The presence of Merkel cell polyomavirus is associated with deregulated expression of BRAF and Bcl-2 genes in non-small cell lung cancer.

Polyomaviruses such as BK virus (BKV), JC virus (JCV) and Merkel cell polyomavirus (MCPyV) are typically nononcogenic, although they have been detected in a variety of human neoplasms. The aim of our study was to determine the frequency of the most common polyomaviruses MCPyV, BKV and JCV as well as the gene expression profile of genes involved in oncogenesis including K-ras, BRAF, RKIP, Bax, Bcl-2, p53 and RB1 in a cohort of non-small cell lung cancer (NSCLC) patients. Real-time and nested polymerase chain reaction (PCR) were used to assess the presence of polyomaviruses DNA in tissue biopsies from 110 patients with primary NSCLC and 14 tissue specimens from macroscopically healthy sites of their lung. Real-time PCR was also used to determine the mRNA expression of K-ras, BRAF, RKIP, Bax, Bcl-2, p53 and RB1 in selected samples. Results showed that ten NSCLC specimens were positive for the presence of MCPyV DNA (10/110, 9.1%), whereas no control sample was tested positive for the virus. The MCPyV-positive samples were predominantly obtained from male smokers (9/10). BKV and JCV DNA were not detected either in lung tissues biopsies or the control specimens. Interestingly, gene expression analysis revealed increased mRNA and protein expression of BRAF gene in association with BRAF phosphorylation in the MCPyV-positive samples, whereas Bcl-2 gene expression was downregulated in the same type of samples. The detected MCPyV prevalence in NSCLC in combination with the deregulated expression of BRAF and Bcl-2 genes suggests that these events are likely to contribute to the pathogenesis of NSCLC.

Vaccination against human papillomavirus in childhood: the next rubella analogue?

Direct comparisons between different vaccination programmes can reveal new targets and solve challenges that have been faced and managed in the past during similar health interventions. In the rubella vaccination programme both boys and girls were included in order to ensure that women of childbearing age are effectively protected. For human papillomavirus (HPV) vaccination, at the moment only girls have been included into the scheme. The aspect of vaccinating both boys and girls against HPV, similarly to the rubella paradigm, would interrupt "high-risk" HPVs transmission from males to females and vice versa ensuring further elimination of HPV. The new generation of HPV vaccines is expected to cost less and this will contribute to the possible introduction of HPV vaccine in both males and females.

George N. Papanicolaou (1883-1962): Fifty years after the death of a great doctor, scientist and humanitarian.

Fifty years have passed since the death of Dr George Nicholas Papanicolaou, who was born in Kyme at the island of Euboea in Greece in 1883 and became known for his innovative revolutionary invention of the Pap smear test performed at the Cornell University Medical College in the USA. To date, even after the introduction of HPV vaccination into the clinical practice, Dr George Papanicolaou's method remains an essential component of the prevention strategy against cancer and has resulted in a 70% decrease in cervical cancer mortality over the last 60 years. This article, which presents briefly his biography, is dedicated to him on the occasion of the 50th anniversary of his death.

Use of loop-mediated isothermal amplification of DNA for the rapid detection of Mycobacterium tuberculosis in clinical specimens.

Loop-mediated isothermal amplification (LAMP) is a recently developed molecular method that has been successfully implemented in the detection of Mycobacterium tuberculosis in clinical specimens. LAMP has several advantages, such as rapidity, high sensitivity, ease of application and cost-effectiveness. As a result, it is anticipated that its use for the detection of M. tuberculosis is likely to become widespread, especially in low-resource countries. The present review aimed to present this method and all of the available information on its implementation in the detection of M. tuberculosis in clinical specimens.

mRNA expression of G(1)-phase cell cycle regulatory molecules in bladder cancer.

PURPOSE: Cell cycle regulation, which is important for normal cellular proliferation, is controlled by a complex network of intracellular proteins, with cyclins, cyclin-dependent kinases (CDKs) and cyclin-dependent kinase inhibitors (CD-KIs) playing a central role. This equilibrium is interrupted in cancer cells, resulting in uncontrolled cellular proliferation. METHODS: In the present study we examined, by means of semi-quantitative RT-PCR, the expression of G(1)-phase cell cycle regulators MDM2, E2F1, Cyclin D1 (CCND1), CDK4, p19(INK4D), p21(WAF1/CIP1) and p27(KIP1) in a series of 32 bladder cancer specimens paired with adjacent normal tissues. RESULTS: Cyclin D1 was overexpressed in 10/32 (31.2%) and downregulated in 8/32 (25.0%) bladder cancer specimens. Additionally, p21 was overexpressed in 9/32 (28.1%) and downregulated in 10/32 (31.3%) cancer samples. On the contrary, MDM2, E2F1, CDK4, p19 and p27 expression was normal in the majority of malignant specimens. Further statistical analysis revealed significant associations between increased p21 levels and bladder cancer patients with no exposure to chemicals (p=0.048), as well as with patients with no artificial sweetener intake (p=0.012), and between increased Cyclin D1 levels and study subjects with no artificial sweetener intake (p=0.012). CONCLUSION: Based on these results, we conclude that Cyclin D1 and p21 mRNA deregulation seems to be an important event in bladder carcinogenesis. However, further studies are needed, in order to determine whether these two cell cycle regulators can be used as markers for the early detection of bladder cancer and to monitor its progression and recurrence.

Expression profile of Rho kinases in urinary bladder cancer.

PURPOSE: To investigate the expression of RhoA, RhoB, RhoC, Rac1 and Cdc42 kinases in urothelial cell carcinoma (UCC) of the urinary bladder and determine the expression profile of 107 Rho-associated genes, including GTPases, GDIs, GAPs and GEFs. METHODS: Rho expression was investigated using microarrays, qPCR and Western blotting in 77 UCC specimens with paired normal urothelium. Computational analysis was also performed on Gene Expression Omnibus datasets. Further microarray analysis was carried out for the expression profiling of the Rho-associated genes. RESULTS: RhoB mRNA and protein levels were significantly lower in UCC, suggesting a tumour-suppressor role. On the contrary, mRNA of RhoC and protein levels of RhoA, RhoC and Cdc42, respectively, were significantly higher in UCC vs. normal tissue. High Cdc42 mRNA levels correlated with worse overall survival (p=0.027), whereas high RhoB mRNA levels correlated both with better overall (p=0.0258) and cancer-specific (p=0.0272) survival. Computational analysis verified the expression profile of Rho kinases among superficial UCCs, muscle-invasive UCCs and normal tissues. CONCLUSION: The majority of the Rho-related genes showed over-expression in UCC vs. normal tissue. Alterations in RhoA, RhoB, RhoC, Rac1 and Cdc42 expression play a significant role in the genesis and progression of UCC of the urinary bladder.

Viral DNA detection and RAS mutations in actinic keratosis and nonmelanoma skin cancers.

BACKGROUND: Actinic keratosis (AK) is a well-established precancerous skin lesion that has the potential to progress to squamous cell carcinoma (SCC). Basal cell carcinoma (BCC) is a locally aggressive slowly growing tumour that rarely metastasizes. A number of viruses have been proposed to play a role in the development of nonmelanoma skin cancers (NMSC), but the most plausible evidence to date suggests that cutaneous human papillomavirus (HPV) is the key instigating factor. OBJECTIVES: To evaluate the prevalence of HPV, cytomegalovirus (CMV), herpes simplex virus (HSV) and Epstein-Barr virus (EBV) and investigate their relationship with the presence of RAS gene mutations in cutaneous lesions obtained from nonimmunosuppressed patients. METHODS: HPV, CMV, HSV and EBV detection was performed using polymerase chain reaction (PCR) in skin biopsies (26 AK, 12 SCC and 15 BCC samples) that were collected from immunocompetent patients. The RAS mutation incidence was also investigated in all cutaneous lesions by use of PCR/restriction fragment length polymorphism and direct DNA sequencing. RESULTS: Seventeen out of 53 (32%) skin lesions were found to be positive for HPV DNA. The highest incidences of HPV infection were five of 15 (33%) in BCC and four of 12 (33%) in SCC specimens. The HPV incidence was eight of 26 (31%) in AK and eight of 53 (15%) in normal skin tissue. Twelve out of 53 (23%) skin lesions were CMV-positive. The highest incidence of CMV infection was six of 15 (40%), observed in BCC specimens. The CMV incidence was two of 26 (8%) in AK and four of 12 (33%) in SCC. No normal skin biopsy was found to be positive for CMV. All cutaneous samples were negative for HSV and EBV DNA, as assessed by our PCR-based assays. Only three samples, one AK (4%), one BCC (6%) and one SCC (8%), were found to carry a G>T transversion at the second position of HRAS codon 12. Both HRAS mutant SCC and BCC biopsies were HPV- and CMV-positive, as well. CONCLUSIONS: HPV DNA is detected in NMSC, AK and normal skin biopsies. Our results also indicate that CMV is involved in NMSC at higher levels than in premalignant lesions, whereas the virus was not detected in normal skin biopsies. HSV and EBV do not appear to be involved in the pathogenesis of cutaneous lesions. Moreover, we suggest that the HRAS codon 12 mutation is not a very common event in AK or NMSC. Finally, both viral infection and HRAS activation appear to represent independent factors in the aetiology of NMSC, samples of which were obtained from immunocompetent patients.

Reduced placental prolyl hydroxylase 3 mRNA expression in pregnancies affected by fetal growth restriction.

OBJECTIVE: To investigate the role of the hypoxia-inducible factor (HIF) pathway in fetal growth restriction (FGR). DESIGN: A case-control study. SETTING: Research laboratory and gynaecology clinic. SAMPLE: Twenty placentas from normal pregnancies and 20 from FGR pregnancies. METHODS: RNA extraction, cDNA synthesis, quantitative real-time polymerase chain reaction (qRT-PCR) assay, statistical analysis. MAIN OUTCOME MEASURES: mRNA expression of HIF-1α, HIF-2α and HIF-ß (ARNT), along with prolyl hydroxylase domain 3 (PHD3), which leads to proteasomal degradation of HIF-α subunits. RESULTS: No statistically significant differences in the transcription levels of ARNT and HIF-2α were found between FGR and normal placentas. By contrast, PHD3 and HIF-1α mRNA were downregulated in FGR placentas. PHD3 mRNA expression was associated with gestational age at delivery (P = 0.008), birthweight centile (P = 0.029) and abnormal umbilical artery (UA) Doppler measurements (P = 0.034). CONCLUSIONS: As PHD3 regulates the HIF-mediated hypoxic response in FGR, we deduce that fetal adaptation to hypoxia ranges from impaired to adequate, as observed by the gradient of PHD3 downregulation in relation to the severity of FGR.

Resistance status and evolution trends of Klebsiella pneumoniae isolates in a university hospital in Greece: ineffectiveness of carbapenems and increasing resistance to colistin.

BACKGROUND: Due to its increased non-susceptibility rates, Klebsiella pneumoniae has emerged as one of the most problematic pathogens. METHODS: The level of resistance to 25 antimicrobials of K. pneumoniae isolates from a teaching hospital in Greece and the evolution trends during 2 decades were examined. RESULTS: A statistically significant increase in non-susceptibility rates was found for almost all antimicrobials examined. During 2008, the isolates presented non-susceptibility rates to aminoglycosides >50% and to quinolones >60%. Nowadays, 1 out of 10 isolates is non-susceptible to colistin. Moreover, the isolates non-susceptible to imipenem were almost doubled between 2007 (29%) and 2008 (50%). Among the imipenem-resistant isolates, 1 out of 4 was also resistant to colistin. CONCLUSION: The effectiveness of carbapenems has been compromised and the increase in resistance to colistin is rapid and steep.

Examining the discovery of the human retrovirus.

Retroviruses have been found in many bird and animal species where they often cause various types of cancer. Dr. Robert Gallo's contribution to the field of retrovirology and the link he established between RNA viruses and cancer has been significant. Historical aspects of his discoveries in the area of human retroviruses are presented and an attempt is made to focus attention on his outstanding role.

Activation of RAS family genes in urothelial carcinoma.

PURPOSE: Bladder cancer is the fifth most common malignancy in men in Western society. We determined RAS codon 12 and 13 point mutations and evaluated mRNA expression levels in transitional cell carcinoma cases. MATERIALS AND METHODS: Samples from 30 human bladder cancers and 30 normal tissues were analyzed by polymerase chain reaction/restriction fragment length polymorphism and direct sequencing to determine the occurrence of mutations in codons 12 and 13 of RAS family genes. Moreover, we used real-time reverse transcriptase-polymerase chain reaction to evaluate the expression profile of RAS genes in bladder cancer specimens compared to that in adjacent normal tissues. RESULTS: Overall H-RAS mutations in codon 12 were observed in 9 tumor samples (30%). Two of the 9 patients (22%) had invasive bladder cancer and 7 (77%) had noninvasive bladder cancer. One H-RAS mutation (11%) was homozygous and the remaining 89% were heterozygous. All samples were WT for K and N-RAS oncogenes. Moreover, 23 of 30 samples (77%) showed over expression in at least 1 RAS family gene compared to adjacent normal tissue. K and N-RAS had the highest levels of over expression in bladder cancer specimens (50%), whereas 27% of transitional cell carcinomas demonstrated H-RAS over expression relative to paired normal tissues. CONCLUSIONS: Our results underline the importance of H-RAS activation in human bladder cancer by codon 12 mutations. Moreover, they provide evidence that increased expression of all 3 RAS genes is a common event in bladder cancer that is associated with disease development.

Deregulation of the tumour suppressor genes p14(ARF), p15(INK4b), p16(INK4a) and p53 in basal cell carcinoma.

BACKGROUND: Basal cell carcinoma (BCC) is a locally aggressive slowly growing tumour that rarely metastasizes and is mostly seen in older members of the population. OBJECTIVES: To determine the involvement of the tumour suppressor genes p14(ARF), p15(INK4b), p16(INK4a) and p53 in BCC. METHODS: We investigated the integrity of the CDKN2A locus in 15 BCC samples by analysing the presence of allelic imbalance/loss of heterozygosity (LOH). Moreover, we studied the mRNA expression levels of the tumour suppressor genes p14(ARF), p15(INK4b), p16(INK4a) and p53 in the BCC samples and compared them with mRNA levels in the corresponding normal tissue. The presence of mutations was examined by sequencing for exons 1a and 2 of p16(INK4a). RESULTS: We found LOH in one BCC sample for the marker D9S1748. A polymorphism (G442A) of exon 2 was detected in three cases. p14(ARF), p15(INK4b) and p53 presented high expression levels, whereas p16(INK4a) exhibited low mRNA levels compared with the corresponding normal tissue. Significant correlations were detected among the genes studied. CONCLUSIONS: Our results demonstrate a different expression profile between p16(INK4a) and p14(ARF), p15(INK4b) and p53 in BCC. Moreover, we found a low percentage of LOH and of a polymorphic sequence variant (Ala148Thr) for the CDKN2A locus.

Matrix metalloproteinases and their inhibitors as markers of inflammation and fibrosis in chronic liver disease (Review).

Chronic liver disease (CLD) is a cause of morbidity and mortality worldwide, due to haemodynamic and metabolic complications of liver cirrhosis. During CLD the extracellular matrix undergoes a process of remodelling, leading to new collagen formation and deposition. Tissue remodelling is regulated by fine molecular mechanisms, involving proteases, inhibitors and growth factors. The major role in matrix degradation is played by matrix metalloproteinases (MMPs), a class of zinc and calcium-dependent enzymes, and their tissue inhibitors (TIMPs). Along with the progress in diagnostic techniques, leading to more precise and less invasive methods, the concept of monitoring has gained importance for the clinical management of CLD. At the present state of our knowledge, liver biopsy still represents an essential procedure for staging liver disease. However, despite its importance, liver biopsy presents some limitations: the risk of a disease underestimation is the most significant one, as hepatic lesions are often irregularly located within the liver. Parallel to the limitations of liver biopsy, clinical needs for an early identification of progressive fibrosis require additional non-invasive techniques to be developed. In this review we discuss the major problems concerning this important clinical necessity. Moreover, we focus on the role of MMPs and TIMPs in the pathogenesis of CLD, as well as their possible use as non-invasive serum markers for inflammation and fibrosis in this pathology.

Genomic instability, mutations and expression analysis of the tumour suppressor genes p14(ARF), p15(INK4b), p16(INK4a) and p53 in actinic keratosis.

Actinic keratosis (AK) is a well-established pre-cancerous skin lesion that has the potential to progress to squamous cell carcinoma (SCC). We investigated the involvement of the CDKN2A, CDKN2B and p53 genes in AK and in the progression of AK to SCC. Mutational analysis on exons 1a, 1b and 2 of the CDKN2A locus and exon 1 of the CDKN2B locus as well as allelic imbalance was performed in 26 AK specimens. Expression levels of the genes p14(ARF), p15(INK4b), p16(INK4a) and p53 were examined in 16 AKs and 12 SCCs by real-time RT-PCR. A previously described polymorphism of p16(INK4a) (Ala148Thr) was detected at an allelic frequency of 12%. Six samples carried novel mutations at codon 71 of the CDKN2A locus and one sample presented an additional mutation at codon 65. Two AK samples carried a not-previously described non-UV type missense mutation at codon 184 (Val184Glu) of exon 1b in the p14(ARF) gene. Regarding the CDKN2B locus a new mutation at codon 50 (Ala50Thr) and another at codon 24 (Arg24Arg), were detected. Microsatellite instability (MSI) was found in 15% of AKs in at least one marker, indicating that genetic instability has some implication in the development of AK. Down-regulation of p16(INK4a) and p53 mRNA levels was noted in SCC compared to AK. TSGs expression levels in sun-exposed morphologically normal-appearing skin, suggests that abnormal growth stimuli might exist in these tissues as well. Furthermore, we suggest a possible role of p15(INK4b), independently from the intracellular pathway mediated by p16(INK4a), and of p14(ARF) in AK development, as well as in the progression of AK to SCC. The deregulation of the expression profiles of the CDKN2A, CDKN2B and p53 genes may, independently of mutations and LOH at 9p21, play a significant role in AK and progression of AK to SCC.

High-risk human papilloma viruses (HPVs) were not detected in the benign skin lesions of a small number of children.

AIM: Human papilloma virus (HPV) can be transmitted via sexual as well as nonsexual routes. Recently, 'high-risk' HPVs were detected in the oral mucosa of children in whose cases there was no suspicion of sexual abuse. This implies that HPV 16 and 18 have additional nonsexual modes of transmission in childhood, such as vertical transmission and autoinoculation. METHODS: Using polymerase chain reaction (PCR) assays, we examined the skin tissues of 12 children with benign skin lesions, aged between 6 and 13 years, for the presence of HPV. RESULTS: Among 12 biopsy skin specimens, no 'high-risk' HPV DNA was detected. Specific PCRs for HPV DNA 16 and 18 were also negative. CONCLUSION: This preliminary case-control study indicates the absence of mucosal 'high-risk' HPV types in the benign skin lesions of children.

The dose response principle from philosophy to modern toxicology: The impact of ancient philosophy and medicine in modern toxicology science.

Since ancient times the concept of dose response, from a toxicological perspective, has been a matter of concern. Already by the 8th century BC and over the years, many enlightened people have attempted to interpret this phenomenon, observing and coming across its results and practical implementation through exposure to chemical substances, either from natural or synthetic sources. Nowadays, the environmental exposure of human populations to chemicals in terms of quantity and quality might differ. Nevertheless, dose response still remains an issue joining hands with scientific and technological progress. The aim of the present review is not only to briefly recount the history of the dose response concept, from ancient time theories to novel approaches, but also to draw the outline of challenges and requirements toxicology science needs to fulfill.

Common mental disorders and association with telomere length.

Telomeres are repeated 5'-TTAGGG-3' sequences at the end of chromosomes, which maintain genomic stability. Their length is related to a number of diseases that affect humans. Apart from cancer, cardiovascular diseases, diabetes and other, telomere length has been associated with chronic diseases. Chronic mental illness includes various types of mental disorders with the most common being depression, schizophrenia and stress-anxiety. The aim of this review is to summarize the current state of knowledge on the role of telomeres in these disorders and to compare telomere length variations in patients receiving medication and patients not taking treatment. Most studies report reduced telomere length in patients suffering from mental disorders, compared to the general population. Since the factors that can affect telomere length are various, more experiments and investigations are required to understand the general impact of different factors on telomere length.

Polymorphisms of Cx(3)CR1 and CXCR6 receptors in relation to HAART therapy of HIV type 1 patients.

The chemokine polymorphisms CXCR6-3E/K, In1.1T/C, H7 haplotype, CX(3)CR1-V249I, and CX(3)CR1-T280M have been shown to affect the course of HIV infection. We studied their influence on immunologic and virologic response to HAART in a group of 143 HIV-1 patients. We performed Kaplan-Meier analysis using the following end-point criteria: (1) time from HAART initiation to undetectable viral load (VL < 50 copies/ml), (2) maximum duration of viral suppression, (3) time from HAART administration until CD4 elevation above 200 cells/microl for patients with baseline CD4 below 200 cells/microl and above 500 cells/microl for patients with baseline CD4 between 200 and 500 cells/microl, respectively, and (4) time from HAART initiation until CD4 reduction below baseline values. Our results revealed an improved immunologic response to HAART in patients with the CX(3)CR1-249I or CX(3)CR1-280M allele. On the contrary, patients with initial VL suppression due to HAART showed a faster virologic failure in the presence of the CXCR6-3K allele. The In1.1T/C polymorphism and H7 haplotype did not reveal any specific effect on HAART response.

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 Warthin's 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 Warthin's 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.

Highly conserved sequence of exon 15 BRAF gene and KRAS codon 12 mutation among Greek patients with colorectal cancer.

BACKGROUND: The RAS/RAF/MEK/MAP kinase pathway is essential to intracellular signaling transduction regulating cell proliferation, differentiation and death. We investigated the occurrence of exon 15 BRAF and KRAS codon 12 mutations among Greek patients with colorectal cancer. METHODS: Sixty-one samples from patients with sporadic colorectal adenocarcinomas were studied for exon 15 BRAF mutations. DNA from surgically resected specimens was analyzed by a combination of polymerase chain reaction and direct sequencing. KRAS codon 12 mutational analysis was technically possible in 58 samples (58/61) by a combination of polymerase chain reaction and restriction fragment length polymorphism. RESULTS: No exon 15 BRAF mutations were detected in any of the colon cancer specimens. The frequency of KRAS codon 12 mutations was 29.3% (17/58). Patients aged < or = 70 years more frequently presented carcinomas harboring KRAS codon 12 mutations than patients aged >70 years (p=0.028). Patients between 61 and 70 years of age were more likely to be carriers of this mutation (p=0.040). CONCLUSIONS: Despite the limited study sample, our data suggest that BRAF mutations might be present less frequently than KRAS mutations in Greek patients with colorectal carcinomas. Further research involving larger patient series will be necessary to confirm these findings and to assess possible ethnic, environmental and lifestyle influences on BRAF and KRAS mutagenesis.