A genome-wide map of single nucleotide polymorphisms (SNP) and a pattern of linkage disequilibrium (LD) between their alleles are being established in three main ethnic groups. An important question is the applicability of such maps to different populations within a main ethnic group. Therefore, we have developed high-resolution SNP, haplotype and LD maps of vitamin D receptor gene region in large samples from five populations. Comparative analysis reveals that the LD patterns are identical in all four European populations tested with two small regions of 1.3 and 5.7 kb at which LD is disrupted completely resulting in three block-like regions over which there is significant and extensive LD. In an African population the pattern is similar, but two additional LD-breaking spots are also apparent. This LD pattern suggests combined action of recombination hotspots and founder effects, but cannot be explained by random recombination and genetic drift alone. Direct comparison indicates that the tag SNPs selected in one European population effectively predict the non-tag SNPs in the other Europeans, but not in the Gambians, for this region.

OBJECTIVETo study whether modification of LDL by methylglyoxal (MG), a potent arginine-directed glycating agent that is increased in diabetes, is associated with increased atherogenicity.RESEARCH DESIGN AND METHODSHuman LDL was isolated and modified by MG in vitro to minimal extent (MGmin-LDL) as occurs in vivo. Atherogenic characteristics of MGmin-LDL were characterized: particle size, proteoglycan-binding, susceptibility to aggregation, LDL and non-LDL receptor–binding, and aortal deposition. The major site of modification of apolipoprotein B100 (apoB100) modification was investigated by mass spectrometric peptide mapping.RESULTSMGmin-LDL contained 1.6 molar equivalents of MG modification—mostly hydroimidazolone—as found in vivo. MGmin-LDL had decreased particle size, increased binding to proteoglycans, and increased aggregation in vitro. Cell culture studies showed that MGmin-LDL was bound by the LDL receptor but not by the scavenger receptor and had increased binding affinity for cell surface heparan sulfate–containing proteoglycan. Radiotracer studies in rats showed that MGmin-LDL had a similar fractional clearance rate in plasma to unmodified LDL but increased partitioning onto the aortal wall. Mass spectrometry peptide mapping identified arginine-18 as the hotspot site of apoB100 modification in MGmin-LDL. A computed structural model predicted that MG modification of apoB100 induces distortion, increasing exposure of the N-terminal proteoglycan–binding domain on the surface of LDL. This likely mediates particle remodeling and increases proteoglycan binding.CONCLUSIONSMG modification of LDL forms small, dense LDL with increased atherogenicity that provides a new route to atherogenic LDL and may explain the escalation of cardiovascular risk in diabetes and the cardioprotective effect of metformin.

Background and aims: Genetic association between Crohn's disease (CD) and OCTN1 (SLC22A4) C1672T/OCTN2 (SLC22A5) G2207C variants in IBD5 has recently been reported. These genes encode solute carriers and the association was suggested to be distinct from the background IBD5 risk haplotype. There have been conflicting reports of the association between markers in the IBD5 region and ulcerative colitis (UC) and interaction (epistasis) between this locus and CARD15. Our aim was to ascertain the contribution of OCTN variants to UC and CD in a large independent UK dataset, to seek genetic evidence that the OCTN association is distinct from the IBD5 risk haplotype and to identify interactions between the IBD5 and CARD15 loci. Methods: A total of 1104 unrelated Caucasian subjects with inflammatory bowel disease (IBD) (496 CD, 512 UC, 96 indeterminate) and 750 ethnically matched controls were genotyped for three single nucleotide polymorphisms (SNPs) in the CD associated genes (OCTN1+1672, OCTN22207, and IGR2230), and two flanking IBD5 tagging SNPs, IGR2096 and IGR3096. Data were analysed by logistic regression methods within STATA. Results: OCTN variants were as strongly associated with UC and IBD overall as they were with CD (p = 0.0001; OR 1.3 (95% confidence interval 1.1-1.5)). OCTN variants were in tight linkage disequilibrium with the extended IBD5 risk haplotype D9 0.79 and 0.88, and r 2 = 0.62 and 0.72 for IGR2096 and 3096, respectively. There was no deviation from a multiplicative model of interaction between CARD15 and IBD5 on the penetrance scale. Conclusions: The OCTN variants were associated with susceptibility to IBD overall. The effect was equally strong in UC and CD. Although OCTN variants may account for the increased risk of IBD associated with IBD5, a role for other candidate genes within this extended haplotype was not excluded. There was no statistical evidence of interaction between CARD15 and either OCTN or IBD5 variants in susceptibility to IBD.

Background The poly(ADP-ribose) polymerase (PARP) inhibitor olaparib potentiated radiation and temozolomide (TMZ) chemotherapy in preclinical glioblastoma models but brain penetration was poor. Clinically, PARP inhibitors exacerbate the hematological side effects of TMZ. The OPARATIC trial was conducted to measure penetration of recurrent glioblastoma by olaparib and assess the safety and tolerability of its combination with TMZ. Methods Preclinical pharmacokinetic studies evaluated olaparib tissue distribution in rats and tumor-bearing mice. Adult patients with recurrent glioblastoma received various doses and schedules of olaparib and low-dose TMZ in a 3 + 3 design. Suitable patients received olaparib prior to neurosurgical resection; olaparib concentrations in plasma, tumor core and tumor margin specimens were measured by mass spectrometry. A dose expansion cohort tested tolerability and efficacy of the recommended phase II dose (RP2D). Radiosensitizing effects of olaparib were measured by clonogenic survival in glioblastoma cell lines. Results Olaparib was a substrate for multidrug resistance protein 1 and showed no brain penetration in rats but was detected in orthotopic glioblastoma xenografts. Clinically, olaparib was detected in 71/71 tumor core specimens (27 patients; median, 496 nM) and 21/21 tumor margin specimens (9 patients; median, 512.3 nM). Olaparib exacerbated TMZ-related hematological toxicity, necessitating intermittent dosing. RP2D was olaparib 150 mg (3 days/week) with TMZ 75 mg/m2 daily for 42 days. Fourteen (36%) of 39 evaluable patients were progression free at 6 months. Olaparib radiosensitized 6 glioblastoma cell lines at clinically relevant concentrations of 100 and 500 nM. Conclusion Olaparib reliably penetrates recurrent glioblastoma at radiosensitizing concentrations, supporting further clinical development and highlighting the need for better preclinical models.

The delivery of peptide drugs to the brain is challenging, principally due to the blood brain barrier and the low metabolic stability of peptides. Exclusive delivery to the brain with no peripheral exposure has hitherto not been demonstrated with brain quantification data. Here we show that polymer nanoparticles encapsulating leucine-enkephalin hydrochloride (LENK) are able to transport LENK exclusively to the brain via the intranasal route, with no peripheral exposure and nanoparticle localisation is observed within the brain parenchyma. Animals dosed with LENK nanoparticles (NM0127) showed a strong anti-nociceptive response in multiple assays of evoked and on going pain whereas animals dosed intranasally with LENK alone were unresponsive. Animals did not develop tolerance to the anti-hyperalgesic activity of NM0127 and NM0127 was active in morphine tolerant animals. A microparticulate formulation of clustered nanoparticles was prepared to satisfy regulatory requirements for nasal dosage forms and the polymer nanoparticles alone were found to be biocompatible, via the nasal route, on chronic dosing.

Background/Objectives:Decreased plasma concentration of high-density lipoprotein cholesterol (HDL-C) is a risk factor linked to increased risk of cardiovascular disease (CVD). Decreased anti-atherogenic properties of HDL are also implicated in increased CVD risk. The cause is unknown but has been linked to impaired glucose tolerance. The aim of this study was to quantify the modification of HDL by methylglyoxal and related dicarbonyls in healthy people and patients with type 2 diabetes characterise structural, functional and physiological consequences of the modification and predict the importance in high CVD risk groups.Subjects/Methods:Major fractions of HDL, HDL2 and HDL3 were isolated from healthy human subjects and patients with type 2 diabetes and fractions modified by methylglyoxal and related dicarbonyl metabolites quantified. HDL2 and HDL3 were glycated by methylglyoxal to minimum extent in vitro and molecular, functional and physiological characteristics were determined. A one-compartment model of HDL plasma clearance was produced including formation and clearance of dicarbonyl-modified HDL.Results:HDL modified by methylglyoxal and related dicarbonyl metabolites accounted for 2.6% HDL and increased to 4.5% in patients with type 2 diabetes mellitus (T2DM). HDL2 and HDL3 were modified by methylglyoxal to similar extents in vitro. Methylglyoxal modification induced re-structuring of the HDL particles, decreasing stability and plasma half-life in vivo. It occurred at sites of apolipoprotein A-1 in HDL linked to membrane fusion, intramolecular bonding and ligand binding. Kinetic modelling of methylglyoxal modification of HDL predicted a negative correlation of plasma HDL-C with methylglyoxal-modified HDL. This was validated clinically. It also predicted that dicarbonyl modification produces 2–6% decrease in total plasma HDL and 5–13% decrease in functional HDL clinically.Conclusions:These results suggest that methylglyoxal modification of HDL accelerates its degradation and impairs its functionality in vivo, likely contributing to increased risk of CVD—particularly in high CVD risk groups.

Vitamin D is known to modulate the immune system, and its administration has been associated with reduced risk of type 1 diabetes. Vitamin D acts via its receptor (VDR). Four single nucleotide polymorphisms (SNPs) of the VDR gene have been commonly studied, and evidence of association with type 1 diabetes has been reported previously. We sequenced the VDR gene region and developed its SNP map. Here we analyzed association of the 98 VDR SNPs in up to 3,763 type 1 diabetic families. First, we genotyped all 98 SNPs in a minimum of 458 U.K. families with two affected offspring. We further tested eight SNPs, including four SNPs associated with P < 0.05 in the first set and the four commonly studied SNPs, in up to 3,305 additional families from the U.K., Finland, Norway, Romania, and U.S. We only found weak evidence of association (P ‫؍‬ 0.02-0.05) of the rs4303288, rs12721366, and rs2544043 SNPs. We then tested these three SNPs in an independent set of 1,587 patients and 1,827 control subjects from the U.K. and found no evidence of association. Overall, our results indicate that common sequence variation in the VDR gene has no major effect in type 1 diabetes in the populations tested. Diabetes 53: 2709 -2712, 2004 T he molecular mechanisms underlying type 1 diabetes are only partly understood. It develops as a result of a complex interaction of many genetic and environmental factors leading to the immune destruction of the insulin-producing ␤-cells (1). Three disease loci have been identified so far that contribute to the etiology of type 1 diabetes, the HLA complex, the variable number of tandem repeats locus located in the promoter region of the insulin (INS) gene, and the cytotoxic T-cell-associated antigen-4 gene (CTLA4) (1,2).1␣,25-dihydroxyvitamin D 3 , the hormonally active form of vitamin D, has been successfully used to prevent autoimmune insulitis and reduce diabetes incidence in the mouse model of type 1 diabetes, as well as in animal models of other autoimmune diseases (3-6). In humans, population studies suggest that vitamin D supplementation in early childhood decreases type 1 diabetes incidence (7,8), raising hopes that it may be used as a type 1 diabetes preventive treatment. Vitamin D has been long known to play a central role in bone and mineral metabolism. Now it is widely recognized to regulate growth and differentiation in many target tissues and act as a modulator in the immune system (9). Its effects are mediated by the vitamin D receptor (VDR), a member of the nuclear receptor superfamily of transcriptional regulators. VDR is found in Ͼ30 different tissues, including islet cells of the pancreas, circulating monocytes, dendritic cells, and activated Tcells (9). Upon binding 1␣,25-dihydroxyvitamin D 3 , VDR regulates gene expression by direct interaction with specific sequence elements in the promoter region of hormone-responsive target genes. In the immune system, 1␣,25-dihydroxyvitamin D 3 was shown to suppress production of the interleukin (IL)-12, IL-2, tumor necrosis factor-␣, and interf...

Increased renal clearance of thiamine (vitamin B1) occurs in experimental and clinical diabetes producing thiamine insufficiency mediated by impaired tubular re-uptake and linked to the development of diabetic nephropathy. We studied the mechanism of impaired renal re-uptake of thiamine in diabetes. Expression of thiamine transporter proteins THTR-1 and THTR-2 in normal human kidney sections examined by immunohistochemistry showed intense polarised staining of the apical, luminal membranes in proximal tubules for THTR-1 and THTR-2 of the cortex and uniform, diffuse staining throughout cells of the collecting duct for THTR-1 and THTR-2 of the medulla. Human primary proximal tubule epithelial cells were incubated with low and high glucose concentration, 5 and 26 mmol/l, respectively. In high glucose concentration there was decreased expression of THTR-1 and THTR-2 (transporter mRNA: −76% and −53% respectively, p<0.001; transporter protein −77% and −83% respectively, p<0.05), concomitant with decreased expression of transcription factor specificity protein-1. High glucose concentration also produced a 37% decrease in apical to basolateral transport of thiamine transport across cell monolayers. Intensification of glycemic control corrected increased fractional excretion of thiamine in experimental diabetes. We conclude that glucose-induced decreased expression of thiamine transporters in the tubular epithelium may mediate renal mishandling of thiamine in diabetes. This is a novel mechanism of thiamine insufficiency linked to diabetic nephropathy.