Rapid multiplex analysis for the factor V Leiden and prothrombin G20210A mutations associated with hereditary thrombophilia

Thromboembolic episodes are common events and affect approximately one in 1,000 persons annually. Pulmonary embolism alone accounts for 50,000 to 100,000 deaths per year in the United States with > 50% of those being elderly persons. Resistance to activated protein C is the most common inherited disorder associated with hereditary thrombophilia. A missense mutation has been identified in the gene coding for coagulation factor V (codon 506) which renders this procoagulant factor resistant to inactivation by activated protein C resulting in an increased risk for venous thrombosis. Recently, a second polymorphism was identified in the prothrombin gene (factor II) which is also associated with increased risk for venous thrombosis. Because of the high prevalence of these two mutations in the general population as well as in specific patient populations, the ability readily to detect these two mutations must be feasible. In this study, we evaluated 303 patients for the prothrombin mutatin (G20210A) which were previously tested for the factor V mutation using established polymerase chain reaction-mediated restriction fragment length polymorphism assays. In these patients, 30 (9.9%) were found to be heterozygous for the factor V Leiden mutation with no homozygous mutants identified. Twenty individuals (6.6%) were heterozygous for the prothrombin G20210A mutation, and we identified two individuals (0.66%) who were homozygous for the 20210A allele. Of the total 303 individuals screened, two were double heterozygotes for both the factor V Leiden and the prothrombin gene mutations. We also describe a multiplex polymerase chain reaction-mediated restriction fragment length polymorphism assay for detecting both mutations in a single-tube double-enzyme digestion reaction making identification of these two mutations easily achievable.     

Phenotype and genotype of advanced premalignant head and neck lesions after chemopreventive therapy

BACKGROUND: The goal of chemoprevention is to reduce the risk of cancer development by reversing or blocking the tumorigenic process through the use of pharmacologic or natural agents. To determine the potential role of genetic alterations in assessing cancer risk and in evaluating the efficacy of chemopreventive agents, we studied 22 patients with advanced premalignant lesions of the head and neck who were part of a prospective cancer prevention trial that is investigating a regimen of 13-cis-retinoic acid, interferon alfa, and alpha-tocopherol administered for 12 months or until disease progression. METHODS: We used polymerase chain reaction analysis of microsatellite DNA sequences in cells from precancerous lesions to determine the frequencies of genetic alterations--namely, loss of heterozygosity (LOH) and microsatellite instability--at chromosomal loci that are commonly deleted in head and neck cancer. RESULTS: Prior to treatment, 17 (81%) of 21, eight (44%) of 18, and eight (42%) of 19 patients who were informative (i.e., heterozygous) at chromosomes 9p21, 3p14, and 17p13, respectively, exhibited LOH in at least one of their lesion biopsy specimens. Among nine patients who exhibited LOH at chromosome 9p21 in pretreatment biopsy specimens and who had completed at least 5 months of therapy, the genetic loss persisted in eight--including three of the four patients who exhibited complete histologic responses (i.e., no evidence of dysplasia in their biopsy specimens). IMPLICATION: Our data suggest that clinical and histologic assessments of the response to chemopreventive agents may be insufficient to determine their efficacy and that critical genetic alterations could be used as independent biomarkers to augment the ability to evaluate the efficacy of such agents.     

Biotransformation of an antihypertensive arylalkylamine analogue in the rat

1. The excretion and metabolism of N-[2-(3,4-dimethoxyphenyl)ethyl]-5-methoxy-N,alpha-dimethyl-2-(phenyl ethynyl) benzenepropanamine (RWJ-26240) in the Wistar rat has been investigated after a single oral dose of 14C-RWJ-26240 (50 mg/kg free base). 2. Plasma samples were obtained for 24 h after dosing and urine and faecal samples were collected over 8 days, and they accounted for 0.9 and 96% of the dose, respectively. 3. Representative samples of plasma, urine and faecal samples were purified for metabolite isolation and identification using HPLC, tlc, mass spectra (CI and EI), 1H-NMR and derivatization. 4. Unchanged RWJ-26240 plus 11 metabolites were identified and accounted for > 80% of the sample radioactivity. 5. Four metabolic pathways for RWJ-26240 are proposed; namely (1) N-demethylation, (2) O-demethylation, (3) phenyl hydroxylation and (4) N-dealkylation. Pathways 1-3 appeared to be quantitatively more important.     

The effect of minimum luminal nutrition on mucosal cellularity and immunity of the gut

Many catabolic patients can only consume small volumes of enteral nutrients. The aim of this study was to evaluate markers of cellularity and immunity in the small intestine of rats randomized to receive 6 days of parenteral nutrition, 25% enteral and 75% parenteral nutrition (i.e. minimum luminal nutrition) or enteral nutrition. The same glutamine-enriched solution was used for both parenteral and enteral nutrition. Enteral nutrition was associated with the least amount of jejunal atrophy (P<0.01), with the results from the minimum luminal nutrition group approximating those of the parenteral nutrition group. Parenteral nutrition was associated with the greatest number of CD2+ cells (P< 0.05) and the lowest CD4/CD8 cell ratio (P< 0.01) in the jejunal mucosa. In essence, we failed to demonstrate that there are any appreciable benefits associated with the enteral consumption of 25% of a nutrient load.     

Mode matches in hydrophobic free energy eigenfunctions predict peptide-protein interactions

OBJECTIVE: To find out whether concentrations of albumin (reflecting nutritional state), C-reactive protein (reflecting an acute phase reaction) or plasma protease inhibitors (reflecting ongoing proteolysis) are good predictors of postoperative complications, and whether other biochemical tests may improve diagnostic accuracy. DESIGN: Retrospective study. SETTING: University hospital, Sweden. SUBJECTS: 260 patients undergoing elective surgery for malignant (n = 149) or benign (n = 111) disease. MAIN OUTCOME MEASURES: Preoperative biochemical plasma measurements and postoperative complications. RESULTS: 192 patients recovered uneventfully and 35 had minor and 33 major postoperative complications. An increased plasma C-reactive protein concentration preoperatively, as well as a reduced albumin concentration, predicted the risk of developing major postoperative complications. Measurement of plasma protease inhibitors (C1-esterase inhibitor, alpha-2-macroglobulin and antithrombin III), specific biochemical studies of microheterogeneity, or comparison of quantitative and functional concentrations of the inhibitors gave no additional information. CONCLUSION: One measurement of the C-reactive protein and albumin concentrations preoperatively will identify patients at risk of developing severe postoperative complications.     

Dysregulated cyclin D1 expression early in head and neck tumorigenesis: in vivo evidence for an association with subsequent gene amplification

Cyclin D1 proto-oncogene is a key regulator of the mammalian cell-cycle acting at the restriction point in late G1. Amplification of the cyclin D1 locus, located on chromosome 11q13, as well as cyclin D1 protein overexpression have been reported in several human malignancies. The purpose of this study was to evaluate cyclin D1 gene copy status and protein expression during the multistep process of head and neck tumorigenesis, using a combination of fluorescence in situ hybridization and immunohistochemistry techniques. From 29 selected patients presenting with head and neck squamous carcinoma and whose tumor cytospins had been previously screened for presence (16 cases) or absence (13 cases) of amplification at the 11q13 band, we analysed 46 paraffin-embedded tissue specimens that demonstrated, besides the primary tumor, the presence of contiguous adjacent normal tissue and/or premalignant lesions. Of the 16 amplified cases, nine demonstrated a continuous progression from premalignant to invasive carcinoma and seven (77.7%) of these cases showed cyclin D1 gene amplification in premalignant lesions prior to development of invasive carcinoma. Increased cyclin D1 protein expression was observed in all 16 amplified tumors and five of the 13 (38.4%) non-amplified tumors. Interestingly, dysregulated cyclin D1 expression was also found in the premalignant lesions adjacent to all 16 amplified tumors, and it appeared to precede cyclin D1 gene amplification. In contrast no dysregulated expression was detected in the premalignant lesions of the non-amplified tumors. In conclusion, these findings provide strong evidence for early dysregulation of cyclin D1 expression during the tumorigenesis process and suggest that dysregulated increased expression precedes and possibly enables gene amplification.     

Amplification of fluorescent in situ hybridisation signals in formalin fixed paraffin wax embedded sections of colon tumour using biotinylated tyramide

Fluorescent in situ hybridisation (FISH) is a powerful tool for the evaluation of chromosomal alterations in formalin fixed paraffin wax embedded sections of colorectal cancer. However, initial experiments using a two-step detection system for digoxigenin labelled chromosome specific centromeric probes resulted in a complete lack of hybridisation signal from a number of colorectal tumour sections. This was due to high levels of background autofluorescence observed in this tissue, which masked any relatively weak hybridisations present. To overcome this problem, a biotinylated tyramide mediated amplification system was incorporated into the FISH detection protocol. This involves the use of horseradish peroxidase to activate the biotinylated tyramide, resulting in the deposition of a large number of biotin molecules at the site of bound peroxidase, which corresponds directly to the location of hybridised probe. Final detection was by means of a streptavidin-FITC conjugate. Using this technique, a panel of 11 colorectal tumour samples studied to date have shown strong, specific hybridisation signals to the nucleus of tumour cells. Amplification of FISH signals by biotinylated tyramide has the potential to improve weak hybridisation signals in cells from numerous sources, using a variety of probe types, including single copy gene probes as well as centromere specific probes.     

Assessment of mitral regurgitation severity by Doppler color flow mapping of the vena contracta

BACKGROUND: Although Doppler color flow mapping is widely used to assess the severity of mitral regurgitation (MR), a simple, accurate, and quantitative marker of MR by color flow mapping remains elusive. We hypothesized that vena contracta width by color flow mapping would accurately predict the severity of MR. METHODS AND RESULTS: We studied 80 patients with MR. Vena contracta width was measured in multiple views with zoom mode and nonstandard angulation to optimize its visualization. Flow volumes across the left ventricular outflow tract and mitral annulus were calculated by pulsed-Doppler technique to determine regurgitant volume. Effective regurgitant orifice area was calculated by dividing the regurgitant volume by the continuous-wave Doppler velocity-time integral of the MR jet. The cause of MR was ischemia in 24, dilated cardiomyopathy in 34 mitral valve prolapse in 12, endocarditis in 2, rheumatic disease in 2, mitral annular calcification in 1, and uncertain in 5. Regurgitant volumes ranged from 2 to 191 mL. Regurgitant orifice area ranged from 0.01 to 1.47 cm2. Single-plane vena contracta width from the parasternal long-axis view correlated well with regurgitant volume (r = .85, SEE = 20 mL) and regurgitant orifice area (r = .86, SEE = 0.15 cm2). Biplane vena contracta width from apical views correlated well with regurgitant volume (r = .85, SEE = 19 mL) and regurgitant orifice area (r = .88, SEE = 0.14 cm2). A biplane vena contracta width > or = 0.5 cm was always associated with a regurgitant volume > 60 mL and a regurgitant orifice area > 0.4 cm2. A biplane vena contracta width < or = 0.3 cm predicted a regurgitant volume < 60 mL and a regurgitant orifice area < 0.4 cm2 in 24 of 29 patients. No other parameter, including jet area, left atrial size, pulmonary flow reversal, or semiquantitative MR grade, correlated significantly with regurgitant volume or regurgitant orifice area in a multivariate analysis. CONCLUSIONS: Our results demonstrate that careful color flow mapping of the vena contracta of the MR jet provides a simple quantitative assessment of MR that correlates well with quantitative Doppler techniques.     

Doppler spectrum analysis versus direct femoral artery pressure measurement for rapid assessment of aortoiliac obstruction

Adequate patient selection is required to limit the clinical workload and improve the cost-effectiveness of noninvasive hemodynamic evaluation of the aortoiliac system. In a prospective blinded fashion the traditional invasive technique of direct femoral artery pressure measurements and the computerized Doppler spectrum analysis of blood flow velocities in the common femoral artery were studied. Both tests for rapid assessment of aortoiliac obstruction were compared with duplex ultrasonographic imaging, using a peak systolic velocity ratio of 2.5 to demonstrate stenoses of 50% or more. In a series of 17 consecutive patients (34 aortoiliac segments) with suspected aortoiliac obstructive disease, a good level of agreement (kappa = 0.6) was found for both methods when compared with duplex scanning. Analysis of deviations from the duplex registrations indicated an overestimation of the pathologic cases using femoral artery pressure measurements and an underestimation using Doppler spectrum analysis of blood flow velocities in the common femoral artery. Both methods were well tolerated, but femoral artery pressure measurements had a higher technical failure rate. Because of its noninvasive character and its feasibility the Doppler technique is preferred for the selection of patients for more extensive duplex sonographic investigation.