be used as a marker for diagnosis and management of diseases. We evaluated the effects of age, sex and diurnal rhythm on serum concentration of Cu, Fe, Zn and their carrier proteins. The subjects (N=336) were participants of the randomized multidisciplinary Berlin Aging Study (BASE), stratified for age (70–103 yrs) and sex. There is no diurnal variation for the carrier proteins coeruloplasmin, transferrin and albumin. The age-related decline of these proteins is not significant. Not only serum Fe but also Zn levels undergo a progressive decrease during the day. In the aged the serum concentration of both trace elements decreased, but the most important changes are diurnal variations. To avoid mistakes in the interpretation of clinical findings, so called time-quantified reference values are recommended.

Rationale: Autoantibodies directed against the beta1-adrenoceptor (beta1-AABs) have been proposed to drive the pathogenesis of idiopathic dilated cardiomyoparthy (DCM), Chagas' cardiomyopathy, and peripartum cardiomyopathy. For disease treatment, aptamers that bind and neutralize beta1-AABs could be significant. Objective: We determined whether oligonucleotide-aptamers, selected to target human beta1-AABs directed against the second extracellular loop of the beta1-AAB, can neutralize these AABs and modulate their function in vitro. Methods and Results: Using Monolex technology, we identified an ssDNA aptamer that targets human beta1-AABs. The neutralization potential of this aptamer against beta1-AABs isolated from patients with DCM, Chagas' cardiomyopathy, and peripartum cardiomyopathy was analyzed using cultured neonatal rat cardiomyocytes by monitoring beta1-AAB induced cell toxicity and chronotropic cell responses. Aptamer addition reduced beta1-AAB induced cell toxicity and neutralized chonotropic beta1-AAB function in a dose-dependent manner. In the presence of aptamer neutralized beta1-AABs, cells remained fully responsive to agonists and antagonists, such as isoprenaline and bisoprolol. Both aptamer pretreated with a complementary (antisense) aptamer and a control scrambled-sequence aptamer were ineffective at beta1-AAB neutralization. Beta1-AABs directed against the first extracellular loop of the beta1-receptor and AABs directed against other G-protein coupled receptors were not affected by the selected aptamer. Conclusions: A specific aptamer that can neutralize cardiomyopathy associated human beta1-AABs in vitro has been identified and characterized, providing a framework for future in vivo testing of this treatment option in animal experiments.

A highly sensitive surface-enhanced Raman (SERS)-based method for detection of influenza viral nucleoproteins is described. The intrinsic SERS spectrum of the aptamer–nucleoprotein complex provides direct evidence of binding between a polyvalent anti-influenza aptamer and the nucleoproteins of three influenza strains.

In the near future, Computer Assisted Diagnosis (CAD) which is well known in the area of mammography might be used to support clinical experts in the diagnosis of images derived from imaging modalities such as endoscopy. In the recent past, a few first approaches for computer assisted endoscopy have been presented already. These systems use a video signal as an input that is provided by the endoscopes video processor. Despite the advent of high-definition systems most standard endoscopy systems today still provide only analog video signals. These signals consist of interlaced images that can not be used in a CAD approach without deinterlacing. Of course, there are many different deinterlacing approaches known today. But most of them are specializations of some basic approaches. In this paper we present four basic deinterlacing approaches. We have used a database of non-interlaced images which have been degraded by artificial interlacing and afterwards processed by these approaches. The database contains regions of interest (ROI) of clinical relevance for the diagnosis of abnormalities in the esophagus. We compared the classification rates on these ROIs on the original images and after the deinterlacing. The results show that the deinterlacing has an impact on the classification rates. The Bobbing approach and the Motion Compensation approach achieved the best classification results in most cases.

Contains fulltext : 89367.pdf (Publisher’s version ) (Closed access) OBJECTIVES: Premature activation of pancreatic digestive enzymes is considered as a major factor in the pathogenesis of pancreatitis. Genetic alterations of different pancreatic zymogens or their inhibitors have been associated with chronic pancreatitis (CP). METHODS: We sequenced all 12 GP2 exons in 380 German CP patients and in 182 German control subjects. In addition, we analyzed exon 3 of GP2 in 803 further CP patients and 1780 controls originating from Germany, the Netherlands, and India by targeted DNA sequencing. RESULTS: We detected 12 nonsynonymous and 6 synonymous exonic variants. All nonsynonymous changes with exception of c.220C>T (p.R74X) and c.502_503delG (p.G168fsX174) in exon 3 and c.541C>T (p.R181X) in exon 4 were missense mutations and predominantly located in exon 3. All nonsynonymous variants were found in single cases only, with exception of 2 alterations, c.355A>G (p.M119V) and c.409G>A (p. A137T), both located in exon 3. To elucidate the role of these 2 exon 3 variants, we investigated additional patients and controls. The frequency of these variants was similar between patients and controls regardless of ethnic background or cause of CP. CONCLUSIONS: Our data suggest that GP2 alterations do not alter the risk for the development of CP. 01 maart 2010

Contains fulltext : 81167.pdf (Publisher’s version ) (Closed access) OBJECTIVES: Chronic pancreatitis (CP) and pancreatic adenocarcinoma (pCA) are associated with risk factors such as alcohol intake and tobacco smoking. Microsomal epoxide hydrolase (EPHX1) is a phase II detoxifying enzyme capable of tobacco-borne toxicant inactivation. We studied the role of the EPHX1 c.337T>C (p.Y113H) variant, whichleads to altered enzyme activity, in pancreatic diseases. METHODS: We genotyped 2391 patients by melting curve analysis. We enrolled 367 patients with pCA, 341 patients with alcoholic CP (aCP), 431 patients with idiopathic CP or hereditary pancreatitis, 192 patients with acute pancreatitis, and 679 controls of German descent. We replicated data in 77 patients with aCP and 304 controls from The Netherlands. RESULTS: In German patients with aCP, Y113 was more common than in controls (allele frequencies, 0.73 vs 0.68; risk ratio, 1.21 [95% confidence interval, 1.05-1.39]). However, we could not confirm this association in the Dutch population (allele frequencies, 0.62 vs 0.68, P=not significant). In total, Y113 frequency was 0.71 in aCP and 0.68 in controls (P = not significant). Allele frequencies did not differ in the other disease groups (acute pancreatitis, 0.69; idiopathic CP or hereditary pancreatitis, 0.68; pCA, 0.68; and control, 0.68). CONCLUSIONS: The EPHX1 Y113H variant is not associated with pancreatic diseases indicating that EPHX1 does not play a significant role in the initiation of pancreatic inflammation or cancer.

Although the conditions for in vitro cultivation of adult stem cells and tissue are easily standardized, little is known about the optimal conditions for biointegration after transfer of the tissue graft, playing an important role in the treatment of defects especially soft-tissue skin injuries. To examine the influence of the microenvironment, we investigated the doubling time of primary epithelial cells in relation to the culture medium. Serum from patients of different age groups (n = 15,20 years; n = 9,20 years; and fetal calf serum) was pooled independently of age and added to culture medium of epithelial cells from a skin donor (10%). Number of cells was counted in vitro after 1 and 4 days of cultivation using a photometric extinction test. Results were plotted using quotient for calculating cell proliferation ([T4 -T1]:T1). Statistical significance was calculated by Wilcoxon test. Highest proliferation rate was achieved by cultivating the cells in the heterological serum admixture. Homologous serum admixtures in the cell cultures of20 donators yielded a significantly higher proliferation rate than adult serum (P0.01). High regenerative capacity of skin in children has, thus far, mainly been attributed to the high plasticity of the cellular structures. Our study shows for the first time that the age-dependent regenerative capacity in vitro is also influenced by age-dependent humoral factors. In vivo cells from older patients may thus be transferred into an altogether suboptimal microenvironment. Responsible humoral factors should be more closely examined to optimize the clinical management of cellular transplants.

For many years, scientists around the world have studied water, the basis of all life on our planet. Expecting to reach disruptive benefits similar to the huge value created by doping silicon and glass fibers, some scientists have been relentlessly searching for ways to dope water and bring about a quantum leap in life sciences similar to those seen in microelectronics and communications. Although early failures such as the now infamous “polywater” discovery discouraged and disappointed some in the scientific community, these temporary setbacks have not halted the effort. The quest to dope water and to create a revolutionary material upon which to base a new generation of aqueous material useful for therapeutics has continued unabated. Recent developments in nanotechnology, which focuses on materials at their nanometer scale, are enabling scientists to experiment with and understand a new class of water-based nanotechnology materials that are poised to become the first generation of “dopedwater” materials.

Contains fulltext : 71092.pdf (Publisher’s version ) (Closed access) BACKGROUND: A recent study reported that the c.30T>A (p.Cys10Ter; rs2043211) variant, in the CARD8 (TUCAN) gene, is associated with Crohn's disease (CD). The aim of this study was to analyze the frequency of p.C10X in 3 independent European (IBD) cohorts from Germany, Hungary, and the Netherlands. METHODS: We included a European IBD cohort of 921 patients and compared the p.C10X genotype frequency to 832 healthy controls. The 3 study populations analyzed were: (1) Germany [CD, n = 317; ulcerative colitis (UC), n = 180], (2) Hungary (CD, n = 149; UC, n = 119), and (3) the Netherlands (CD, n = 156). Subtyping analysis was performed in respect to NOD2 variants (p.Arg702Trp, p.Gly908Arg, c.3020insC) and to clinical characteristics. Ethnically matched controls were included (German, n = 413; Hungarian, n = 202; Dutch, n = 217). RESULTS: We observed no significant difference in p.C10X genotype frequency in either patients with CD or patients with UC compared with controls in all 3 cohorts. Conversely to the initial association study, we found a trend toward lower frequencies of the suggestive risk wild type in CD from the Netherlands compared with controls (P = 0.14). We found neither evidence for genetic interactions between p.C10X and NOD2 nor the C10X variant to be associated with a CD or UC phenotype. CONCLUSIONS: Analyzing 3 independent European IBD cohorts, we found no evidence that the C10X variant in CARD8 confers susceptibility for CD.

Contains fulltext : 69611.pdf (Publisher’s version ) (Closed access) Chronic pancreatitis is a persistent inflammatory disease of the pancreas, in which the digestive protease trypsin has a fundamental pathogenetic role. Here we have analyzed the gene encoding the trypsin-degrading enzyme chymotrypsin C (CTRC) in German subjects with idiopathic or hereditary chronic pancreatitis. Two alterations in this gene, p.R254W and p.K247_R254del, were significantly overrepresented in the pancreatitis group, being present in 30 of 901 (3.3%) affected individuals but only 21 of 2,804 (0.7%) controls (odds ratio (OR) = 4.6; confidence interval (CI) = 2.6-8.0; P = 1.3 x 10(-7)). A replication study identified these two variants in 10 of 348 (2.9%) individuals with alcoholic chronic pancreatitis but only 3 of 432 (0.7%) subjects with alcoholic liver disease (OR = 4.2; CI = 1.2-15.5; P = 0.02). CTRC variants were also found in 10 of 71 (14.1%) Indian subjects with tropical pancreatitis but only 1 of 84 (1.2%) healthy controls (OR = 13.6; CI = 1.7-109.2; P = 0.0028). Functional analysis of the CTRC variants showed impaired activity and/or reduced secretion. The results indicate that loss-of-function alterations in CTRC predispose to pancreatitis by diminishing its protective trypsin-degrading activity.

Item does not contain fulltext UDP-glucuronosyltransferases (UGT) catalyze the glucuronidation of various compounds and thus inactivate toxic substrates. Genetic variations reducing the activity of UGT1A7 have been associated with various gastrointestinal cancers. Most recently, the UGT1A7*3 allele has been reported as a significant risk factor for pancreatic disorders, but we could not confirm these data. This study focused on the possible causes for the noted discrepancy. UGT1A7 genotypes were assessed in 37 samples, which were previously analyzed for UGT1A7 polymorphisms by others. We determined genotypes by melting curve analysis and by DNA sequencing. Additionally, we produced UGT1A7*1 and *3 constructs with or without a mutation at position - 57 of UGT1A7 and analyzed various combinations of these constructs. In 14/37 samples UGT1A7 genotyping results differed. The discrepancy could be explained by polymerase chain reaction bias owing to an unbalanced allelic amplification which was caused by a -57T>G variant located within the sequence of the chosen primer template in previous studies. Our findings indicate that most of the previously reported genetic associations between UGT1A7 and gastrointestinal cancers are based on primer-dependent genotyping errors.

Background As a new class of therapeutic and diagnostic reagents, more than fifteen years ago RNA and DNA aptamers were identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps. Here we report the application of a new one-step selection method (MonoLEX) to acquire high-affinity DNA aptamers binding Vaccinia virus used as a model organism for complex target structures. Results The selection against complete Vaccinia virus particles resulted in a 64-base DNA aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy and real-time PCR, following an aptamer blotting assay. The same oligonucleotide showed the ability to inhibit in vitro infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner. Conclusion The MonoLEX method is a straightforward procedure as demonstrated here for the identification of a high-affinity DNA aptamer binding Vaccinia virus. MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers. Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method.

Aim: To report the data from a multidisciplinary bad breath consultation in Germany. Materials and methods: In this cross sectional study, 407 patients attending a bad breath consultation were examined by a specially trained dentist, with an ENT-specialist, an internist, and a psychologist on call. Results: All patients reported suffering from bad breath but only 72.1% showed detectable signs of breath malodour. Within this group, 92.7% revealed an oral cause, 7.3% revealed an extra-oral cause. Within the group without malodour, 76.3% had received prior diagnostics and treatments from other doctors, whereby 36% had received one or more gastroscopies and 14% had undergone an ENT operation. In only ten cases had an organoleptic evaluation of the putative malodour been performed. Conclusion: Our data reveal that breath malodour is mainly of oral origin and that patients with pseudo-halitosis are frequently not diagnosed correctly by doctors, resulting in a considerable amount of over-treatment.

The aim of the study was to develop an in vitro microbial-based caries model to test potentially caries-preventive agents.In this model, cariogenic Streptococcus mutans biofilms are grown on tooth samples within a reaction chamber hermetically surrounded by a bacteria-tight glove box allowing the manipulation of specimens during operation. The specimens were mounted in two rows on the inner and outer rims of a specimen turntable passing several inlet pipes transporting all necessary media. Using 64 lower incisors in 4 experiments, a 10 ppm NaF solution and an experimental potentially caries-preventive glycan solution were tested compared to a water control. The mean lesion depth was determined by confocal laser scanning microscopy.The depths of the caries-like lesions showed no statistically significant difference irrespective of whether the specimens were mounted on the inner or outer rim of the specimen turntable. As expected, the NaF solution inhibited the development of caries-like lesions almost completely. The experimental glycan solution revealed a statistically significantly lower demineralization depth compared to the control and a significantly higher depth compared to the NaF group. The system could be operated over a period of more than 9 weeks without unintentional contamination and the manipulation of the tooth specimens could be accomplished.In conclusion, our in vitro system is suitable for testing potential caries-preventive agents in a reproducible way by using whole tooth samples and offers full access together with the possibility of manipulating the specimens during operation.

Contains fulltext : 50765.pdf (Publisher’s version ) (Closed access) Keratin 8 (KRT8) is one of the major intermediate filament proteins expressed in single-layered epithelia of the gastrointestinal tract. Transgenic mice over-expressing human KRT8 display pancreatic mononuclear infiltration, interstitial fibrosis and dysplasia of acinar cells resulting in exocrine pancreatic insufficiency. These experimental data are in accordance with a recent report describing an association between KRT8 variations and chronic pancreatitis. This prompted us to investigate KRT8 polymorphisms in patients with pancreatic disorders. The KRT8 Y54H and G62C polymorphisms were assessed in a cohort of patients with acute and chronic pancreatitis of various aetiologies or pancreatic cancer originating from Austria (n=16), the Czech Republic (n=90), Germany (n=1698), Great Britain (n=36), India (n=60), Italy (n=143), the Netherlands (n=128), Romania (n=3), Spain (n=133), and Switzerland (n=129). We also studied 4,234 control subjects from these countries and 1,492 control subjects originating from Benin, Cameroon, Ethiopia, Ecuador, and Turkey. Polymorphisms were analysed by melting curve analysis with fluorescence resonance energy transfer probes. The frequency of G62C did not differ between patients with acute or chronic pancreatitis, pancreatic adenocarcinoma and control individuals. The frequency of G62C varied in European populations from 0.4 to 3.8%, showing a northwest to southeast decline. The Y54H alteration was not detected in any of the 2,436 patients. Only 3/4,580 (0.07%) European, Turkish and Indian control subjects were heterozygous for Y54H in contrast to 34/951 (3.6%) control subjects of African descent. Our data suggest that the KRT8 alterations, Y54H and G62C, do not predispose patients to the development of pancreatitis or pancreatic cancer.

The cytoskeleton comprises three filamentous systems: microfilaments, intermediate filaments, and microtubules. In epithelial cells, type I keratins such as keratin 18 (KRT18) and type II keratins such as keratin 8 (KRT8) polymerise to form the intermediate filaments. KRT18 and KRT8 represent the major keratins expressed in single-layered epithelia of the gastrointestinal tract including liver and pancreas.1 Animal studies suggest KRT8 and KRT18 have a hepatoprotective role against mechanical and toxic injury.2 Transgenic mice overexpressing mutant KRT18 display fragile hepatocytes with disrupted cytoskeleton filaments.3 These mice developed chronic hepatitis and were more susceptible to liver injury in comparison to mice overexpressing wild type KRT18.4 The viability of KRT8 null mice depends on the genetic background of the different mouse strains suggesting further genetic factors contribute to the resultant phenotype. For instance, in one mouse strain KRT8 -deficient mice died during embryonic development due to extensive liver haemorrhage.5 However, in another strain 55% of the KRT8 -deficient mice had a normal life expectancy but developed signs of inflammatory bowel disease and in some cases a mild inflammation of the liver.6 A recent report emphasises the importance of Keratin 8 for the formation of an intact placental barrier function for the viability of KRT8 -deficient embryos. These findings argue in favour of an extraembryonic defect responsible for lethality of these embryos.7 Furthermore, KRT8 null mice showed an abnormal histological liver architecture and were more vulnerable to liver damage after exposure to hepatotoxic substances compared to wild type mice.8–10 The above mentioned results support the hypothesis that keratin mutations might predispose humans to liver disease. Indeed, Ku et al described an association between two KRT8 mutations and cryptogenic cirrhosis. A heterozygous single base substitution involving a Gly to Cys at codon 62 (G62C) was found …

There is evidence that the quantity of antigen load is crucial for the activation of IgA immune responses. In order to investigate the relevance of these findings in aggressive periodontitis, salivary antibody responses were measured during non-surgical and antibiotic treatment. Twenty-one patients with generalized aggressive periodontitis were monitored for total salivary IgA and IgA reactive to Porphyromonas gingivalis in resting and stimulated whole saliva. Non-surgical treatment included full-mouth professional tooth cleaning and subgingival scaling and root planing (SRP) under local anesthesia. Patients were recalled at 3 months and 6 months following systemic antibiotic treatment. Non-parametric statistics showed significant improvements in the clinical parameters in all patients. Between baseline and 4 wk following SRP, median concentrations of total IgA decreased both in resting (-46%) and in stimulated (-33%) saliva. The P. gingivalis-specific IgA activity showed a twofold increase at 4 wk after SRP. In addition to these changes, periodontal treatment of aggressive periodontitis did not appear to affect salivary IgA, and there were no significant correlations of IgA to the clinical parameters. In conclusion, salivary IgA responses during periodontal treatment were not found to have a diagnostic or prognostic significance.

Tropical calcific pancreatitis (TCP) is a chronic pancreatitis unique to developing countries in tropical regions. The cause of TCP is obscure. Whereas environmental factors, such as protein energy malnutrition and ingestion of cassava, have been implicated, a genetic predisposition to the disease also may be important. In the present study we report on mutations in the serine protease inhibitor, Kazal type 1 (SPINK1) gene in north Indian patients with TCP.We studied 66 unrelated TCP patients (44 men, 49 with diabetes, and 6 with family history of TCP), 25 relatives, and 92 healthy control subjects. Samples were analyzed for SPINK1 variants (-53CT, L14P, N34S, P55S, and 272TC) and cationic trypsinogen (PRSS1) variants (A16V, K23R, N29I, and R122H) by melting curve analysis.Twenty-nine patients (44%) carried the N34S missense mutation, of whom 9 (14%) were homozygotes. In contrast, only 2 (2.2%) control subjects were N34S heterozygotes (prevalence ratio 20.2; 95% confidence interval 5.0-81.8; P0.0001 vs. TCP). The severity of pancreatitis did not differ between TCP patients with or without N34S, or among those heterozygous or homozygous for N34S. Among TCP patients with or without diabetes, the frequency of N34S carriers (43% vs. 47%) and N34S homozygotes (14% vs. 12%) was similar.TCP is highly associated with the SPINK1 N34S mutation. The high prevalence of N34S in TCP patients with and without diabetes suggests that these 2 subtypes have a similar genetic predisposition. The genetic predisposition to TCP resembles, at least in part, the idiopathic chronic pancreatitis found in industrialized countries.

The pathogenesis of chronic pancreatitis (CP) is poorly understood. Genetic studies revealed mutations in the cationic trypsinogen gene and an increased frequency of cystic fibrosis gene mutations in patients with CP. Recently, a point mutation (N34S) in the gene encoding the serine protease inhibitor, Kazal type 1 (SPINK1), was found in approximately 20% of patients with CP. The aim of our study was to determine the frequency of the N34S SPINKI gene mutation in a well-defined patient cohort with idiopathic CP (ICP) and to compare the incidence with healthy controls. In addition, we investigated the impact of this mutation on the long-term course of CP.Fourteen patients with early-onset and four patients with late-onset CP of our well-defined pancreatitis cohort were enrolled in the present study, and 397 healthy individuals served as a control population. Coding exonic and the flanking intronic sequences of SPINK1 were investigated by direct DNA sequencing. The mutations found were confirmed by melting curve analysis. In addition, the N34S mutation was detected by analyzing the DNA fragments generated by digestion with restriction enzyme TspR I. Clinical data of patients with the N34S mutation were compared with those without mutations.The N34S mutation was detected in six of 14 (43%) patients with early-onset ICP. One patient was homozygous, and five patients were heterozygous for this mutation. The N34S mutation in a heterozygous state was found in four of 397 healthy controls (1.0%). The different allele frequency observed (seven of 28 vs four of 794) was significant (odds ratio = 66, 95% CI = 18-242, p0.0001). The clinical course was similar in patients with a mutation compared with those without a mutation. No other SPINKI mutations were detected. The N34S mutation was not found in patients with late-onset ICP.Our results indicate that the N34S mutation in the SPINKI gene is strongly associated with ICP, especially with the early-onset type. The natural course is similar in patients with mutations compared with SPINK1 mutation-negative patients. The N34S mutation may easily be screened for by restriction digestion with TspR I.

Periodontal bacteria are known to invade the systemic circulation. Chronic low-level bacteremia and a systemic inflammatory response have been suggested as a pathogenetic link between periodontal disease and atherosclerosis. The purpose of this study was to examine the systemic effect of a non-surgical therapy on white blood cell count (WBC count) and differential blood count in smoking and non-smoking generalized aggressive periodontitis (GAP) patients.27 adult periodontitis patients (13 smokers and 14 non-smokers) with previously untreated GAP were subjected to 3 sessions of oral hygiene procedure. Afterwards, the patients were treated by scaling and root planing under local anaesthesia. Periodontal examinations were performed after supragingival pretreatment and three months after subgingival therapy. Pocket probing depth (PPD) and relative attachment level (RAL) were measured with Florida probe and disc probe. Accompanying clinical evaluation venous blood samples were taken to analyse the WBC counts and differential blood counts. For statistical analysis non-parametric tests were utilized.No clinical or demographic differences were found between smokers (n=13) and non-smokers (n=14). PPD, bleeding on probing (BoP) and suppuration improved significantly after therapy both in smokers and non-smokers. Following periodontal treatment WBC counts, neutrophil and platelet counts decreased significantly in non-smokers (por =0.004), while in smokers only platelet counts were significantly reduced (p=0.006). Non-smokers showed a significantly higher reduction of WBC counts (p=0.005) and neutrophils (p=0.001) compared to smokers.The results indicate that a therapeutical intervention may have a systemic effect on the blood count in GAP patients. This effect seems to differ between smokers and non-smokers.

· Background: Many successful pigment epithelium transplantation studies involving pink-eyed Royal College of Surgeons (RCS) dystrophic rats showed highly pigmented transplanted cells forming a double layer with slightly pigmented cells, attached to Bruch’s membrane. Since it is not clear whether transplanted pigmented cells can displace retinal pigment epithelial (RPE) host cells from Bruch’s membrane, we suggested that RPE cells of RCS dystrophic rats can phagocytize melanin granules, possibly derived from perished transplanted cells. · Methods: In a series of three experiments, RPE cells of nine pink-eyed, 2\(\)-month-old RCS dystrophic rats were isolated by trypsinization and mechanical dissection and cultivated in Dulbecco’s modified Eagles’ medium. These cells were then fed with melanin granules, isolated from bovine RPE cells, double-trypsinized after phagocytosis and viewed by light and electron microscopy. We also transplanted iris pigment epithelial (IPE) cells of 20-day-old Long-Evans rats into the subretinal space of pink-eyed RCS dystrophic rats of the same age, shown in light-microscopic photography after 42 days. · Results: Living RPE cells were heavily pigmented after feeding with isolated melanin granules in all three experiments as viewed by light microscopy. In addition, we identified melanin granules phagocytized by dystrophic RPE cells in electron microscopy. After transplantation of pigmented IPE cells into the subretinal space of pink-eyed RCS dystrophic rats’ eyes, a layer of slightly pigmented cells was seen on Bruch’s membrane below the transplanted IPE cells, shown in light microscopy. · Conclusion: We have shown by phagocytosis assay that dystrophic RPE cells can take up melanin granules in vitro. Our results assume that pigmented cells in transplantation studies, found as a monolayer, attached to Bruch’s membrane, cannot automatically be identified as transplanted cells. Instead, the possibility of perished transplanted cells serving as melanin donors for RPE host cells must be taken into consideration.

Cell-free human immunodeficiency virus type 1 (HIV-1) can be taken up and released by a monolayer of primary human gingival cells and remain infectious for CD4 + cells. Virus-sized latex particles covalently coated with purified native HIV-1 envelope glycoprotein gp120 are also transported through the primary epithelial cells. This process is significantly stimulated by increasing the intracellular cyclic AMP (cAMP) concentration. Inhibition experiments with mannan and α- methyl -mannopyranoside indicated that mannosyl groups are involved in the interaction between gp120 and gingival cells. An increase of cellular oligomannosyl receptors by incubation with the mannosidase inhibitor deoxymannojirimycin augmented transcellular transport of the gp120-coated particles. The results suggest that infectious HIV can penetrate gingival epithelia by a cAMP-dependent transport mechanism involving interaction of the lectin-like domain of gp120 and mannosyl residues on glycoproteins on the mucosal surface. Penetration of HIV could be inhibited by soluble glycoconjugates present in oral mucins.

In old age a large part of the variance in cognitive performance in population samples is explained by normal aging; in addition many subjects over 80 years are demented and therefore dementia also explains a part of cognitive variability. The question is whether the different factors for dementia (such as ApoE4, external atrophy parameter of the cranial computer tomography [cCT], education, sex or serum zinc level) influence the relation between age or dementia and Mini Mental State (MMSE) performance. In an epidemiological study data were analyzed of N = 239 subjects for the above factors. Most statistically significant variables of the MMSE do not change the amount of the partial correlation coefficient between the parameters age or dementia and MMSE. The external atrophy, however, diminishes the magnitude of the partial correlation between age and MMSE. In contrast the dementia-MMSE relation is unchanged. This points to a generally similar factor structure of cognitive aging and dementia in old age, but differences exist with respect to the importance of the external atrophy parameter of the brain. Most factors investigated explain separate parts of variance of cognitive performance in old age.

The apolipoprotein E4 allele has been reported to be associated with late onset Alzheimer's disease. Here we report the relation of several neuropsychological test parameters and the diagnosis of dementia to the apolipoprotein E polymorphism in an epidemiological sample of 477 subjects aged 70–103 years. The apolipoprotein E4 allele was found to be associated with reduced performance in several sensitive neuropsychological memory tests and with diagnosis of dementia only in the oldest subjects (>84 years). The association with dementia in this population based sample was much weaker than previously described and became only significant in a logistic regression analysis when age was included in the model.