CFTR, SPINK1, CTRC and PRSS1 variants in chronic pancreatitis: is the role of mutated CFTR overestimated?

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<0.0001). Severe cystic fibrosis (CF)-causing variants increased the risk of developing CP 2.9-fold, and mild CF-causing variants 4.5-fold (p<0.0001 for both). Combined CF-causing variants increased CP risk 3.4-fold (p<0.0001), while non-CF-causing variants displayed a 1.5-fold over-representation in patients (p=0.14). CFTR compound heterozygous status with variant classes CF-causing severe and mild represented an OR of 16.1 (p<0.0001). Notably, only 9/660 (1.4%) patients were compound heterozygotes in this category. Trans-heterozygosity increased CP risk, with an OR of 38.7, with 43/660 (6.5%) patients and 3/1667 (0.2%) controls being trans-heterozygous (p<0.0001). 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.

Computer-assisted diagnosis (CAD) in colposcopy: evaluation of a pilot study.

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.

Computer-assisted diagnosis in colposcopy: results of a preliminary experiment?

PURPOSE: Diagnosis of cervical intraepithelial neoplasia (CIN) is currently based on the histological result of an aiming biopsy. This preliminary study investigated whether diagnostics for CIN can potentially be improved using semiautomatic colposcopic image analysis. METHODS: 198 women with unremarkable or abnormal smears underwent colposcopy examinations. 375 regions of interest (ROIs) were manually marked on digital screen shots of the streaming documentation, which we provided during our colposcopic examinations (39 normal findings, 41 CIN I, and 118 CIN II-III). These ROIs were classified into two groups (211 regions with normal findings and CIN I, and 164 regions with CIN II-III). We developed a prototypical computer-assisted diagnostic (CAD) device based on image-processing methods to automatically characterize the color, texture, and granulation of the ROIs. RESULTS: Using n-fold cross-validation, the CAD system achieved a maximum diagnostic accuracy of 80% (sensitivity 85% and specificity 75%) corresponding to a correct assignment of abnormal or unremarkable findings. CONCLUSIONS: The CAD system may be able to play a supportive role in the further diagnosis of CIN, potentially paving the way for new and enhanced developments in colposcopy-based diagnosis.

The role of epoxide hydrolase Y113H gene variant in pancreatic diseases.

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.

Impact of microenvironment on the growth of primary human epidermal cells.

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 of <20 donators yielded a significantly higher proliferation rate than adult serum (P < 0.01). 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.

The SPINK1 N34S variant is associated with acute pancreatitis.

OBJECTIVE: 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. METHODS: 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. RESULTS: The c.101A>G (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; P<0.001). 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.163C>T (p.P55S) variant did not differ between patients and controls. CONCLUSION: 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.

No association of the CARD8 (TUCAN) c.30T>A (p.C10X) variant with Crohn's disease: a study in 3 independent European cohorts.

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.

An evaluation and comparison of the performance of state of the art approaches for the detection of spiculated masses in mammograms.

Mammography is the standard examination method for the early detection of breast cancer. In the last decade, computer assisted detection systems have been developed that assist the physician in the detection of suspicious regions in mammograms. However, recent clinical studies indicate that state of the art CAD systems might have a negative impact on the accuracy of screening mammography. Therefore, besides additional clinical studies, better evaluations of state of the art detection approaches are necessary. In this contribution three methods for the detection of spiculated masses in mammograms are evaluated and compared. All three of them are based on gradient orientation images. To detect masses, the methods use circular neighbourhoods with different sizes around a single pixel. The number of orientations in every neighbourhood is used by every method in different ways to form a result. The main contribution is the first fair comparison of the performance of different detection approaches for spiculated masses. Furthermore, a novel gradient direction analysis is introduced. The analysis is an extension to the three approaches, which increases the performance for one of the three approaches.

Cultured epithelial autografts in the treatment of facial skin defects: clinical outcome.

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 cm2. 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.

Keratin 8 sequence variants in patients with pancreatitis and pancreatic cancer.

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.

Tropical calcific pancreatitis: strong association with SPINK1 trypsin inhibitor mutations.

BACKGROUND & AIMS: 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. METHODS: 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 (-53C>T, L14P, N34S, P55S, and 272T>C) and cationic trypsinogen (PRSS1) variants (A16V, K23R, N29I, and R122H) by melting curve analysis. RESULTS: 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; P < 0.0001 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. CONCLUSIONS: 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.

Mutations of the serine protease inhibitor, Kazal type 1 gene, in patients with idiopathic chronic pancreatitis.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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, p < 0.0001). 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. CONCLUSIONS: 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.

White blood cell count in generalized aggressive periodontitis after non-surgical therapy.

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< or =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.