P53 codon 72 polymorphism and correlation with ovarian and endometrial cancer in Greek women.

The polymorphism of codon 72 in the p53 tumour suppressor gene has been associated in the last decade with the risk of developing various neoplasias. An influence of this polymorphism on ovarian and endometrial cancer has also been suggested. We examined the genotype frequency of this polymorphism in archival samples from 56 patients with endometrial neoplasias and 51 patients with ovarian neoplasias. Cervical smears from 30 healthy, human papillomavirus (HPV)-negative women with normal cytology and colposcopy, served as control sample. Women with ovarian neoplasias, especially adenocarcinomas, had Arg/Arg more often than healthy controls [odds ratio (OR) 4.16 at P = 0.0058]. No statistically significant difference was found between women with endometrial cancer and controls. Differentiation of ovarian tumours did not appear to be associated in a statistically significant manner with the genotype, whereas a positive linear trend of Arg/Arg towards poor differentiation was noted in endometrial malignancies (mainly endometrioid adenocarcinomas). Our results suggest that homozygous arginine at codon 72 of p53 may represent a risk factor for developing ovarian malignancies and may affect the differentiation of endometrial cancer. Further studies need to be carried out in order to establish the clinical use of this polymorphism for risk assessment and possibly outcome prediction of ovarian and endometrial neoplasias.

Methylenetetrahydrofolate reductase polymorphism C677T is not associated to the risk of cervical dysplasia.

The aim of the study was to explore a possible association between methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and cervical neoplasia. A total of 229 women were subjected to cytologic and colposcopic evaluation. Ninety-one of them were found to be normal, and served as the control group, while the other 138 of them had present or past histologically proven cervical pathology (patients group). All patients and controls were investigated for the MTHFR C677T polymorphism. Statistical analysis between the groups of cases with cervical intraepithelial neoplasia or invasive cervical cancer and the control group did not reveal any statistically significant difference in the frequency of the MTHFR C677T polymorphism.

p53 codon 72 polymorphism and risk of intra-epithelial and invasive cervical neoplasia in Greek women.

In 1998, Storey and co-workers suggested that individuals homozygous for arginine (Arg) at codon 72 of the p53 gene are about seven times more susceptible to human papillomavirus (HPV)-related carcinogenesis than heterozygotes. Since then, several studies from Northern Europe, Japan and the USA have failed to demonstrate a similar correlation. By contrast, a study in Brazil as well as one recent study in Italian and Swedish populations showed strong positive associations. We examined the frequency of p53 codon 72 polymorphism in samples from both invasive and intra-epithelial cervical neoplasias (CIN), and compared them with samples from healthy controls. All 88 samples came from women with a Greek ethnic background. Tissue specimens were collected from archival material with histologically diagnosed low-grade CIN (LGCIN), high-grade CIN (HGCIN) or cervical cancer (CxCa). As a control, we used cellular material newly collected by cytobrush from the cervices of 30 healthy women with normal cytological and colposcopical examinations. p53 Arg homozygosity (Arg/Arg) alone was associated with four-, six- or eight-fold increased risks for LGCIN, HGCIN or invasive cancer, respectively. The frequency of the p53Arg/Arg genotype and of the proline (Pro) allele showed significant linear trends according to the degree of severity of the lesion (P = 0.0007 and P = 0.0009, respectively). Exclusion of the ten HPV16/18-negative cases did not substantially alter the Arg/Arg frequency among the groups nor the significant linear trend. Our results confirm the initial findings of Storey and co-workers, as well as the data of the Brazilian and the recent European study, but do not accord with those of the other aforementioned studies. Variations in ethnic background, laboratory performance, verification of the HPV status, definition of controls, and sample size are the most plausible explanations for this controversy. In all our samples, the distribution of the p53 alleles fits the Hardy-Weinberg equilibrium and the 0.48 frequency of the Pro allele in our controls accords well with the percentages previously reported for different ethnic groups as characteristic of the assumed north-south cline. Some authors assert that the discrepancy in the results could not be attributed to differences in the methods; however, the Brazilian study emphasized the effect of inter-laboratory variation in detecting the association between p53 polymorphism and cervical cancer. Regarding the control group, our samples were only from women with a cytologically and colposcopically benign cervical epithelium. We think that simply choosing 'normal volunteers' for collecting control DNA blood samples without knowing the status of their cervical epithelium is indeed a possible source of bias. Finally, it is very unlikely that loss of heterozygosity at the p53 locus could be a factor interfering with the allelotype distribution. Our present small study results, which suggest a biologically relevant association, provide strong evidence that homozygous arginine at codon 72 of p53 may confer a higher susceptibility to HPV-associated intra-epithelial and invasive cervical neoplasia.

Factor V leiden and prothrombin G20210A mutations, but not methylenetetrahydrofolate reductase C677T, are associated with recurrent miscarriages.

The aim of this study was to investigate the relationship between recurrent miscarriages and factor V Leiden, prothrombin G20210A and C677T methylenetetrahydrofolate reductase (MTHFR) mutations. In this case-control study the prevalence of factor V Leiden, prothrombin G20210A and C677T methylenetetrahydrofolate reductase mutations was determined in a consecutive series of 80 recurrent miscarriage patients and 100 controls. Fifteen of 80 recurrent miscarriage patients and four out of 100 controls carried the factor V Leiden mutation (19 versus 4%, P = 0.003, odds ratio 5.5, 95% confidence interval (CI): 1.7-17). Seven of 80 recurrent miscarriage patients and two of 100 controls were carriers of the prothrombin G20210A mutation (9 versus 2%, P = 0.038, odds ratio 4.6, 95% CI: 0.9-23.2). Six of 80 recurrent miscarriage women and 15 of 100 controls were homozygotes for the C677T MTHFR mutation (8 versus 15%, P = 0.134, odds ratio: 0.4, 95% CI: 0.1-1.2). Our results suggest that the presence of factor V Leiden and prothrombin G20210A polymorphism, but not MTHFR C677T homozygosity, could be additional risk factors for recurrent miscarriages. Furthermore, it was suggested that the prevalence of factor V Leiden and prothrombin G20210A mutations is more prominent in second trimester, primary fetal losses and it is independent of the existence of additional pathology predisposing to recurrent fetal losses.

Application of polymerase chain reaction for detection of Legionella pneumophila in serum samples.

OBJECTIVE: To apply the polymerase chain reaction (PCR) to serum samples for the rapid diagnosis of Legionnaire's disease using the L5SL9 and L5SR93 primers designed to generate a 104-base-pair (bp) fragment from the 5S RNA gene of Legionella spp. The amplified product was detected by electrophoresis and by hybridization with the L5S-1-specific probe. METHODS: Single specimens of serum obtained from 24 patients with confirmed legionellosis, at different stages of their disease, were tested by PCR. Additionally, 10 serum samples from patients with no clinical symptoms of pneumonia and 10 samples from patients suffering from pneumonia caused by Mycoplasma pneumoniae, Coxiella burnetii or Chlamydia psittaci were also tested as controls in order to determine the specificity of the method. RESULTS: Of the 24 examined serum samples, the amplified products from 12 hybridized with the L5S-1 probe (sensitivity 50%). None of the negative controls was positive after PCR. No correlation was found between the day of illness and the positivity in the test. CONCLUSIONS: The PCR technique could be applied as a diagnostic tool for the rapid diagnosis of legionellosis in serum samples after modification, mainly to improve its sensitivity.

Incidence of human papillomavirus 6, 11, 16, 18 and 33 in normal oral mucosa of a Greek population.

The polymerase chain reaction (PCR) was applied for the detection of human papillomavirus (HPV) infection in samples obtained from the clinically normal mucosa of the oral cavity of 169 asymptomatic subjects in northern Greece. Of the subjects, 9.5% were found to be infected with HPV. Typing of HPV by Southern blot hybridization revealed that 2.4%, 0%, 0%, 4.1%, 0.6% of the subjects were infected with HPV 16, 18, 33, 6 and 11, respectively.

Factor V Leiden in Greek thrombophilic patients: relationship with activated protein C resistance test and levels of thrombin-antithrombin complex and prothrombin fragment 1 + 2.

We studied 172 Greek patients (72 men aged 44.0 +/- 16.7 years and 100 women aged 46.5 +/- 14.1 years) with an unexplained thrombophilic tendency. One hundred and four apparently healthy persons (63 men aged 34.2 +/- 10.0 years and 41 women aged 37.1 +/- 13.3 years) were included as a control group. We performed the activated protein C resistance (APC-r) test using a clotting test (Chromogenix kit), detection of factor V Leiden using polymerase chain reaction (PCR)-restriction fragment length polymorphisms and measurement of thrombin-antithrombin complexes (TAT) and prothrombin fragment 1 + 2 (F1 + 2) levels with an immunoenzymatic assay. The normal range for the APC-r test (> 2.12) was determined from the controls. The factor V Leiden mutation was found in 31.9% of all the patients tested, in 28.1% of the unrelated patients with documented thrombophilic tendency of unknown origin and in 4.8% of the healthy controls. The APC-r test had a sensitivity of 0.42 and a specificity of 0.91 for the detection of factor V Leiden. Furthermore, we found no significant difference in levels of TAT and F1 + 2 between patients with and without the mutation and there was no correlation between aPC-r values and levels of TAT and F1 + 2.

Rapid typing of 4 VNTR loci, 3'ApoB, MCT118,St14 and YNZ22 by the polymerase chain reaction of a Greek sample.

Allelic data from a Greek sample of unrelated individuals for the D17S30, 3'ApoB, D1S80 and DXS52 loci were obtained by the PCR and subsequent analysis with agarose gel electrophoresis. The distribution of observed genotypes is in agreement with expected values according to the Hardy-Weinberg equilibrium. An heterozygosity of at least 76% was demonstrated with 11-13 alleles for each locus. The use of the combination of the four loci could be a powerful tool for paternity testing and individual discrimination, in combination with the fact that PCR needs a minimal amount of DNA and is a very quick and sensitive method.

Epidemiology of human papillomavirus infection in Greek asymptomatic women.

In an attempt to estimate the prevalence of human papilloma virus (HPV) positivity among asymptomatic, cytologically normal Greek women, and the possible associations between HPV infection and other demographic, sexual, behavioural and sociological parameters, we undertook an epidemiological study of 226 clinically normal women from an outpatient gynaecological clinic in Northern Greece. The polymerase chain reaction was used for detection of HPV DNA and dot blot hybridization analysis for HPV typing (only for the high-risk types 16 and 18). Eighty-two of the 226 women examined (36.3%) were positive for HPV DNA, 6.6% (15/226) were positive for HPV-16 DNA and only 1.3% (3/226) were positive for HPV-18 DNA. From all epidemiological correlates, age and residence showed a negative correlation with risk of HPV infection, whereas use of contraceptive intrauterine device, class II or III result of the last Papanicolaou cytological examination, history of painful inflammatory disease of inner genitals and frequent washing of the genital area, particularly during the menstrual period, were positively correlated with increased risk of HPV infection. No association was found between HPV DNA positivity and other well-known risk factors for cervical cancer, confirming the observations of other authors that sexual behaviour, a significant risk factor for cervical cancer, is not inevitably correlated with risk of HPV infection.

Detection of human papillomavirus using the polymerase chain reaction and typing for HPV16 and 18 in the cervical smears of Greek women.

The polymerase chain reaction (PCR) was applied for the detection of human papilloma virus (HPV) infection, in samples obtained from the uterine cervices of 202 asymptomatic women with normal cytology in Northern Greece. About 41.8% of the women with microscopically and cytologically normal cervices were found to be infected with HPV. Typing of HPV revealed that 6.9% and 1.5% of the women were infected with HPV16 and HPV18, respectively.

Toxicity of Bacillus thuringiensis to laboratory populations of the olive fruit fly (Dacus oleae).

A survey of Bacillus thuringiensis recovered from the environments of olive groves in Greece was carried out. Of 80 soil samples, 24 were found to contain B. thuringiensis with parasporal crystal inclusions; these were tested for toxicity against the olive fruit fly (Dacus oleae). Mortality levels of larvae caused by the different isolates varied from 7 to 87%. Higher levels of mortality were observed if a mixture of relatively pure crystals and spores was used compared with the mortality resulting from either fraction alone. We were able to show that the toxicity of the most active isolate is likely to be specific for D. oleae.

Effect of interferon on the accumulation of RNA transcripts of genes coding for cellular and viral proteins.

Using plasmids containing sequences complementary to the genes that code for oligo-2',5'-adenylate synthetase, actin and H4 histone, we have shown that although interferon does not affect the accumulation of RNA transcripts of the actin and H4 histone genes, it activates the accumulation of RNA transcripts of the oligo-2',5'-adenylate synthetase gene. However, interferon inhibits the accumulation of RNA transcripts of simian virus 40 (SV40) genes in SV40-infected cells.

Induction of 2',5' oligo(A) synthetase in Vero cells.

2',5' oligo(A) synthetase can be induced in Vero cells with interferon; maximal levels are reached after treatment for 24 hours. This induction is a result of increased accumulation of RNA transcripts of the specific gene.