Morphology of the Vasculature and Blood Supply of the Brown Adipose Tissue Examined in an Animal Model by Micro-CT.

We performed micro-CT imaging of the vascular blood supply in the interscapular area of the brown adipose tissue in three mice with the use of intravascular contrast agent Aurovist™. Resulting 3D data rendering was then adapted into 2D resolution with visualization using false color system and grayscale images. These were then studied for the automatic quantification of the blood vessel density within this area. We found the highest most occurring density within arterioles or venules representing smaller blood vessels whereas with the increase of the vessel diameters a lower percentage rate of their presence was observed in the sample. Our study shows that micro-CT scanning in combination with Aurovist™ contrast is suitable for anatomical studies of interscapular area of brown adipose tissue blood vessel supply.

Hepcidin expression in adipose tissue increases during cardiac surgery.

Hepcidin, a key regulator of iron metabolism, plays a crucial role in the pathogenesis of anemia of chronic disease. Although it is produced mainly in the liver, its recently described expression in adipose tissue has been shown to be enhanced in massive obesity due to chronic low-grade inflammation. Our objective was to study the changes in hepcidin expression in adipose tissue during acute-phase reaction. We measured hepcidin mRNA expression from isolated subcutaneous and epicardial adipose tissue at the beginning and at the end of the surgery. The expression of mRNAs for hepcidin and other iron-related genes (transferrin receptor 1, divalent metal transporter 1, ferritin, ferroportin) were measured by real-time RT-PCR. Hepcidin expression significantly increased at the end of the surgery in subcutaneous but not in epicardial adipose tissue. Apart from the increased levels of cytokines, the parameters of iron metabolism showed typical inflammation-induced changes. We suggest that acute inflammatory changes could affect the regulation of hepcidin expression in subcutaneous adipose tissue and thus possibly contribute to inflammation-induced systemic changes of iron metabolism.

[The influence of very-low-calorie diet on soluble adhesion molecules and their gene expression in adipose tissue of obese women]. / Vliv nízkokalorické diety na sérové koncentrace a expresi cytoadhezivních molekul v tukové tkáni u obézních zen.

BACKGROUND: Adhesion molecules (AM) are proteins expressed on the endothelial surface that play an important role in development of endothelial dysfunction. Higher concentrations of AM were found in patients with atherosclerosis, obesity or type 2 diabetes mellitus. The aim of our study was to measure serum concentrations and gene expression of ICAM-1 (intercellular adhesion molecule 1), VCAM-1 (vascular adhesion molecule 1) and E-selectin in subcutaneous adipose tissue samples obtained by needle biopsy from obese women and healthy controls and to evaluate the effect of 3-weeks very-low-calorie diet (VLCD) on these parameters. METHODS AND RESULTS: 20 obese women (BMI 46.2 +/- 9.7 kg/m2) and 13 lean control women (BMI 23.8 +/- 2.3 kg/m2) were included into the study. Gene expression of AM in subcutaneous adipose tissue was measured using RT-PCR, serum AM levels were measured by multiplex immunoanalysis. At the baseline, serum E-selectin concentrations were higher in obese women compared to controls (24.4 +/- 2.3 vs. 15 +/- 1,5 ng/ml, p < 0,05). 3 weeks of VLCD significantly decreased BMI and serum E-selectin levels. Baseline mRNA expression of E-selectin, ICAM-1 and VCAM-1 in subcutaneous adipose tissue was lower in obese relative to lean women (p < 0.05). Weight reduction increased ICAM-1 and VCAM-1 gene expression (p < 0.05). CONCLUSIONS: Our results suggest that the subcutaneous adipose tissue is not the major source of the studied soluble adhesion molecules in obese women and that the regulation of AM local gene expression in subcutaneous adipose tissue probably differs from its circulating levels.

Multimarker real-time reverse transcription-PCR for quantitative detection of melanoma-associated antigens: a novel possible staging method.

BACKGROUND: Detection of melanoma cells in peripheral blood is a promising method for monitoring haematogenous spread of melanoma cells. It enables us to detect early metastasis and to better stratify candidates for adjuvant immunotherapy. Inconsistent data on the sensitivity and clinical relevance of this method have been reported. STUDY DESIGN: We developed a multimarker real-time reverse transcription-PCR (RT-PCR) for quantification of five melanoma markers: Melan-A, gp100, MAGE-3, MIA and tyrosinase. In this prospective study, 65 patients with resected cutaneous melanoma stage IIB-III were screened. Peripheral blood samples were collected every 3 months for the following 18 months, and circulating melanoma cells were examined and compared with clinical staging results. RESULTS: Eighteen patients relapsed during the trial and showed different types of melanoma progression. All these patients experienced statistically significant tumour marker elevation in the period from 0 to 9 months before the disease progression. MAGE-3 was the most sensitive progression marker. In patients with progression, we observed three concordant positive markers in 39% of cases, two concordant positive markers in 28%, and finally one marker in 33%. CONCLUSIONS: This report describes a multiple-marker real-time RT-PCR, which is able to provide quantitative data on melanoma markers in the peripheral blood of melanoma patients. Measurement of the studied molecular markers in our hands represents a prognostic factor and a useful method for early detection of metastasis and treatment response of melanoma patients.

Increased angiotensinogen production in epicardial adipose tissue during cardiac surgery: possible role in a postoperative insulin resistance.

Critical illness induces among other events production of proinflammatory cytokines that in turn interfere with insulin signaling cascade and induce insulin resistance on a postreceptor level. Recently, local renin-angiotensin system of adipose tissue has been suggested as a possible contributor to the development of insulin resistance in patients with obesity. The aim of our study was to determine local changes of the renin-angiotensin system of subcutaneous and epicardial adipose tissue during a major cardiac surgery, which may serve as a model of an acute stress potentially affecting endocrine function of adipose tissue. Ten patients undergoing elective cardiac surgery were included into the study. Blood samples and samples of subcutaneous and epicardial adipose tissue were collected at the beginning and at the end of the surgery. Blood glucose, serum insulin and adiponectin levels were measured and mRNA for angiotensinogen, angiotensin-converting enzyme and angiotensin II type 1 receptor were determined in adipose tissue samples using RT PCR. Cardiac surgery significantly increased both insulin and blood glucose levels suggesting the development of insulin resistance, while serum adiponectin levels did not change. Expression of angiotensinogen mRNA significantly increased in epicardial adipose tissue at the end of surgery relative to baseline but remained unchanged in subcutaneous adipose tissue. Fat expression of angiotensin-converting enzyme and type 1 receptor for angiotensin II were not affected by surgery. Our study suggests that increased angiotensinogen production in epicardial adipose tissue may contribute to the development of postoperative insulin resistance.

[Pilot study to evaluate blood glucose control by a model predictive control algorithm with variable sampling rate vs. routine glucose management protocol in peri- and postoperative period in cardiac surgery patients]. / Pilotní studie pro srovnání pocítacového algoritmu s variabilním intervalem merení glykémie a standardního protokolu pro intenzivní inzulínovou terapii v peri- a pooperacním období u kardiochirurgických pacientu.

BACKGROUND: Increased blood glucose levels are frequently observed in critically ill patients. Recent studies have shown that the normalization of glycemia by intensive insulin therapy decreases mortality, length of the hospitalization and number of complications. METHODS AND RESULTS: The aim of this pilot study was to compare blood glucose control by an automated model predictive control algorithm with variable sampling rate (eMPC) with routine glucose management protocol (RP) in peri- and postoperative period in cardiac surgery patients. 20 patients were included into this study (14 men and 6 women, mean age 68 +/- 10 let, BMI 28.3 +/- 5.0 kg/m2). 10 patients were randomized for treatment using eMPC algorithm and 10 patients for routine protocol. All patients underwent elective cardiac surgery and were treated with continuous insulin infusion to maintain glycemia in target range 4.4-6.1 mmol/l. The study duration was 24 hours. Mean blood glucose was significantly lower in eMPC vs. RP group (5.80 +/- 0.45 vs. 7.23 +/- 0.84 mmol/l, p < 0.05). Percentage of time in target range was significantly higher in eMPC vs. RP group (67.6 +/- 8.7% vs. 27.6 +/- 15.8%, p < 0.05). Percentage of time above the target range was higher in RP vs. eMPC group. Average insulin infusion rate was higher in eMPC vs. RP group (4.18 +/- 1.19 vs. 3.24 +/- 1.43 IU/hour, p < 0.05). Average sampling interval was significantly shorter in eMPC vs. RP group (1.51 +/- 0.24 vs. 2.03 +/- 0.16 hour, p < 0.05). No severe hypoglycaemia in either group occurred during the study. CONCLUSIONS: The results of our pilot study suggest that eMPC algorithm is more effective in maintaining euglycemia in peri- and post-operative period in patients after cardiac surgery and comparably safe as compared to RP.

Influence of PPAR-alpha agonist fenofibrate on insulin sensitivity and selected adipose tissue-derived hormones in obese women with type 2 diabetes.

PPAR-alpha agonists improve insulin sensitivity in rodent models of obesity/insulin resistance, but their effects on insulin sensitivity in humans are less clear. We measured insulin sensitivity by hyperinsulinemic-isoglycemic clamp in 10 obese females with type 2 diabetes before and after three months of treatment with PPAR-alpha agonist fenofibrate and studied the possible role of the changes in endocrine function of adipose tissue in the metabolic effects of fenofibrate. At baseline, body mass index, serum glucose, triglycerides, glycated hemoglobin and atherogenic index were significantly elevated in obese women with type 2 diabetes, while serum HDL cholesterol and adiponectin concentrations were significantly lower than in the control group (n=10). No differences were found in serum resistin levels between obese and control group. Fenofibrate treatment decreased serum triglyceride concentrations, while both blood glucose and glycated hemoglobin increased after three months of fenofibrate administration. Serum adiponectin or resistin concentrations were not significantly affected by fenofibrate treatment. All parameters of insulin sensitivity as measured by hyperinsulinemic-isoglycemic clamp were significantly lower in an obese diabetic group compared to the control group before treatment and were not affected by fenofibrate administration. We conclude that administration of PPAR-alpha agonist fenofibrate for three months did not significantly affect insulin sensitivity or resistin and adiponectin concentrations in obese subjects with type 2 diabetes mellitus. The lack of insulin-sensitizing effects of fenofibrate in humans relative to rodents could be due to a generally lower PPAR-alpha expression in human liver and muscle.

[The treatment of hyperglycaemia in critically ill patients: comparison of standard protocol and computer algorithm]. / Lécba hyperglykemie u kriticky nemocných pacientu: srovnání standardního protokolu a pocítacového algoritmu.

INTRODUCTION: Hyperglycemia is commonly observed in patients hospitalized on intensive care units. It is has been demonstrated that normalization of blood glucose level using intensive insulin therapy significantly improves prognosis of these patients. The aim of our study was comparison of standard protocol of intensive insulin therapy used on cardiac surgery ICU in General University Hospital in Prague and computer algorithm MPC (Model Predictive Control). PATIENTS AND METHODS: 20 patients with glycaemia higher than 6.7 mmol/l at the time of admission to ICU were included into the study, 10 subjects were randomized for standard treatment, 10 for treatment with MPC algorithm. Glycaemia was measured hourly during 48 hours, insulin infusion was rate was adjusted hourly in MPC algorithm or in 1-2 hours in standard protocol group. RESULTS: Blood glucose levels were in the target range significantly longer in MPC relative to standard protocol group (26.3 +/- 2.1 hrs vs 20.3 +/- 2.5 hrs). Mean blood glucose was also lower using MPC algorithm (6.47 +/- 0.11 vs 6.72 +/- 0.23 mmol/l). On the contrary the target range was established faster using standard protocol (8.9 +/- 1.2 vs 10.3 +/- 0.9 hrs), duration of hyperglycaemia was the same in both groups (7.3 +/- 1.9 in standard protocol vs 7.3 +/- 1.3 hrs in MPC algorithm). Average 48-hours insulin dose was higher in MPC than standard protocol group (230.2 +/- 38.8 vs 199.1 +/- 27.8 IU/48 hrs). 2 hypoglycaemic episodes occured in 2 patients in standard protocol group. CONCLUSIONS: Our results show that the use of MPC algorithm result in more effective blood glucose control in critically ill patients than standard protocol.

[Monitoring of glucose concentration in critical patients, comparing arterial blood glucose concentrations and interstitial glucose concentration measured by microdialysis technique]. / Monitorování glykemie u kriticky nemocných pacientu: srovnání arteriálních a intersticiálníh hladin glukózy merených pomocí mikrodialýzy tukové tkáne.

INTRODUCTION: Recent studies have shown that normalization of blood glucose in critically ill patients by intensive insulin therapy significantly decreases their mortality and morbidity. The aim of our study was to compare interstitial glucose concentrations in subcutaneous adipose tissue (measured by microdialysis technique) and arterial blood glucose concentrations to test the suitability of subcutaneous adipose tissue for long-term placement of biosensors for glucose measurement in critically ill patients. PATIENTS AND METHODS: 20 patients (16 men and 4 women) after cardiac surgery hospitalized at postoperative intensive care unit were included into the study. Mean age was 68 +/- 10 years, BMI was 28.3 +/- 3.9 year. Only patients with glycemia higher than 6.7 mmol/l at a time of admission to the ICU were included. Samples for measurement of interstitial glucose concentrations were collected in 60 minutes intervals during 48 hours using microdialysis of the subcutaneous adipose tissue. Perfusion fluid was 5% mannitol, perfusion rate was 1 microl/min. Arterial blood glucose concentration was measured in 60 minutes intervals, absolute concentrations of interstitial glucose were calculated using ionic reference technique. RESULTS: Mean arterial glucose concentration during the study was 6.7 +/- 0.56 mmol/l, absolute concentration of glucose in interstitial fluid was 3.55 +/- 0.58 mmol/l. Mean correlation coefficient between arterial and interstitial concentrations was 0.77 +/- 0.15. CONCLUSION: Our study demonstrated good correlation between interstitial glucose concentrations in subcutaneous adipose tissue and arterial blood glucose concentrations in post-cardiac surgery patients. Further studies are needed to evaluate this relationship in patients with more severely disturbed perfusion of subcutaneous adipose tissue.

The influence of very-low-calorie-diet on serum leptin, soluble leptin receptor, adiponectin and resistin levels in obese women.

The aim of our study was to determine whether adipocyte-derived hormones leptin, adiponectin and resistin contribute to the improvement of insulin sensitivity after very-low calorie diet (VLCD). Therefore, serum levels of these hormones were measured in fourteen obese females before and after three weeks VLCD and in seventeen age- and sex-matched healthy controls. Body mass index, HOMA index, serum insulin and leptin levels in obese women before VLCD were significantly higher than in control group (BMI 48.01+/-2.02 vs. 21.38+/-0.42 kg/m(2), HOMA 10.72+/-2.03 vs. 4.69+/-0.42, insulin 38.63+/-5.10 vs. 18.76+/-1.90 microIU/ml, leptin 77.87+/-8.98 vs. 8.82+/-1.52 ng/ml). In contrast, serum adiponectin and soluble leptin receptors levels were significantly lower in obese women before VLCD than in the control group. No differences were found in serum glucose and resistin levels between the obese group before VLCD and the control group. VLCD significantly decreased BMI, HOMA index, serum glucose, insulin and leptin levels and increased soluble leptin receptor levels. The changes in serum adiponectin and resistin levels in obese women after VLCD did not reach statistical significance. We conclude that leptin and soluble leptin receptor levels were affected by VLCD while adiponectin and resistin concentrations were not. Therefore, other mechanisms rather than changes in the endocrine function of the adipose tissue are probably involved in the VLCD-induced improvement of insulin sensitivity.

Adipocyte-derived hormones in heroin addicts: the influence of methadone maintenance treatment.

Heroin addiction markedly affects the nutritional and metabolic status and frequently leads to malnutrition. The aim of our study was to compare circulating concentration of adipose tissue-derived hormones leptin, adiponectin and resistin in 12 patients with heroin addiction before and after one-year methadone maintenance treatment with the group of 20 age- and body mass index-matched healthy subjects. Basal serum leptin and adiponectin levels in heroin addicts were significantly decreased (3.4+/-0.4 vs. 4.5+/-0.6 ng/ml and 18.9+/-3.3 vs. 33.9+/-3.1 ng/microl, respectively; p 0.05) while serum resistin concentrations were increased compared to healthy subjects (10.1+/-1.2 vs. 4.6+/-0.3 ng/ml; p 0.05). Moreover, positive correlation of serum leptin levels with body mass index was lost in the addicts in contrast to control group. One year of methadone maintenance treatment normalized serum leptin, but not serum adiponectin and resistin concentrations. In conclusion, circulating concentrations of leptin, adiponectin and resistin are markedly altered in patients with chronic heroin addiction. These alterations appear to be relatively independent of nutritional status and insulin sensitivity.

[Detection of tumor circulating cells in patients with ocular melanoma]. / Prukaz cirkulujících nádorových bunek u pacientu s ocním melanomem.

The authors investigated a group of 51 patients (29 men and 22 women) with intraocular melanoma: 41 patients with melanoma of the chorioid, 10 patients with melanoma of the ciliary body. They evaluated the clinical and pathological finding according to the TNM classification recommended by UICC (International Union Against Cancer). In all investigated patients they assessed circulating tumour cells (melanocytes) in the peripheral blood stream based on the detection of mRNA tyrosinase and marker MART 1. When evaluating the presence of markers according to the diagnosis irrespective of time, they found in patients in the clinical stage of T2 choroidal melanoma a 19% positivity of different markers and very rare a concurrent positivity of both markers. Patients in the clinical stage T3 had a 51% positivity of one marker and 34% concurrent positivity of both markers. In melanoma of the ciliary body evidence of individual markers was positive in 17% and only in 11% both markers were positive concurrently. On comparison of therapeutic procedures from the aspect of development in time in patients treated by brachytherapy only rare positivity was found at the time of administration the radioactive plaque, following an eight-month interval after brachytherapy the positivity of markers increased to 28%. On evaluation of markers of choroidal melanoma and ciliary body melanoma resolved by enucleation had their positivity at the time of operation was 36%, and during check-ups up to one year or longer it persisted at similar levels. Concurrent presence of both markers before this operation was rare, during postoperative check-up examinations it was within a range of 23 and 33%. The presence of both markers was repeatedly proved in five patients with chooidal melanoma after enucleation of the eye, in four of them in direct correlation with a metastatic process.

[Comparison study of the HUMFGA and HTPO STR system in the Saxon and Czech population]. / Srovnávací populacní studie saského a ceského HUMFGA a HTPO STR systému.

High variability of repetitive DNA in a non-coding region of human genome affords possibilities for vestige identification and determination of their origin. For analysis of DNA many a time obtained from decomposed material, usually in a very small amount and sometimes partially degraded, polymerase chain reaction is considered as optimal technique. For identification of individuals is used an Amplified Fragment Length Polymorphisms (AmpFLP), which present repetitions 10 to 70 base pairs long in an amplification products of 170 to 1200 bp. In legal medicine PCR systems using tetrameric repetition polymorphism are widely used. These systems are highly sensitive, but in comparison to AmpFLP systems, imperceptibly influenced by nucleic acid degradation in cases of old or partially degraded samples. Aim of our study was to compare frequency data for 2 STR-systems, FGA and HTPO, both after PCR amplification, on 200 samples of Czech and 228 of German population. In FGA system of Czech population 14 different alleles and 44 genotypes were determined, while German population provided 13 different alleles and 47 genotypes. Furthermore, frequency of alleles and heterozygosity rate were determined. Heterozygosity rate for Czech samples was 85%, for German ones 87.1%. Alleles 21 and 22 were most frequent (approx. 20%) between both Czech and German population, followed by alleles 20, 23 and 24 (14-12%). Besides these data, three different transient alleles were detected. In HTPO systems of both population groups 12 different genotypes, consisting of 5 alleles, were determined. Genotypes 8/8 and 8/11 observed in 30% were the most frequent ones in both population groups. Heterozygosity rate for Czech samples was 62.5%, for German ones 65%. Results were statistically analyzed. In both population groups, no significant difference from Hardy-Weinberg's balance was found. Additionally, statistic parameters for forensic effectivity evaluation were constructed. Comparison of both populations using homogeneity tests provided non-significant difference. FGA system provides extraordinary high forensic exploitativeness and therefore is fully acceptable for vestige identification and determining of their origin. For significant determination of transient alleles analysis of fluorescein-labelled fragments in denaturing gel system should follow, using automated laser detection of fluorescence. Polymorphism of HTPO-system is significantly lower compared to FGA-system, but relatively small PCR product required as well as simple detection and imperceptible influence of nucleic acid degradation allow the use of HTPO for analysis of highly degraded DNA.

[Principles of gene therapy]. / Principy genové terapie.

Treatment of disease by gene transfer into the genome of target host cells has become possible. This article presents current clinical applications of gene therapy and describes possible corrections of genetic defects.

[Vectors used in gene therapy]. / Vektory pouzívané v genové terapii.

The ability to direct gene transfer to particular target cells is important to achieve a therapeutic effect. In this review we describe the development of methods for gene delivery to specific cell types.

Molecular aspects of malignant melanoma. Role of protooncogenes and oncosuppressor genes.

This review summarizes current knowledge of oncogenes and tumour-suppressor genes in development and progression of malignant melanoma.

[Recent findings on the effect and role of oncogenes in carcinogenesis]. / Novejsí poznatky o vlivu a úcasti onkogenu v kancerogenezi.

The tumourous phenotype of cells may be induced by physical, chemical and biological factors. All have the same target, the cell genoma where they can produce minor (e.g. point mutations) or major changes (e.g. reconstruction--chromosomal aberrations, or gene insertion--oncoretroviruses). At present it may be assumed that these factors participate in the activation of proto-oncogenes (cellular oncogenes, c-onc) or in the inactivation of yet not well recognized "tumour suppressor" genes, so-called "anti-oncogenes". So far it was found that deletion of both alleles of "suppressor" genes causes the development of several recessive hereditary tumours (retinoblastoma, Wilms tumours etc.). Activation of proto-oncogenes (by point mutation, amplification, translocation) was found in a number of advanced primary tumours and in metastases. Probably activation of proto-oncogenes will play a more important part in the development of "acquired" tumours and in their progression. The problem of the mutual relationship of proto-oncogenes and "anti-oncogenes" remains so far obscure and controversial.

G-banding chromosome studies of acute lymphoblastic Lewis rat leukemia (KPH-Lw-I).

Changes of karyotype in spontaneous acute lymphoblastic Lewis rat leukemia have been studied by conventional Giemsa staining method and by G- and C-banding techniques. Comparing with previously published normal findings in first passages on rats, an increasing number of breaks, gaps and fragments in the 5th and 14th passages has been proved. Chromosomal investigation performed after two-year transplantation of leukemia on syngenic animals revealed pseudodiploid karyotype of leukemic lymphoblasts with the persistence of cell line 42, XX, del 2, -7, -18, +2 mar, that remained unchanged up to the latest examination in April 1984. Remarkable stability of chromosomal changes such as del 2 and 7/18 translocation might indicate possible insertion sites of transforming (viral) agent and/or localization of putative oncogenes.

Atherosclerotic aneurysms of the superficial femoral artery: a literature review and report of six additional cases.

Six patients with seven superficial femoral artery aneurysms are described, and additional cases in the literature are reviewed. Superficial femoral artery aneurysms are found in elderly patients with advanced atherosclerosis. These patients usually present with signs and symptoms of a high mass, often with local expansion and rupture. Proximal and distal ligation with vein bypass grafting was, for the authors, a satisfactory method of treatment. Patients with this lesion should be screened for possible abdominal aortic aneurysms, present in 33% of the authors' patients, and for other peripheral aneurysms, present in 50% of the authors' patients. Superficial femoral artery aneurysms should be surgically repaired, because when untreated, they tend to rupture and occasionally serve as a source of emboli.