Distinct methylation patterns in genes that affect mitochondrial function are associated with kidney disease in blood-derived DNA from individuals with Type 1 diabetes.

AIMS: Epigenetic modifications, such as DNA methylation, can influence the risk of developing kidney disease. We studied methylation profiles in genes related to mitochondrial function to assess whether differences in these epigenetic features were associated with diabetic kidney disease in people with Type 1 diabetes. METHODS: A case-control association study was undertaken (n = 196 individuals with diabetic kidney disease vs. n = 246 individuals without renal disease). Participants were White and diagnosed with Type 1 diabetes before 31 years of age. Genes that encode mitochondrial proteins (n = 780) were downloaded from mitoproteome.org. DNA methylation profiles from blood-derived DNA were generated using the Illumina Infinium HumanMethylation450 (262 samples) and Illumina Infinium HumanMethylation27 (192 samples) arrays. Beta values (ß) were calculated and quality control was conducted, including evaluating blind duplicate DNA samples. RESULTS: Fifty-four Cytosine-phosphate-Guanine probes across 51 unique genes were significantly associated (P ≤ 10(-8) ) with diabetic kidney disease across both the 450K and the 27K methylation arrays. A subanalysis, employing the 450K array, identified 755 Cytosine-phosphate-Guanine probes in 374 genes that were significantly associated (P ≤ 10(-8) ) with end-stage renal disease. Forty-six of the top-ranked variants for diabetic kidney disease were also identified as being differentially methylated in individuals with end-stage renal disease. The largest change in methylation (Δß = 0.2) was observed for cg03169527 in the TAMM41 gene, chromosome 3p25.2. Three genes, PMPCB, TSFM and AUH, were observed with differential methylation at multiple Cytosine-phosphate-Guanine sites each (P < 10(-12) ). CONCLUSIONS: Differential methylation in genes that influence mitochondrial function are associated with kidney disease in individuals with Type 1 diabetes.

Genetic risk factors affecting mitochondrial function are associated with kidney disease in people with Type 1 diabetes.

AIM: To evaluate the association with diabetic kidney disease of single nucleotide polymorphisms (SNPs) that may contribute to mitochondrial dysfunction. METHODS: The mitochondrial genome and 1039 nuclear genes that are integral to mitochondrial function were investigated using a case (n = 823 individuals with diabetic kidney disease) vs. control (n = 903 individuals with diabetes and no renal disease) approach. All people included in the analysis were of white European origin and were diagnosed with Type 1 diabetes before the age of 31 years. Replication was conducted in 5093 people with similar phenotypes to those of the discovery collection. Association analyses were performed using the plink genetic analysis toolset, with adjustment for relevant covariates. RESULTS: A total of 25 SNPs were evaluated in the mitochondrial genome, but none were significantly associated with diabetic kidney disease or end-stage renal disease. A total of 38 SNPs in nuclear genes influencing mitochondrial function were nominally associated with diabetic kidney disease and 16 SNPS were associated with end-stage renal disease, secondary to diabetic kidney disease, with meta-analyses confirming the same direction of effect. Three independent signals (seven SNPs) were common to the replication data for both phenotypes with Type 1 diabetes and persistent proteinuria or end-stage renal disease. CONCLUSIONS: Our results suggest that SNPs in nuclear genes that influence mitochondrial function are significantly associated with diabetic kidney disease in a white European population.

Local drug delivery with a self-contained, programmable, microfluidic system.

The development and optimization of many new drug therapies requires long-term local delivery with controlled, but variable dosage. Current methods for chronic drug delivery have limited utility because they either cannot deliver drugs locally to a specific organ or tissue, do not permit changes in delivery rate in situ, or cannot be used in clinical trials in an untethered, wearable configuration. Here, we describe a small, self-contained system for liquid-phase drug delivery. This system enables studies lasting several months and infusion rates can be programmed and modified remotely. A commercial miniature pump is integrated with microfabricated components to generate ultralow flow rates and stroke volumes. Solutions are delivered in pulses as small as 370 nL, with pulses delivered at any interval of 1 min or longer. A unique feature of the system is the ability to infuse and immediately withdraw liquid, resulting in zero net volume transfer while compounds are exchanged by mixing and diffusion with endogenous fluid. We present in vitro results demonstrating repeatability of the delivered pulse volume for nearly 3 months. Furthermore, we present in vivo results in an otology application, infusing into the cochlea of a guinea pig a glutamate receptor antagonist, which causes localized and reversible changes in auditory sensitivity.

Neuropathy progression in Charcot-Marie-Tooth disease type 1A.

OBJECTIVE: To determine the rate of disease progression in Charcot-Marie-Tooth disease type 1A (CMT1A). BACKGROUND: CMT1A is the most common inherited peripheral neuropathy, affecting approximately 1:5,000 people irrespective of ethnic background or gender. There is no cure for CMT1A. Clinical trials are being initiated that use the CMT Neuropathy Score (CMTNS), a composite score based on patient symptoms, signs, and neurophysiologic abnormalities, as the primary outcome variable. The sensitivity of the CMTNS or any other score to change over time, as a measure of CMT1A progression, has yet to be determined. METHODS: We determined the CMTNS as well as the Neuropathy Impairment Score (NIS) on 72 patients followed for up to 8 years. The rate of disease progression was evaluated for the CMTNS and NIS using mixed effects linear regression models, adjusting for age and gender. RESULTS: Both CMTNS and NIS showed changes over time. The CMTNS increased an average of 0.686 points per year (95% CI 0.461 to 0.911, p

CMT1X phenotypes represent loss of GJB1 gene function.

OBJECTIVE: To investigate possible genotype-phenotype correlations and to evaluate the natural history of patients with Charcot-Marie-Tooth disease type 1X (CMT1X). BACKGROUND: CMT1X is caused by over 260 distinct mutations in the gap junction beta 1 (GJB1) gene, located on the X chromosome, which encodes the gap junction protein connexin 32 (Cx32). The natural history of CMT1X is poorly understood, and it remains unknown whether particular mutations cause more severe neuropathies through abnormal gain-of-function mechanisms. METHODS: We evaluated 73 male patients with CMT1X, who each have 1 of 28 different GJB1 mutations predicted to affect nearly all domains of Cx32. Disability was evaluated quantitatively by the CMT Neuropathy Score (CMTNS) as well as by the CMT Symptom Score (CMTSS) and the CMT Examination Score (CMTES), which are both based on the CMTNS. Patients were also evaluated by neurophysiology. RESULTS: In all patients, disability increased with age, and the degree of disability was comparable with that observed in patients with a documented GJB1 deletion. Disability correlated with a loss of motor units as assessed by motor unit number estimates. CONCLUSIONS: Taken together, these data suggest that most GJB1 mutations cause neuropathy by a loss of normal connexin 32 function. Therefore, treatment of male patients with Charcot-Marie-Tooth disease type 1X may prove amenable to gene replacement strategies.

Accelerated HER-2 degradation enhances ovarian tumor recognition by CTL. Implications for tumor immunogenicity.

We investigated the ubiquitination and degradation of a tumor antigen, the HER-2/neu (HER-2) protooncogene product which is overexpressed in epithelial cancers. HER-2 degradation was investigated in the ovarian tumor line, SKOV3.A2, that constitutively overexpressed long-life HER-2. We used as agonist geldanamycin (GA), which initiated downmodulation of HER-2 from the cell surface. HER-2 was polyubiquitinated and degraded faster in the presence than in the absence of GA. GA did not decrease HLA-A2 expression. Presentation of the immunodominant cytotoxic T lymphocyte (CTL) epitope, E75 (369-377) from SKOV.A2 was inhibited by proteasome inhibitors, such as LLnL but was enhanced by cysteine protease inhibitors such as E64, indicating that both the proteasome and cysteine proteases are involved in epitope formation but have different effects. Enhanced tumor recognition was not an immediate or early effect of GA treatment, but was evident after 20 h of GA treatment. In contrast, 20 h GA treatment did not increase tumor sensitivity to LAK cell lysis. Twenty hour GA-treated SKOV3.A2 cells expressed an unstable HER-2 protein synthesized in the presence of GA, of faster electrophoretic mobility than control HER-2. This suggested that the newly synthesized HER-2 in the presence of GA was the main source of epitopes recognized by CTL. Twenty hour GA-treated SKOV3.A2 cells were better inducers of CTL activity directed to a number of HER-2 CTL epitopes, in peripheral blood mononuclear cells compared with control untreated SKOV3.A2 cells. Thus, induction of HER-2 protein instability enhanced the sensitivity of tumor for CTL lysis. Increased HER-2 CTL epitopes presentation may have implications for overcoming the poor immuno-genicity of human tumors, and design of epitope precursors for cancer vaccination.

Continuing care after discharge from hospital for stoma patients.

The purpose of this study was to monitor the progress of patients given a permanent colostomy for colorectal carcinoma and to evaluate the need for nursing interventions or referral. A pretested semistructured interview schedule was used. Interviews were conducted at 1 week, 1 month, 6 months and 1 year after discharge. Complete data sets were obtained from 112 patients. In this study it was found that survival was strongly related to Dukes' staging system. More than half of those surviving to 1 year suffered fatigue, one in 10 had severe pain and one in five had parastomal hernia. At each interview approximately one in four people required intervention and one in 10 were referred. This study demonstrates the need for home visits and sustained patient contact. High priority should be given to a full benefit analysis of screening programmes, including the considerable costs of aftercare.

Equal care for all.

Fifty years after the NHS was launched to offer health care for all, there are still many inequalities, particularly for clients from ethnic minorities. This article discusses these inequalities in the light of an increase in stoma formation and bowel disease in Walsall's Asian community. The author recommends a number of proactive measures that trusts and nurses can use to improve uptake of services by ethnic minority patients.

Dental casualties during Canadian UN operations in Somalia.

The Canadian Forces Dental Services (CFDS) provides dental support to Canadian Forces (CF) on selected United Nations (UN) duties. This study investigated the nature of dental care sought by and provided to 1,000 Canadian regular force UN peacekeepers by CFDS from January to May 1993, during a 149 day UN mission to Somalia. Care was categorized as either emergency, urgency, or routine. Emergency and urgency care represented 10 per cent and 25 per cent of the 269 patient encounters, respectively. The dental casualty rate (per 1,000 tropps per year) was 232.

Ontario dentists: 1. Radiologic practices and opinions.

In February 1991, about 10 per cent of Ontario's general dentists were polled via a mail survey to gather information about their radiological practices and opinions. Responses were received from 413 out of 537 general practitioners, for a response rate of 77 per cent. A large majority of dentists (86 per cent) have no written office policy and/or protocol for selecting patients for X-ray examination, and just over half (54 per cent) primarily use clinical experience to select patients for these examinations. In the majority of practices (57 per cent), radiographs of asymptomatic recall patients are taken after both their history and clinical examination are complete. A similar percentage (56 per cent) of dentists are opposed to explicit guidelines for radiological examinations.

Ontario dentists: 2. Bitewing utilization and restorative treatment decisions.

Guidelines for the selection of patients requiring radiological examination have recently been published in Canada and the United States. The purpose of this paper, the second in a three-part series on the results of a questionnaire to Ontario general practitioners, is to compare bitewing use in Ontario against the U.S. guidelines. This questionnaire determined the frequency of recall bitewing examination for low and high caries risk patients, as well as the factors that influence the diagnostic and treatment decisions of the responding dentists. For example, 49 to 78 per cent of dentists order recall bitewings--in accordance with U.S. guidelines--for their patients in individual low caries risk categories. Similarly, 49 to 88 per cent of dentists order recall bitewings for individual patients in high caries risk categories, which is also consistent with the U.S. guidelines. However, across all low and high caries risk groups of patients, the percentages of dentists who followed the recommended guidelines were 34 and 15 per cent, respectively. The responding dentists' estimates of the length of time required for caries to progress through enamel are indirectly associated with the interval prescribed for recall bitewing examination.

Ontario dentists: 3. Radiographs prescribed in general practice.

In February 1991, a mail survey was used to poll a sample consisting of about 10 per cent of Ontario's general dentists. The data obtained provided information about the radiographs prescribed by dentists for five different patient types, which were described to the respondents. The per cent agreement between the radiographic procedures prescribed by Ontario dentists and the ADA-approved Center for Devices and Radiological Health (CDRH) guidelines ranged from three per cent to 79 per cent, depending on patient type and disease risk. For each patient and risk type, there was considerable variation in the radiographs prescribed.

Determination of chlorinated phenols in surface-treated lumber by HPLC.

A solution of internal standard in acetonitrile is used to extract samples of wood from lumber which had been commercially treated with chlorinated phenols. The tetrachlorophenol(TCP) and pentachlorophenol( PCP) were analytically separated from each other and from the other wood extractive compounds using a high performance liquid chromatograph (HPLC). The ultraviolet (UV) absorptions of the TCP, PCP, and internal standard were automatically measured as they eluted. The UV absorption peaks were integrated, and the amounts of TCP and PCP present were calculated with a dedicated microcomputer. Compared with the method previously used, this method is faster (up to 144 samples per 48 hours), has the same accuracy and precision, and it is linear over the concentration range used.

Echocardiographic evaluation of left-to-right shunt in ventricular septal defect and persistent ductus arteriosus.

Forty-five patients with either a ventricular septal defect or a persistent ductus arteriosus were assessed by echocardiography and cardiac catheterisation. Left atrial dimension was expressed either as a function of the body surface area (LAD cm per m2 BSA), or as a multiple of the aortic root dimension (LAD/AR), and was compared with the shunt size as determined by oximetry. A highly significant (P less than 0-001) regression relation was found for the group as a whole. A significant relation also existed for each individual group: ventricular septal defect, ventricular septal defect with pulmonary hypertension, and persistent ductus arteriosus. Regression equations were derived for the whole group. The value of echocardiography is in separating large from small shunts and in adding a dimension to the follow-up of the individual patient.