Vitamin D, bone mineral density and risk of fracture in people with intellectual disabilities.

BACKGROUND: People with intellectual disabilities (IDs) have very high rates of osteoporosis and fractures, to which their widespread vitamin D deficiency and other factors could contribute. We aimed to assess in people with IDs previously treated for vitamin D deficiency (1) long-term adherence to vitamin D supplementation and (2) bone mineral density (BMD), as an indicator for risk of fractures, according to vitamin D supplementation and other factors. METHOD: We recorded height, weight, medical, pharmacological, dietary and lifestyle assessment. Blood sample were taken for vitamin D and related analytes. dual-energy X-ray absorptiometry for BMD was performed. RESULTS: Of 51 study participants (mean [standard deviation, SD] age 51.5 [13.6] years, 57% male), 41 (80.4%) were taking vitamin D and 10 were not. Mean [SD] serum vitamin D was 81.3 [21.3] vs. 25.2 [10.2] nmol/L (P < 0.0001), respectively. Thirty-six participants underwent a dual-energy X-ray absorptiometry scan, which showed osteoporosis in 23.7% and osteopenia in 52.6%. Participants on vitamin D had higher BMD than those who were not, a statistically significant difference when confounders (lack of mobility and hypogonadism) were removed. BMD was significantly different according to mobility, particularly in wheelchair users, in whom hip BMD was 33% lower (P < 0.0001) than in participants with normal mobility. Participants still taking vitamin D showed a 6.1% increase in BMD at the spine (P = 0.003) after mean [SD] 7.4 [1.5] years vitamin D treatment. CONCLUSIONS: In people with IDs and previous vitamin D deficiency, BMD increases on long-term vitamin D supplementation. However, additional strategies must be considered for osteoporosis and fracture prevention in this population.

Sex-related differences in coronary and carotid vessel geometry, plaque composition and shear stress obtained from imaging.

Atherosclerosis manifests itself differently in men and women with respect to plaque initiation, progression and plaque composition. The observed delay in plaque progression in women is thought to be related to the hormonal status of women. Also features associated with the vulnerability of plaques to rupture seem to be less frequently present in women compared to men. Current invasive and non-invasive imaging modalities allow for visualization of plaque size, composition and high risk vulnerable plaque features. Moreover, image based modeling gives access to local shear stress and shear stress-related plaque growth. In this review, current knowledge on sex-related differences in plaque size, composition, high risk plaque features and shear stress related plaque growth in carotid and coronary arteries obtained from imaging are summarized.

Renal defects in KCNE1 knockout mice are mimicked by chromanol 293B in vivo: identification of a KCNE1-regulated K+ conductance in the proximal tubule.

KCNE1 is a protein of low molecular mass that is known to regulate the chromanol 293B and clofilium-sensitive K+ channel, KCNQ1, in a number of tissues. Previous work on the kidney of KCNE1 and KCNQ1 knockout mice has revealed that these animals have different renal phenotypes, suggesting that KCNE1 may not regulate KCNQ1 in the renal system. In the current study, in vivo clearance approaches and whole cell voltage-clamp recordings from isolated renal proximal tubules were used to examine the physiological role of KCNE1. Data from wild-type mice were compared to those from KCNE1 knockout mice. In clearance studies the KCNE1 knockout mice had an increased fractional excretion of Na+, Cl−, HCO3(−) and water. This profile was mimicked in wild-type mice by infusion of chromanol 293B, while chromanol was without effect in KCNE1 knockout animals. Clofilium also increased the fractional excretion of Na+, Cl− and water, but this was observed in both wild-type and knockout mice, suggesting that KCNE1 was regulating a chromanol-sensitive but clofilium-insensitive pathway. In whole cell voltage clamp recordings from proximal tubules, a chromanol-sensitive, K+-selective conductance was identified that was absent in tubules from knockout animals. The properties of this conductance were not consistent with its being mediated by KCNQ1, suggesting that KCNE1 regulates another K+ channel in the renal proximal tubule. Taken together these data suggest that KCNE1 regulates a K+-selective conductance in the renal proximal tubule that plays a relatively minor role in driving the transport of Na+, Cl− and HCO3(−).

Contribution of Fcgamma receptor IIIA gene 158V/F polymorphism and copy number variation to the risk of ACPA-positive rheumatoid arthritis.

BACKGROUND: Fcgamma receptors (FcgammaRs) are potent immune modulators. FcgammaR genes encompass a complex region, polymorphic by both single nucleotide polymorphisms (SNPs) and copy number variation (CNV). The heterogeneity of rheumatoid arthritis (RA) combined with the genetic complexity of FcgammaR genes may be the cause of inconsistent findings in previous RA studies on FcgammaR SNPs. There is increasing evidence that anti-citrullinated peptide antibody (ACPA)-positive RA and ACPA-negative RA have a different genetic background. OBJECTIVE: To investigate whether FcgammaRIIIA 158V/F SNP associates differently with ACPA-positive and ACPA-negative RA and to assess if the FcgammaRIIIA gene CNV affects the association of the FcgammaRIIIA 158V/F SNP with RA and whether the FcgammaRIIIA gene CNV confers risk for RA. METHODS: 945 patients with RA and 388 healthy controls, all Dutch-Caucasians, were included in the study. FcgammaRIIIA 158V/F SNP was genotyped using Sequenom. CNV of the FcgammaRIIIA gene was determined in 456 patients with RA and 285 controls using multiplex ligation-dependent probe amplification. Associations between genotypes and RA were analysed, stratifying for the presence/absence of ACPA and CNV. RESULTS: In all patients with RA the FcgammaRIIIA 158V/F SNP was not associated with RA. In ACPA-positive RA (n = 358), the VV genotype was more prevalent in cases than in controls (18.4% vs 13.2%, OR = 1.5, p = 0.05). After stratification for CNV the VV genotype was associated with RA in general (n = 426) (OR = 1.6, 95% CI 0.97 to 2.6, p = 0.05) and with ACPA-positive RA (n = 135) (OR = 2.1, 95% CI 1.2 to 3.8, p = 0.009) but not with ACPA-negative RA. The distribution of CNV was not significantly different between patients with RA and controls. CONCLUSION: The FcgammaRIIIA 158 VV genotype confers risk for ACPA-positive RA; this association increased slightly after correction for CNV of the FcgammaRIIIA gene. CNV itself is not associated with RA susceptibility.

Antigen array for serological diagnosis and novel allergen identification in severe equine asthma.

Severe equine asthma (sEA), which closely resembles human asthma, is a debilitating and performance-limiting allergic respiratory disorder which affects 14% of horses in the Northern Hemisphere and is associated with increased allergen-specific immunoglobulin E (IgE) against a range of environmental proteins. A comprehensive microarray platform was developed to enable the simultaneous detection of allergen-specific equine IgE in serum against a wide range of putative allergenic proteins. The microarray revealed a plethora of novel pollen, bacteria, mould and arthropod proteins significant in the aetiology of sEA. Moreover, the analyses revealed an association between sEA-affected horses and IgE antibodies specific for proteins derived from latex, which has traditionally been ubiquitous to the horse's environment in the form of riding surfaces and race tracks. Further work is required to establish the involvement of latex proteins in sEA as a potential risk factor. This work demonstrates a novel and rapid approach to sEA diagnosis, providing a platform for tailored management and the development of allergen-specific immunotherapy.

In vitro and in vivo analysis of expression cassettes designed for vascular gene transfer.

Increasing the level and duration of transgene expression and restricting expression to vascular cells are important goals for clinically useful gene therapy vectors. We evaluated several promoters, enhancers and introns in endothelial, smooth muscle and liver cells in tissue culture and in vivo, comparing local delivery to the carotid artery with intravenous delivery to the liver. A 1800-bp fragment of the oxidized LDL receptor (LOX-1) promoter showed highest in vivo activity in the carotid artery, achieving 39% the activity of the reference cytomegalovirus promoter, with 188-fold greater specificity for carotid artery over liver. An enhancer from the Tie2 gene in combination with the intracellular adhesion molecule-2 promoter improved endothelial specificity of plasmid vectors, increased the expression from adenoviral vectors in cultured endothelial cells and doubled the specificity for carotid artery over liver in vivo. Adding a short intron to expression cassettes increased expression in both endothelial and smooth muscle cells in vitro; however, the eNOS enhancer failed to consistently increase the expression or endothelial specificity of the vector. In conclusion, elements from the LOX-1 promoter and Tie2 enhancer together with an intron can be used to improve vectors for vascular gene transfer.

Copy number variation and mosaicism.

Mosaicism is the presence of cells within an organism that have a different genetic composition despite deriving from a single zygote. The consequence of this depends on the number and type of cells that are affected as well as the specific DNA involved. There are several diseases where mosaicism is known to occur, but the currently observed frequency is presumably an underestimation due to the difficulty of detecting changes in only a percentage of cells. Recent advances in technology have provided a greater insight into the frequency and mechanisms of mosaicism in all forms. This review will cover the different techniques that can be used for detecting copy number variation (CNV) in mosaic form, and describe some of the insights that different diseases have given on the true frequencies and mechanisms responsible for somatic rearrangements. It will conclude with a discussion of the implications of the recent description of CNV in identical twins, and what remains to be elucidated in the world of mosaic CNV.

Methods to detect CNVs in the human genome.

The detection of quantitative changes in genomic DNA, i.e. deletions and duplications or Copy Number Variants (CNVs), has recently gained considerable interest. First, detailed analysis of the human genome showed a surprising amount of CNVs, involving thousands of genes. Second, it was realised that the detection of CNVs as a cause of genetic disease was often neglected, but should be an essential part of a complete screening strategy. In both cases new efficient CNV screening methods, covering the entire range from specific loci to genome-wide, were behind these developments. This paper will briefly review the methods that are available to detect CNVs, discuss their strong and weak points, show some new developments and look ahead. Methods covered include microscopy, fluorescence in situ hybridization (including fiber-FISH), Southern blotting, PCR-based methods (including MLPA), array technology and massive parallel sequencing. In addition, we will show some new developments, including a 1400-plex CNV bead assay, fast-MLPA (from DNA to result in approximately 6 h) and a simple Melting Curve Analysis assay to confirm potential CNVs. Using the 1400-plex CNV bead assay, targeting selected chromosomal regions only, we detected confirmed rearrangements in 9% of 320 mental retardation patients studied.

Avian genomics in the 21st century.

The chicken has long been an important model organism for developmental biology, as well as a major source of protein with billions of birds used in meat and egg production each year. Chicken genomics has been transformed in recent years, with the characterisation of large EST collections and most recently with the assembly of the chicken genome sequence. As the first livestock genome to be fully sequenced it leads the way for others to follow--with zebra finch later this year. The genome sequence and the availability of three million genetic polymorphisms are expected to aid the identification of genes that control traits of importance in poultry. As the first bird genome to be sequenced it is a model for the remaining 9,600 species thought to exist today. Many of the features of avian biology and organisation of the chicken genome make it an ideal model organism for phylogenetics and embryology, along with applications in agriculture and medicine. The availability of new tools such as whole-genome gene expression arrays and SNP panels, coupled with information resources on the genes and proteins are likely to enhance this position.

Variation of CNV distribution in five different ethnic populations.

Recent studies have revealed a new type of variation in the human genome encompassing relatively large genomic segments ( approximately 100 kb-2.5 Mb), commonly referred to as copy number variation (CNV). The full nature and extent of CNV and its frequency in different ethnic populations is still largely unknown. In this study we surveyed a set of 12 CNVs previously detected by array-CGH. More than 300 individuals from five different ethnic populations, including three distinct European, one Asian and one African population, were tested for the occurrence of CNV using multiplex ligation-dependent probe amplification (MLPA). Seven of these loci indeed showed CNV, i.e., showed copy numbers that deviated from the population median. More precise estimations of the actual genomic copy numbers for (part of) the NSF gene locus, revealed copy numbers ranging from two to at least seven. Additionally, significant inter-population differences in the distribution of these copy numbers were observed. These data suggest that insight into absolute DNA copy numbers for loci exhibiting CNV is required to determine their potential contribution to normal phenotypic variation and, in addition, disease susceptibility.

Duplications in the DMD gene.

The detection of duplications in Duchenne (DMD)/Becker Muscular Dystrophy (BMD) has long been a neglected issue. However, recent technological advancements have significantly simplified screening for such rearrangements. We report here the detection and analysis of 118 duplications in the DMD gene of DMD/BMD patients. In an unselected patient series the duplication frequency was 7%. In patients already screened for deletions and point mutations, duplications were detected in 87% of cases. There were four complex, noncontiguous rearrangements, with two also involving a partial triplication. In one of the few cases where RNA was analyzed, a seemingly contiguous duplication turned out to be a duplication/deletion case generating a transcript with an unexpected single-exon deletion and an initially undetected duplication. These findings indicate that for clinical diagnosis, duplications should be treated with special care, and without further analysis the reading frame rule should not be applied. As with deletions, duplications occur nonrandomly but with a dramatically different distribution. Duplication frequency is highest near the 5' end of the gene, with a duplication of exon 2 being the single most common duplication identified. Analysis of the extent of 11 exon 2 duplications revealed two intron 2 recombination hotspots. Sequencing four of the breakpoints showed that they did not arise from unequal sister chromatid exchange, but more likely from synthesis-dependent nonhomologous end joining. There appear to be fundamental differences therefore in the origin of deletions and duplications in the DMD gene.

In vitro efficacy of AdTRAIL gene therapy of bladder cancer is enhanced by trichostatin A-mediated restoration of CAR expression and downregulation of cFLIP and Bcl-XL.

Current therapies for bladder cancer are suboptimal and adenoviral gene therapy has been explored as an alternative treatment. In this study, we evaluated the in vitro efficacy of an adenovirus expressing TNF-related apoptosis-inducing ligand (AdTRAIL). At low concentrations of virus, T24 cells were more resistant to AdTRAIL-induced apoptosis than 5637 bladder carcinoma cells. Resistance in T24 cells correlated with poor infectivity and lack of surface expression of coxsackie and adenovirus receptor (CAR). Pretreatment with low concentrations of the histone deacetylase inhibitor trichostatin A, restored CAR expression in T24 cells, which facilitated viral infection and resulted in apoptosis at low concentrations of AdTRAIL. In addition, trichostatin A reduced the expression of Bcl-X(L) and cFLIP resulting in increased sensitivity to recombinant TRAIL. Overexpression of cFLIP inhibited TRAIL-mediated killing in trichostatin A pretreated cells, indicating that downregulation of this antiapoptotic protein is required for sensitization. Therefore, trichostatin A can enhance the efficacy of AdTRAIL by restoring CAR expression and by generating a more pro-apoptotic phenotype that would facilitate bystander activity of TRAIL. Combination of histone deacetylase inhibitors with intravesical AdTRAIL gene therapy may be a novel treatment strategy for bladder cancer.

Copy number variation in the genome; the human DMD gene as an example.

Recent developments have yielded new technologies that have greatly simplified the detection of deletions and duplications, i.e., copy number variants (CNVs). These technologies can be used to screen for CNVs in and around specific genomic regions, as well as genome-wide. Several genome-wide studies have demonstrated that CNV in the human genome is widespread and may include millions of nucleotides. One of the questions that emerge is which sequences, structures and/or processes are involved in their generation. Using as an example the human DMD gene, mutations in which cause Duchenne and Becker muscular dystrophy, we review the current data, determine the deletion and duplication profile across the gene and summarize the information that has been collected regarding their origin. In addition we discuss the methods most frequently used for their detection, in particular MAPH and MLPA.

Nine unknown rearrangements in 16p13.3 and 11p15.4 causing alpha- and beta-thalassaemia characterised by high resolution multiplex ligation-dependent probe amplification.

BACKGROUND: Approximately 80% of the alpha- and 10% of the beta-thalassaemias are caused by genomic deletions involving the alpha- and beta-globin gene clusters on chromosomes 16p13.3 and 11p15.5, respectively. Gap-PCR, Southern blot analysis, and fluorescent in situ hybridisation are commonly used to identify these deletions; however, many deletions go undetected using conventional techniques. METHODS: Patient samples for which no abnormalities had been found using conventional DNA techniques were analysed by a three colour multiplex ligation-dependent probe amplification assay. Two sets of 35 and 50 probes, covering a region of 700 kb of the alpha- and 500 kb of the beta-globin gene cluster, respectively, were designed to detect rearrangements in the alpha- and beta-globin gene clusters. RESULTS: In 19 out of 38 patient samples, we found 11 different alpha-thalassaemia deletions, six of which were not previously described. Two novel deletions leaving the alpha-globin gene cluster intact were found to cause a complete downregulation of the downstream alpha-genes. Similarly, 31 out of 51 patient samples were found to carry 10 different deletions involving the beta-globin gene cluster, three of which were not previously described. One involves the deletion of the locus control region leaving the beta-globin gene cluster intact. CONCLUSIONS: These deletions, which are not easily detected by conventional techniques, may have clinical implications during pregnancy ranging from mild to life threatening microcytic haemolytic anaemia in neonates. The approach as described here is a rapid and sensitive method for high resolution analysis of the globin gene clusters and for any region of the genome.

Unique glycosignature for intervertebral disc and articular cartilage cells and tissues in immaturity and maturity.

In this study, on/off markers for intervertebral disc (IVD) and articular cartilage (AC) cells (chondrocytes) and distinct glycoprofiles of cell and tissue-types were identified from immaturity to maturity. Three and eleven month-old ovine IVD and AC tissues were histochemically profiled with a panel of lectins and antibodies. Relationships between tissue and cell types were analysed by hierarchical clustering. Chondroitin sulfate (CS) composition of annulus fibrosus (AF), nucleus pulposus (NP) and AC tissues was determined by HPLC analysis. Clear on/off cell type markers were identified, which enabled the discrimination of chondrocytes, AF and NP cells. AF and NP cells were distinguishable using MAA, SNA-I, SBA and WFA lectins, which bound to both NP cells and chondrocytes but not AF cells. Chondrocytes were distinguished from NP and AF cells with a specific binding of LTA and PNA lectins to chondrocytes. Each tissue showed a unique CS composition with a distinct switch in sulfation pattern in AF and NP tissues upon disc maturity while cartilage maintained the same sulfation pattern over time. In conclusion, distinct glycoprofiles for cell and tissue-types across age groups were identified in addition to altered CS composition and sulfation patterns for tissue types upon maturity.

Acute effect of fructose intake from sugar-sweetened beverages on plasma uric acid: a randomised controlled trial.

BACKGROUND/OBJECTIVE: Excessive fructose intake has been linked to hyperuricaemia. Our aim was to test whether 355 and 600 ml of commercial sugar-sweetened soft drinks would acutely raise plasma uric acid. SUBJECTS/METHODS: Forty-one participants were randomised to a control group or an intervention group. The control group consumed 600 ml of fructose and 600 ml of glucose beverages. The soft drink group consumed 355 and 600 ml of beverages in random order. The control beverages were matched for fructose content with 600 ml of soft drink (26.7 g). Blood samples were collected at baseline, 30 and 60 min and analysed for plasma uric acid. RESULTS: Plasma uric acid concentrations were 13 (95% confidence interval: (CI): 3, 23) and 17 µmol/l (95% CI: 6, 28) higher 30 and 60 min after consumption of 600 ml of soft drink compared with the glucose control. The corresponding values for the fructose beverage were 22 (95% CI: 16, 29) and 23 µmol/l (95% CI: 14, 33). There was no significant difference in the increase in uric acid following the 600-ml soft drink compared with the fructose control at 30 min (6 µmol/l; 95% CI: -4, 15) or 60 min (5 µmol/l; 95% CI: -7, 17). There was no difference in the uric-acid-raising effect between the 355 and 600 ml volumes at 30 min (-1 µmol/l; 95% CI: -9, 6) or 60 min (-5 µmol/l; 95% CI: -10, 1). CONCLUSION: Small and transient increases in plasma uric acid are likely after consumption of sucrose-sweetened commercially available single-serve soft drinks in volumes as small as 355 ml.

Identification of novel RNA secondary structures within the hepatitis C virus genome reveals a cooperative involvement in genome packaging.

The specific packaging of the hepatitis C virus (HCV) genome is hypothesised to be driven by Core-RNA interactions. To identify the regions of the viral genome involved in this process, we used SELEX (systematic evolution of ligands by exponential enrichment) to identify RNA aptamers which bind specifically to Core in vitro. Comparison of these aptamers to multiple HCV genomes revealed the presence of a conserved terminal loop motif within short RNA stem-loop structures. We postulated that interactions of these motifs, as well as sub-motifs which were present in HCV genomes at statistically significant levels, with the Core protein may drive virion assembly. We mutated 8 of these predicted motifs within the HCV infectious molecular clone JFH-1, thereby producing a range of mutant viruses predicted to possess altered RNA secondary structures. RNA replication and viral titre were unaltered in viruses possessing only one mutated structure. However, infectivity titres were decreased in viruses possessing a higher number of mutated regions. This work thus identified multiple novel RNA motifs which appear to contribute to genome packaging. We suggest that these structures act as cooperative packaging signals to drive specific RNA encapsidation during HCV assembly.

Selective targeting of gene transfer to vascular endothelial cells by use of peptides isolated by phage display.

BACKGROUND: Gene transfer to vascular cells is a highly inefficient and nonselective process, defined by the lack of specific cell-surface receptors for both nonviral and viral gene delivery vectors. METHODS AND RESULTS: We used filamentous phage display to isolate a panel of peptides that have the ability to bind selectively and efficiently to quiescent human umbilical vein endothelial cells (HUVECs) with reduced or negligible binding to nonendothelial cells, including vascular smooth muscle cells and hepatocytes. By direct biopanning on HUVECs and a second approach involving preclearing steps before panning on HUVECs, we isolated and sequenced 140 individual phages and identified 59 peptides. We selected 7 candidates for further investigation by secondary screening of homogeneous phages on a panel of cell types. Using adenovirus-mediated gene transfer as a model gene delivery system, we cloned the peptide SIGYPLP and the positive control peptide KKKKKKK upstream of the S11e single-chain Fv ("adenobody") directed against the knob domain of the adenovirus to create fusion proteins. Adenovirus-mediated gene transfer via fiber-dependent infection was blocked with S11e, whereas inclusion of the KKKKKKK peptide retargeted gene transfer. The peptide SIGYPLP, however, retargeted gene delivery specifically to endothelial cells with a significantly enhanced efficiency over nontargeted adenovirus and without transduction of nontarget cells. CONCLUSIONS: Our study demonstrates the feasibility of using small, novel peptides isolated via phage display to target gene delivery specifically and efficiently to HUVECs and highlights their use for retargeting both viral and nonviral gene transfer to vascular endothelial cells for future clinical applications.

ATP-sensitive potassium channels and efaroxan-induced insulin release in the electrofusion-derived BRIN-BD11 beta-cell line.

The properties of ATP-sensitive K+ (K(ATP)) channels were explored in the electrofusion-derived, glucose-responsive, insulin-secreting cell line BRIN-BD11 using patch-clamp techniques. In intact cells, K(ATP) channels were inhibited by glucose, the sulfonylurea tolbutamide, and the imidazoline compounds efaroxan and phentolamine. Each of these agents initiated insulin secretion and potentiated the actions of glucose. K(ATP) channels were blocked by ATP in a concentration-dependent manner and activated by ADP in the presence of ATP. In both intact cells and excised inside-out patches, the K(ATP) channel agonists diazoxide and pinacidil activated channels, and both compounds inhibited insulin secretion evoked by glucose, tolbutamide, and imidazolines. The mechanisms of action of imidazolines were examined in more detail. Pre-exposure of BRIN-BD11 cells to either efaroxan or phentolamine selectively inhibited imidazoline-induced insulin secretion but not the secretory responses of cells to glucose, tolbutamide, or a depolarizing concentration of KCl. These conditions did not result in the loss of depolarization-dependent rises in intracellular Ca2+ ([Ca2+]i), K(ATP) channel operation, or the actions of either ATP or efaroxan on K(ATP) channels. Desensitization of the imidazoline receptor following exposure to high concentrations of efaroxan, however, was found to result in an increase in SUR1 protein expression and, as a consequence, an upregulation of K(ATP) channel density. Our data provide 1) the first characterization of K(ATP) channels in BRIN-BD11 cells, a novel insulin-secreting cell line produced by electrofusion techniques, and 2) a further analysis of the role of imidazolines in the control of insulin release.

The expression and function of cadherin-mediated cell-to-cell adhesion in human embryonal carcinoma cells.

Human embryonal carcinoma (EC) cells typically require high cell densities to maintain their characteristic phenotype; they are generally subject to differentiation when cultured at low cell densities, marked by changes in morphology and expression of the surface antigen, SSEA-1. To test whether cadherin mediated cell-to-cell adhesion may be responsible for maintaining an EC phenotype we ascertained that human EC cells generally express E- and P-cadherins, and are subject to cadherin mediated, Ca2+ dependent aggregation. However, in the NTERA2 human EC cell line, inhibition of cadherin mediated adhesion by culture in low levels of Ca2+ did not result in the changes typically seen under low cell density conditions. Low Ca2+ levels also did not affect the pattern of differentiation in these cells following induction with retinoic acid. Therefore, cadherin-mediated cell adhesion does not appear to play a role in maintaining an EC phenotype. On the other hand, culture at both low cell density and in the absence of Ca2+ did result in changes in the patterns of cadherin expression suggesting a feedback regulatory effect of cell-to-cell adhesion. Further, lithium which inhibits the cytoplasmic kinase GSK3beta and hence influences beta-catenin levels did cause differentiation of NTERA2 cells. However, consideration of the phenotype of the resultant cells suggested that this effect may be because of lithium mimicking activation of a Wnt signalling pathway, rather than an effect on signalling consequent upon cadherin mediated cell to cell adhesion.