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.

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.

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.

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.

[Is cancer a hereditary or a degenerative disease?]. / Is kanker een erfelijke of een verworven aandoening?

All malignancies, whether they are solid tumors or leukemias, always originate from modifications of the genetic information of the cells. In most cases these changes occur in a single cell, which will then generate a whole series of malignant cells through cell division. In these cases the cancer is sporadic and therefore not hereditary. In a small percentage of the cases the DNA defects are inherited from one of the parents. All cells of the body will then carry this defect, but only a few tissues will become cancerous later in life based on this defect. These forms of cancer, such as some forms of breastca, colonca, thyroidca a.o. are hereditary. Nevertheless the malignant process will not be initiated in all individuals who carry these defects and the age at onset cannot be predicted with precision. In some malignancies, whether sporadic or hereditary, the first gene that carries a defect will be essential for normal cellular function. As a result, the malignant behavior of the cells becomes irreversible. Gradually, additional defects will accumulate in other genes in these cells leading to an increasing malignant behavior of the cancer. In other cases the consequences of the first defect remain limited but the accumulation of additional defects in other genes is facilitated resulting in an increased aggressive behavior of the cells. The environmental factors that cause or facilitate the occurrence of the first DNA defect in the cells, or that facilitate the initiation of hereditary cancers remain largely unknown. Chance plays of course an important role since we accumulate continuously defects in our DNA. Nevertheless evidence exists for the presence of components in our atmosphere, our food or beverages that facilitate the accumulation of defects in our DNA. The identification of these components provides an important starting point for improved prevention. In the mean time, making genetic tests available to individuals at risk for hereditary cancers remains an important, be it delicate task, since they can potentially influence their chances of survival. The improving insights into the mechanisms involved in the pathogenesis of cancers guarantees a better and more efficient treatment in the future.

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.

The C-terminal part of the R-domain, but not the PDZ binding motif, of CFTR is involved in interaction with Ca(2+)-activated Cl- channels.

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) inhibits Ca(2+)-activated Cl- channels (CaCC) by an unknown mechanism. This inhibition does not require CFTR activation (activity-independent inhibition), but is potentiated when CFTR is activated (activity-dependent inhibition). In this study, we evaluated, in endothelial cells, possible structural determinants for this interaction. Bovine pulmonary artery endothelium (CPAE) cells, which do not express CFTR, were transfected transiently with three hybrid CFTR constructs. The functional interaction between CaCC and CFTR was assessed using the patch-clamp technique in the whole-cell configuration. CaCC was stimulated by application of adenosine 5'-triphosphate (ATP) to the bath solution. CFTR currents were evoked by application of a forskolin/3-isobutyl-l-methylxanthine (IBMX) cocktail. The inhibitory effect of CFTR was conserved when the PDZ (PSD-95/Discs large/ZO-1) binding motif was deleted (CFTR-delta PDZ). In contrast, both the CFTR activity-independent and -dependent inhibition of CaCC were abolished when the C-terminal part of the regulatory (R)-domain of CFTR was deleted (CFTR-delta R780-830). The activity-dependent inhibition of CaCC, but not the activity-independent inhibition, could be rescued by introducing the multiple drug resistance (MDR)-1 mini-linker in place of the deletion (CFTR-delta R-linker). It is concluded that the C-terminal part of the R-domain is an important determinant for CFTR-CaCC interaction.

Adhesion of Porphyromonas gingivalis strains to cultured epithelial cells from patients with a history of chronic adult periodontitis or from patients less susceptible to periodontitis.

BACKGROUND: The present study aimed to explain the interindividual variation in periodontitis susceptibility by differences in the initial adhesion rate of Porphyromonas gingivalis to the pocket epithelium of these individuals, and/or by inter-P. gingivalis strain differences in association capacity (adhesion and internalization). METHODS: Adhesion assays were performed on epithelial monolayers (cultured in vitro from pocket epithelium belonging to patients who were less or more susceptible to chronic adult periodontitis) using 11 genetically different clinical strains of P. gingivalis. RESULTS: Both the disease category (less susceptible versus susceptible) and the interstrain variation were found to have a significant effect (both P <0.05) on the initial bacterial association. The chronic adult periodontitis group showed significantly more association of P. gingivalis when compared to less susceptible patients (4.2 x 10(6) versus 3.5 x 10(6)). Also, the interstrain variation was significant, with strains Pg 4 and 5 representing the least and best associating bacteria (1.8 x 10(6) colony forming units for Pg 4, 9 x 10(6) for Pg 5). CONCLUSIONS: These results indicate that periodontitis susceptibility is influenced by both the interindividual differences in pocket epithelium (allowing more adhesion of P. gingivalis) or by the strain type by which the patient is infected (intra-species differences in adhesion capacity).

Functional interaction between TRP4 and CFTR in mouse aorta endothelial cells.

BACKGROUND: This study describes the functional interaction between the putative Ca2+ channel TRP4 and the cystic fibrosis transmembrane conductance regulator, CFTR, in mouse aorta endothelium (MAEC). RESULTS: MAEC cells express CFTR transcripts as shown by RT-PCR analysis. Application of a phosphorylating cocktail activated a Cl- current with characteristics similar to those of CFTR mediated currents in other cells types (slow activation by cAMP, absence of rectification, block by glibenclamide). The current is present in trp4 +/+ MAEC, but not in trp4 -/- cells, although the expression of CFTR seems unchanged in the trp4 deficient cells as judged from RT-PCR analysis. CONCLUSIONS: It is concluded that TRP4 is necessary for CFTR activation in endothelium, possibly by providing a scaffold for the formation of functional CFTR channels.

Functional integrity of the vesicle transporting machinery is required for complete activation of cFTR expressed in xenopus laevis oocytes.

We expressed the human cystic fibrosis transmembrane conductance regulator (CFTR) in oocytes of the South African clawed frog Xenopus laevis. We performed simultaneous and continuous recording of membrane current (Im), conductance (Gm) and capacitance (Cm), the latter being a direct measure of membrane surface area. A cAMP-cocktail containing cAMP and isobutylmethylxanthine (IBMX) increased all parameters, demonstrating that CFTR activation was partly achieved by exocytotic delivery and insertion of preformed CFTR molecules into the plasma membrane. CFTR currents after cAMP-cocktail were correlated with the capacitance of the oocytes: oocytes with larger Cm exhibited larger currents. Expression of CFTR itself did not change the Cm of the oocytes. However, activation of CFTR with cAMP-cocktail increased Im and Gm 15- and 20-fold, respectively while membrane surface area increased by about 7%, indicating the functional insertion of preformed CFTR into the plasma membrane. While cAMP-cocktail yielded maximal CFTR stimulation, IBMX alone, but not caffeine or theophylline, was sufficient to stimulate more than half of the increases in Im and Gm as observed with cAMP-cocktail. Since Cm was not significantly stimulated by IBMX, we conclude that IBMX alone activated the CFTR channels already present in the oocyte membrane. CFTR stimulation by cAMP-cocktail was independent of external Ca2+ and ATP had no additional activating potency. The role of protein trafficking in the activation of CFTR evoked by increases of cytoplasmic cAMP was assessed by measuring the effects of brefeldin A (BFA), nocodazole and primaquine on the bioelectric parameters and membrane surface area. All these compounds that interfere with the protein trafficking machinery at different stages prevented the translocation of CFTR from intracellular pools to the plasma membrane. These data confirm and extend our previous observations that CFTR expressed in Xenopus laevis oocytes is activated via dual pathways including direct activation of CFTR already present in the membrane and exocytotic insertion of preformed CFTR channels into the membrane. Furthermore, we show that complete activation of CFTR requires an intact protein trafficking machinery.

Different activation mechanisms of cystic fibrosis transmembrane conductance regulator expressed in Xenopus laevis oocytes.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP sensitive Cl- channel that is defective in cystic fibrosis (CF). The most frequent mutation, namely deltaF508-CFTR, accounts for 66% of CF. Here we show that cAMP-activation of CFTR occurs via at least two distinct pathways: activation of CFTR molecules already present in the plasma membrane and protein kinase A (PKA)-mediated vesicular transport of new CFTR molecules to the plasma membrane and functional insertion into the membrane. We investigated the mechanisms that are responsible for these activation pathways using the Xenopus laevis oocytes expression system. We expressed CFTR and recorded continuously membrane current (Im), conductance (Gm) and capacitance (Cm), which is a direct measure of membrane surface area. Expression of CFTR alone did not change the plasma membrane surface area. However, activation of CFTR with cAMP increased Im, Gm and Cm while deltaF508-CFTR-expressing oocytes showed no response on cAMP. Inhibition of protein kinase A or buffering intracellular Ca2+ abolished the cAMP-induced increase in Cm while increases of Im and Gm were still present. ATP or the xanthine derivative 8-cyclopentyl-1,3-dipropylxanthine (CPX) did not further activate CFTR. Insertion of pre-formed CFTR into the plasma membrane could be prevented by compounds that interfere with intracellular transport mechanisms such as primaquine, brefeldin A, nocodazole. From these data we conclude that cAMP activates CFTR by at least two distinct pathways: activation of CFTR already present in the plasma membrane and exocytotic delivery of new CFTR molecules to the oocyte membrane and functional insertion into it.