[Detection of regulatory protein p16/INK4A in the dysplastic cervical squamous cell epithelium is a diagnostic tool for carcinoma prevention]. / Detekcia regulacného proteínu p16/INK4A v dysplastickom dlazdicovom epiteli krcka maternice ako diagnostický nástroj prevencie karcinómu.

Parallel sections from 423 randomly selected blocks representing biopsies of 178 women with the diagnosis of cervical dysplasia and/or erosion were stained for p16 polypeptide. The p16/INK4A (inhibitory kinase 4) protein is a cellular division regulator, expression of which increases in the presence of oncoprotein E7, encoded by human papillomavirus (HPV). Expression of p16 protein was seen in the nuclei and cytoplasm of dysplastic squamous epithelium cells as well as in carcinoma cells. In 16.6% of erosion cases, the p16 antigen was present in the basal and suprabasal layer of the surrounding squamous epithelium revealing features of CIN I/LSIL. In CIN I/LSIL as classified by HE staining, the p16 antigen was found in 65 out of 80 (81%) cases. The p16 protein was typically seen in dysplastic basal and suprabasal cells encompassing a confluent layer in the lowest third segment of stratified epithelium. In CIN II and CIN III grouped as HSIL, the positive rate of p16 antigen presence was 95% (in 45 cases out of 47) and/or 100% (in each of 27 cases), respectively. The typical sign of p16 antigen distribution in HSIL was its staining over two thirds and/or throughout the whole dysplastic epithelium. Extensive staining for p16 antigen was registered within nuclei as well as cytoplasm of neoplastic cells in all 6 cervical squamous cell carcinomas, which were examined in many sections when being used as positive controls. Based on our experience, we consider the p16 antigen staining a helpful tool indicating dysplastic cells and estimating their extent.

[Factor V Leiden and the Slovak population]. / Faktor V Leiden a slovenská populace.

The resistance to activated protein C (APC-resistance) based on the presence of factor V Leiden (F V Leiden) is the most frequent thrombophilic condition in the white race population. It contributes to the origin of thrombosis especially in the venous part of blood vessels. Significant geographic differences have been detected within Europe. The aim of this retrospective study was to determine the frequency in the occurrence of F V Leiden: 1. in healthy (asymptomatic) Slovak population, 2. in their consanguineously unrelated members with thrombosis and 3. in patients with myocardial infarction (IM) without or with other known risk factors of this disease (nicotinism, obesity, hypertension, dyslipoproteinemia, diabetes mellitus), respectively. The detection of FV Leiden was made by molecular biology methods. The occurrence in a group of 152 healthy individuals was four % (6 persons) and this frequency corresponds to the geographic localization of the Slovak Republic in Europe. In a group of 349 patients with thrombosis in anamnesis, FV Leiden was detected in 103 persons (29.5%). The occurrence was higher than the usually reported incidence in these patients (20%). Likewise, in a group of 35 patients with IM without risk factors in anamnesis, the occurrence of FV Leiden (8.6%) was significantly higher in comparison with healthy population and the incidence further increased significantly in a group of 41 patients with IM and the presence of at least one risk factor (14.6%). The authors therefore suppose an active role of the Leiden mutation of FV gene in the pathogenesis of this disease.

Cytotoxic T lymphocyte antigen 4 (CTLA-4) dimorphism in patients with systemic lupus erythematosus.

OBJECTIVE: Genetic susceptibility to systemic lupus erythematosus (SLE) is conferred not only by various genes within the major histocompatibility complex (MHC) region, but also by several other non-MHC linked genes. The negatively signalling molecule CTLA-4 is involved in establishing and maintaining of peripheral T cell tolerance, which controls T cell activation and reactivity. Its attenuating action helps to prevent an inappropriate initiation of T cell responses to self antigens and to terminate ongoing T cell responses. We tested if there was an association between CTLA-4 and SLE, a disease with B and T cell hyperreactivity and impaired peripheral T cell tolerance. METHODS: Using the polymerase chain reaction--restriction fragment length polymorphism method with Bbv I digestion, we assessed an exon 1 transition dimorphism (49 A/G) of the CTLA-4 gene in 102 SLE patients and in 76 healthy controls. RESULTS: The distribution of CTLA-4 exon 1 genotypes in the SLE group was significantly different from that in the controls (chi 2 = 6.178, p < 0.05). 17.6% of the SLE patients were G/G homozygotes compared to 5.3% of the controls; 36.3% were A/G heterozygotes vs 40.8% of controls; and 46.1% were A/A homozygotes vs 53.9% of the controls. The frequency of the G allele was significantly higher in SLE patients (35.8%) than in controls (25.7%; chi 2 = 4.142, p = 0.042). CONCLUSION: Our results indicate that the non-MHC linked CTLA-4 gene could confer susceptibility in SLE, as it does in various other autoimmune diseases (Hashimoto thyroiditis, Graves' disease, IDDM).

Association between systemic lupus erythematosus and insertion/deletion polymorphism of the angiotensin converting enzyme (ACE) gene.

OBJECTIVE: ACE takes part in the renin-angiotensin and kallikrein-kininogen systems by creating angiotensin-II and inactivating bradykinin. ACE gene insertion/deletion polymorphism is associated with the level of circulating enzymes--subjects with the DD genotype have higher levels of circulating ACE than subjects with the II genotype and show an increased tendency towards impaired vascular function and structure. Patients with systemic lupus erythematosus (SLE) suffer from differentially expressed vascular pathology. We attempted to determine whether the type of ACE polymorphism could contribute to this pathology. METHODS: 101 SLE patients fulfilling the ACR criteria were investigated. The I/D polymorphism was ascertained by PCR, followed by electrophoresis of the amplified fragments and UV visualization. RESULTS: The frequency of the D allele was higher in the SLE group (0.623) than in the controls (0.520) (chi 2 test, p < 0.025). The distribution of the ACE genotype in SLE group was different from that in the control group (p < 0.05). An association between the DD genotype and visceral damage (p < 0.006) was observed. CONCLUSION: Our results suggest that in the multifactorially determined vascular pathology of SLE, changes associated with I/D polymorphism could influence vessel wall inflammation (monocyte adhesion and activation with cytokine release, T-lymphocyte metabolism), a tendency towards vascular impairment (neointimal proliferation, vasospasm, platelet activation, myocyte proliferation) and lead to the subsequent ischemia. The ACE gene could serve as the visceral damage indicator in SLE.

[Endothelial dysfunction and lipid profiles: analysis of the EDO Study]. / Endotelová dysfunkcia a lipidový profil: analýzy stúdie EDO.

On the basis of experimental as well as clinical observations, endothelial dysfunction is (defined as impaired or absent endothelium-dependent relaxation) considered to be an important factor in the atherogenesis. Serum lipids abnormalities have been accepted as an epidemiological risk factor of atherosclerosis. In vitro, experimental as well as epidemiological studies revealed the fact, that lipoprotein oxidation plays an important role in atherogenesis. Recently invented non-invasive methods to test and measure the endothelial function in vivo opened the opportunity to study the influence of different serum lipids on the endothelial function directly. Therefore, we decided to employ this non-invasive method for studying the endothelial function and observe the influence of various levels of plasma lipids and lipoprotein oxidation on the endothelial function of arteries in middle-aged men, since they are the most endangered part of population. In our study we used a method of measuring the diameter of a. radialis by high-resolution ultrasound (Sonoline 450, Siemens, Japan) and further mathematical and statistical analysis of functional as well as relative vasodilation reserve followed these measurements. Blood samples were taken within 24 hours of ultrasonography to study serum lipids (total cholesterol, HDL, triglycerides) and parameters of oxidation/antioxidation (superoxid dismutase--SOD, malondialdehyde--MDA). Sixty men, 25-45 years old, from an area of basically the same level of pollution were examined. We found a negative correlation between FVDR and TCH (p = 0.01), FVDR and Tg (p = 0.002) and FVDR a TCH/HDL (p = 0.015). Positive correlation exists (p < 0.001) between TCH, Tg levels and TCH/HDL ratio and MDA level in all cases. Analysing further data from the EDO Study, we can conclude, that increased plasma lipids are more likely to be oxidized, which, in turn, is the probable reason of endothelium-dependent vasodilation impairment.

[Is PLA1/A2 gene polymorphism of platelet membrane glycoprotein IIIa a risk factor for myocardial infarct?]. / Je PLA1/A2 polymorfizmus génu pre trombocytárny membránový glykoproteín IIIa rizikovým faktorom infarktu myokardu?

BACKGROUND: The PlA1/A2 polymorphism of the human platelet membrane glycoprotein IIIa gene cause T-->C transition in the exon ii (position 1565) resulting in the leucine-->proline substitution in amino-acid sequence. This polymorphism was shown to be associated with increased risk of myocardial infarction (MI). AIM: To test genetic parameters of the PlA1/A2 polymorphism in our population and to assess the relation between mutant PlA2 allele and MI. METHODS: DNA was isolated from peripheral blood, collected from 40 patients with MI and with present risk factors (hypertension, hypercholesterolaemia, diabetes, obesity, smoking...), 33 patients with MI without risk factors, 34 controls with equivalent average age to both groups of the MI-patients, 58 control probands without MI in their family history and 33 healthy controls randomly recruited. After PCR amplification the resulting 267 bp fragment was digested with the restriction endonuclease NciI and subfragments were separated electrophoretically in 12% polyacrylamide gel. RESULTS: The frequency of the PlA2 allele was 0.121 in patients with MI without risk factors, 0.205 in patients with present risk factors, 0.162 in controls of the equivalent average age to the MI-patients, 0.172 in controls without MI in their family history and 0.20 in healthy controls randomly recruited. Genotype frequencies were in all groups in genetic equilibrium. Although the groups differed significantly (p < 0.01) in serum concentrations of total cholesterol, HDL-cholesterol, LDL-cholesterol, apolipoprotein A-1, apolipoprotein B and malondialdehyde, no significant differences in the serum concentrations of these metabolites between A1/A1, A1/A2 and A2/A2 genotypes were observed. CONCLUSIONS: PLA1/A2 polymorphism is associated with MI, however not as a dominant risk factor, but as a part of environmentally influencable multigene system. There is no relation between genotypes of the PLA1/A2 polymorphism and the lipoproteins plasma concentrations. (Tab. 4, Ref. 17.)

[Analysis of phenotype resistance to activated protein C (APC resistance)]. / Analýza fenotypu rezistence na aktivovaný protein C (APC-rezistence).

The laboratory diagnosis of resistance to activated C protein (APC-resistance) involves examination of the phenotype and genotype of this thrombophilia. For examination of the phenotype coagulation and chromogenic tests are used. Their essence is examination in the presence and absence of exogenous APC. While the result of the original coagulation examination of APC-resistance which uses the APTT principle is influenced by a number of factors, the sensitivity and specificity of the modification of this examination (dilution of the examined plasma sample by FV deficient plasma before making the test) in relation to detection of FV Leiden is almost 100% and eliminates the majority of limitations of the original examination. The chromogenic assessment of APC-resistance has similar advantages, however, it cannot differentiate between the heterozygous and homozygous form of FV Leiden. During examination of the genotype of subjects with APC-resistance the mutation of FV Leiden is detected in as many as 90%. The group of subjects with the phenotype of APC-resistance comprises in particular subjects with acquired APC-resistance caused by conditions which lead to a disbalance between procoagulation and anticoagulation proteins of haemostasis which influence the reactions of the applied laboratory examinations. The acquired phenotype of APC-resistance can be also associated with an increased risk of thrombosis and the clinical manifestations of this thrombophilia resemble the classical, FV Leiden conditioned APC resistance. Rarely also congenital causes of the phenotype of APC-resistance are encountered caused by another mutation than the Leiden mutation of gene FV. The concurrent examination of the patient's plasma with the original and modified coagulation test makes it possible to assess the inborn cause of APC-resistance (positive finding also in modified examination). The presence of FV Leiden is then confirmed by examination of the genotype by the polymerase chain reaction.