Deletion of CD73 in mice leads to aortic valve dysfunction.

Aortic stenosis is known to involve inflammation and thrombosis. Changes in activity of extracellular enzyme - ecto-5'-nucleotidase (referred also as CD73) can alter inflammatory and thrombotic responses. This study aimed to evaluate the effect of CD73 deletion in mice on development of aortic valve dysfunction and to compare it to the effect of high-fat diet. Four groups of mice (normal-diet Wild Type (WT), high-fat diet WT, normal diet CD73-/-, high-fat diet CD73-/-) were maintained for 15weeks followed by echocardiographic analysis of aortic valve function, measurement of aortic surface activities of nucleotide catabolism enzymes as well as alkaline phosphatase activity, mineral composition and histology of aortic valve leaflets. CD73-/- knock out led to an increase in peak aortic flow (1.06±0.26m/s) compared to WT (0.79±0.26m/s) indicating obstruction. Highest values of peak aortic flow (1.26±0.31m/s) were observed in high-fat diet CD73-/- mice. Histological analysis showed morphological changes in CD73-/- including thickening and accumulation of dark deposits, proved to be melanin. Concentrations of Ca2+, Mg2+ and PO43- in valve leaflets were elevated in CD73-/- mice. Alkaline phosphatase (ALP) activity was enhanced after ATP treatment and reduced after adenosine treatment in aortas incubated in osteogenic medium. AMP hydrolysis in CD73-/- was below 10% of WT. Activity of ecto-adenosine deaminase (eADA), responsible for adenosine deamination, in the CD73-/- was 40% lower when compared to WT. Deletion of CD73 in mice leads to aortic valve dysfunction similar to that induced by high-fat diet suggesting important role of this surface protein in maintaining heart valve integrity.

Clinical value of digital image analysis in the diagnosis of urinary bladder cancer, particularly in aggressive tumors: a preliminary report.

The aim of the project was to evaluate the clinical value of a computer analysis of cytological specimen images obtained from urine and bladder washing samples. Three sample types (voided urine, catheterized urine and bladder washing) from 59 patients with primary or recurrent tumor were analyzed. All patients underwent cystoscopy and biopsy or resection. The histological results were compared with the results of the image analyzing computer system of collected urine samples. The consistency between the computer diagnosis and the clinical or histological diagnosis both in the presence and absence of cancer was as follows: 77% for voided urine samples, 72.5% for catheterized urine samples and 78% for bladder washing samples. The specificity of the method at the standard pathology level was 71%, and the sensitivity was 83%. The positive and negative predictive values (PPV and NPV) were 87.5% and 63% respectively. The sensitivity for G3 or CIS or T2 or T3 tumors reached nearly 100%. Computer analysis of urine provided correct diagnoses in cancer and control patients with the sensitivity of 83% and specificity of 71% and gave excellent results in aggressive tumors such as T2, T3, G3 and in CIS.

A personalised approach to prostate cancer screening based on genotyping of risk founder alleles.

BACKGROUND: To evaluate whether genotyping for 18 prostate cancer founder variants is helpful in identifying high-risk individuals and for determining optimal screening regimens. METHODS: A serum PSA level was measured and a digital rectal examination (DRE) was performed on 2907 unaffected men aged 40-90. Three hundred and twenty-three men with an elevated PSA (≥4 ng ml⁻¹) or an abnormal DRE underwent a prostate biopsy. All men were genotyped for three founder alleles in BRCA1 (5382insC, 4153delA and C61G), for four alleles in CHEK2 (1100delC, IVS2+1G>A, del5395 and I157T), for one allele in NBS1 (657del5), for one allele in HOXB13 (G84E), and for nine low-risk single-nucleotide polymorphisms (SNPs). RESULTS: On the basis of an elevated PSA or an abnormal DRE, prostate cancer was diagnosed in 135 of 2907 men (4.6%). In men with a CHEK2 missense mutation I157T, the cancer detection rate among men with an elevated PSA or an abnormal DRE was much higher (10.2%, P=0.0008). The cancer detection rate rose with the number of SNP risk genotypes observed from 1.2% for men with no variant to 8.6% for men who carried six or more variants (P=0.04). No single variant was helpful on its own in predicting the presence of prostate cancer, however, the combination of all rare mutations and SNPs improved predictive power (area under the curve=0.59; P=0.03). CONCLUSION: These results suggest that testing for germline CHEK2 mutations improves the ability to predict the presence of prostate cancer in screened men, however, the clinical utility of incorporating DNA variants in the screening process is marginal.

An inherited NBN mutation is associated with poor prognosis prostate cancer.

BACKGROUND: To establish the contribution of eight founder alleles in three DNA damage repair genes (BRCA1, CHEK2 and NBS1) to prostate cancer in Poland, and to measure the impact of these variants on survival among patients. METHODS: Three thousand seven hundred fifty men with prostate cancer and 3956 cancer-free controls were genotyped for three founder alleles in BRCA1 (5382insC, 4153delA, C61G), four alleles in CHEK2 (1100delC, IVS2+1G>A, del5395, I157T), and one allele in NBS1 (657del5). RESULTS: The NBS1 mutation was detected in 53 of 3750 unselected cases compared with 23 of 3956 (0.6%) controls (odds ratio (OR)=2.5; P=0.0003). A CHEK2 mutation was seen in 383 (10.2%) unselected cases and in 228 (5.8%) controls (OR=1.9; P<0.0001). Mutation of BRCA1 (three mutations combined) was not associated with the risk of prostate cancer (OR=0.9; P=0.8). In a subgroup analysis, the 4153delA mutation was associated with early-onset (age ≤ 60 years) prostate cancer (OR=20.3, P=0.004). The mean follow-up was 54 months. Mortality was significantly worse for carriers of a NBS1 mutation than for non-carriers (HR=1.85; P=0.008). The 5-year survival for men with an NBS1 mutation was 49%, compared with 72% for mutation-negative cases. CONCLUSION: A mutation in NBS1 predisposes to aggressive prostate cancer. These data are relevant to the prospect of adapting personalised medicine to prostate cancer prevention and treatment.

Protection of mouse heart against hypoxic damage by AMP deaminase inhibition.

Clinical observation in patients with heart disease indicates that reduced activity of AMP deaminase could be protective in heart failure and ischemic heart disease. This study evaluated the effect of 3-[2-(3-carboxy-4-bromo-5,6,7,8-tetrahydronaphthyl)ethyl]-3,6,7,8-tetrahydroimidazo [4,5-d][1,3]diazepin-8-ol, an AMP deaminase inhibitor (AMPDI) in the mouse heart subjected to hypoxia. ApoE/LDLR knock-out mice were subjected to reduced oxygen tension in breathing air. AMPDI was infused before hypoxia in the treated group. We observed amelioration of elcetrocardiographic changes during hypoxia in the treated group that are consistent with a protective effect.

Biological efficiency of AMP deaminase inhibitor: 3-[2-(3-carboxy-4-bromo-5,6,7,8-tetrahydronaphthyl)ethyl]-3,6,7,8-tetrahydroimidazo[4,5]-[1,3]diazepin-8-OL.

AMP deaminase could be a potential target for treatment of heart disease but experimental evaluation of this concept is difficult due to limited availability of inhibitors with proven efficiency in biological systems. This study evaluated the effect of 3-[2-(3-carboxy-4-bromo-5,6,7,8-tetrahydronaphthyl)ethyl]-3,6,7,8-tetrahydroimidazo [4,5-d][1,3]diazepin-8-ol, an AMP deaminase inhibitor (AMPDI) on the pathways of nucleotide metabolism in perfused rat heart. We show that AMPDI at 0.3 mM concentration effectively inhibits AMP deaminase in this experimental model.

Pharmacological inhibition of AMP-deaminase in rat cardiac myocytes.

Because mutation of AMP deaminase 1 gene leading to reduced AMP deaminase activity may result in protection of cardiac function in patients with heart disease, inhibitors of AMP deaminase (AMPD) may have therapeutic applications. This study evaluated the effect of a specific inhibitor of AMP deaminase 3-[2-(3-carboxy-4-bromo-5,6,7,8-tetrahydronaphthyl)ethyl]-3,6,7,8-tetrahydroimidazo [4,5-d][1,3]diazepin-8-ol (AMPDI) on the isolated human enzyme and on nucleotide catabolism in rat cardiomyocytes. AMPDI effectively inhibited isolated human AMPD with an IC(50) = 0.5 micro M. AMPDI was much less effective with isolated cardiomyocytes (IC(50) = 0.5 mM). AMPDI is a very effective inhibitor of AMPD that despite lower efficiency in the cell system examined could be useful for in vivo studies.

Modulation of AMP deaminase in rat hearts subjected to ischemia and reperfusion by purine riboside.

Changes in AMP deaminase (AMPD) activity influence heart function and progression of heart disease, but the underlying mechanism is unknown. We evaluated the effect of purine riboside (Purr) on the activity of AMPD in perfused rat hearts and in isolated rat cardiomyocytes. Brief perfusion of the pre-ischemic heart with 200 micro M Purr resulted in activation of AMPD, more pronounced degradation of the adenine nucleotides, and reduced recovery of the adenine nucleotide pool during reperfusion. Brief incubation of rat cardiomyocytes with 200 micro M Purr also activated AMPD, while prolonged exposure resulted in enzyme inhibition. We conclude that Purr activates AMPD, whereas metabolites of this compound may inhibit the enzyme.

A large germline deletion in the Chek2 kinase gene is associated with an increased risk of prostate cancer.

BACKGROUND: Germline mutations in the Chek2 kinase gene (CHEK2) have been associated with a range of cancer types. Recently, a large deletion of exons 9 and 10 of CHEK2 was identified in several unrelated patients with breast cancer of Czech or Slovak origin. The geographical and ethnic extent of this founder allele has not yet been determined. PARTICIPANTS AND METHODS: We assayed for the presence of this deletion, and of three other CHEK2 founder mutations, in 1864 patients with prostate cancer and 5496 controls from Poland. RESULTS: The deletion was detected in 24 of 5496 (0.4%) controls from the general population, and is the most common CHEK2 truncating founder allele in Polish patients. The deletion was identified in 15 of 1864 (0.8%) men with unselected prostate cancer (OR 1.9; 95% CI 0.97 to 3.5; p = 0.09) and in 4 of 249 men with familial prostate cancer (OR 3.7; 95% CI 1.3 to 10.8; p = 0.03). These ORs were similar to those associated with the other truncating mutations (IVS2+1G-->A, 1100delC). CONCLUSION: A large deletion of exons 9 and 10 of CHEK2 confers an increased risk of prostate cancer in Polish men. The del5395 founder deletion might be present in other Slavic populations, including Ukraine, Belarus, Russia, Baltic and Balkan countries. It will be of interest to see to what extent this deletion is responsible for the burden of prostate cancer in other populations.

Changes in the content of health-promoting compounds and antioxidant activity of broccoli after domestic processing.

The effect of water- and steam-cooking on the content of vitamin C, polyphenols, carotenoids, tocopherols and glucosinolates, as well as on the antioxidant activity of broccoli, are reported. Flavonoids, phenolic acids, vitamins C and E, beta-carotene, lutein, and glucosinolates in domestically processed broccoli were quantified using high-performance liquid chromatography (HPLC) methods; total polyphenols were determined with Folin-Ciocalteu reagent. The antioxidant capacities of broccoli extracts were evaluated using the Trolox equivalent antioxidant capacity (TEAC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) methods. The results indicated that steam-cooking of broccoli results in an increase in polyphenols, as well as the main glucosinolates and their total content as compared with fresh broccoli, whereas cooking in water has the opposite effect. Steam-cooking of broccoli has no influence on vitamin C, whereas cooking in water significantly lowers its content. Both, water- and steam-cooking of broccoli results in an increase in beta-carotene, lutein, and alpha- and gamma-tocopherols as compared with fresh broccoli. Similar effects of steaming and water-cooking of broccoli on their antioxidant activity were observed.

Purine metabolism in pigs and humans and its implications for xenotransplantation.

We compared concentrations of nucleotide substrates and activities of enzymes of nucleotide metabolism in pig and human blood, heart, and kidney. The most important difference was lower ecto-5-nucleotidase (ESN) activity in both pig hearts and kidney. Furthermore, higher hypoxanthine, inosine, adenine, and uracil, but lower uridine and uric acid concentrations were observed in pig blood as compared to human. A twofold increase in UTP concentration has been observed in pig hearts following 4 h perfusion with human blood. Purine metabolism is an important target for genetic and pharmacological manipulation during xenotransplantations.

Maternal and fetal outcome of lupus pregnancy: a prospective study of 29 pregnancies.

The aim of this study was to analyse pregestational and pregnancy risk factors for adverse fetal and maternal outcome in lupus pregnancy. Twenty women with systemic lupus erythematosus (SLE) (29 pregnancies) were prospectively evaluated. Mean patient age was 29.5+/-4.7 years, and mean disease duration, 6.3+/-6.5 years. Twenty-two pregnancies (75.9%) ended in live births; preterm delivery occurred in 17.4%, intrauterine growth restriction in 50%, preeclampsia in 3.7%, and gestational hypertension in 8%. Six pregnancies (20.7%) ended in spontaneous abortions. Adverse live-birth outcome was significantly associated with low pregestational serum albumin level, elevated gestational anti-dsDNA antibody, and diabetes mellitus. Spontaneous abortion was directly associated with low levels of pregestational serum albumin, positive anticardiolipin IgA, anti-beta2-glycoprotein I IgM, and anti-La antibodies, and inversely associated with number of patients' children. Postgestational lupus flare-up was noted in six pregnancies. Risk factors included high pregestational SLE Disease Activity Index (SLEDAI), lower serum albumin, elevated serum antibody to dsDNA, proteinuria, and use of prednisone and hydroxychloroquine. We conclude that despite high rate of obstetrical complications and postpartum lupus flare-up, pregnancy poses low risk for the majority of women with SLE.

Minimal requirements for IgE-mediated regulation of surface Fc epsilon RI.

The IgE-FcepsilonRI network plays a central role in allergic inflammation. IgE levels control cell surface levels of FcepsilonRI and, in turn, FcepsilonRI levels modulate the intensity of effector responses. Treatment of allergic patients with anti-IgE Abs has been shown to induce a decrease in FcepsilonRI expression on basophils and a decrease in Ag-triggered histamine release. However, the mechanisms underlying IgE-mediated regulation of FcepsilonRI expression remain unclear. Here, we designed an in vitro model system to establish the minimal cellular requirements for regulation of FcepsilonRI by IgE. Using this system, we demonstrate that transcriptional regulation, hemopoietic-specific factors, and signaling are not required for IgE-mediated increases in FcepsilonRI expression. IgE binding to the alpha-chain is the minimal requirement for the induction of FcepsilonRI up-regulation. The rate of up-regulation is independent of the baseline level of expression. The mechanism of this up-regulation is the result of a combination of three factors: 1) stabilization of the receptor at the cell surface, which prevents receptor internalization and degradation; 2) use of a preformed pool of receptor comprising recycled and recently synthesized receptors; and 3) continued basal level of protein synthesis. It is possible that in vivo additional factors contribute to modulate the basic regulatory mechanism described here.

Outcome predictors of high-energy transurethral microwave thermotherapy.

PURPOSE: Despite the good results of high-energy transurethral microwave thermotherapy (TUMT) for treatment of benign prostatic hyperplasia (BPH), it still is difficult to predict the response to treatment on an individual basis. In addition to clinical baseline parameters, histologic parameters seem to play a role in response variance after TUMT. High-energy TUMT has become widely accepted as a minimally invasive outpatient treatment in patients with lower urinary tract symptoms and BPH. Most patients benefit substantially from targeted microwave thermotherapy; however, little is known about optimal patient selection and the most relevant outcome parameters. MATERIALS AND METHODS: We evaluated Medline-based studies published between 1989 and 2000, including 900 patients suffering from lower urinary tract symptoms due to BPH who were undergoing TUMT. We evaluated outcome predictors for TUMT, such as histopathological parameters, prostate-specific antigen, and volume. RESULTS: Histologic and clinical outcome parameters were identified. Patient-to-patient differences in stromal-to-epithelial ratio of prostate tissue did affect outcomes. Poor responders to TUMT seemed to have a higher vessel density and a lower epithelial/stromal ratio. Relatively more abundant epithelial cells in the prostate tissue may lead to more favorable outcomes. Use of higher energy, patients with higher grade of obstruction, younger patient age, larger prostate volume (>25 mL), and higher prostate-specific antigen levels seemed to be associated with a better response to TUMT. CONCLUSIONS: New energy protocols could help tailor treatment to the individual needs of each patient. Nomograms based on volume, age, and pressure-flow parameters could assist in making clinical recommendations and identifying treatment responders; however, the total amount of energy appeared to have a high impact on the prediction of response.

Laparoscopic operative technique for adrenal tumors.

BACKGROUND AND OBJECTIVES: Laparoscopy has acquired an unquestionable position in surgical practice as a diagnostic and operative tool. Recently, the laparoscopic approach has become a valuable option for adrenalectomy. This paper reports, in detail, our experience of laparoscopic adrenalectomy performed for adrenal tumors. METHODS: We performed 12 laparoscopic adrenalectomies from October 29, 1997 to October 31, 1998. The technique of laparoscopic adrenalectomy is described thoroughly in all relevant details for either left or right-sided adrenal lesions. RESULTS: The presented technique of laparoscopic adrenalectomy in all 12 cases provided good and relatively simple exposure of the immediate operative area. All relevant vascular elements were safely controlled, adrenal tumors could be successfully removed, and adequate hemostasis was achieved. No intraoperative or postoperative complications were observed. CONCLUSIONS: Laparoscopic adrenalectomy is a safe alternative to open surgery and is preferred for most patients because of shorter postoperative hospital stay and less postoperative discomfort.

Perceptions of the benefits and costs associated with condom use and unprotected sex among late adolescent college students.

To assess the differential effects of the perceived benefits and costs associated with both condom use and unprotected sex on sexual risk behaviors, data were collected from 704 ethnically diverse male and female sexually experienced late adolescent college students (aged 17-25). Perceived benefits and costs for condom use and perceived benefits and costs for unprotected sex were measured separately through an anonymous self-report survey. In addition, participants completed measures of self-efficacy for practicing safer sex and temptation for unsafe sex in various situations, and three measures of sexual risk-taking (stage of change for condom use, consistency of condom use during the past month, and whether or not a condom was used for the last act of intercourse). Univariate analyses indicated that benefits and costs of condom use, benefits of unprotected sex, self-efficacy and situational temptation were all related to sexual risk-taking. Gender differences were identified, with females reporting more benefits of condom use and costs of unprotected sex, fewer benefits of unprotected sex and costs of condom use, greater self-efficacy for practicing safer sex, and less situational temptation for unsafe sex. Multivariate analyses indicated that sexual risk behaviors were most related to situational temptation, self-efficacy for safer sex, and perceived benefits of unprotected sex. The results suggest that, among late adolescents, perceived benefits of the unhealthy behavior (unprotected sex) were better determinants of sexual risk-taking than were perceived benefits (or costs) associated with the healthy behavior (condom use). Perceived costs associated with unprotected sex were unrelated to sexual behaviors. These findings support previous work identifying adolescents as more driven by their perceptions of the positive benefits associated with risky behaviors, rather than knowledge of the costs or dangers involved in risk-taking.

Prevalence of IgE-mediated food allergy among children with atopic dermatitis.

OBJECTIVE: There is a growing body of clinical and laboratory evidence to support the notion that food allergy plays a role in the pathogenesis of atopic dermatitis (AD). However, the incidence of IgE-mediated food allergy in children with AD is not well established. DESIGN: A prospective study to determine the prevalence of IgE-mediated food hypersensitivity among patients referred to a university-based dermatologist for evaluation of AD. SETTING: University hospital pediatric dermatology clinic. PATIENTS: A total of 63 patients with AD were recruited (35 male; 32 white, 24 African-American, 7 Asian). METHODS: Patients were assigned an AD symptom score (SCORAD) and were screened for food-specific serum IgE antibodies to six foods (milk, egg, wheat, soy, peanut, fish) known to be the most allergenic in children. The levels of food-specific serum IgE were determined by the CAP System fluoroscein-enzyme immunoassay (CAP); patients with a value >/=0.7 kIUa/L were invited for an additional allergy evaluation. Those with CAP values below the cutoff were considered not food allergic. Patients were considered to be allergic if they met one of the following criteria for at least one food: 1) reaction on food challenge; 2) CAP value more than the 95% confidence interval predictive for a reaction; 3) convincing history of an acute significant (hives, respiratory symptoms) reaction after the isolated ingestion of a food to which there was a positive CAP or prick skin test. RESULTS: A total of 63 patients (median age, 2.8 years; median SCORAD, 41.1) were recruited; 22 had negative CAP values (without a significant difference in age or SCORAD score, compared with the 41 with positive specific IgE values). Further allergy evaluation was offered to the 41 remaining patients; 10 were lost to follow-up and 31 were evaluated further. Of these, 19 underwent a total of 50 food challenges (36 double-blind, placebo-controlled, and 14 open), with 11 patients experiencing 18 positive challenges (94% with skin reactions). Additionally, 6 patients had a convincing history with a predictive level of IgE; 5 had a convincing history with positive, indeterminate levels of IgE; and 1 had predictive levels of IgE (to egg and peanut) without a history of an acute reaction. Overall, 23/63 (37%; 95% confidence interval, 25% to 50%) had clinically significant IgE-mediated food hypersensitivity without a significant difference in age or symptom score between those with or without food allergy. CONCLUSIONS: Approximately one third of children with refractory, moderate-severe AD have IgE-mediated clinical reactivity to food proteins. The prevalence of food allergy in this population is significantly higher than that in the general population, and an evaluation for food allergy should be considered in these patients.