A luminescent europium hairpin for DNA photosensing in the visible, based on trimetallic bis-intercalators.

Luminescence monitoring of DNA intercalator complexes is important for assessing their localisation and targeting: We report herein a luminescent hetero-trimetallic complex with europium as a luminescent reporter and two attached platinum acetylide terpyridyl units as the DNA recognition units. The ligand, based on a bisamide derivative of diethylenetriaminepentaacetic acid functionalized with two ethynyl groups, provides a backbone to anchor two platinum terpyridyl units, Pt-tpy, leading to the hairpin-shaped heterometallic complex 1. We also prepared a related mono-nuclear platinum complex 2 to compare its intercalation properties with 1. Linear dichroism, UV-visible and melting experiments show the ability of both complexes to interact with calf thymus DNA, with linear dichroism confirming intercalation and demonstrating the expected greater DNA stiffening by the bis-intercalator 1. Importantly, the tri-metallic complex 1 shows a three-fold enhancement in europium luminescence upon addition of calf thymus DNA; other mono-intercalator lanthanide designs have commonly shown a decrease in emission on binding. The ability of the complex to monitor DNA interactions gives the potential use as a luminescence switch in sensing experiments and highlights the design of heterometallic bis-intercalator complexes as an effective approach for DNA-responsive sensitisation of a lanthanide luminescence signal.

Association between ICAM-1 Gly-Arg polymorphism and renal parenchymal scarring following childhood urinary tract infection.

Renal parenchymal scarring (RPS) following urinary tract infection (UTI) is an important cause of renal morbidity in children. Studies have shown that the intensity of the inflammatory response following infection is related to the risk of RPS. However, genetic variability in this response has not been studied. Adhesion molecules play a crucial role in leucocyte recruitment following infection, and polymorphisms have been reported in the genes for key cell adhesion molecules. We have investigated the possibility that children who develop RPS following UTI may exhibit altered genotype or allele frequencies for polymorphisms of the intercellular adhesion molecule-1 (ICAM-1) (exons 4 and 6), E-selectin (exons 2 and 4), platelet endothelial cell adhesion molecule-1 (PECAM-1) (exon 3) and CD11b (3'UTR) genes, which may predict outcome of UTI. DNA was isolated from 99 children shown to have developed RPS, 43 children with no evidence of scarring (NS) following UTI and 170 healthy controls. Genotyping was performed by restriction fragment length polymorphism (RFLP) analysis. When the RPS group was compared with the NS group, there was a significant reduction in the frequency of the ICAM-1 exon 4 A allele (10.6 vs. 21.3%, respectively, chi2 = 6.01, P = 0.014). There was no significant difference in either allele or genotype frequency for any of the other polymorphisms studied. These data suggest that the A allele of the ICAM-1 exon 4 polymorphism may protect against the risk of RPS following UTI and may participate in the regulation of the inflammatory response following UTI.

A PCR-RFLP typing method for adhesion molecule gene polymorphisms and allele frequencies in a normal UK population.

We report simple and reproducible PCR-RFLP typing methods for the polymorphisms in the ICAM-1, E-selectin and PECAM-1 genes. The genotype and allele frequencies detected in a normal UK population did not deviate significantly from the Hardy-Weinberg equilibrium; neither did they differ from frequencies previously reported using SSP or SSCP methods.

Plasma and urinary soluble adhesion molecule expression is increased during first documented acute pyelonephritis.

BACKGROUND: The degree of inflammatory reaction and leucocyte trafficking during acute pyelonephritis has been related to the risk of developing renal parenchymal scarring. Adhesion molecules play a central role in leucocyte recruitment during inflammation. AIMS: (1) To determine whether circulating and urinary concentrations of E-selectin and intercellular adhesion molecule 1 (ICAM-1) were abnormal during first documented acute pyelonephritis; (2) to investigate whether circulating or urinary concentrations were predictive for the development of abnormalities on DMSA imaging. METHODS: Plasma and urine samples were collected from 40 children with a first episode of acute pyelonephritis within one week of infection (acute sample) and at six weeks (late sample). Control samples were collected from 21 healthy age matched controls and 18 age matched controls with febrile illness not secondary to urinary tract infection. RESULTS: Plasma and urinary sE-selectin were higher in acute samples (median 176.3 ng/ml and 0.12 ng/mmol respectively) compared with late (97.8 ng/ml and 0.029 ng/mmol) and both control (65.6 ng/ml and 0 ng/mmol) and febrile control (urine 0 ng/mmol) samples. Plasma sICAM-1 was higher in acute samples (428 ng/ml) than controls (365.2 ng/ml), and acute sICAM-1 urine concentrations were higher than febrile control concentrations (3.2 v 0.7 ng/mmol). No correlations were detected between sE-selectin or sICAM-1 and acute or late DMSA scan changes. CONCLUSION: Plasma and urinary sE-selectin and sICAM-1 are significantly increased during acute pyelonephritis, though no correlation exists between the presence of high plasma or urine concentrations and DMSA scan changes, both during acute infection and six weeks post-infection.

Endothelial expression of nitric oxide synthases and nitrotyrosine in systemic sclerosis skin.

Although a multisystem disease, systemic sclerosis (SSc) most commonly affects the skin. The skin lesion is characterized by progressive changes, chief amongst which are vascular abnormalities, including endothelial cell (EC) injury and death, and dermal fibrosis. The pathogenesis of the vascular changes, and their relationship to dermal fibrosis, is poorly understood. The purpose of this study was to examine the potential role of nitric oxide (NO)-related free radical production, as part of an assessment of mechanisms leading to endothelial damage. Histologically graded skin biopsies from 33 patients with SSc (ten grade 0, ten grade 1, eight grade 2, and five grade 3) and eight healthy controls were reacted with antibodies against constitutive (eNOS) and inducible (iNOS) forms of nitric oxide synthase and nitrotyrosine. The degree of staining was assessed using a semi-quantitative system and a staining score was developed for the ECs of different vessel types in different areas of dermis at all grades. In biopsies from patients with SSc, superficial microvessel ECs showed a peak of eNOS expression in grade 1 skin which fell as the grade increased. By contrast, iNOS staining increased with the grade of skin lesion, a pattern paralleled by endothelial nitrotyrosine expression. From these findings, it is concluded that a metabolic switch occurs in dermal ECs from endothelial to cytokine inducible forms of NOS during the progression of the skin lesion of SSc. iNOS is a potent inducer of NO production which, in turn, can mediate NO free radical production. At a time of development of the SSc skin lesion when previous studies report evidence of EC damage, the cells express immunodetectable nitrotyrosine, a marker of NO-mediated free radical injury. The data suggest a role for iNOS-induced NO production in EC damage in SSc.

TGF beta--a role in systemic sclerosis?

Systemic sclerosis (SSc) is a multisystem connective tissue disorder in which there is progressive fibrosis. Transforming growth factor beta (TGF beta) has wide-ranging cellular actions. It is a potent chemoattractant for human dermal fibroblasts, from which it may induce synthesis of collagen, which suggests that it may have a central role to play in the pathogenesis of SSc. This is supported to some extent by in vitro studies. SSc fibroblasts produce more collagens and fibronectin than normal fibroblasts and elevated TIMP levels have been observed, all of which could be explained on the basis of TGF beta stimulation of fibroblasts. Some studies have suggested that fibroblasts are the source of TGF beta. However, the serum of patients with SSc is cytotoxic to endothelial cells, which could culminate in TGF beta synthesis by them, with secondary fibroblast stimulation. The role of TGF beta remains elusive, although it would seem an ideal candidate as a mediator of fibrosis in systemic sclerosis.