Effect of 5-year continuous positive airway pressure treatment on the lipid profile of patients with obstructive sleep apnea: A pilot study.

Continuous positive airway pressure (CPAP) provides a well-documented symptomatic relief for most patients with obstructive sleep apnea (OSA); however, its effect on dyslipidaemia remains contradictory. The aim of this longitudinal pilot study was to investigate the effect of long-term CPAP treatment on the lipid profile of patients with severe OSA. Fasting serum levels of total cholesterol (TC), low- and high-density lipoprotein cholesterol (LDL-C and HDL-C) and triglyceride (TG) were longitudinally measured in 33 OSA patients with an apnea-hypopnea index (AHI) of ≥30 events/hr, at the time of diagnosis (baseline) and at control visits following fixed-pressure CPAP treatment. Compared to baseline values, even as short as a 2-month CPAP therapy resulted in a significant decrease of both TC and LDL-C levels (TC, 5.62 ± 0.22 vs. 5.18 ± 0.21 mmol/L; LDL-C, 3.52 ± 0.19 vs. 3.19 ± 0.2 mmol/L; p < 0.05 for each). These lipid fractions exhibited similar improvements at 6 months and after 5 years of CPAP treatment (TC, 5.1 ± 0.17 mmol/L; LDL-C, 2.86 ± 0.16 mmol/L; p < 0.01 for each). The reduction in lipid levels was greater in younger patients and/or in those who had higher body mass index (BMI) (p < 0.05). There were no significant correlations between AHI and lipid levels (p > 0.05); BMI showed a weak negative association with HDL-C fraction (BMI, r = -0.263, p < 0.05). CPAP therapy had neither short- nor long-term effects on TG and HDL-C levels (p > 0.05). CPAP therapy has a rapid and long-lasting beneficial effect on the lipid profile of patients with severe OSA.

Monitoring Antioxidant Enzyme Activity during Exacerbations of Chronic Obstructive Pulmonary Disease.

Superoxide dismutases (SODs) and catalase (CAT) have been implicated as major antioxidant enzymes of the human lungs. In this study, we investigated whether activities of these enzymes are altered in the airways of patients hospitalized with acute exacerbation of chronic obstructive pulmonary disease (AECOPD). SOD and CAT activities were measured in the sputum, exhaled breath condensate, and serum of 36 COPD patients experiencing a severe exacerbation. Measurements were performed using colorimetric assays in samples collected at the time of hospital admission and at the time of hospital discharge following treatment of AECOPD. For comparison, antioxidants were also assessed in 24 stable COPD patients and 23 healthy control subjects. SOD and CAT activities in sputum were significantly increased in patients with AECOPD compared to those with stable disease (SOD: 0.142 [0.053-0.81] vs. 0.038 [0.002-0.146] U/mL, p < 0.01; CAT: 48.7 [18.7-72.6] vs. 10.2 [2.9-40.6] nmol/min/mL, p < 0.05), while treatment of exacerbation led to a decrease in enzyme activities (SOD: 0.094 [0.046-0.45] U/mL, p < 0.05; CAT: 28.0 [7.3-60.4] nmol/min/mL, p < 0.005). No changes were observed in the serum (p > 0.05). Both SOD and CAT activities significantly correlated with sputum neutrophil and lymphocyte cell counts in patients with AECOPD. Moreover, SOD and CAT values correlated with each other and also with sputum malondialdehyde, an established marker for oxidative stress. Our data demonstrate that sputum antioxidant activity is elevated during COPD exacerbation and suggest that activation of SODs and CAT is an integral part of the human defense mechanism against the increased oxidant production associated with AECOPD.

Complex analysis of multiple single nucleotide polymorphisms as putative risk factors of tooth agenesis in the Hungarian population.

OBJECTIVES: The role was studied of multiple single nucleotide polymorphisms in tooth agenesis in the Hungarian population using a complex approach. METHODS: Eight SNPs, PAX9 -912 C/T, PAX9 -1031 A/G, MSX1 3755 A/G, FGFR1 T/C rs881301, IRF6 T/C rs764093, AXIN2-8150 A/G, AXIN2-8434 A/G and AXIN2-30224 C/T, were studied in 192 hypodontia and 17 oligodontia cases and in 260 healthy volunteers. Case-control analysis was performed to test both allelic and genotypic associations as well as associations at the level of haplotypes. Multivariate exploratory Bayesian network-based multi-level analysis of relevance (BN-BMLA) as well as logistic regression analysis were performed. RESULTS: Conventional statistics showed that PAX9 SNP -912 C/T and the MSX1 SNP changed the incidence of hypodontia, although after Bonferroni correction for multiple hypothesis testing, the effects were only borderline tendencies. Using a statistical analysis better suited for handling multiple hypotheses, the BN-BMLA, PAX9 SNPs clearly showed a synergistic effect. This was confirmed by other multivariate analyses and it remained significant after corrections for multiple hypothesis testing (p < 0.0025). The PAX9-1031-A-PAX9-912-T haplotype was the most relevant combination causing hypodontia. Interaction was weaker between PAX9 and MSX1, while other SNPs had no joint effect on hypodontia. CONCLUSION: This complex analysis shows the important role of PAX9 and MSX1 SNPs and of their interactions in tooth agenesis, while IRF6, FGFR1 and AXIN2 SNPs had no detectable role in the Hungarian population. These results also reveal that risk factors in hypodontia need to be identified in various populations, since there is considerable variability among them.

Sputum Cytokine Profiling in COPD: Comparison Between Stable Disease and Exacerbation.

Purpose: Cytokines are extracellular signaling proteins that have been widely implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Here, we investigated cytokine expression both at the mRNA and protein level in the sputum of healthy individuals, stable COPD patients, and those experiencing a severe acute exacerbation (AECOPD) requiring hospitalization. Patients and Methods: Sputum was collected in 19 healthy controls, 25 clinically stable COPD patients, and 31 patients with AECOPD. In AECOPD patients sample collection was performed both at the time of hospital admission and at discharge following treatment. Sputum supernatant was analyzed by an antibody microarray detecting 120 cytokines simultaneously, while the mRNA expression of 14 selected cytokines in sputum cells was investigated by real-time PCR (qPCR). Results: Proteomic analysis identified interleukin (IL)-6 and growth-regulated oncogene (GRO)α as the only sputum cytokines that were differentially expressed between stable COPD patients and healthy controls. At the onset of AECOPD, several cytokines exhibited altered sputum expression compared to stable COPD. Recovery from AECOPD induced significant changes in the sputum cytokine protein profile; however, the length of hospitalization was insufficient for most cytokines to return to stable levels. With regard to gene expression analysis by qPCR, we found that bone morphogenetic protein (BMP)-4 was up-regulated, while IL-1α, monokine-induced by interferon-γ (MIG), and BMP-6 were down-regulated at the mRNA level in patients with AECOPD compared to stable disease. Conclusion: The sputum cytokine signature of AECOPD differs from that of stable COPD. Protein level changes are asynchronous with changes in gene expression at the mRNA level in AECOPD. The observation that the levels of most cytokines do not stabilize with acute treatment of AECOPD suggests a prolonged effect of exacerbation on the status of COPD patients.

Predictive Value of Exhaled Nitric Oxide and Blood Eosinophil Count in the Assessment of Airway Eosinophilia in COPD.

Purpose: Fractional exhaled nitric oxide (FENO50) level and peripheral blood eosinophil count may serve as indicators of airway eosinophilia. The aim of this study was to estimate the diagnostic value of these markers for detecting airway eosinophilia in patients with stable chronic obstructive pulmonary disease (COPD) and those experiencing an acute exacerbation (AECOPD). Patients and Methods: FENO50 levels, sputum and blood eosinophil counts were assessed in 53 clinically stable ex-smoker COPD patients and 67 ex-smoker COPD patients experiencing a severe exacerbation. In AECOPD, clinical variables were measured at the time of hospital admission and discharge following treatment. Results: In stable COPD, blood eosinophil count but not FENO50 level was found to be a good predictor of airway eosinophilia (area under the receiver operating characteristic curve [ROC AUC]: ≥0.82). The sensitivity and the specificity of the test ranged between 75% and 98%, the negative predictive value (NPV) was high (>90%). In AECOPD, FENO50 was predictive for airway eosinophilia (ROC AUC: >0.8) with high NPV (>88%), but with lower sensitivity and specificity (64-70%). In contrast, the predictive accuracy of blood eosinophil count for airway eosinophilia in AECOPD was modest (ROC AUC: 0.54-0.63). The combined use of the two markers provided only limited additional benefit. Correlation analyses supported ROC curve findings. Conclusion: In stable COPD the peripheral blood eosinophil count, while in AECOPD the FENO50 level is a good surrogate marker of airway eosinophilia.

Effect of 5-year continuous positive airway pressure treatment on MMPs and TIMPs: implications for OSA comorbidities.

Continuous positive airway pressure (CPAP) treatment results in nearly complete remission of symptoms of obstructive sleep apnoea (OSA); however, its effect on OSA comorbidities including cardiovascular diseases remains contradictory. Here we investigated the short- and long-term effect of CPAP treatment on matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in patients with severe OSA. Serum levels of 7 MMPs and 3 TIMPs were followed in OSA patients (n = 28) with an apnoea-hypopnoea index of ≥30 events/h at the time of diagnosis and at control visits (2 months, 6 months and 5 years) after initiation of fixed-pressure CPAP treatment. The first few months of CPAP therapy resulted in significant decrease of MMP-8 and MMP-9 levels (MMP-8: 146 (79-237) vs. 287 (170-560) pg/mL; MMP-9: 10.1 (7.1-14.1) vs. 12.7 (10.4-15.6) ng/mL, p < 0.05 for each at 2 months), while the rest of the panel remained unchanged as compared to baseline values. In contrast, at 5 years, despite of uninterrupted CPAP treatment and excellent adherence the levels of MMP-8, MMP-9 and TIMPs significantly increased (p < 0.05). Our data suggest that initiation of CPAP therapy leads to a decrease in the level of key MMPs in the short-term; however, this effect is not sustained over the long-term.

Elimination of bacterial DNA during RNA isolation from sputum: Bashing bead vortexing is preferable over prolonged DNase treatment.

Sputum often contains large amounts of contaminating bacterial DNA that, if not eliminated during RNA isolation, may interfere with gene expression studies. During RNA isolation only repeated DNase treatment can effectively remove contaminating bacterial DNA from samples, but this compromises RNA quality. In this study we tested alternative methods to facilitate the removal of DNA and improve the quality of RNA obtained. Sputum samples obtained from patients with chronic obstructive pulmonary disease were processed with dithiothreitol and subjected to various RNA isolation methods, yet with modified protocols. Modifications included prolonged DNase treatment or vortexing of sputum cells in the presence of beads prior to RNA isolation. Bacterial DNA contamination was tested by PCR using universal bacterial primers, while RNA quality was assessed by real-time PCR using GAPDH primers for amplicons of different length. We found that the RNeasy Plus Mini kit equipped with the gDNA eliminator spin column was able to completely eliminate bacterial DNA, if sputum cells were lysed in the presence of bashing beads. Notably, compared with the standard protocol, the modified procedure yielded better quality RNA as well, as indicated by improved threshold profiles of qPCR. Bead vortexing of cells was less effective when combined with other RNA isolation methods, and the repeated DNase treatment needed to completely remove contaminating DNA from the samples reduced the quality of RNA markedly. Bead vortexing in combination with certain RNA extraction methods greatly facilitates the isolation of sputum RNA that is free of contaminating bacterial DNA, and is suitable for downstream applications.

Different expression of occludin and ZO-1 in primary and metastatic liver tumors.

Tight junction (TJ) components were found to be correlated with carcinogenesis and tumor development. TJs are composed of three main integral membrane proteins; occludin, claudins and JAMs. Alteration of the TJ protein expression may play an important role in the process of cell dissociation, which is among the first steps of tumor invasion and metastasis. Reduced expression of ZO-1 has been reported to be associated with invasion of several tumors. The aim of the present study was to detect differences between occludin and ZO-1 expression in normal liver samples, HCCs and colorectal liver metastases. Expression of occludin and ZO-1 was analysed in 25 surgically removed human hepatocellular carcinomas (HCC) and 25 human colorectal liver metastases. Gene expression levels were measured by real-time RT PCR, protein expression was determined by immunohistochemistry, comparing tumors with the surrounding nontumorous parenchyma and with seven normal liver samples. Occludin and ZO-1 mRNAs showed significant downregulation in HCCs in comparison with normal liver and were also downregulated in the metastases when compared with normal liver. Occludin and ZO-1 proteins were weakly expressed on hepatocytes in normal liver, while strong expression was found on bile canaliculi. In HCCs occludin and ZO-1 did not show immunopositivity on tumor cells, while colorectal metastatic tumors revealed high levels of these molecules. HCCs and metastases are characterized by markedly different protein expression pattern of occludin and ZO-1, which phenomenon might be attributed to the different histogenesis of these tumors.

Comparison of the expression of agrin, a basement membrane heparan sulfate proteoglycan, in cholangiocarcinoma and hepatocellular carcinoma.

Heparan sulfate proteoglycans mediate cell adhesion and control the activities of numerous growth and motility factors. They play a critical role in carcinogenesis and tumor progression. Agrin is a large multidomain heparan sulfate proteoglycan associated with basement membranes in several tissues. The expression of agrin in the liver has recently been described under physiologic and pathologic conditions. However, little is known about its role in malignancies. We aimed to study the mRNA and protein expression of agrin in cholangiocarcinoma (CC) and focused on the differences between CC and hepatocellular carcinoma (HCC). Eighty surgically removed liver specimens were studied by immunohistochemistry. Representative samples were used for immunoblotting. mRNA expression was measured in 32 samples by real-time polymerase chain reaction. By immunohistochemistry, agrin was seen around bile ducts and blood vessels within the portal areas in the normal liver. Although no expression was found within the hepatic lobules, agrin was deposited in the neovascular basement membrane in HCCs. Agrin was abundant in the tumor-specific basement membrane in well-differentiated areas of CCs, whereas with immunostaining, it was fragmented, decreased, or it even disappeared in less differentiated areas and sites of infiltration. By real-time polymerase chain reaction, up-regulation of agrin expression was measured in HCCs compared with that in the normal liver. CC samples showed an even higher expression of agrin. Immunoblotting confirmed these findings. Our results indicate that agrin might play an important role in neoangiogenesis in human HCC, being a part of the newly formed vasculature. In CC, however, agrin might be involved in tumor progression.

Distinct claudin expression profile in histologic subtypes of lung cancer.

PURPOSE: Claudins are the main constituents of tight junctions. Little is known about their expression and localization in the normal bronchial epithelium and in lung cancer. PATIENTS AND METHODS: One hundred four lung cancer tissue blocks were studied including 46 adenocarcinomas (ADC), 30 squamous cell carcinomas (SCC), 15 small cell lung cancers (SCLC), 8 typical and 5 atypical carcinoids. All slides contained normal bronchial mucosa as well. Immunohistochemistry using antibodies against claudins-1, -2, -3, -4, and -7 proteins, as well as semi-quantitative estimation were performed. RT-PCR analysis was also carried out in 22 immunohistochemically representative tumor samples. RESULTS: Normal bronchial epithelial cells expressed all the examined claudin proteins. When compared, SCLCs and carcinoids showed striking differences in regard to claudins-1, -3, and -4 expressions (p<0.0001, p<0.0001, and p<0.0004, respectively), whereas ADCs and SCCs revealed significant differences in claudins-3, -4, and -7 expressions (p<0.0001, p<0.0001, and p<0.0053, respectively). However, comparison of ADCs with SCLCs revealed significant difference only in claudin-2 expression (p<0.0002). The comparison of ADCs and carcinoids resulted in significant differences regarding claudins-1, -3, and -4 expressions (p<0.0006, p<0.0001, and p<0.0001, respectively). SCCs and SCLCs varied in respect to claudin-2, -3, and -4 expressions (p<0.0009, p<0.0001, and p<0.0019, respectively), whereas SCCs and carcinoids showed different claudins-1 and -4 expressions (p<0.0076 and p<0.0045, respectively). RT-PCR analysis revealed parallel changes in the mRNA and protein expression of certain claudins. CONCLUSIONS: The observed distinct claudin expression profile within the non-small cell lung cancer group, further, the marked differences between SCLCs and carcinoids may have differential diagnostic impact, and the overexpression of certain claudins might have therapeutic implications.

Detection of bladder cancer from the urine using fluorescence in situ hybridization technique.

The authors report on their first experiences with the UroVysion fluorescence in situ hybridization (FISH) kit developed for the detection of bladder cancer. This new non-invasive diagnostic application of the FISH technique in the field of urology was elaborated to replace cystoscopy. The special urine examination method detects genetic alterations of the urothelial cells found in the urine, using fluorescent directlabeled DNA probes binding to the peri-centromeric regions of chromosomes 3, 7 and 17 as well as on the 9p21 locus. We aimed to evaluate the utility of UroVysion test in the light of the histological diagnosis. Urine samples from 43 bladder cancer patients and 12 patients with no or benign alterations were studied using a new application of FISH technique: the UroVysion reagent kit. The obtained FISH results were compared with the histological findings of the transurethral surgical resection specimens. The study rated the specificity and sensitivity of the technique 100% and 87%, respectively. Therefore, the technique could well fit into the diagnostic process of bladder carcinomas. Statistical analyses showed significant correlation between tumor progression and the severity of the genetic alterations detected by this FISH technique. Furthermore, positive correlation was found between tumor grade and the proportion of tumor cells showing genetic abnormality. The noninvasiveness, the robustness of evaluation and the high specificity/sensitivity are all in favor of this technique. The disadvantages are the higher costs of the technical background and the required future clinical studies to determine whether this technique can replace cystoscopy.

Claudin-1 and claudin-2 differentiate fetal and embryonal components in human hepatoblastoma.

Claudins (CLDNs), a family of transmembrane proteins, are major constituents of tight junctions (TJs). They have been shown to be differentially regulated in malignant tumors and play a role in carcinogenesis and progression. We aimed to explain the molecular mechanism underlying the main epithelial components of hepatoblastomas (HBs) based on the composition of TJs. Fourteen formalin-fixed, paraffin-embedded surgical resection specimens were analyzed by immunohistochemistry for CLDN-1, -2, -3, -4, -7; proliferating cell nuclear antigen (PCNA); Ki-67; beta-catenin; cytokeratin-7 (CK-7); and hepatocyte-specific antigen; messenger RNA was isolated for real-time reverse transcriptase polymerase chain reaction analysis of the CLDNs from dissected fetal and embryonal cell types. Significantly increased protein and messenger RNA expression of CLDN-1 and -2 was detected in the fetal compared with the embryonal component. Both cell types displayed negative or weak immunostainings for CLDN-3, -4, and -7. Hepatocyte-specific antigen was dominantly expressed in the fetal component. PCNA and Ki-67 labeling indices were significantly higher in embryonal compared with fetal cells. beta-catenin cytoplasmic/nuclear immunoreaction was frequent, although not showing significant differences between fetal and embryonal cells. Mutational analysis of beta-catenin detected mutation in two cases. Our results suggest that increased expression of CLDN-1 and -2 characterizes the more differentiated fetal component in HBs and is a reliable marker for differentiating fetal and embryonal cell types in HBs. The results proved that the embryonal and fetal components of HBs differ in such important feature as the protein composition of TJs. The expression of CLDN-1 and -2 is inversely correlated with cell proliferation. The more aggressive, rapidly proliferating embryonal phenotype is associated with the decrease/loss of CLDN-1 and -2. However, there are no data indicating association with the nuclear translocation of beta-catenin.

Claudin-1, -3 and -4 proteins and mRNA expression in benign and malignant breast lesions: a research study.

INTRODUCTION: We compared levels of protein and mRNA expression of three members of the claudin (CLDN) family in malignant breast tumours and benign lesions. METHODS: Altogether, 56 sections from 52 surgically resected breast specimens were analyzed for CLDN1, CLDN3 and CLDN4 expression by immunohistochemistry. mRNA was also analyzed using real-time PCR in 17 of the 52 cases. RESULTS: CLDNs were rarely observed exclusively at tight junction structures. CLDN1 was present in the membrane of normal duct cells and in some of the cell membranes from ductal carcinoma in situ, and was frequently observed in eight out of nine areas of apocrine metaplasia, whereas invasive tumours were negative for CLDN1 or it was present in a scattered distribution among such tumour cells (in 36/39 malignant tumours). CLDN3 was present in 49 of the 56 sections and CLDN4 was present in all 56 tissue sections. However, CLDN4 was highly positive in normal epithelial cells and was decreased or absent in 17 out of 21 ductal carcinoma grade 1, in special types of breast carcinoma (mucinous, papillary, tubular) and in areas of apocrine metaplasia. CLDN1 mRNA was downregulated by 12-fold in the sample (tumour) group as compared with the control group using GAPDH as the reference gene. CLDN3 and CLDN4 mRNA exhibited no difference in expression between invasive tumours and surrounding tissue. CONCLUSIONS: The significant loss of CLDN1 protein in breast cancer cells suggests that CLDN1 may play a role in invasion and metastasis. The loss of CLDN4 expression in areas of apocrine metaplasia and in the majority of grade 1 invasive carcinomas also suggests a particular role for this protein in mammary glandular cell differentiation and carcinogenesis.

Increased expression of claudins in cervical squamous intraepithelial neoplasia and invasive carcinoma.

Claudins (CLDNs), of which 24 types have been identified, are integral transmembrane proteins of the tight junctions that are critical for maintaining cell adhesion and polarity. They also act as selective barriers. Cells and tissues are characterized by individual CLDN patterns; the composition and levels of expression change during differentiation and tumor formation. Alterations in the expression of individual CLDNs have been detected in several carcinomas and shown to be related to progression and invasion; however, their role in carcinogenesis is controversial. Using a panel of polyclonal (CLDNs 1, 3, and 7) and monoclonal (CLDNs 2 and 4) antibodies, CLDN pattern and expression were studied by immunohistochemistry in 105 cervical tissue specimens, including normal epithelia (n = 20), cervical intraepithelial neoplasias (CINs; CIN 1/2, n = 27; CIN 3, n = 10), carcinoma in situ (CIS, n = 15), and 33 squamous keratinizing and nonkeratinizing invasive carcinomas. No CLDN 3 was observed in normal or intraepithelial neoplastic cells, but significantly increased expression of CLDNs 1, 2, 4, and 7 was detected in the CIN/CIS lesions and invasive carcinomas compared with the normal tissues (P < .001) and reduced reactivity of CLDNs 1 and 2 was observed in invasive cervical cancers compared with CIN 3/CIS (P = .0001) and of CLDNs 2, 4, and 7 compared with CIN 1/2. These results indicate increased expression of CLDNs in the early phase of carcinogenesis in intraepithelial lesions, which decreases during progression to invasive disease. Expression of CLDN 1 was strongest in premalignant stages; thus, it may serve as a good diagnostic marker for the detection of CIN.

Claudin expression in Barrett's esophagus and adenocarcinoma.

Claudins (CLDNs) are key molecules in cell adhesion, polarity, and control of paracellular solute transport. Several studies suggested that changes in claudin pattern have a role in cancer development. This study aimed to detect alterations in CLDN 1, 2, 3, 4, and 7 expression patterns in Barrett's esophagus (BE) and adenocarcinoma (ACC) compared with that in foveolar epithelium (FOV), normal squamous epithelium (SQ), and squamous cell carcinoma (SQCC). One hundred twenty five surgically or endoscopically removed, paraffin-embedded cases were studied by immunohistochemistry and analyzed statistically. BE, ACC, and FOV were dissected from 30 paraffin-embedded samples for further mRNA expression analysis. CLDN 7 was the dominating type in all epithelia and carcinomas, but its expression did not differ in normal and altered tissues. CLDN 1 expression was significantly increased in SQCC compared with that in SQ. CLDNs 3 and 4 were significantly elevated both in BE and ACC compared with that in FOV. CLDN 2 expression increased significantly in ACCs compared with that in BE. This is the first report proving similarities and differences regarding claudin expression pattern in BE and ACC compared with that in FOV and SQ. Our data prove a close link in CLDN pattern between BE and ACC, adding further evidence that BE is an alteration preceding esophageal ACC.

Changes of cell adhesion and extracellular matrix (ECM) components in cervical intraepithelial neoplasia.

Cell-cell and cell-extracellular matrix interaction is crucial in tumor progression. Tight junction (TJ) proteins as occludin and claudins (CLDNs) play important role in this process together with several extracellular matrix components, as syndecan. Our previous work suggested significant changes in the expression of claudins even in the early stages of cervical carcinogenesis. The aim of our present work was to study the expression of occludin and syndecan-1, as compared to CLDNs, in early phases of cervical carcinogenesis. Paraffin sections of 50 samples were studied by immunohistochemistry, including cervical intraepithelial neoplasias (CINI-II-III), in situ carcinomas (CIS) and normal cervical samples. Occludin and CLDN-2 were found colocalized in the basal layer, while syndecan-1 and CLDN-1, -4 and -7 were coexpressed in the parabasal and intermedier layers in normal epithelia. Intensity of occludin staining decreased in CIN/CIS lesions, although it was more extended towards the upper epithelial layers with inverse relation with grades, as seen in the case of CLDN-2 expression. CLDN-1, -2, -4 and -7 were detected in the entire epithelium in CIN, showing decrease in CIS. The progression of CIN was associated with reduced syndecan-1 expression, in contrast to CLDN-1, -4 and -7 which increased toward CIS. The obtained data suggest that significant changes occur in the composition of cell adhesion complexes even in early stages of cervical carcinogenesis. The pattern of expression is characteristic for the alteration, the changes in the different components, however, are not parallel with each other.

The expression of five different claudins in invasive breast carcinomas: comparison of pT1pN1 and pT1pN0 tumors.

The evaluation of the role of claudins (CLDNs) in breast carcinogenesis has recently begun. We investigated the expression of CLDNs 1, 2, 3, 4, and 7 in pT1pN0 and pT1pN1 invasive ductal breast carcinomas. Tissue arrays of 30-30 pT1pN0 and pT1pN1 invasive ductal breast carcinomas of different grades were constructed, and the expression of CLDN 1, 2, 3, 4, and 7 proteins was analyzed using standard and immunofluorescent immunohistochemistry. The results were evaluated by light and confocal microscopy. Regarding CLDN 1, 4, and 7 expressions, differences were noted between normal and tumor cells and also between tumors of different grades, while no remarkable differences were noted between pT1pN0 and pT1pN1 tumors. CLDNs 1 and 7 were found to be downregulated in tumor cells compared to the normal epithelium, while CLDN 4 expression was decreased in grade 1 tumors. CLDN 7 protein was abundant in normal epithelia, and the staining decreased in grade 3 tumors. There were no differences between normal and neoplastic cells regarding CLDN 2 and 3 expressions. As a preliminary result, our observations suggest that the analyzed CLDNs do not promote tumor metastasis. On the basis of our findings, it seems that CLDN 1, CLDN 4, and CLDN 7 may rather have an important role in tumorigenesis or in cell-to-cell adhesion.

Effect of formalin, acetone, and RNAlater fixatives on tissue preservation and different size amplicons by real-time PCR from paraffin-embedded tissue.

RNA recovered from paraffin-embedded tissue has been reported to be a suitable substrate for polymerase chain reaction. During tissue fixation and paraffin embedding, RNA undergoes degradation, but with certain restrictions, it can be used for gene expression studies. At the same time, formalin-fixed, paraffin embedded histopathology archives contain an unestimable collection, which could be analyzed to investigate changes in mRNA expression in pathologic processes. To decide for future tissue conservation of pathology samples, it would be reasonable to satisfy both histologic and molecular biologic needs. The effect of three different fixation methods, RNAlater (SIGMA R 0901, St Louis, MO), acetone, and formalin, were compared by histology, immunohistochemistry, and real-time PCR. To assess tissue structure preservation and antigenicity, hematoxylin-eosin staining and immunohistochemistry were performed; to assess RNA quality, RNA was extracted and the transcription of different amplicon sizes (121, 225, 406 bp for GAPDH; 166, 310, 536 bp for beta globin) were examined on human endometrium samples. The most adequate tissue preservation was found in case of formalin fixation, while there were no significant differences in the three fixatives' yields for various size real-time PCR amplicons. Longer amplicons (above approximately 225 bp) have limited use for gene expression studies, while shorter amplicons could give more reliable results.

Viral hepatitis: new data on hepatitis C infection.

Viral hepatitis (VH) is almost as old as human beings, at least as old as known human history. However, the natural history and the epidemiology of the disease has undergone changes during the centuries and even recently we have been facing several new aspects. The estimated global prevalence is around 3-5%, which means that approximately 400 million patients are infected with hepatitis B virus and that there are 170 million infections with hepatitis C virus. The mortality figures are projected to show a 2- to 3-fold increase over the next two decades as hepatitis C virus-infected patients develop cirrhosis, which makes this the leading indication for liver transplantation. These data point to the importance of VH being a significant public health problem worldwide. The list of hepatotropic viruses is well known, including hepatitis A (HAV), B (HBV), C (HCV), D (HDV), E (HEV), G (HGV) and F (HFV). HGV and HFV are excluded from the present review, mainly because they are questionable in relation to the causation of liver disease. Our knowledge of HAV, HBV, HDV and HEV has accumulated over the last decade, so the present discussion is focused on HCV, which is currently generating considerable concern and controversy, and is the leading cause of chronic liver disease worldwide. The main questions to be discussed, are: the characterization of the agents' viral genotypes/subtypes, the viral-cell interaction, the pathogenesis of VH, the extrahepatic manifestations of viral infection and hepatocarcinogenesis.