ELIMINATE: a PCR record-based macroelimination project for systematic recall of HCV-RNA-positive persons in Austria.

BACKGROUND AND AIMS: Micro-elimination projects targeted to specific hepatitis C virus (HCV) risk populations have been successful. Systematic identification of persons with HCV viremia, regardless of risk group, based on already available laboratory records may represent an effective macroelimination approach to achieve global HCV elimination. METHODS: Persons with a last positive HCV-RNA PCR result between 2008-2020 in the reference virology laboratories in eastern Austria were identified. First, (i) we described their demographic characteristics, (ii) we systematically recalled persons to the respective centers and (iii) started antiviral treatment if HCV-RNA viremia was confirmed, and (iv) recorded sustained virologic response (SVR). This interim report includes the preliminary results from 8 participating centers. RESULTS: During the study period 22,682 persons underwent HCV-RNA PCR testing, 11,216 (49.4%) were positive at any point in time, and 6006 (26.5%) showed detectable HCV-RNA at the last PCR test, suggesting ongoing HCV viremia. At the time of this interim report, 2546/6006 HCV-RNA PCR(+) persons were evaluated: 443/2546 (17.4%) had died, 852/2546 (33.5%) had invalid contact data, and 547/2546 (21.5%) had achieved SVR between data retrieval and recall. Contact could be established in 236/704 (33.5%) of the remaining target population with 97/236 (41.1%) presenting at the clinic for treatment evaluation. Ultimately, 71/236 (30.1%) started antiviral treatment and SVR was documented in 47/71 (66.2%). CONCLUSION: This ELIMINATE project based on systematic assessment of HCV-RNA PCR-records, identified 6006 persons with potential persisting HCV viremia. Invalid contact data and missed visits for treatment evaluation were the main barriers towards HCV elimination within this project. Importantly, many subjects with HCV viremia lost to follow-up were successfully linked to care and started antiviral treatment.

Performance study of the anterior nasal AMP SARS-CoV-2 rapid antigen test in comparison with nasopharyngeal rRT-PCR.

Introduction: The gold standard for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection is real-time reverse transcription PCR (rRT-PCR), which is expensive, has a long turnaround time and requires special equipment and trained personnel. Nasopharyngeal swabs are uncomfortable, not suitable for certain patient groups and do not allow self-testing. Convenient, well-tolerated rapid antigen tests (RATs) for SARS-CoV-2 detection are called for. Gap statement: More real-life performance data on anterior nasal RATs are required. Aim: We set out to evaluate the anterior nasal AMP RAT in comparison with rRT-PCR in a hospital cohort. Methodology: The study included 175 patients, either hospitalized in a coronavirus disease 2019 (COVID-19) ward or screened in a preadmittance outpatient clinic. Two swabs were collected per patient: an anterior nasal one for the RAT and a combined naso-/oropharyngeal one for the rRT-PCR. Sixty-five patients (37%) were rRT-PCR-positive [cycle threshold (C t) <40]. Results: The anterior nasal AMP RAT showed an overall sensitivity and specificity of 29.2 % (18.6-41.8, 95 % CI) and 100.0 % (96.7-100.0, 95 % CI) respectively. In patients with a C t value <25, <30 and <33, higher sensitivities were observed. Time since symptom onset was significantly higher in patients with a false-negative RAT (P=0.02). Conclusion: The anterior nasal AMP RAT showed low sensitivities in this cohort, especially in patients with a longer time since symptom onset. Further knowledge concerning the viral load and antigen expression over time and in different swabbing locations is needed to outline the usage time frame for SARS-CoV-2 RAT.

Standard blood laboratory values as a clinical support tool to distinguish between SARS-CoV-2 positive and negative patients.

Standard blood laboratory parameters may have diagnostic potential, if polymerase-chain-reaction (PCR) tests are not available on time. We evaluated standard blood laboratory parameters of 655 COVID-19 patients suspected to be infected with SARS-CoV-2, who underwent PCR testing in one of five hospitals in Vienna, Austria. We compared laboratory parameters, clinical characteristics, and outcomes between positive and negative PCR-tested patients and evaluated the ability of those parameters to distinguish between groups. Of the 590 patients (20-100 years, 276 females and 314 males), 208 were PCR-positive. Positive compared to negative PCR-tested patients had significantly lower levels of leukocytes, neutrophils, basophils, eosinophils, lymphocytes, neutrophil-to-lymphocyte ratio, monocytes, and thrombocytes; while significantly higher levels were detected with erythrocytes, hemoglobin, hematocrit, C-reactive-protein, ferritin, activated-partial-thromboplastin-time, alanine-aminotransferase, aspartate-aminotransferase, lipase, creatine-kinase, and lactate-dehydrogenase. From all blood parameters, eosinophils, ferritin, leukocytes, and erythrocytes showed the highest ability to distinguish between COVID-19 positive and negative patients (area-under-curve, AUC: 72.3-79.4%). The AUC of our model was 0.915 (95% confidence intervals, 0.876-0.955). Leukopenia, eosinopenia, elevated erythrocytes, and hemoglobin were among the strongest markers regarding accuracy, sensitivity, specificity, positive and negative predictive value, positive and negative likelihood ratio, and post-test probabilities. Our findings suggest that especially leukopenia, eosinopenia, and elevated hemoglobin are helpful to distinguish between COVID-19 positive and negative tested patients.

Leukocyte telomere length is linked to vascular risk factors not to Alzheimer's disease in the VITA study.

Association of telomere shortening with overall dementia or Alzheimer's disease is described controversially and the pathophysiologic relevance is unclear. Whether patients, suffering from pure probable Alzheimer's disease or pure vascular dementia, have shorter leukocyte telomeres than cognitively healthy controls was determined. Leukocyte telomere lengths (LTLs) of 597 participants of the VITA study (longitudinal community-based age-cohort [mean 75.7 (±0.45) years] study: 243 male; 578 non-demented at baseline) were compared with different aspects of cognition, risk factors of dementia and survival. LTLs of 264 persons cognitively healthy at baseline (mild cognitive impaired excluded) and all follow-ups (mean = 5643 bp, SD = 736) did not show any difference to LTLs of 43 incident pure possible (mean = 5548 bp; SD = 666) or 34 incident pure probable Alzheimer's diseases (mean = 5712 bp; SD = 695; post hoc Dunnett test: MD = -95; SE = 119; p = 0.67 and MD =+68.3; SE = 132; p = 0.84, res.). 264 stably cognitively healthy showed a trend to longer telomeres than 6 incident vascular dementias (mean = 5643 bp, SD = 736 vs mean = 5101 bp, SD = 510; t test: T = 1.8; df = 268; p = 0.07). Males (n = 243; mean = 5470 bp; SD = 684) had significantly shorter telomeres than females (n = 354; mean = 5686 bp; SD = 714; t test: T = -3.7; df = 595; p = 0.0001) and died significantly earlier (113.7 vs 130.1 months: Log Rank Chi square = 12.2; p = 0.0001). Shorter telomeres were associated with prevalence of more than one vascular risk factor (n = 587; mean = 5728; SD = 723 vs mean = 5533; SD = 691; t test: T = 3.1; df = 576; p = 0.002) and, as a trend, with poorer survival (Cox Regression: Wald = 4.9; p = 0.026; OR = 0.98; 95% CI 0.96-0.99). In 75.7 years old persons, no association of LTL with incident pure Alzheimer's disease was found. Significantly shorter telomeres were associated with sum of vascular risk factors, males and early mortality in males. Exclusion of mixed dementias may improve the search for risk factors more specific for Alzheimer's disease.

Evaluation of Gene Expression Patterns in Micrografts Demonstrate Induction of Catagen-Like Processes During Storage.

BACKGROUND: Alterations of gene expression patterns may contribute to the commonly observed transient reduction of hair shaft elongation in hair restoration surgery. OBJECTIVE: To elucidate the molecular causes, we evaluated changes in gene expression patterns in hair follicle micrografts during storage. MATERIALS AND METHODS: Micrografts with different amounts of adjacent connective tissue (regular, skinny, and chubby) were stored for different periods, and the expression of key genes was determined: dermal papilla (DP): FGF7, alkaline phosphatase (ALP), versican; outer root sheath: Krt15; inner root sheath: Krt 25; cuticula: Krt85; Henle layer: filaggrin; genes related to apoptosis and growth/differentiation: Caspase 3, Ovol1, and Foxo1. RESULTS: A significant decrease in FGF7 (4 × 10 fold) was observed after 4 hours, with further decrease after 48 hours. A significant decrease of versican (0.35 fold) and ALP (0.004 fold) was observed after 24 hours of storage. No differences relating to adjacent connective tissue were observed. No changes of different keratins genes or genes related to growth/differentiation and apoptosis were observed. CONCLUSION: These data clearly demonstrate a reduction in the specific function of cells in the DP, which seem to be the causative for the induction of hair follicle cycling during micrograft preparation and storage.

Validation of kappa free light chains as a diagnostic biomarker in multiple sclerosis and clinically isolated syndrome: A multicenter study.

BACKGROUND: Kappa free light chains (KFLCs) have been proposed as a diagnostic biomarker in patients with clinically isolated syndrome (CIS) and multiple sclerosis (MS). OBJECTIVE: The objective of this paper is to validate the diagnostic accuracy of intrathecal KFLC synthesis in a multicenter study. METHODS: KFLCs were measured by nephelometry under blinded conditions in cerebrospinal fluid (CSF) and serum sample pairs of patients with CIS (n = 60), MS (n = 60) and other neurological diseases (n = 60) from four different MS centers. The upper normal limit for intrathecal KFLC concentrations depending on blood-CSF barrier function was previously calculated in a cohort of 420 control patients. RESULTS: Diagnostic sensitivity of intrathecal KFLC synthesis, IgG synthesis according to Reiber, IgG index and oligoclonal bands (OCBs) was 95%, 72%, 73% and 93% in patients with MS and 82%, 47%, 43% and 72% in patients with CIS. Specificity of intrathecal KFLC synthesis was 95% and 98% for all other measures. CONCLUSION: These findings further support the diagnostic value of intrathecal KFLC synthesis in CIS and MS patients and demonstrate a valid, easier and rater-independent alternative to OCB detection.

Impact of visceral fat and pro-inflammatory factors on the pathogenesis of age-related macular degeneration.

PURPOSE: Previous studies have indicated that the immune system is involved in the pathogenesis of the AMD. Increased visceral fat, in addition, has a pro-inflammatory effect on the organism by producing or influencing different kinds of inflammatory factors. The aim of this study is to determine the relationship of body fat distribution in patients with age-related macula degeneration in comparison to a control group in the Austrian population. METHODS: In this case-control study, body weight and height, and body mass index (BMI) were measured for each subject in 54 patients with exudative AMD and compared to 46 gender- and age-matched healthy control subjects. Body composition and abdominal fat areas were measured using dual-energy X-ray absorptiometry (DEXA). Data on age, gender distribution, smoking history and systemic diseases, respectively, were compared. The inflammatory markers CRP, tumour necrosis factor-alpha (TNF-alpha), leptin, amyloid A, amyloid beta and interleukin-6 (IL-6) were assayed by ELISA (R&D). RESULTS: DEXA revealed central-abdominal-to-total body fat ratio of 0.073 +/- 0.011 in AMD patients compared to 0.061 +/- 0.013 in the controls (p <0.001; d = 0.98). The calculation of BMI has provided a significant result (p =0.045). U-test results for Aß1-42, IL-6, SAA and CRP each were significant (p < 0.05), with higher values in AMD patients. Leptin, TNF-alpha and Aß1-40 showed no significant differences between the groups. CONCLUSION: Our results suggest that abdominal fat distribution is significantly associated with age-related macular degeneration. Analysis of patients with exudative AMD revealed higher levels of CRP, amyloid ß1-42, IL-6 and amyloid alpha.

Extra fructose in the growth medium fuels lipogenesis of adipocytes.

Fructose in excessive amounts exerts negative effects on insulin sensitivity, blood pressure, and liver metabolism. These adverse outcomes were attributed to its disturbances of key metabolic pathways in the liver. Recently, possible consequences of high fructose levels directly on adipocytes in vivo have been considered. We have cultured adipocytes in growth media containing 1 g/L fructose additionally to glucose and monitored the cells fate. Cells developed lipid vesicles much earlier with fructose and showed altered kinetics of the expression of mRNAs involved in lipogenesis and hexose uptake. Adiponectin secretion, too, peaked earlier in fructose containing media than in media with glucose only. From these data it can be speculated that similar effects of fructose containing diets happen in vivo also. Apart from toxic action on liver cells, adipocytes might be stimulated to take up extra fructose and generate new lipid vesicles, further dysregulating energy homeostasis.

Spectrum of pathogens of in-patient children and youths with community acquired pneumonia: a 3 year survey of a community hospital in Vienna, Austria.

BACKGROUND: No actual data are available on the epidemiology and morbidity of community acquired pneumonia (CAP) in youths and children in Vienna, Austria. OBJECTIVE: The objective was to determine the epidemiology of CAP and morbidity of children hospitalized due to CAP in a tertiary care facility. METHODS: During three winter seasons youths and children hospitalized due to CAP were enrolled. Testing for viral and bacterial pathogens of pneumonia was performed in a routine clinical setting. Blood cultures were performed; respiratory viruses, Mycoplasma pneumoniae and Chlamydia pneumoniae were searched for by an established Real Time polymerase chain reaction (PCR) panel. Clinical signs and indices of inflammation were documented. RESULTS: Out of 279 children and youths with CAP a causative agent could be detected in 190 (68 %). Viruses and bacteria were diagnosed in 107 (57 %) and 58 patients (30 %), respectively. Co-infection was found in 20 patients (10 %), Mycoplasma pneumoniae or Clamydia pneumoniae in 16 cases (8 %). In seven patients blood cultures were positive. C-reactive protein (CRP) was significantly higher in children with positive Streptococcus pneumoniae antigen (SPAG) than with viral infection and/or co-infection. Clinical parameters showed no statistically significant differences. C. pneumoniae and M. pneumoniae were only diagnosed in children and youths with 5 years and older. CONCLUSIONS: Testing for pathogens in CAP in clinical routine achieves a high recovery rate. Blood cultures are rarely helpful, but the molecular testing for viruses seemed to be helpful to establish the diagnosis.

Optimized quantification of lymphocyte subsets by use of CD7 and CD33.

Identification and quantification of lymphocyte subsets is based on the expression of specific cell surface antigens. As only a minority of these structures is lineage-restricted gating strategies were established, which should permit to include a maximum of lymphocytes, to reach a high purity within the gate and to avoid specific loss of subsets. Two problems remain: First, the incomplete removal of nonlymphoid cells when CD14 is used to exclude them from the lymphocyte gate. Second, the lack of a restricted marker to identify NK cells that are usually defined as CD3(-) /CD16/56(+) lymphocytes, though contaminating monocyte subsets share the expression of CD16, respectively, CD56. This study demonstrates the advantage of CD33 over CD14 at the creation of a pure lymphocyte gate, because CD33 enables the exclusion of all monocyte subpopulations as well as basophils and granulocytes. Independent of the applied NK cell marker mean purity was significantly higher, when CD33 was used (P < 0.001). For the identification of NK cells, CD7 was compared with CD16/56 and with single stained CD56. CD7 and CD16/56 exhibited as equivalent in various CD33 settings (P ≥ 0.173), whereas the mean proportion of CD56(+) NK cells was significantly lower (P ≤ 0.008). Use of CD14 entailed a significantly higher amount of CD3(-) /CD16/56(+) cells than of CD3(-) /CD7(+) cells (P = 0.008) because of the remaining CD14(-) /CD16(+) monocytes. As CD7 is restricted to T cells and NK cells in peripheral blood, misclassification of contaminating monocytes is avoided and CD7(+) NK cells can be identified by lack of CD3. Applying this new selection of mAbs, we reached a mean purity of ≥99.50% within the revised lymphocyte gate. As gates can be set very broadly, high inclusivity and high purity are not mutually exclusive. We propose the adoption of CD7 and CD33 for the quantification of lymphocyte subsets.

Implication of CD21, CD35, and CD55 in the pathogenesis of age-related macular degeneration.

PURPOSE: To determine a possible implication of CD21, CD35, and CD55 in the pathogenesis of age-related macular degeneration (AMD) by assessing the difference in expression rates of these factors on AMD patients and a control group. DESIGN: Case-control study. METHODS: Fifty unrelated AMD patients and 48 unrelated sex- and age-matched control subjects participated in this case-control study. Samples of fresh EDTA-blood were stained and flow cytometry was chosen to measure fluorescence emissions. The association between exudative AMD and CD21, CD35, and CD55 was evaluated from all patients who completed the study. RESULTS: Our study shows CD35 to be expressed in a significantly higher frequency in AMD patients on monocytes (P = .00586), lymphocytes (P = .000605), and granulocytes (P < .000033). In contrast, the expression rate of CD21 (P > .05) and CD55 (P > .05) are similar in both groups. CONCLUSION: More regulative factors of the complement system are involved in pathogenesis of AMD. Our study underlines the key role of the complement system in AMD and shows the involvement of the whole immune system through more regulative factors.

Circulating retinol-binding protein 4 and metabolic syndrome in the elderly.

BACKGROUND: Retinol-binding protein (RBP) 4, a human adipokine that specifically binds to retinol, has been reported to provide a link between obesity and insulin resistance. Plasma RBP4 concentration may be under the influence of age and obesity, but only a few studies has investigated this link in elderly individuals. Consequently, we tested the correlation between RBP4 concentrations and type 2 diabetes/metabolic syndrome (MetS) components in a large population based cohort study (VITA) of elderly [1, 2]. Using a single birth cohort, this investigation could exclude the influence of age. METHODS: We evaluated the correlation of RBP4 with type 2 diabetes and MetS components including Body Mass Index (BMI), blood pressure, lipid parameters, fasting glucose insulin, homeostasis model assessment insulin resistance (HOMA-IR), and smoking in exclusively 75-76 year old participants (N = 232). RESULTS: In the present study, RBP4 concentrations were associated with type 2 diabetes and metabolic syndrome (MetS) components. Of all the individual components of metabolic syndrome that were associated with RBP4 concentrations, the correlations of RBP4 with serum triglycerides and a negative correlation with HDL were the strongest ones observed in our study cohort (p<0.0001). CONCLUSIONS: RBP4 plays a role in biological mechanisms that are responsible for insulin resistance and development of type 2 diabetes.

Genetic cardiovascular risk factors and age-related macular degeneration.

PURPOSE: To investigate the association between genetic cardiovascular risk factors and exudative age-related macular degeneration (AMD) in a White Austrian population. METHODS: Seventy-five unrelated AMD patients and 75 unrelated healthy, sex- and age-matched control patients were genotyped for the following 19 single nucleotide polymorphisms (SNPs) in 14 different genes: blood coagulation factor V (FV) R506Q, factor II (prothrombin) G20210A and factor XIII (FXIII) V34L; 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T, A1298C; plasminogen activator inhibitor 1 (PAI-1) 4G/5G; endothelial protein C receptor (EPCR) 4600 A>G (A3 haplotype), 4678 G>C (A1 haplotype); apolipoprotein B (ApoB) R3500Q; apolipoprotein E (ApoE) E2/E3/E4; ß-fibrinogen -455 G>A; human platelet antigen 1 (HPA1) a/b; angiotensin-converting enzyme (ACE) I/D; endothelial nitric oxide synthase (eNOS) 786 T>C, 894 G>T; lymphotoxin alpha (LTA) 804 C>A and 9p21 rs10757278. Genotyping was carried out by polymerase chain reaction (PCR) followed by reverse hybridization (CVD StripAssays; ViennaLab Diagnostics, Vienna, Austria). RESULTS: No statistically significant association could be observed between AMD and the investigated genetic risk factors for cardiovascular disease (CVD). All factors seem to be uniformly distributed in the two groups of AMD patients and healthy controls. Two variables -ß-fibrinogen: -455 G>A (p = 0.0786) and apolipoprotein E4 (p = 0.0636) - were not as far from association as the others. CONCLUSION: Our data show that the 19 tested CVD risk markers do not play a significant role in AMD. ß-Fibrinogen and apolipoprotein E4 should be examined in a larger cohort.

Serum VEGF and CFH in exudative age-related macular degeneration.

PURPOSE: To determine serum vascular endothelial growth factor 165 (VEGF165) levels and the association of the complement factor H gene (CFH) Y402H polymorphism in patients with exudative age-related macular degeneration (AMD) in comparison to unaffected control subjects. METHODS: Sixty-six AMD patients and 66 healthy age- and gender-matched controls were included in this case-control study. The serum VEGF165 was assayed by ELISA (R&D). Genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism analysis. Chi-squared tests were used regarding the polymorphism, a t-test regarding the VEGF-levels. RESULTS: Levels of serum VEGF165 were similar in both groups (p-value = 0.2112). Genotype frequency differed significantly between patients with exudative AMD and the healthy control group (p = 0.003136). The serum VEGF165 levels were similar irrespective of the presence of the CFH Y402H polymorphism (p = 0.4113) and independent of the specific genotype (p = 0.9634). CONCLUSION: In the present study, exudative AMD is not associated to serum VEGF165 levels; furthermore, our data does not establish a statistical link between VEGF165 and the CFH Y402H polymorphism.

VKORC1 -1639G>A and CYP2C9*3 are the major genetic predictors of phenprocoumon dose requirement.

PURPOSE: Phenprocoumon, similar to other coumarin-derived anticoagulants, is associated with a large variation in the individual dose requirement to achieve stable anticoagulation. Polymorphisms in the vitamin K epoxide reductase complex subunit 1 (VKORC1) and the liver enzyme cytochrome P450 2C9 (CYP2C9) effectively account for the variability in warfarin and acenocoumarol response but are less well-defined pharmacogenetic predictors in phenprocoumon therapy. METHODS: A retrospective study was performed on 185 outpatients attending anticoagulation clinics in Austria and Germany. These patients were genotyped for the VKORC1 -1639G>A and 3730G>A polymorphisms as well as for the CYP2C9 *2 and *3 polymorphisms using a reverse hybridisation-based teststrip assay. RESULTS: The VKORC1 -1639A allele, which was present at a frequency of 41.4% in the study cohort, significantly reduced the mean weekly phenprocoumon dose by 3 mg (19%) in the heterozygous and by 6.7 mg (43%) in the homozygous state compared to wild-type carriers (15.5 +/- 6.8 mg, p < 0.0001). A stepwise multiple regression analysis revealed that VKORC1 -1639G>A, age and CYP2C9*3 were the major independent determinants of phenprocoumon dose, accounting for 14.2, 9.1 and 4.7% of its variability, respectively (p A genotype had no additional predictive power for individual dose variability. CONCLUSION: Similar to warfarin and acenocoumarol, the VKORC1 -1639G>A polymorphism had the highest impact on the maintenance dose of phenprocoumon. The factor age was the second most important predictor and explained a greater percentage of the variability than CYP2C9 genotype.

Diagnosis of infection with human herpes viruses in routine laboratory practice.

BACKGROUND: The potential for faster detection of human herpes viruses using PCR compared to other methods is undisputed. However, because of fear of contamination, the clinical implication of nucleic amplification methods in routine laboratories is not widespread. Herpes viruses cause a wide spectrum of diseases and can cause morbidity and mortality in immune-compromised patients. Using real-time PCR, most of the problems associated with PCR (contamination, cumbersome detection, and rather expensive tests) are solved, and a rapid, economical, and--most importantly--closed system is at hand. METHODS: We evaluated work procedures in our laboratory that enable the routine diagnosis of viral infections with high accuracy and rapid turn-around time. In parallel, inherent problems usually associated with PCR testing, especially cross-contamination could be suppressed to a minimum. The start of the work flow process begins with an automated nucleic acid extraction procedure that yields high quality DNA. A common--internally and externally controlled--PCR program for all six viruses allows rapid sample turn around. RESULTS: In all, 7500 analyses for human herpes virus infection were performed in the last 5 years. Results for various different specimens were produced within 24 h. Contamination occurred rarely and could be ameliorated easily. The use of internal controls identified rare PCR-inhibited samples. The detection limits for our assays are markedly below the clinically relevant range. CONCLUSIONS: Our workflow allowed rapid, cost-efficient, and labor saving routine diagnostic detection of viral infections.

Activation of the beta-catenin signaling pathway and its impact on RPE cell cycle.

PURPOSE: To investigate the effect of EGF, IGF-1, and VEGF on ARPE19 cell proliferation and differentiation. METHODS: The gene expression of RPE-specific differentiation and proliferation markers and the transcriptional and translational activity of beta-catenin signaling markers were measured by flow cytometry and RT-PCR. RESULTS: The data showed a significant decrease in RPE65, CRALBP, and cytokeratin 18 in ARPE-19 cells stimulated with EGF and IGF-1. In addition, a significant decrease in GSK-3beta and beta-catenin was observed that was paralleled by an increase in cyclin D1 expression. Cell cycle studies revealed an increase in ARPE cells in the S-G(2)/M-phase after treatment with EGF or IGF-1. VEGF, on the other hand, led to a reduction in cyclin D1 and to an increase in GSK 3beta and beta-catenin expression which was paralleled by an increase in RPE-specific differentiation markers. CONCLUSIONS: The data demonstrate the induction of proliferation by EGF and IGF-1 and upregulation of the beta-catenin signaling pathway in ARPE-19 cells. The data suggest that activation of the beta-catenin signaling pathway may be key in activating ARPE-19 cells by different growth factors.

Correlated downregulation of estrogen receptor beta and the circadian clock gene Per1 in human colorectal cancer.

There is a growing body of evidence that disturbed circadian clock gene expression is associated with tumor development and tumor progression. Based on our initial experiments demonstrating decreased period 1 (Per1) expression in colon cancer, we evaluated clock gene and estrogen receptor (ER) alpha/beta expression in colon cancer cells of primary colorectal tumors and adjacent normal colon mucosa (NM) by real-time RT-PCR. Analysis of gene expression in G(2) and G(3) colorectal tumors revealed a decrease of Per1 mRNA compared with paired NM (G(2): 0.52-fold; P = n.s. and G(3): 0.48-fold; P = 0.03). A significant gender specific difference of Per1 expression was observed in G(2) tumors as compared with NM (female: 0.38-fold; P = 0.004 vs. male: 0.73-fold; P = n.s.). Expression of CLOCK was significantly elevated in G(2) tumors of male patients (1.63-fold, P = 0.01). The expression of ER-beta was significantly decreased in G(2) and G(3) tumors (G(2): 0.32-fold; P = 0.003 and 0.27; P = 0.001). No significant gender specific differences of ER-beta reduction in tumors were observed. A significant correlation between the decrease of Per1 and ER-beta in colorectal tumors (r = 0.61; P < 0.001) was found. No changes in gene expression were detected for ER-alpha and Per2. Our data demonstrate a correlated decrease of Per1 and ER-beta in colorectal tumors, mediated probably by epigenetic mechanisms. The observed gender differences in the expression of CLOCK and Per1 in G(2) tumors might suggest a gender-specific, distinctive role of the cellular clock in colorectal tumorigenesis.