Expression of genes encoding kinin receptors in peripheral blood mononuclear cells from patients with acute coronary syndromes.

BACKGROUND: Inflammation plays a critical role in all stages of atherogenesis, including plaque destabilization leading to the rupture and local thrombosis, clinically manifested as unstable angina (UA) or myocardial infarction (MI). Recent data report enhanced expression of numerous pro-inflammatory genes in patients with acute coronary syndrome (ACS) both in plaque and in inflammatory cells. Kinins are peptides involved in vasodilation, vascular permeability, pain and inflammation. Their effects are mediated by two receptors, B1 and B2. As the role of kinins in ACS is not clear, the aim of the study was to assess the expression of the genes encoding kinin receptors in patients with ACS. METHODS: The study was carried out on 40 patients with ACS and 10 age-matched healthy subjects (control (C)). To evaluate gene expression of B1 and B2 kinin receptors, total mRNA was extracted from peripheral blood mononuclear cells and the number of mRNA copies was assessed by quantitative reverse transcriptase-polymerase chain reaction. RESULTS: In patients with MI and UA, the B1 receptor (B1R)/B2 receptor (B2R) ratio was inversed compared with healthy subjects (C group) (MI vs C: 1.54 +/- 0.39 vs 0.36 +/- 0.04; P < 0.01; UA vs C: 2.13 +/- 0.98 vs 0.36 +/- 0.04; P < 0.05 respectively). B2R gene mRNA level was markedly lower in MI group versus C group (24 216 +/- 5409 copies/microg vs 39 908 +/- 5309 copies/microg; P < 0.05). The difference in B1R gene expression between MI and C group was negligible. We have not observed differences in studied genes expression between UA and C groups. CONCLUSION: Patients with ACS show inverted B1R/B2R ratio. Such disturbance in kinin signalling may reflect increased activation of circulating mononuclears, which are important participants of atherosclerotic plaque development and eventually rupture.

Differential diagnosis of gingival hyperplasia based on IFN-gamma-stimulated gene expression using RT-PCR.

Epulus is a benign gingival tumour of unknown aetiopathogenesis. Classification is inconsistent, and standard management strategies are lacking. Epuli are generally believed to be inflammatory rather than neoplastic lesions. The literature does not present any molecular analysis of the tumour characteristics. The purpose of the present study was to compare benign (epulus) and malignant (cancer) gingival hyperplasias with regard to the activity of the genes of apoptosis, proliferation, and inflammation using RT-PCR. The investigation involved 70 patients with epuli and 15 patients with gingival squamous cell carcinoma. Each subject had specimens collected from the tumour, tissue margin (incision line), and healthy tissue. Molecular investigations by RT-PCR were used to evaluate expression levels of the genes associated with apoptosis (Bcl-2, Bax, Bcl-2/Bax), proliferation (H3 histone), and inflammatory processes (IFN-gamma, IFNgamma-R1, IFN-gammaR2, IFN-gammaR1/IFN-gammaR2). Correlations have been disclosed between apoptosis and proliferation genes expression in giant cell epuli and high-differentiated gingival squamous cell carcinoma. In RT-PCR molecular analysis, giant cell epulus shows characteristics of a neoplastic lesion, while other epulus types seem to be inflammatory tumours.

Preliminary report of expression of bax in oral cavity pathologies.

Bax is considered one of major effectors of apoptosis--programmed cell death. Immunohistochemical analysis of in vitro patterns of bax expression was mostly investigated in mammalian cell lines and tissues. The present study is the first in vivo molecular analysis of bax expression in oral cavity pathologies. The study population consisted of 45 patients with hyperplasia, neoplasm in situ malignancy, and carcinoma. Biopsies were taken from incision line, tumour section, and healthy tissue. bax expression was investigated depending on the site of biopsy material sampling and final histopathology result. No statistically significant difference was demonstrated in bax expression between four hyperplasia subgroups. However, statistically significant differences in bax expression were found between the three basic study groups (P = 0.001). Statistically significant differences in bax expression were demonstrated depending on tissue collection site (P = 0.0002). We conclude that differences in bax expression may play a role in the pathogenesis of neoplastic disease.

TGF-beta1 mRNA expression in liver biopsy specimens and TGF-beta1 serum levels in patients with chronic hepatitis C before and after antiviral therapy.

BACKGROUND AND OBJECTIVE: Transforming growth factor (TGF)-beta1 is the best-characterized profibrogenic cytokine. TGF-beta1 increases the production of extracellular matrix proteins and their receptors and inhibits the synthesis of matrix degrading proteolytic enzymes. We undertook this study to simultaneously evaluate the effect of interferon alpha 2b plus ribavirin therapy on TGF-beta1 daily serum levels and on mRNA TGF-beta1 expression in liver biopsy specimens from 60 patients with chronic hepatitis C. METHODS: Serum levels of TGF-beta1 were measured by ELISA. The levels of the RNAs in liver biopsy specimens were measured by quantitative reverse transcriptase polymerase chain reaction. After treatment, patients were divided into two groups: 34 responders [undetectable hepatitis C virus (HCV)-RNA, normal ALT levels, decrease in histology activity index compared with pretreatment liver biopsy] and 26 non-responders (detectable HCV-RNA, elevated ALT levels, no decrease in the histology activity index). RESULTS AND DISCUSSION: In patients with hepatitis C, the 'responders' to the antiviral treatment showed significant decreases in both mean daily serum TGF-beta1 levels and mRNA TGF-beta1 expression in the liver biopsy specimens. The 'non-responders' serum TGF-beta1 concentrations did not change significantly, but the mRNA TGF-beta1 expression did. CONCLUSION: Both serum TGF-beta1 concentration and mRNA TGF-beta1 expression in liver biopsy specimens may be useful as prognostic markers in patients with hepatitis C undergoing antiviral therapy.

Human telomerase RNA as endogenous control in endometrial tissue.

Telomerase is a reverse transcriptase that adds repetitive telomere sequences to the end of chromosomes, which is thought to be essential for cellular immortality and oncogenesis. The enzyme consists of three subunits: human telomerase reverse transcriptase (hTERT), human telomerase RNA (hTR), and telomerase protein 1 (TP1). The hTERT subunit determines the activity of telomerase as an enzyme and is detected in most human tumors and regenerative cells. But many studies have revealed that hTR and TP1 are expressed constitutively. This results suggest that the hTR and TP1 subunits may be potentially good markers of endogenous RNA control. Endometrial dating was determined from the pathomorphology of the endometrium and classified into normal proliferative endometrium, endometrial hyperplasia (simple, complex, and atypical), and endometrial adenocarcinoma. The analysis of the expression of the hTERT, TP1, and hTR telomerase subunits was performed by a quantitative polymerase chain reaction method, based on fluorescent TaqMan methodology (ABI Prism 7,700 Sequence Detection System) capable of measuring fluorescence in real time. The aim of the study was an analysis of the expression profiles of genes encoding hTR and TP1 telomerase subunits in normal endometrium, endometrial hyperplasia, and adenocarcinoma for the estimation of the possibility of once application in endogenous RNA control of gene analysis in the endometrium. The nonparametric Mann-Whitney U test and analysis of variance Friedman test were used to evaluate the variation in telomerase subunit mRNA level between normal endometrium, and endometrial hyperplasia and adenocarcinoma. The results confirm the hTR subunit expression as a good marker of endogenous control in quantitative analysis of gene transcription in endometrial tissue. TP1 subunit transcriptions have not been detected constitutively in our study.

Sequence analysis of proviral DNA of porcine endogenous retroviruses.

Among all species analyzed, the domestic pig seems to be the most appropriate organ donor for xenotransplantation. Porcine endogenous retroviruses (PERVs) are present in genomes of all pigs and are capable of infecting human cells in vitro thus posing a serious threat for xenotransplantation procedures. Despite the abundant distribution of PERVs integrated with porcine genome, the majority of PERV proviral DNA is not capable of expressing viral proteins unless seriously mutated. The aim of the study was to analyze PERV genome for mutations. The study was performed on blood samples from 146 pigs. Long-range polymerase chain reaction (Long-PCR) was performed with primer sets designed within long terminal repeats (LTRs). Long-PCR products of different molecular weights were obtained: 530 bp (33.1% of individuals), 580 bp (76.7%), 933 bp (100%), and 2900 bp (59.8%). Amplimers of 7200 bp were absent in 12.8% of individuals, indicating the lack of intact proviral DNA. Sequence analysis showed that most PERV proviral DNA was significantly mutated, thus suggesting the inability to express functional viral RNA; however, it cannot be ruled out that compensatory recombination processes could occur enabling replication of defective proviruses.

Human cytomegalovirus DNA level in patients with idiopathic pulmonary fibrosis.

The objectives of the study were to estimate human cytomegalovirus (HCMV) DNA copy number in broncho-alveolar lavage cells, blood leukocytes, and serum of patients with idiopathic pulmonary fibrosis (IPF). The study groups consisted of 16 patients, newly diagnosed with IPF and never treated, (mean age 40.9 +/-11.0 yr; F/M-7/9) and in 16 adult healthy volunteers (mean age 36.8 +/-6.4 yr; F/M-4/12) used as controls. The HCMV DNA copy number was calculated by a Q-PCR method using TaqMan ABI PRISM 7700. We found that the prevalence of the HCMV DNA positive subjects in the patient group (75%) did not differ significantly from that in the control group (69%). We also found that in both patient and control groups the mean HCMV DNA copy number in BAL cells was significantly higher than that in blood leukocytes (log10=2.7 vs. 1.2 for patients and 2.8 vs. 0.9 for controls, respectively). However, a higher HCMV DNA copy number in blood serum was observed in IPF patients than in controls (log10=3.2 vs. 2.0, respectively). We conclude that the lungs play an important role in the human pathobiology of cytomegalovirus sustenance.

Smooth muscle cell apoptosis in primary varicose veins.

OBJECTIVES: One of the important factors responsible for vessel wall remodelling is programmed cell death. In the paper the role of smooth muscle cell (SMC) apoptosis in primary varicose veins (PVV) is investigated. MATERIAL AND METHODS: Vein specimens were obtained from 40 patients with PVV. In each case proximal and distal (upper crural) great saphenous veins (GSV) were harvested. Morphometric computer assessed quantitative evaluation of SMCs, collagen and elastin content was carried out. Apoptotic cells were detected by TUNEL assay. The levels of p53, BAX, BCLl-2 and p21 mRNA expression were assessed by real time RT-QPCR and the presence of respective proteins in the vessel wall was confirmed by immunohistochemistry. RESULTS: In the proximal GSV segments a significant increase of p53, p21 and BCL-2 mRNA levels was found in PVV patients. In the distal segments BAX and BCL-2 expression levels were higher. Taking into account the patient age, elevated p53 mRNA expression level was noticed in the distal incompetent GSVs of young PVV patients. In this group a statistically significant increase in the apoptotic index (APIx) within the vein media was found which correlated positively with p53 mRNA expression level. There was no increase of the apoptotic activity in elderly patients that led to the structural changes increase. In proximal GSV segments, despite SMC amount reduction or presence of structural changes in perivalvular wall region, no increase of the APIx with was noticed. CONCLUSIONS: P53-related apoptosis is one of the regulatory mechanisms of vein wall homeostasis maintenance. During varicose vein development its activation is related to the early stages of the disease. In the further course, the down-regulation of the SMC apoptosis within the vein media leads to the structural changes increase. The reduction of the SMC population corresponding to an increase of p21 expression in proximal saphenous vein segments suggests that the cell cycle disturbances may lead to the 'weakness' of the proximal GSV wall. Valve injury is not the only factor leading to the varicose veins occurrence.

Genetic disregulation of gene coding tumor necrosis factor alpha receptors (TNFalpha Rs) in colorectal cancer cells.

The expression of TNF ligand by malignant cells might be a mechanism for tumour immune escape. Genetic disregulation of gene coding TNF receptors was observed in neoplastic disease by an increased number of receptors on tumour cells and ligand-receptor activity. It might cause tumour proliferation and metastatic potential. Structure of TNF receptors influences TNF activity in vivo and structure of TNF R2 gene may suggest post-transcription modification based on alternative splicing. The aim of the study was to analyse the expression of gene coding TNF receptors R2 and R2/R7 (without exon 7) by estimation of mRNA expression of colorectal cancer cells in comparison with surrounding tissue free from neoplastic infiltration and searched for differently spliced TNFalphaR2/R7 isoforms. The study included fifty four patients with histopathologically confirmed adenocarcinoma (Stage III according to the AJC TNM Classification). Tissue samples removed from the tumour region were obtained from colorectal cancer patients undergoing surgical treatment. The samples were divided into two parts. The first one--was routinely examined histopathologically, the second one--was used for RNA extraction and the number of TNF and its receptors mRNA copies were subsequently quantified. The TNF and TNFRII genes expression were estimated based on the number of mRNA copies on 1 microg total RNA. The presence of TNFR2 and TNFR2/R7 isoforms in tumour, normal and metastatic cells was observed. The highest number of mRNA TNF copies and over expressed TNF genes were investigated and significantly noticed in metastatic cells (lymph nodes). The decreased number of TNFR2/R7 mRNA copies in metastatic lymph nodes secondarily influenced the decreased TNF soluble receptors' concentration. In conclusion, the genetic disregulation observed in neoplastic disease usually concerns dysfunction of cytokines receptor genes.

The delay of anoikis due to the inhibition of protein tyrosine dephosphorylation enables the maintenance of normal rat colonocyte primary culture.

Apoptosis induced by detachment of cells from the extracellular matrix (anoikis) appears to be one of the main obstacles in attempts to establish long-term primary culture of normal colonocytes. In the present study, the dynamics of molecular events related to apoptosis of isolated normal rat colonocytes was investigated. The whole colonic crypts were isolated using collagenase/dispase digestion technique. DNA fragmentation typical for the apoptosis and the apoptotic morphology of cells were observed already at the end of their isolation. Considerable increase in caspase-3 activity was noted during the first two hours of cell cultivation. Delaying of apoptosis by treatment of cells with sodium orthovanadate, the specific protein tyrosine phosphatase inhibitor, was found to be possible. It may facilitate long-term culture of intestinal epithelial cells.

Biological activity of Desulfovibrio desulfuricans lipopolysaccharides evaluated via interleukin-8 secretion by Caco-2 cells.

BACKGROUND: Although Desulfovibrio desulfuricans species, besides existing in the natural environment, is also found in the human digestive tract, no information is currently available on its role in the intestinal ecosystem and its activity in regard to the intestinal mucosa. Bacterial products (lipopolysaccharides, LPSs) are generally known for their ability to trigger inflammatory response by stimulating cytokine expression, such as interleukin-8 (IL-8). METHODS: Colonic Caco-2 cells were exposed to LPSs isolated from the soil type and intestinal wild strains of D. desulfuricans bacteria. The amount of IL-8 secreted was measured by ELISA. The effects of sodium butyrate and cell preincubation with sodium butyrate on the IL-8 secretion in response to LPSs were also analysed. RESULTS: LPSs from D. desulfuricans down-regulated IL-8 secretion by the cells. Incubation of these cells with butyrate alone resulted in a dose-dependent stimulation of IL-8 release. Butyrate also modulated IL-8 secretion by cells stimulated with LPSs. CONCLUSIONS: Our findings suggest the lack of inflammatory response of intestinal mucosa in the presence of LPSs of D. desulfuricans. This response can be conditioned by the natural bacterial product, butyrate, which exerts a stimulatory effect on the IL-8 secretion and modulates its release in response to LPSs.

The implications of genetic mutations in the sodium channel gene (SCN5A).

Mutations in sodium channel alpha-subunit gene (SCN5A) result in multiple arrhythmic syndromes, including long QT3 (LQT3), Brugada syndrome (BS), an inherited cardiac conduction defect, sudden unexpected nocturnal death syndrome (SUNDS) and sudden infant death syndrome (SIDS), constituting a spectrum of disease entities termed Na+ channelopathies. These diseases are allelic disorders, if not the same disease with variable penetrance and variable modifiers worldwide. Interestingly, death occurs during sleep in all of these disorders, suggesting a common mechanism. To date, mutational analyses have revealed about 103 distinct mutations in SCN5A, of which at least more than 30 mutations are associated with LQT3, whereas the rest of the mutations are affiliated with the remaining sodium channel disorders. The majority of these mutations are missense. However, other types such as deletions, insertions, frameshifts, nonsense and splice-donor errors have also been reported.

Electron spin resonance investigations of human retinal pigment epithelium melanosomes from young and old donors.

Electron spin resonance (ESR) examinations of human retinal pigment epithelium melanosomes isolated from eyes of young and old donors were carried out. The examined ESR signal was a single line, which is characteristic for free radicals of eumelanin o-semiquinones. The content of free radicals related to melanosomes dry weight for samples from older donors (ages over 45 years) were higher than for sample from younger donors (between 14 and 22 years). Simultaneously, the content of free radicals calculated for one melanosome is constant and does not depend on age. The homogeneous broadening of the recorded ESR lines shows that there are no isolated spin packets in all investigated melanin samples. Slow spin-lattice (T1 approximately 10(-5) s) and fast spin-spin (T2 approximately 10(-8) s) relaxation processes occur in these samples. Saturation of the ESR lines at low microwave power was measured. High concentration of free radicals in melanosome samples was responsible for the fast spin-spin relaxation process.

Genetic disregulation of gene coding tumor necrosis factor alpha receptors (TNF alpha Rs) in follicular thyroid cancer--preliminary report.

UNLABELLED: TNF alpha receptors participate in programmed cell death. TNF R2 efficiently assists TNF R7 effects by ligand passing. Structure of TNF alpha receptors influences TNF activity in vivo and the structure of TNF R2 gene may suggest post-transcription modification based on alternative splicing. The aim of the study was to analyse the expression of gene coding TNF alpha receptors R2 and R7 by estimation of mRNA expression of differentiated thyroid carcinomas in comparison to surrounding tissue free from neoplastic infiltration and search for differently spliced TNF alpha R2/R7 isophorms. The study included seven patients with histopathologically confirmed follicular thyroid cancer. Tissue samples removed from tumor region were obtained from the follicular thyroid cancer patients undergoing surgical treatment. The samples were divided into two parts. The first one was routinely examined histopathologically, the second was used for RNA extraction and the number of TNF and its receptors mRNA copies were subsequently quantified. RESULTS: 1) The presence of TNF alpha expression was observed in all examined samples, in contrast to TNF R1 expression; 2) The high level of TNF alpha expression was noted both for typical and sought TNF R2/R7 isoforms and 3) A considerable number of samples displayed higher levels of TNF R2 isoforms than TNF R2/R7 mRNA expression. Genetic disregulation observed in neoplastic disease usually concerns dysfunction of cytokines receptor genes.

Efficiency of lipofection of adherent cells is limited by apoptosis.

Stability of gene expression and transfection efficiency plays the main role in the application of gene transfer method. In somatic cell gene delivery, expression of the gene product is limited by the function of the cell to which it is delivered. In the present study analyzing the lipofected adherent cells, we have shown that lower level of transgene: beta-galactosidase activity at later time period correlated with decrease in cell viability, which was shown to be due to apoptosis. Apoptosis following DNA uptake occurred only when DNA was present during lipofection.

The effect of prolonged ischaemia and cryopreservation on the cell viability of human aortic and femoral artery allografts.

The viability of the human arterial allograft cells depends on the time and method of vessel procurement and storage. In this study, an evaluation of the effect of the duration of 4 degrees C ischaemia and cryopreservation on human aortic and femoral artery allograft viability was performed. After the isolation of arterial wall cells, the identification of cultured cells was performed using mRNA analysis for estimation of smooth-muscle markers of differentiation: desmin and heavy-caldesmon. The viability of cells from the medial layer of the aortic wall ranged from 74 to 90% (61-79% for femoral arteries). Cold ischaemia time (from harvesting until the beginning of the preparation) is a statistically significant factor influencing smooth muscle cell viability. Smooth muscle cells represented the majority of live cell population.

Positive and negative strands of HCV-RNA in sera and peripheral blood mononuclear cells of chronically hemodialyzed patients.

BACKGROUND: Hepatitis C virus (HCV) RNA can be detected in sera and peripheral blood mononuclear cells (PBMC) of patients undergoing chronic hemodialysis. However, the natural course of HCV infection in this group of patients is not fully known. Although the exact mechanism of HCV replication is not completely explained, there is evidence that HCV replicate through synthesis of complementary negative (-) RNA strand, whereas positive (+) RNA strand serves as a template. Thus, the detection of negative HCV-RNA strand can be regarded as a marker of ongoing viral replication. MATERIAL AND METHODS: We investigated the prevalence of (+) and (-) strands of HCV-RNAs in sera and PBMC of 45 chronically hemodialyzed patients using PCR methods. We also determined HCV genotypes and their subtypes by Inno-LIPA method. RESULTS: Eight (17.8%) of analyzed patients were anti-HCV positive. In this group, we detected HCV-RNA (+) strands in sera and PBMC in 2 and 4 cases respectively, whereas HCV-RNA (-) ones were found in PBMC of 4 patients. Among the remaining 37 anti-HCV negative patients we found HCV-RNA positive strands in sera and PBMC in 2 and 3 cases respectively, whereas HCV-RNA negative strand was present in PBMC in one of them. CONCLUSIONS: Our results indicate that HCV actively replicate in PBMC in chronically hemodialyzed patients. In number of patients HCV-RNAs can be detected only in PBMC without concomitant presence of viremia or anti-HCV in sera. We did not find any correlation between genotypes of HCV and presence of HCV-RNAs strands in PBMC of the patients.

[The assessment of expression of kininogen and bradykinin receptors genes in different vulvar pathologies by QPCR (TaqMan)]. / Ocena ekspresji genów kodujacych kininogen oraz receptory kinin w róznych stanach patologii sromu metoda QPCR (TaqMan).

OBJECTIVES: In different vulvar pathologies inflammatory process and pain are often observed. In these processes, kinins, released from kininogen, play an important role. Their effects are mediated by at least two types of bradykinin receptors--B1 and B2. B1 receptor appears in certain pathological states, B2 is widely distributed in normal tissues. The expression of genes coding kininogen, B1 and B2 receptors can be a very sensitive marker of tissue pathology. DESIGN: In the present study, the analysis of expression of genes coding kininogen, B1 and B2 was performed. The relation between the analysed genes expression and the pathology stage was analysed. MATERIALS AND METHODS: The specimens from condylomata accuminata, vulvar cancer and surgical margin were analysed. The number of DNA and mRNA copies of beta-actine, kininogen, B1 and B2 were examined basing on Q-PCR standard curves for beta-actine by use of Perkin Elmer-kit and the sequence detector ABI PRISM 7700-Taq Man application. RESULTS: In condylomata accuminata the high expression of mRNA of kininogen, B1 and B2 was found, while in vulvar cancer tissue, the expression of analysed genes was low. In the tissue from the tumour center, the lowest kinin genes expression was stated. CONCLUSIONS: The absence of kininogen and B2 mRNA expression characterised vulvar cancer tissue. The profile of expression of kininogen and its receptor genes can be a useful marker in the assessment of vulvar cancer surgical margin.

[The quantitative analysis of E6 HPV and P53 genes expression by QPCR (TaqMan) in the assessment of surgical treatment range in vulvar carcinoma induced by HTV infection]. / Ilosciowa analiza ekspresji genów E6 HPV 18 i P53 metoda QPCR (TaqMan) w ocenie marginesu operacyjnego w raku sromu indukowanym infekcja HPV.

OBJECTIVES: Human Papilloma Virus (HPV) infections especially 16 and 18 are risk factors for squamous cell vulvar cancer. E6 protein of HPV joins the tumor suppressor protein P53 and promotes its degradation. This is one of the possible mechanisms of viral oncogenes action. DESIGN: In this study the quantitative analysis of mRNA copies E6 HPV 18 and mRNA P53 expression in vulvar cancer tissue was performed. The expression of analysed genes was applied in the assessment of surgical treatment range. MATERIALS AND METHODS: The specimens from a 26 year old woman with vulvar squamous cell cancer stage II FIGO treated surgically modo Way were analysed. The number of DNA and mRNA copies E6 HPV and P53 were examined basing on Q-PCR standard curves for beta-actine by use of Perkin Elmer-kit and the sequence detector ABI PRISM 7700-Taq Man application. RESULTS: The overexpression of mRNA E6 HPV and P53 in analysed specimens was found. The highest number of mRNA copies in cancer tissue was ascertained. In lichen sclerosus and lymphonoduli tissue lower number of analysed copies was found. CONCLUSION: The quantitative analysis of E6 HPV and P53 genes expression can be useful in the assessment of surgical treatment range in vulvar cancer and can also be used as a prognostic marker.

[Prenatal diagnostics of Toxoplasma gondii invasion by quantitative method of PCR-TaqMann]. / Prenatalna diagnostyka zarazenia pierwotniakiem Toxoplasma gondii metoda ilosciowej polimerazowej reakcji lancuchowej PCR-TaqMann.

Routine serological diagnosis of toxoplasmosis provides high sensitivity, but not high specificity. The high sensivity combined with high specifity offered by PCR-TaqMann as well as the degree of infection led us to investigate the presence and levels of T. gondii DNA in amniotic fluid, maternal and neonatal blood in cases of pregnancy where infection with this agent was suspected. Samples of amniotic fluid and blood were taken from pregnant women. Postnatal blood samples were also taken from their infants. Presence and levels of toxoplasma DNA was investigated using PCR-TaqMann. PCR products were detected by electrophoresis on polyacrylamide gel. The PCR-TaqMann test is highly sensitive, specific and useful method allowing detection of the parasite genome and assessement of its level.