G. Sourvinos

Overexpression of the Tpl-2/Cot oncogene in human breast cancer

Tpl-2/Cot proto-oncogene encodes a serine threonine kinase and was initially cloned as a provirus insertion site in MoMuLV-induced T cell lymphomas in rats. Tpl-2 locus was also shown to be affected by provirus insertion in MMTV-induced mammary carcinomas in mice. The involvement of Tpl-2 in 35 human breast paired tumour specimens versus their corresponding adjacent normal tissue was evaluated. Tpl-2 was found overexpressed in 14 of the 35 breast tumours tested using a semi-quantitative RT – PCR method. Gene amplification was detected in eight out of the 14 specimens overexpressing Tpl-2, suggesting the increased number of copies of Tpl-2 gene as a possible mechanism for Tpl-2 overexpression. Significant association was found between the overexpression of Tpl-2 and stage I of the tumours, indicating that this molecular alteration may be an early event in the development of the disease. Furthermore, overexpression of Tpl-2 was associated with positive progesterone receptor status of the samples. This is the first report on the Tpl-2 oncogene linked to human breast tumours suggesting that it may be a key molecule for the study of human breast cancer.

Microsatellite instability and loss of heterozygosity in human pterygia.

AIMS/BACKGROUND Pterygium is a common benign lesion of the corneo-conjunctival limbus. Although environmental factors, such as ultraviolet irradiation, have been suggested as the main causative factor in the development of the disease, however, the aetiopathology of pterygium remains obscure. In this study the possibility of detecting genetic alterations in the microsatellite DNA of the pterygium was investigated. METHODS Fifteen specimens were assessed for loss of heterozygosity (LOH) and microsatellite instability (MI) by seven microsatellite markers on four chromosomal arms. RESULTS Nine (60%) pterygia exhibited genetic alterations. Eight specimens (53%) exhibited LOH, while two specimens (13%) MI in at least one marker. 17q11.2-q21 is a commonly deleted region, as the frequency of LOH at this region is significantly high (47%). CONCLUSION This finding indicates the existence of tumour suppressor genes in this region implicated in the disease without excluding the presence of other tumour suppressor genes in the other chromosomal regions that were examined. MI was apparent in only a few specimens but it is indeed a detectable phenomenon, suggesting that decreased fidelity in DNA replication and repair may be associated with the development of pterygium. Detection of LOH and MI, two events taking place in tumour cells or in premalignant cells, constitutes strong evidence that there must be transformed cells in the pterygial tissue and it should be considered to be a neoplastic benign lesion.

Evaluation of loss of heterozygosity and microsatellite instability in human pterygium: clinical correlations

AIMS To evaluate the incidence of loss of heterozygosity (LOH) and microsatellite instability (MI) in pterygia and their possible correlation with clinical variables. METHODS 50 pterygia, blood, and conjunctival specimens were obtained. A personal and family history was recorded for each patient. Amplification of 15 microsatellite markers at regions 17p, 17q, 13q, 9p, and 9q was performed using the polymerase chain reaction. The electrophoretic pattern of DNA from pterygia was compared with the respective pattern from blood and conjunctiva. RESULTS LOH incidence was the highest at 9p (48%), followed by 17q (42%). Only three cases displayed MI. LOH incidence at individual markers was positively correlated with recurrence (D9S59, p=0.11 and D9S270, p=0.16), family history of neoplasia (D13S175, p=0.09), altitude of present residence ( D9S112, p=0.1), duration of the existence of pterygium (D9S144, p=0.06), and inversely correlated with age (D9S59, p=0.09). Concerning chromosome arms, LOH was positively correlated with the altitude of present residence (13q and 17p, p=0.03) and duration of the existence of pterygium (13q and 17p, p=0.09). CONCLUSIONS LOH is a common event whereas MI is a very uncommon one at the examined markers in pterygium, indicating the presence of putative tumour suppressor genes implicated in the aetiopathogenesis of the disease. The fact that LOH at 9q31–33 was more frequent in recurrent pterygia and also correlated with known risk factors such as young age and high altitude of residence, implies a possible predictive value of this finding for postoperative recurrence.

Exhaled Breath Condensate 8-Isoprostane, Clinical Parameters, Radiological Indices and Airway Inflammation in COPD

Background: Exhaled breath condensate (EBC) 8-isoprostane levels were found increased in chronic obstructive pulmonary disease. However, the relation between EBC 8-isoprostane and parameters which have a known predictive value in COPD, remains vastly unknown, and so does subsequently its clinical value. Objectives: To investigate the relationship between 8-isoprostane level in EBC and clinical parameters, radiological indices and airway inflammation in COPD patients. Materials and Methods: We studied 18 COPD patients (all ex-smokers) and 12 healthy controls (5 ex-smokers and 7 never-smokers). All patients underwent clinical evaluation, sputum induction, high-resolution computed tomography (HRCT) of the thorax and EBC 8-isoprostane measurement. 8-Isoprostane levels were correlated with markers that reflect disease severity, such as dyspnea severity, FEV1 (%pred), emphysema changes and bronchiectasis in HRCT. Emphysema was quantified as the percentage of lung area with attenuation values < –950 Hounsfield units. Results: 8-Isoprostane levels were significantly elevated in EBC of patients with COPD [mean (SE) 18.1 (2) vs. 5.6 (0.7) pg/ml, p = 0.0001], irrespective of lung function impairment. 8-Isoprostane levels were correlated with emphysema score in HRCT (r2 = 0.43, p = 0.001) as well as with Medical Research Council dyspnea scale score (rho = 0.61, p = 0.005). Conclusion: Our findings suggest that EBC 8-isoprostane levels may reflect the extension of lung emphysema in COPD patients. In this respect, further investigation is required in order to evaluate the possible role of EBC 8-isoprostane in assessing disease progress in COPD patients.

Prevalence of BK virus and human papillomavirus in human prostate cancer

Polyomaviruses such as the BK virus (BKV), JC virus (JCV) and SV40, as well as the human papillomaviruses (HPV) are frequently detected throughout human populations, causing subclinical persistent infections and inducing oncogenesis in human and other cell lines. To test the involvement of these viruses in prostate tumorigenesis, we investigated the prevalence of BKV, JCV and HPV in a series of human prostatic malignancies. Forty-two samples of diagnosed prostatic malignancies were tested using standard polymerase chain reaction (PCR) protocols. Differentiation between BKV and JCV among the polyomavirus-positive samples was achieved after sequencing analysis of the PCR products. Reconstitution of BKV in vitro was performed and indirect immunofluorescence for the large T-antigen of the virus was applied to confirm the production of progeny virus. Detection and typing of HPV was carried out by PCR. The overall prevalence of polyomaviruses was 19% in the prostate cancer cases. Sequencing analysis of the polyomavirus-positive specimens revealed the presence of BKV in all samples. Reconstitution of the BKV from the BKV-positive prostate samples was successfully achieved in cell culture and progeny viral particles were obtained, confirming the presence of the virus in the human biopsies. HPV was detected in 4.8% of the samples, however, no HPV-11, HPV-16, HPV-18 or HPV-33 types were identified. BKV was frequently detected and could play a relevant role in the development and progression of human prostate cancer, whereas HPV does not seem to be implicated in this type of human neoplasia.

High-risk human papillomavirus (HPV) in parotid lesions

Although several studies have reported that oropharyngeal infection with HPV may predispose to tumorigenesis, little is known about the etiological factors of salivary gland tumors and the presence of HPV. We studied 9 parotid lesions for HPV infection including an oncocytoma, an acinic cell carcinoma, a high-grade adenocarcinoma, a low-grade polymorphous adenocarcinoma, a Warthins tumor and 2 pleomorphic adenomas, a lymphoepithelial cyst and a lipoma of the parotid gland. DNA was extracted from formalin-fixed and paraffin-embedded tissue sections. Solution PCR for HPV detection was performed using the GP5+/GP6+ primers, while HPV typing was carried out by multiplex PCR for HPV6, 11, 16, 18, and 33; positive samples were recorfirmed by PCR with specific primers for each type. Quantitative real-time PCR for the high-risk HPV genotypes 16, 18, 31, 33, 35, 52, 58 and 67 was also performed to quantitate the viral load. Finally, in situ PCR was employed with HPV16-specific primers by direct-detection method. Seven of the 9 parotid lesions were HPV positive while 6 of these 7 had been infected by HPV16 and/or HPV18 oncogenic types. High viral load of highrisk genotypes of HPV was found in the oncocytoma, in one of the pleomorphic adenomas, and in the Warthins tumor. Finally, in situ PCR indicated that HPV16 amplification occurred in the salivary gland tumors. This is the first time that highrisk HPV genotypes are detected in these histological types of parotid lesions, suggesting the possible involvement of the virus in the disease.

Microsatellite DNA instability and loss of heterozygosity in bronchial asthma.

Genetic alterations, such as loss of heterozygosity (LOH) or microsatellite instability (MI), have been reported in both malignant and benign disorders. In order to identify loci of deoxyribonucleic acid (DNA) mutation in asthma, MI and LOH were studied in sputum cells. DNA was extracted from cells in the sputum and blood cells of 22 patients with moderate asthma. Cells were analysed for MI and LOH using 18 polymorphic markers on chromosome 5q, 6p, 11q, 14q. Microsatellite analysis was also performed in six healthy subjects. None of the healthy individuals exhibited any genetic alteration. Genetic alterations were found in 16 of 22 asthmatic patients (73%). In total, 12 (54.5%) patients exhibited LOH only, one (4.5%) MI only, while three showed both MI and LOH. The highest incidence of LOH and MI was found on chromosome 14q. Mean immunoglobulin E and blood eosinophil levels were significantly higher in asthmatics with three or more genetic alterations. A high incidence of genetic alterations in the deoxyribonucleic acid of the sputum cells was found in asthmatic patients. Further studies are needed to identify the role of loss of heterozygosity and microsatellite instability in the investigation of genetic susceptibility of asthma and thus, in its pathogenesis.

Loss of heterozygosity on chromosomes 1, 2, 8, 9 and 17 in cerebral atherosclerotic plaques.

Objective Atherosclerosis is a fibroproliferative disease which has been attributed to several factors including genetic and molecular alterations. Initial studies have shown genetic alterations at the microsatellite level in the DNA of atherosclerotic plaques. Extending our initial findings, we performed a microsatellite analysis on cerebral atherosclerotic plaques. Methods Twenty-seven cerebral atherosclerotic plaques were assessed for loss of heterozygosity (LOH) and microsatellite instability (MI) using 25 microsatellite markers located on chromosomes 2, 8, 9 and 17. DNA was extracted from the vessels as well as the respective blood from each patient and subjected to polymerase chain reaction. Results Our analyses revealed that specific loci on chromosomes 2, 8, 9 and 17 exhibited a significant incidence of LOH. Forty-six percent of the specimens showed loss of heterozygosity at 2p13–p21, 48% exhibited LOH at 8p12–q11.2, while allelic imbalance was detected in 47% of the cases. The LOH incidence was 39%, 31% and 27% at 17q21, 9q31–34 and 17p13, respectively. Genetic alterations were detected at a higher rate as compared to the corresponding alterations observed in plaques from other vessels. Discussion This is the first microsatellite analysis using atherosclerotic plaques obtained from cerebral vessels. Our results indicate an elevated mutational rate on specific chromosomal loci, suggesting a potential implication of these regions in atherogenesis.

Human papillomavirus in the oral cavity of children and mode of delivery: a retrospective study

Our study aimed to examine the relationship between the presence of human papillomavirus (HPV) in the oral cavity of children and their mode of delivery. We investigated the presence of HPV infection in oral biopsies from 190 children (mean age: 7 years, range: 2–14 years) using the polymerase chain reaction (PCR) technique. Sixteen of 190 children (8.4%) were HPV-positive, with no significant difference between those delivered vaginally and by Caesarean section (C-section). The majority of the HPV-positive children were infected with type 16, whereas in the younger age group HPV type 11 was detected more frequently in children delivered by normal vaginal delivery (NVD) than by C-section. Our findings demonstrate the presence of HPV in the oral cavity of children delivered by both C-section as well as NVD. Further research on the possible modes of transmission of oral HPV infection will enable us to understand the natural history of HPV infection in childhood.

Maternal human papillomavirus (HPV) infection and its possible relationship with neonatal prematurity.

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