Whole Genome Methylation Scanning Based on phi29 Polymerase Amplification.

Identifying differences in DNA methylation is critical to understanding how epigenetics influences gene expression during processes such as development. Here, we propose a method that employs a single, methylation-sensitive restriction endonuclease of choice, to produce discrete pools of methylated and unmethylated DNA from the same sample. A pool of restriction fragments representing unmethylated regions of the genome is first obtained by digestion with a methylation-sensitive endonuclease. The restriction-digested DNA is then concatamerized in the presence of stuffer-adaptor DNA, which prevents interference from originally unmethylated DNA by blocking the ends of the restriction fragments. The concatamerized DNA is amplified by phi29 polymerase to remove methylation marks, and again digested with the same endonuclease to produce a pool of DNA fragments representing methylated portions of the genome. The two pools of DNA fragments thus obtained can be analyzed by end-sequencing or hybridization to a genomic array. In this report we detail a proof of concept experiment that demonstrates the feasibility of our method.

An evaluation of Irish cattle herds with inconclusive serological evidence of bovine brucellosis.

Since 1998, there has been a steady decline in herd restrictions and de-populations in Ireland due to bovine brucellosis. There is concern that the interpretation of laboratory results may become increasingly problematic, as brucellosis prevalence falls in Ireland. Therefore, the purpose of the current study was to evaluate the infection status of Irish herds and animals with inconclusive serological evidence of bovine brucellosis. During 12 months from September 1, 2004, laboratory and observational epidemiological data were collected from all Irish herds where animal testing identified at least one animal with a complement fixation test (CFT) reading greater than zero and/or a positive result to the indirect enzyme-linked immunosorbent assay (iELISA). Due to the observational nature of the study, we have robust estimates of the relative, but not the absolute, performance of the CFT, iELISA and brucellin skin test (BST). Herds were divided into three categories (Group A, B or C) on the basis of test results at initial assessment. A total of 639 herds were enrolled into the study, and observed for at least two years following enrolment. A rising CFT titre, with a CFT reading of 111 International CFT Units (IU) or greater at the subsequent blood test, was generally associated with herds where other evidence of infection was also available. Knowledge of the CFT reading at the initial and a subsequent blood test proved useful in distinguishing false-positive and true-positive brucellosis results. There was poor correlation between the CFT and iELISA results, and between the CFT and BST results. As a result of this study, national policy has been modified to include re-sampling of all animals with CFT readings of 20 IU or greater. This project has also led to a reduction in the number of herds restricted, as well as restriction duration. It has also contributed to a reduction in the number of herds listed for contiguous tests, and therefore the potential for contiguity testing of false positive results.

Intracolonic use of vancomycin for treatment of clostridium difficile colitis in a patient with a diverted colon: report of a case.

Clostridium difficile-associated pseudomembranous colitis (PMC) is a common affliction of postoperative patients. Risk factors include antibiotic therapy, recent surgery, and hospitalization (1,2,3). We present a case of PMC in a diverted colon and its treatment using vancomycin enemas.

Proper timing of blood cardioplegia in infant lambs: superiority of a multiple-dose regimen.

BACKGROUND: In the pediatric and infant age groups, it is unclear whether repeated infusions of blood cardioplegia solution during ischemic arrest are beneficial or detrimental when compared with a single-dose regimen. METHODS: Twenty lambs (aged 6 to 7 weeks) were placed on cardiopulmonary bypass. A miniature glass-tip electrode measured myocardial pH and hydrogen ion concentration, [H+], in the anterior wall. The aorta was clamped for 2 hours. Group S (n = 10) received a single dose of blood cardioplegia solution. Group M (n = 10) received multiple doses of blood cardioplegia solution at 20-minute intervals. RESULTS: The amount of [H+] generated during the cross-clamp period was greater in group S than in group M (39.2 +/- 10.1 nmol/L versus 0.4 +/- 1.4 nmol/L, p < 0.008). The percent increase in the time constant, tau, an index of diastolic relaxation, was more prolonged after cardiopulmonary bypass in group S when compared with group M (51.4% +/- 2.8% versus 6.4% +/- 3.0%, p < 0.0001). Similarly, the percent decrease in end systolic elastance, a measure of systolic contractility, was greater in group S after cardiopulmonary bypass when compared with group M (29.5% +/- 1.4% versus 7.3% +/- 1.3%, p < 0.0001). CONCLUSIONS: In this infant lamb model, multiple doses of blood cardioplegia solution provided superior metabolic preservation and hemodynamic support after 2 hours of aortic clamping when compared with a single-dose regimen.

Quantitative 3-dimensional contrast echocardiographic determination of myocardial mass at risk and residual infarct mass after reperfusion: experimental canine studies with intravenous contrast agent NC100100.

Two-dimensional contrast echocardiography has been shown to enable the evaluation of myocardial perfusion abnormalities. However, its ability to quantify a regional myocardial mass is limited. The goal of this study was to examine the quantitative value of 3-dimensional echocardiography (3DE) in the estimation of myocardial mass at risk, salvaged mass, and residual infarct mass after intravenous injection of contrast. We created acute coronary occlusion, followed by reperfusion in 10 dogs. Three-dimensional echocardiographic data were acquired at the end of each stage, and the perfusion defect mass and dysfunctional mass were measured. The true mass at risk and infarct mass were determined by anatomic methods. The anatomic mass at risk (x) (27.1+/-14.6 g or 23.8%+/-9.7% of the left ventricle [%LV]) correlated well with the 3DE-determined perfusion defect mass (y) during coronary occlusion (y = 0.5x+8.9; r = 0.90; P<.001; mean difference -4.8+/-8.1 g; or y = 0.7x + 6.5; r = 0.83, P<.01; mean difference -0.1+/-5.4 %LV). Good correlation was also found between the anatomic infarct mass (x) (9.3+/-8.1 g or 9.1+/-8.8 %LV) and the 3DE perfusion defect mass after reperfusion (y) (y = 1.2x+1.2; r = 0.93; P<.001; mean difference 2.3+/-4.0 g; or y = 1. 3x, r = 0.98, P <.0001; mean difference 2.7+/-3.7 %LV). The salvaged mass was 13.6 +/-11.0 %LV from anatomic methods and 14.2+/-13.0 %LV by 3DE. To conclude, with the use of intravenous contrast, 3DE could quantify the actual mass at risk during acute ischemia, and in the setting of reperfusion, the residual infarct mass and salvaged mass.

Enhanced visualization of intravascular and left atrial appendage thrombus with the use of a thrombus-targeting ultrasonographic contrast agent (MRX-408A1): In vivo experimental echocardiographic studies.

Echocardiographic evaluation for the recognition of intravascular and left atrial appendage thrombus remains a difficult problem. A thrombus-specific ultrasonographic contrast agent has the potential for an alternative approach for their delineation. The aim of this study was to investigate the usefulness of thrombus-specific contrast agent MRX-408A1 for the detection of acute experimentally created intravascular and intracardiac thrombus. In the first study, we created inferior vena cava thrombus in 9 dogs. With the use of fundamental 2-dimensional echocardiography imaging, we recorded images of the inferior vena cava thrombus at baseline (n = 9), with the thrombus-specific contrast agent MRX-408A1 (n = 9), and with nonspecific contrast agent MRX-113 (n = 6). In the second study, we created a left atrial appendage thrombus in 8 dogs. We imaged left atrial appendage thrombus at baseline and during MRX-113 and MRX-408A1 infusion. Thrombus was successfully created in all dogs in study 1 and in 6 of 8 dogs in study 2. MRX-408A1 produced a visually apparent increase in ultrasonographic contrast enhancement of the thrombus in all cases in which thrombus was found on autopsy. In both studies, MRX-408A1 increased the videointensity of the thrombus significantly compared with baseline images and images obtained during MRX-113 infusion. The size of the visually detectable thrombus on the image was also significantly larger during MRX-408A1 infusion than at baseline and during MRX-113 infusion. These data provide in vivo demonstration of the efficacy of a thrombus-specific contrast agent, MRX-408A1, in the detection of acute intravascular and intracardiac thrombus. It has the potential to improve the diagnostic accuracy of ultrasonography for the detection of acute thrombi at various cardiovascular sites in the clinical setting.

Customer focus: patient, organization and EQuIP in collaboration. Evaluation and Quality Improvement Program.

The Australian Council on Healthcare Standards (ACHS) Evaluation and Quality Improvement Program (EQuIP) calls on healthcare organizations to increase their focus on patients by using leadership to coordinate, and continuous improvement to guide, care delivery. At a large acute care private facility in Melbourne, a program has been developed to create a 'care partnership', characterized by shared decision making, collaboration and conciliation. This program enhances patient care through the coordination of three strategies, a patient communication strategy, an evaluation strategy and a quality improvement strategy. The program has resulted in patient guided reforms such as redesign of a patient information booklet, a hospital-wide discharge planning improvement initiative and a hospital-wide strategy to improve pain management. Through the creation of a care partnership, this program helps an acute care hospital focus its services and processes on one of its key customer groups, patients.

Prevention in Poland: health care system reform.

Despite the political and economic reforms that have swept Eastern Europe in the past 5 years, there has been little change in Poland's health care system. The Ministry of Health and Social Welfare has targeted preventive care as a priority, yet the enactment of legislation to meet this goal has been slow. The process of reform has been hindered by political stagnation, economic crisis, and a lack of delineation of responsibility for implementing the reforms. Despite the delays in reform, recent developments indicate that a realistic, sustainable restructuring of the health care system is possible, with a focus on preventive services. Recent proposals for change have centered on applying national goals to limited geographic areas, with both local and international support. Regional pilot projects to restructure health care delivery at a community level, local health education and disease prevention initiatives, and a national training program for primary care and family physicians and nurses are being planned. Through regionalization, an increase in responsibility for both the physician and the patient, and redefinition of primary health care and the role of family physicians, isolated local movements and pilot projects have shown promise in achieving these goals, even under the current budgetary constraints.

Germline p16 mutations in familial melanoma.

The p16 gene is located in chromosome 9p21, a region that is linked to familial melanoma and homozygously deleted in many tumour cell lines. We describe eight p16 germline substitutions (one nonsense, one splice donor site and six missense) in 13/18 familial melanoma kindreds. Six of these mutations were identified in 33/36 melanoma cases in nine families, whereas two were detected in normal controls and are not disease-related. The melanoma-specific mutations were detected in 9p21-linked, but not in 1p36-linked, families, thereby confirming previous reports of genetic heterogeneity. Functional analyses of these mutations will confirm those causally related to the development of familial melanoma.

Terminal neuroendocrine differentiation of human prostate carcinoma cells in response to increased intracellular cyclic AMP.

Recent clinicopathologic studies have shown that many prostatic adenocarcinomas express focal neuroendocrine differentiation and that neuroendocrine differentiation is most apparent in advanced anaplastic tumors. While studying growth-regulatory signal transduction events in human prostate carcinoma cell lines, we found that in two of four cell lines, the androgen-sensitive line LNCaP and the highly metastatic androgen-independent line PC-3-M, elevation of cAMP through addition of cAMP analogues or phosphodiesterase inhibitors induced a markedly neuronal morphology. Also in LNCaP cells ultrastructural analysis showed that cAMP induced the appearance of neurosecretory cell-like dense-core granules. Phenotypic analysis of untreated LNCaP and PC-3-M cells showed that both cell lines express markers of the neural crest including S-100, chromogranin A, pp60c-src, and neuron-specific enolase as well as the epithelial marker KS1/4 and stage-specific embryonic antigen 4. In PC-3-M cells, cAMP markedly elevated neuron-specific enolase protein and caused an increase in the specific activity of the neuroendocrine marker pp60c-src, and in both cell lines expression of KS1/4 and stage-specific embryonic antigen 4 was down-regulated. In addition to effects on lineage markers, cAMP treatment induced G1 synchronization, growth arrest, and loss of clonogenicity, indicating terminal differentiation. Our data provide direct evidence of plasticity in the lineage commitment of adenocarcinoma of the prostate. We have shown that cell-permeant cAMP analogues can induce terminal differentiation, suggesting that hydrolysis-resistant cyclic nucleotides may present an additional approach to the treatment of advanced prostate cancer.

OKT3 treatment of cardiac allograft rejection.

Since the initial report of the murine monoclonal antibody OKT3 for acute kidney rejection, a significant body of information has been collected regarding the efficacy of OKT3 in reversing acute allograft rejections in kidney, liver, and heart transplant recipients. The use of OKT3 therapy for the reversal of cardiac allograft rejection in patients in whom other therapeutic alternatives have failed or are contraindicated is described. Treatment with OKT3 reversed acute heart rejection in 102 of 113 patients (90%). Complete reversal was achieved in 63 patients, and partial reversal in 39 patients. At 2 years, graft and patient survival rate was 77% and 65%, respectively. No significant differences were noted in reversal rate and in 12-month graft and patient survival rates between those patients experiencing one rejection episode and those patients experiencing two or more rejection episodes. Comparable reversal rates and graft and patient survival rates were achieved in patients whether OKT3 was administered as the primary rejection therapy or as rescue therapy. Adverse events were common in the first 2 days of therapy, but they were well tolerated in most patients. Infectious complications occurred in 49% of patients in whom most infections were not serious. On the basis of this experience, OKT3 appears highly effective in reversing acute cardiac allograft rejection.

H2 excretion after ingestion of complex carbohydrates.

Malabsorption of fermentable material in a variety of foods was assessed by measurement of breath H2 excretion. Breath H2 increased well above that observed in fasting subjects after ingestion of 100 g of carbohydrate in oats, whole wheat, potatoes, corn, and baked beans. Rice caused only a minimal increase in H2 excretion and hamburger was associated with no increase. We estimated the malabsorption of fermentable material by comparing the H2 excretion for 9 h after ingestion of various complex carbohydrates with that after 10 g of lactulose. The mean malabsorption of fermented material after 100-g carbohydrate meals was 20 g for baked beans; 7-10 g for wheat, oats, potatoes, and corn; and 0.9 g for rice. Whole oats or whole wheat resulted in 2-5 times more H2 than did the refined flours. As purified fiber appeared to be a poor substrate for H2 production by fecal homogenates, we conclude that most complex carbohydrates, with the exception of rice, contain a good deal of fermentable material that escapes small bowel absorption and it seems likely that this fermentable material is malabsorbed starch.

Classification criteria for systemic lupus erythematosus. Frequency in normal patients.

One hundred healthy women were examined prospectively during their annual physical examination for any of the preliminary criteria of systemic lupus erythematosus (SLE). No patient had more than three of the criteria; 50 (50%) had none. The preliminary criteria were effective in excluding normal patients. Three of 19 patients taking oral contraceptives had serum antinuclear antibody (ANA), whereas only one of 81 non-pill users had serum ANA (P less than .05). None of these patients with positive ANA had clinical evidence of SLE.