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.

A role for endogenous transforming growth factor beta 1 in Langerhans cell biology: the skin of transforming growth factor beta 1 null mice is devoid of epidermal Langerhans cells.

Transforming growth factor beta 1 (TGF-beta 1) regulates leukocytes and epithelial cells. To determine whether the pleiotropic effects of TGF-beta 1, a cytokine that is produced by both keratinocytes and Langerhans cells (LC), extend to epidermal leukocytes, we characterized LC (the epidermal contingent of the dendritic cell [DC] lineage) and dendritic epidermal T cells (DETC) in TGF-beta 1 null (TGF-beta 1 -/-) mice. I-A+ LC were not detected in epidermal cell suspensions or epidermal sheets prepared from TGF-beta 1 -/- mice, and epidermal cell suspensions were devoid of allostimulatory activity. In contrast, TCR-gamma delta + DETC were normal in number and appearance in TGF-beta 1 -/- mice and, importantly, DETC represented the only leukocytes in the epidermis. Immunolocalization studies revealed CD11c+ DC in lymph nodes from TGF-beta 1 -/- mice, although gp40+ DC were absent. Treatment of TGF-beta 1 -/- mice with rapamycin abrogated the characteristic inflammatory wasting syndrome and prolonged survival indefinitely, but did not result in population of the epidermis with LC. Thus, the LC abnormality in TGF-beta 1 -/- mice is not a consequence of inflammation in skin or other organs, and LC development is not simply delayed in these animals. We conclude that endogenous TGF-beta 1 is essential for normal murine LC development or epidermal localization.

A role for TGFbeta1 in langerhans cell biology. Further characterization of the epidermal Langerhans cell defect in TGFbeta1 null mice.

Previous studies of TGFbeta1 null (-/-) mice indicated that the epidermis was devoid of Langerhans cells (LC) and that the LC deficiency was not secondary to the inflammation that is the dominant feature of the -/- phenotype (Borkowski, T.A., J.J. Letterio, A.G. Farr, and M.C. Udey. 1996. J. Exp. Med. 184:2417-2422). Herein, we demonstrate that dendritic cells could be expanded from the bone marrow of -/- mice and littermate controls. Bone marrow from -/- mice also gave rise to LC after transfer into lethally irradiated recipients. Thus, the LC defect in TGFbeta1 null mice does not result from an absolute deficiency in bone marrow precursors, and paracrine TGFbeta1 production is sufficient for LC development. Several approaches were used to assess the suitability of -/- skin for LC localization. A survey revealed that although a number of cytokine mRNAs were expressed de novo, mRNAs encoding proinflammatory cytokines known to mobilize LC from epidermis (IL-1 and TNFalpha) were not strikingly overrepresented in -/- skin. In addition, bone marrow-derived LC populated full-thickness TGFbeta1 null skin after engraftment onto BALB/c nu/nu recipients. Finally, the skin of transgenic mice expressing a truncated loricrin promoter-driven dominant-negative TGFbeta type II receptor contained normal numbers of LC. Because TGFbeta1 signaling in these mice is disrupted only in keratinocytes and the keratinocyte hyperproliferative component of the TGFbeta1 -/- phenotype is reproduced, these results strongly suggest that the LC defect in TGFbeta1 null mice is not due to an epidermal abnormality but reflects a requirement of murine LC (or their precursors) for TGFbeta1.

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.

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.

Regulation of expression of B7 by murine Langerhans cells: a direct relationship between B7 mRNA levels and the level of surface expression of B7 by Langerhans cells.

Cultured BALB/c epidermal Langerhans cells express high levels of the costimulatory molecule B7 on their surfaces relative to levels expressed on fresh Langerhans cells. Quantitation of relative amounts of B7 mRNA in fresh epidermal cells and cultured epidermal cells following amplification of mRNA signals via reverse transcriptase-polymerase chain reaction, hybridization of PCR products with radiolabeled internal oligonucleotide probes, resolution of hybrids in non-denaturing polyacrylamide gels, and detection by autoradiography revealed dramatically (approximately one thousandfold) higher levels of B7 mRNA in cultured epidermal cells (10-40% I-A+) as compared with fresh epidermal cells (1-4% I-A+). Levels of B7 mRNA in cultured epidermal cells were also substantially greater than those detected in a reference B lymphoma cell line (CH-1). Analysis of B7 mRNA expression in subpopulations of cultured epidermal cells demonstrated that essentially all of the B7 mRNA was present in Langerhans cells; cells bearing I-A and CD45 antigens. Cultured keratinocytes did not contain appreciable amounts of B7 mRNA. These results are consistent with previous data regarding surface expression of B7 by cLC and also demonstrate that fLC are essentially devoid of B7 mRNA and surface protein.

Isolation from calf brain of a polypeptide fraction affecting aminoacylation of tRNA.

The preparation of tRNA obtained from calf brain by three conventional methods exhibits the presence of a slow-migrating fraction in polyacrylamide-gel electrophoresis. This fraction constitutes 2-2.5% of the isolated tRNA and has been identified as a polypeptide of molecular weight of 6000. The aminoacylation with glutamic acid, glycine, leucine and phenylalanine of tRNA devoid of this polypeptide is reduced by half as compared with the initial preparation.

Activity of aminoacyl-tRNA synthetases in experimental hyperthyroidism in muscle tissues of the rabbit.

In cardial and femoral muscles of rabbits specific activities of aminoacyl-tRNA synthetases for twenty amino acids were generally similar, namely the activities towards amino acids and their amides, leucine, isoleucine, histidine, tyrosine, proline and serine were considerably lower than towards the remaining amino acids. Specific activities of most aminoacyl-tRNA synthetases were higher in hyperthyroidism than in euthyreosis, and were higher in femoral muscle than in heart. The response to thyroxine treatment of individual aminoacyl-tRNA synthetases in both kinds of muscles varied with respect to most of the amino acids.

Mitochondrial tRNA in hyperthyroidism.

The mitochondrial tRNA were prepared from liver and brain tissues of thyroxinized and control rabbits. The presence of tRNA for twenty amino acids both in liver and brain mitochondria was revealed. The quantity of radioactive amino acids bound to the mitochondrial tRNA was higher in hyperthyreosis than in control animals but considerable differences between the brain and liver tissues were observed.

Drug addiction in Poland and criminalistic traces.

The authors describe the most popular drugs and their substitutes being used in Poland. The paper also presents the problem of developing and examining criminalistic traces in biological and some non-organic materials.

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.

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.