Art meets science: The Cosmopolitan Chicken Research Project.

The Cosmopolitan Chicken Project is an artistic undertaking of renowned artist Koen Vanmechelen. In this project, the artist interbreeds domestic chickens from different countries aiming at the creation of a true Cosmopolitan Chicken as a symbol for global diversity. The unifying theme is the chicken and the egg, symbols that link scientific, political, philosophical and ethical issues. The Cosmopolitan Chicken Research Project is the scientific component of this artwork. Based on state of the art genomic techniques, the project studies the effect of the crossing of chickens on the genetic diversity. Also, this research is potentially applicable to the human population. The setup of the CC®P is quite different from traditional breeding experiments: starting from the crossbreed of two purebred chickens (Mechelse Koekoek x Poule de Bresse), every generation is crossed with a few animals from another breed. For 26 of these purebred and crossbred populations, genetic diversity was measured (1) under the assumption that populations were sufficiently large to maintain all informative SNP within a generation and (2) under the circumstances of the CCP breeding experiment. Under the first assumption, a steady increase in genetic diversity was witnessed over the consecutive generations, thus indeed indicating the creation of a "Cosmopolitan Chicken Genome". However, under the conditions of the CCP, which reflects the reality within the human population, diversity is seen to fluctuate within given boundaries instead of steadily increasing. A reflection on this might be that this is because, in humans, an evolutionary optimum in genetic diversity is reached. Key words.

The ethical introduction of genome-based information and technologies into public health.

With the human genome project running from 1989 until its completion in 2003, and the incredible advances in sequencing technology and in bioinformatics during the last decade, there has been a shift towards an increase focus on studying common complex disorders which develop due to the interplay of many different genes as well as environmental factors. Although some susceptibility genes have been identified in some populations for disorders such as cancer, diabetes and cardiovascular diseases, the integration of this information into the health care system has proven to be much more problematic than for single gene disorders. Furthermore, with the 1000$ genome supposedly just around the corner, and whole genome sequencing gradually being integrated into research protocols as well as in the clinical context, there is a strong push for the uptake of additional genomic testing. Indeed, the advent of public health genomics, wherein genomics would be integrated in all aspects of health care and public health, should be taken seriously. Although laudable, these advances also bring with them a slew of ethical and social issues that challenge the normative frameworks used in clinical genetics until now. With this in mind, we highlight herein 5 principles that are used as a primer to discuss the ethical introduction of genome-based information and genome-based technologies into public health.

The storage and use of biological tissue samples from minors for research: a focus group study.

Genetic research on pediatric stored tissue samples raises specific ethical questions that differ from those raised when adults are the donors. To investigate opinions on this matter, we conducted 10 focus group discussions. Five focus groups were conducted with adult participants and 5 had teenage participants between 15 and 19 years old. The discussions were analyzed with NVIVO 8 (qualitative research software). We found the following recurrent categories: the requirement that research should not pose any burden on children and that it should benefit other children, the trust people had in the role of parents, the need for information and the growth towards autonomy. Both the adults and teenagers we interviewed thought that the inclusion of tissue samples from minors in research had ethical implications. A major concern was that nontherapeutic research would pose no extra burden on children, which would assume the use of nonintrusive methods of gathering samples and the use of samples that were gathered in a diagnostic context. Participants, however, also understood the necessity of such research. The overall impression was that parents would be the best persons to make decisions on behalf of a small child and that the same parents would engage their children in the decision-making when they grew older. People thought that there was a duty to recontact minors when they reached the age of competence but on a best-effort basis.

An exploratory survey of professionals on the use of stored tissue samples from minors for genetic research.

he ethical aspects of the use of stored tissue samples collected from minors are of topical interest. However, the views of professionals working in the field of genetics have not been investigated in depth anywhere. We conducted a survey among 194 such professionals in Belgium. This list was composed of the members of the High Council for Anthropogenetics, supplemented with all professionals working in the field of genetics that we found on the websites of the eight Belgian centers of human genetics and of the associated university registries. We achieved a response rate of 35.5%. The vast majority (92%) think that research on stored tissue samples is useful. Most respondents stated that parental consent is valid (82.5%), and 76.5% thought that children should also be given the right to assent when they are able to comprehend the implications of the storage of biological samples and of genetic research. Slightly more than half put the age at which young people can understand storage or research rather high: 16-18 years (51 and 53.1%, respectively). Although there is some consensus in the literature that donors should be allowed to give broad consent for future research on their biological samples, only 47.6% in our survey thought that parents should be allowed to consent to any future research on their children's samples. The aim of our study was to give some basis for future ethical reflections and policies on the subject of stored tissue samples from minors for genetic research. We concluded that a large majority of Belgian researchers and clinicians in the field of genetic research think research on stored tissue samples from minors is useful. They also think that parental consent for such research is valid, but that children should be allowed to assent as they grow older.

Quality genetic services for the population, now and in the future.

The possibilities for testing and screening for genes involved in inherited diseases or susceptibility to diseases have increased spectacularly. Combined with a revolution in the availability of sophisticated new technologies for testing, the question arises how will we be able to continue to provide quality services to our customers ? Who will provide these ? Will it be the centres, as we know them today, or will DTC take gradually over this service ? Will the quality criteria, as established today before tests are made available, still be applicable and how will these new services be able to contribute to an increasing and coordinated collection of global information on genetic diversity and on the pathogenic changes in the human genome? As stated in the Bioethics Convention of the European Council and explicited in the recent recommendations from the House of Lords of the UK on Genomic Medicine, we will need a major effort of the European Commission/of our governments, to implement a series of measures which will allow the correct and quality assured introduction into practice of the genetic knowledge that is being generated. Only then will all individuals and the scientific community be able to benefit from our services.

Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice.

It is often challenging for the clinician interested in cystic fibrosis (CF) to interpret molecular genetic results, and to integrate them in the diagnostic process. The limitations of genotyping technology, the choice of mutations to be tested, and the clinical context in which the test is administered can all influence how genetic information is interpreted. This paper describes the conclusions of a consensus conference to address the use and interpretation of CF mutation analysis in clinical settings. Although the diagnosis of CF is usually straightforward, care needs to be exercised in the use and interpretation of genetic tests: genotype information is not the final arbiter of a clinical diagnosis of CF or CF transmembrane conductance regulator (CFTR) protein related disorders. The diagnosis of these conditions is primarily based on the clinical presentation, and is supported by evaluation of CFTR function (sweat testing, nasal potential difference) and genetic analysis. None of these features are sufficient on their own to make a diagnosis of CF or CFTR-related disorders. Broad genotype/phenotype associations are useful in epidemiological studies, but CFTR genotype does not accurately predict individual outcome. The use of CFTR genotype for prediction of prognosis in people with CF at the time of their diagnosis is not recommended. The importance of communication between clinicians and medical genetic laboratories is emphasized. The results of testing and their implications should be reported in a manner understandable to the clinicians caring for CF patients.

Guiding periodontal pocket recolonization: a proof of concept.

The complexity of the periodontal microbiota resembles that of the gastro-intestinal tract, where infectious diseases are treatable via probiotics. In the oropharyngeal region, probiotic or replacement therapies have shown some benefit in the prevention of dental caries, otitis media, and pharyngitis, but their effectiveness in the treatment of periodontitis is unknown. Therefore, this study addressed the hypothesis that the application of selected beneficial bacteria, as an adjunct to scaling and root planing, would inhibit the periodontopathogen recolonization of periodontal pockets. Analysis of the data showed, in a beagle dog model, that when beneficial bacteria were applied in periodontal pockets adjunctively after root planing, subgingival recolonization of periodontopathogens was delayed and reduced, as was the degree of inflammation, at a clinically significant level. The study confirmed the hypothesis and provides a proof of concept for a guided pocket recolonization (GPR) approach in the treatment of periodontitis.

Bacteria interfere with A. actinomycetemcomitans colonization.

It is known that beneficial bacteria can suppress the emergence of pathogenic bacteria, particularly in the gastrointestinal tract. This study examined the potential for a similar suppression of Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans colonization of epithelial cells, due to its potential relevance in periodontal diseases. Seven presumed beneficial bacteria were examined for their ability to interfere, exclude, or displace A. actinomycetemcomitans from epithelial cells in vitro. Streptococcus sanguinis, Streptococcus mitis, and Streptococcus salivarius showed prominent inhibitory effects on either A. actinomycetemcomitans recovery or colonization. These results confirmed the hypothesis that bacterial interactions interfere with A. actinomycetemcomitans colonization of epithelial cells in vitro, and demonstrated the potential beneficial effects of S. mitis, S. salivarius, and S. sanguinis.

Upregulation of Wilms' tumour gene 1 (WT1) in uterine sarcomas.

AIM: Overexpression of Wilms' tumour gene (WT1) has been proven in several tumours. Previous research of our group on the cell cycle of uterine leiomyosarcoma (LMS) and carcinosarcoma (CS) suggested a possible role for WT1. We therefore intended to further explore the expression pattern of WT1 in uterine sarcomas. METHODS: 27 CS, 38 LMS, 15 endometrial stromal sarcomas (ESS) and seven undifferentiated sarcomas (US) were collected. WT1 expression was evaluated by immunohistochemistry (IHC) in 87 samples, by RT-PCR (m-RNA expression) in 23 random selected samples and by Western blotting in 12 samples, separating cytoplasmic and nuclear proteins. A pilot study to detect mutations (exons 7-10) was performed on eight samples. RESULTS: IHC showed WT1 positivity in 12/27 CS, 29/38 LMS, 7/15 ESS and 4/7 US. All-but-one sample had a positive RT-PCR. All Western blottings were positive with more cytoplasmic expression in 9/12 cases. No mutations were found. CONCLUSIONS: WT1 is overexpressed in uterine sarcomas. Since increased levels of mRNA determine the biological role, WT1 might contribute to uterine sarcoma tumour biology.

Presymptomatic and predictive genetic testing in minors: a systematic review of guidelines and position papers.

The objective of this study is to review ethical and clinical guidelines and position papers concerning the presymptomatic and predictive genetic testing of minors. The databases Medline, Philosopher's Index, Biological Abstracts, Web of Science and Google Scholar were searched using keywords relating to the presymptomatic and predictive testing of children. We also searched the websites of the national bioethics committees indexed on the websites of World Health Organization (WHO) and the German Reference Centre for Ethics in the Life Sciences, the websites of the Human Genetics Societies of various nations indexed on the website of the International Federation of Human Genetics Societies and related links and the national medical associations indexed on the website of the World Medical Association. We retrieved 27 different papers dealing with guidelines or position papers that fulfilled our search criteria. They encompassed the period 1991-2005 and originated from 31 different organizations. The main justification for presymptomatic and predictive genetic testing was the direct benefit to the minor through either medical intervention or preventive measures. If there were no urgent medical reasons, all guidelines recommend postponing testing until the child could consent to testing as a competent adolescent or as an adult. Ambiguity existed for childhood-onset disorders for which preventive or therapeutic measures are not available and for the timing of testing for childhood-onset disorders. Although the guidelines covering presymptomatic and predictive genetic testing of minors agree strongly that medical benefit is the main justification for testing, a lack of consensus remains in the case of childhood-onset disorders for which preventive or therapeutic measures are not available.

Analysis of Wnt/Beta catenin signalling in desmoid tumors.

Desmoid tumors are fibromatous lesions occurring both sporadically and in patients with familial adenomatous polyposis (FAP). Because of the association of these tumors with the hereditary colorectal cancer syndrome FAP we set out to define the molecular events driving desmoid tumorigenesis, hypothezising these might be identical to events driving colorectal tumorigenesis. We found that whereas FAP-associated desmoid tumors are caused by germline APC mutations followed by somatic inactivation of the wild-type APC allele, sporadic desmoids are usually characterized by oncogenic mutations in the b-catenin gene, both identical molecular alterations to those found in the vast majority of colorectal cancers. Next we set out to investigate the cellular pathways activated by these mutations, and identified activation of the Wnt signaling pathway in desmoid tumors. Wnt signaling modulates expression of developmental genes and cell fate via beta-catenin, and has been implicated in many cancer types. Currently we are investigating tissue-specific downstream effectors of the Wnt pathway that might be responsible for the behaviour of these invasive fibrous tumors. Our findings also point to a role for this pathway in the regulation of normal myofibroblast proliferation and suggest novel treatments in desmoid tumors and other fibrous proliferative disorders.

Association of tumour necrosis factor alpha variants with the CF pulmonary phenotype.

BACKGROUND: The pulmonary phenotype in patients with cystic fibrosis (CF), even in those with the same CF transmembrane conductance regulator (CFTR) genotype, is variable and must therefore be influenced by secondary genetic factors as well as environmental factors. Possible candidate genes that modulate the CF lung phenotype may include proinflammatory cytokines. One such protein is tumour necrosis factor alpha (TNFalpha), a member of the immune system. METHODS: Three polymorphic loci in the promoter (-851c/t, -308g/a, -238g/a) and one polymorphic locus in intron 1 (+691g ins/del) of the TNFalpha gene were typed by a single nucleotide primer extension assay in CF patients and healthy controls. Spirometric data and first age of infection with Pseudomonas aeruginosa were collected retrospectively from patients' medical records. RESULTS: An association was found between the TNFalpha +691g ins/del polymorphic locus and severity of CF lung disease. Patients heterozygous for +691g ins and +691g del were more likely to have better pulmonary function (mean (SD) forced expiratory volume in 1 second (FEV1) 79.7 (12.8)% predicted) than patients homozygous for +691g ins (mean (SD) FEV1 67.5 (23.0)% predicted; p = 0.008, mean difference 12.2%, 95% CI 3.5 to 21.0). Also, patients heterozygous for +691g ins and +691g del were more likely to have an older first age of infection with P aeruginosa (mean (SD) 11.4 (6.0) years) than patients homozygous for +691g ins (mean (SD) 8.3 (4.6) years; p = 0.018, mean difference 3.1 years, 95% CI 0.5 to 5.6). An association was also found with the -851c/t polymorphic locus. In the group of patients with more severe FEV1% predicted, a higher proportion of patients were homozygous for the -851c allele than in the other group of patients (p = 0.04, likelihood ratio chi2, odds ratio = 2.4). CONCLUSION: TNFalpha polymorphisms are associated with the severity of CF lung disease in Czech and Belgian patients with CF.

Established cell lines used in cystic fibrosis research.

The development of immortalized cell lines has been a significant benefit to the study of human disease, due to limitations in using primary cells and the availability of tissue. The immortalization of cells from cystic fibrosis (CF) patients as well as cells from non-CF individuals from tissues relevant to CF has been critical to enhancing our understanding of the physiological, biochemical and genetic mechanisms underlying CF and for the development of therapeutic strategies designed to manage CF pathology. A comprehensive list of immortalized cells from various tissue and species, with an emphasis on epithelial cells, is presented and discussed here.

Invasion and MMP expression profile in desmoid tumours.

Desmoid tumours are locally invasive soft tissue tumours in which beta-catenin mediated TCF-dependent transcription is activated. The role of soluble factors secreted by the myofibroblastic desmoid tumour, which could stimulate tumour invasiveness, was investigated. Using collagen gel invasion assays, the presence of factors stimulating invasion in desmoid conditioned media (CM) could be established. Since matrix metalloproteinases (MMPs) have been implicated in the process of tumoral invasion, the expression levels of the MMP family members were evaluated. Quantitative reverse transcription-PCR was used to determine the expression levels of MMP1, MMP2, MMP3, MMP7, MMP11, MMP12, MMP13, MMP14 and the inhibitors TIMP1, TIMP2 and TIMP3. Besides overexpression of MMP7, a known TCF-dependent target gene, a striking upregulation of the expression levels of MMP1, MMP3, MMP11, MMP12 and MMP13 in desmoid tumours, compared to unaffected fibroblasts from the same patients, was found. Treating the CM of desmoids with a synthetic and a physiologic MMP inhibitor reduced the invasion-stimulating capacity of the desmoid CM by approximately 50%. These results suggest the involvement of soluble factors, released by the desmoid cells, in stimulating invasion and implicate the MMPs as facilitators of invasion.

Adhesion of Porphyromonas gingivalis to cultured pocket epithelium: mono- and multi-layered.

Adhesion of bacteria to epithelial cells might be influenced by the degree of cell differentiation, as observed in the multi-layering process of epithelial cells. In the present study, the adhesion of a large group of clinical Porphyromonas gingivalis strains (n=11) to in vitro cultured mono- and multi-layers of epithelial cells was examined and compared. The tissue samples originated from 6 patients with chronic adult periodontitis. Porphyromonas gingivalis bacteria adhered more to mono-layers as opposed to the more differentiated multi-layers. Differences between the clinical P. gingivalis strains, however, became obvious only on multi-layers. These partially differentiated cells may also better represent the individual subject variations. Mono-layer cultures, which are simpler to obtain, seem to be less precise. The importance of cell differentiation on bacterial adhesion needs more attention.

Viability of cultured periodontal pocket epithelium cells and Porphyromonas gingivalis association.

OBJECTIVES: Porphyromonas gingivalis, one of the key pathogens in the development of periodontitis, produces a number of virulence factors that might explain its pathogenicity. One of them is the ability to adhere and invade pocket epithelium. The aim of this study was to follow, over time, the association of P. gingivalis and consequent morphological changes of the pocket epithelium cells. MATERIAL AND METHODS: The association capacity of four P. gingivalis serotypes [K1, K2, K4, K- (nonencapsulated)] with in vitro cultured mono-layers from periodontal pocket epithelial cells of patients with periodontitis, was followed by fluorescence microscopy and bacterial culture. The contact time between bacteria and epithelium cells ranged from 45 min to 8 h. The microscopic evaluation allowed differentiation between dead and living cells (bacteria as well as epithelium) and description of the morphological changes after association. RESULTS: A highly significant difference in the number of associating bacteria was found between dead and living epithelium cells, and between non-capsulated and capsulated strains. A significant increase in the proportion of dead pocket epithelium cells was found with prolonged association time. The morphological changes (rounding of the epithelial cell, detachment from the glass cover-slip and loss of intercellular contact) occurred faster for mono-layers inoculated with the non-encapsulated P. gingivalis strain. CONCLUSIONS: This study indicates that dead pocket epithelium cells harbor more P. gingivalis cells, and that a positive correlation exists between contact time and cell death. For the P. ginigvalis species, non-encapsulated strains associate in higher number. As a result, the damage they cause to the host cell seems to occur faster than occurs in encapsulated strains. As such, cell death can be seen as the end-result of bacterial association.

The molecular basis of colorectal cancer.

Colorectal cancers, whether sporadic or hereditary, are caused by a defined set of molecular events. The genes and pathways involved in the earliest steps of tumorigenesis have crucial functions in the regulation of normal crypt homeostasis. Further insight into these pathways can lead to the development of useful prognostic indicators, and target preventive and therapeutic strategies in the management of colorectal cancer. Mutations in the APC/beta-catenin/Tcf-4 pathway lead to important changes in stem cell dynamics, before clinically identifiable lesions appear. Preventive strategies aimed at reversing these changes or therapeutic interventions targeting cell populations with these alterations should be most efficacious.

Linkage and association of HLA class II genes with vitiligo in a Dutch population.

BACKGROUND: Serological typing of HLA has shown discrepancies in HLA associations with vitiligo in different ethnic populations. OBJECTIVES: To perform genotyping of HLA class II genes on a Dutch vitiligo population in order clearly to identify susceptible and protective HLA alleles in vitiligo. METHODS: HLA typing was carried out by amplifying genomic DNA by polymerase chain reaction (PCR) followed by dot-blot hybridization with sequence-specific oligonucleotides (SSO). Fifty Dutch vitiligo probands, and their parents (150 individuals) and 204 healthy controls were studied. RESULTS: Family-based case-control association studies and linkage disequilibrium analysis showed the linkage and association of DRB4*0101 allele with vitiligo (P(c) = 0.0016, relative risk = 2.21). The family-based association study also provided evidence for linkage and association of DQB1*0303 allele with vitiligo (chi(2) = 7.36, P = 0.006). We measured the clinical relevance of the test by calculating the prevalence corrected positive predictive values (PcPPV). The PcPPV of disease for the DRB4*0101 allele was 0.017 and for the DRB4*0101/0101 genotype was 0.0358. In other words, a DRB4*0101/0101 genotype carries a 3.58% risk of developing vitiligo. CONCLUSIONS: Both DRB4*0101 and DQB1*0303 alleles provide significant susceptibility for vitiligo.

Tcf-3 expression and beta-catenin mediated transcriptional activation in aggressive fibromatosis (desmoid tumour).

Aggressive fibromatosis harbours mutations resulting in beta-catenin protein stabilization. Primary cell cultures demonstrate constitutive tcf activation in aggressive fibromatosis. Expression and co-immunoprecipitation studies suggest that beta-catenin binds and activates tcf-3 in this tumour. This is the first demonstration of tcf-3 activation by beta-catenin stabilization in a human neoplastic process.