Publications

2023
Ragkousis A, Kazantzis D, Georgalas I, Theodossiadis P, Kroupis C, Chatziralli I. Association of PON1, APOE and SDF-1 Gene Polymorphisms with Treatment Response to Intravitreal Anti-VEGF Treatment in Patients with Retinal Vein Occlusion. Semin.Ophthalmol. [Internet]. 2023:1 - 8. WebsiteAbstract
PURPOSE: The purpose of this study was to determine whether specific genetic polymorphisms affect the response to intravitreal anti-vascular endothelial growth factor (anti-VEGF) treatment in patients with macular oedema secondary to retinal vein occlusion (RVO). METHODS: Participants in this prospective study were 50 patients with macular oedema secondary to RVO, who were treated with intravitreal ranibizumab or aflibercept, and were followed-up for 12 months after initiation of treatment. Five single nucleotide polymorphisms (SNPs) from three different genes (APOE, PON1, SDF-1) were examined as potential predictors for treatment response to intravitreal anti-VEGF agents. RESULTS: Patients with the LL genotype of the PON1 L55M SNP had significantly higher reduction in central subfield thickness (CST) at month 12 after initiation of intravitreal anti-VEGF treatment (101.63 +/- 56.80 mum in LL vs. 72.44 +/- 39.41 mum in LM vs. 40.25 +/- 19.33 mum in MM, p = .026). Patients with the M allele of the PON1 L55M SNP were significantly associated with lower reduction in CST compared to non-carriers (68.29 +/- 38.77 mum in LM + MM vs. 101.63 +/- 56.80 mum in LL, p = .032). CONCLUSION: PON1 L55M SNP may serve as a promising genetic biomarker for predicting response to intravitreal anti-VEGF treatment in patients with macular oedema due to RVO
Tsounidi D, Tsaousis V, Xenos N, Kroupis C, Moutsatsou P, Christianidis V, Goustouridis D, Raptis I, Kakabakos S, Petrou P. Simultaneous determination of procalcitonin and interleukin-6 in human serum samples with a point-of-care biosensing device. Talanta [Internet]. 2023;258:124403. WebsiteAbstract
The simultaneous determination of two inflammatory diseases biomarkers, namely procalcitonin (PCT) and interleukin-6 (IL-6), in human serum samples employing a Point-of-Care device based on Multi Area Reflectance Spectroscopy is presented. Dual-analyte detection was achieved using silicon chips with two silicon dioxide areas of different thickness, one functionalized with an antibody specific for PCT and the other with an antibody specific for IL-6. The assay included reaction of immobilized capture antibodies with mixtures of PCT and IL-6 calibrators with the biotinylated detection antibodies, streptavidin and biotinylated-BSA. The reader provided for the automated execution of the assay procedure, as well as for the collection and processing of the reflected light spectrum, the shift of which is correlated to analytes concentration in the sample. The assay was completed in 35 min and the detection limits for PCT and IL-6 were 2.0 and 0.01 ng/mL respectively. The dual-analyte assay was characterized by high reproducibility (the intra- and inter-assay coefficients of variation were less than 10% for both analytes) and accuracy (the percent recovery values ranged from 80 to 113% for both analytes). Moreover, the values determined for the two analytes in human serum samples with the assay developed were in good agreement with the values determined for the same samples by clinical laboratory methods. These results support the potential of the proposed biosensing device application for inflammatory biomarkers determination at the Point-of-Need
Vythoulkas D, Lazana I, Kroupis C, Gavriilaki E, Konstantellos I, Bousiou Z, Chondropoulos S, Griniezaki M, Vardi A, Gkirkas K, et al. Endothelial Injury Syndromes after Allogeneic Hematopoietic Stem Cell Transplantation: Angiopetin-2 as a Novel Predictor of the Outcome and the Role of Functional Autoantibodies against Angiotensin II Type 1 and Endothelin A Receptor. Int.J.Mol.Sci. [Internet]. 2023;24(8). WebsiteAbstract
Transplant-associated thrombotic microangiopathy (TMA) occurs in a significant percentage of patients after allogeneic stem cell transplantation (allo-SCT) and is associated with significant morbidity and mortality. The aim of the present study was to examine the association of serum angiopoetin-2 (Ang2) levels and the presence of antibodies against angiotensin II type 1 (AT1R) and ndothelin A Recreptor (ETAR) with the outcome of patients with TMA and/or graft-versus-host disease (GVHD) after allo-SCT. Analysis of our data showed that elevated serum Ang2 levels at the time of TMA diagnosis are significantly associated with increased non-relapse mortality and decreased overall survival. To our knowledge, this is the first study demonstrating an association between raised Ang2 levels and poor outcomes in patients with TMA. Antibodies against AT1R (AT1R-Abs) and ETAR (ETAR-Abs) were detected in 27% and 23% of the patients, respectively, but there was no association between the presence of autoantibodies and the outcome of patients with TMA. However, a significant finding was the strong positive correlation between the presence of AT1R-Abs with the occurrence of chronic fibrotic GVHD, such as scleroderma and cryptogenic organizing pneumonia, raising the possibility of the contribution of autoantibodies in the pathogenesis of fibrotic GVHD manifestations
Gioukaki C, Georgiou A, Gkaralea LE, Kroupis C, Lazaris AC, Alamanis C, Thomopoulou GE. Unravelling the Role of P300 and TMPRSS2 in Prostate Cancer: A Literature Review. Int.J.Mol.Sci. [Internet]. 2023;24(14). WebsiteAbstract
Prostate cancer is one of the most common malignant diseases in men, and it contributes significantly to the increased mortality rate in men worldwide. This study aimed to review the roles of p300 and TMPRSS2 (transmembrane protease, serine 2) in the AR (androgen receptor) pathway as they are closely related to the development and progression of prostate cancer. This paper represents a library-based study conducted by selecting the most suitable, up-to-date scientific published articles from online journals. We focused on articles that use similar techniques, particularly those that use prostate cancer cell lines and immunohistochemical staining to study the molecular impact of p300 and TMPRSS2 in prostate cancer specimens. The TMPRSS2:ERG fusion is considered relevant to prostate cancer, but its association with the development and progression as well as its clinical significance have not been fully elucidated. On the other hand, high p300 levels in prostate cancer biopsies predict larger tumor volumes, extraprostatic extension of disease, and seminal vesicle involvement at prostatectomy, and may be associated with prostate cancer progression after surgery. The inhibition of p300 has been shown to reduce the proliferation of prostate cancer cells with TMPRSS2:ETS (E26 transformation-specific) fusions, and combining p300 inhibitors with other targeted therapies may increase their efficacy. Overall, the interplay between the p300 and TMPRSS2 pathways is an active area of research
Kazantzis D, Sergentanis TN, Machairoudia G, Dimitriou E, Kroupis C, Theodossiadis G, Theodossiadis P, Chatziralli I. Correlation Between Imaging Morphological Findings and Laboratory Biomarkers in Patients with Retinal Vein Occlusion. Ophthalmol.Ther. [Internet]. 2023;12(2):1239 - 1249. WebsiteAbstract
INTRODUCTION: The aim of this study was to investigate the possible correlation between peripheral blood biomarkers and morphological characteristics of retinal imaging in patients with retinal vein occlusion (RVO). METHODS: Participants in this cross-sectional observational study were 65 consecutive patients (65 eyes) with treatment-naive RVO, who underwent spectral-domain optical coherence tomography (SD-OCT) and fundus fluorescein angiography (FFA). In addition, peripheral blood samples were taken to evaluate full blood count and biochemical parameters. The association between imaging characteristics and laboratory parameters was examined. RESULTS: Eyes with subretinal fluid presented significantly higher neutrophil-to-lymphocyte ratios (p = 0.028). Hyperreflective foci on SD-OCT were found to be associated with higher triglyceride levels (p = 0.024). The presence of cysts on SD-OCT was associated with significantly higher triglycerides (p = 0.010). Central subfield thickness (CST) higher than 464 mum was associated with higher lymphocyte count (p = 0.016) and higher urea (p = 0.015). No significant associations were found between laboratory parameters and intraretinal fluid, ellipsoid zone and external limiting membrane condition, or epiretinal membrane and macular ischemia. CONCLUSIONS: Specific imaging morphological characteristics were found to be associated with laboratory parameters in patients with RVO. These findings may help reveal the pathophysiology of RVO and its correlation with the development of specific clinical signs, while they could guide individualized treatment
2022
Stavroulakis G, Methenitis S, Koutroulis G, Xanthis D, Cherouveim E, Kroupis C, Anastasiadis G, Ketselidi K, Vlachopoulou E, Tsolakis C, et al. Exploring the predictors and prognostic significance of exercise-induced cardiac troponin release in master athletes following a 28 km mountain race. The Vamvakou research project. Biomarkers [Internet]. 2022;27(5):418 - 426. WebsiteAbstract
BACKGROUND/OBJECTIVE: Aetiology and significance of exercise-induced troponin release remains a contentious issue. We investigated the effect of a 28 km mountain run on cardiac troponin I (cTnI), in relation to training, performance, nutritional, biochemical and echocardiography variables, in a group of 25 recreational male master athletes. MATERIAL AND METHODS: A comprehensive list of variables related with nutrition, training, performance and echocardiography, was collected pre- and post-race. Twenty-four months later, outcomes regarding cardiovascular events were obtained. RESULTS: Serum cTnI values were increased after the race, with mean values rising from 7.2 +/- 2.2 (before) to 80.0 +/- 33.2 ng/L (post race), (p < 0.001) and 23/25(92%) exceeding Upper Reference limit (50 ng/L). Echocardiography did not reveal significant alterations, or correlations with cTnI values. The percentage difference in hs-cTnI concentrations pre- and post-race correlated positively with age, race-induced changes of selected muscle damage indices, resistance training volume and negatively with endurance capacity and training volume (r: -0.727 to 0.725, p < 0.05). All athletes reported no cardiovascular event during the 24-month period post-race. CONCLUSION: cTnI elevation induced by a 28 km mountain running race was not correlated with echocardiographic, nutritional parameters and was less pronounced in athletes with larger endurance training history, in contrast with resistance training and age
van Schrojenstein LM, Cubukcu HC, Boursier G, Panteghini M, Bernabeu-Andreu FA, Milinkovic N, Mesko BP, Linko S, Brugnoni D, O'Kelly R, et al. An approach for determining allowable between reagent lot variation. Clin.Chem.Lab Med. [Internet]. 2022;60(5):681 - 688. WebsiteAbstract
Clinicians trust medical laboratories to provide reliable results on which they rely for clinical decisions. Laboratories fulfil their responsibility for accurate and consistent results by utilizing an arsenal of approaches, ranging from validation and verification experiments to daily quality control procedures. All these procedures verify, on different moments, that the results of a certain examination procedure have analytical performance characteristics (APC) that meet analytical performance specifications (APS) set for a particular intended use. The APC can in part be determined by estimating the measurement uncertainty component under conditions of within-laboratory precision (u(Rw)), which comprises all components influencing the measurement uncertainty of random sources. To maintain the adequacy of their measurement procedures, laboratories need to distinguish aspects that are manageable vs. those that are not. One of the aspects that may influence u(Rw) is the momentary significant bias caused by shifts in reagent and/or calibrator lots, which, when accepted or unnoticed, become a factor of the APC. In this paper, we postulate a model for allocating a part of allowable u(Rw) to between-reagent lot variation, based on the need for long-term consistency of the measurement variability for that specific measurand. The allocation manages the ratio between short-term and long-term variation and indicates laboratories when to reject or correct certain variations due to reagent lots
Papastefanopoulou V, Stanitsa E, Koros C, Simoudis A, Florou-Hatziyiannidou C, Beratis I, Antonelou R, Andronas N, Voskou P, Angelopoulou E, et al. APOE Allele Frequency in Southern Greece: Exploring the Role of Geographical Gradient in the Greek Population. Geriatrics (Basel) [Internet]. 2022;8(1). WebsiteAbstract
BACKGROUND: the apolipoprotein e4 allele (APOE4) constitutes an established genetic risk factor for Alzheimer's Disease Dementia (ADD). We aimed to explore the frequency of the APOE isoforms in the Greek population of Southern Greece. METHODS: peripheral blood from 175 Greek AD patients, 113 with mild cognitive impairment (MCI), and 75 healthy individuals. DNA isolation was performed with a High Pure PCR Template Kit (Roche), followed by amplification with a real-time qPCR kit (TIB MolBiol) in Roche's Light Cycler PCR platform. RESULTS: APOE4 allele frequency was 20.57% in the ADD group, 17.69% in the MCI group, and 6.67% in the control group. APOE3/3 homozygosity was the most common genotype, while the frequency of APOE4/4 homozygosity was higher in the AD group (8.60%). APOE4 carrier status was associated with higher odds for ADD and MCI (OR: 4.49, 95% CI: [1.90-10.61] and OR: 3.82, 95% CI: [1.59-9.17], respectively). CONCLUSION: this study examines the APOE isoforms and is the first to report a higher APOE frequency in MCI compared with healthy controls in southern Greece. Importantly, we report the occurrence of the APOE4 allele, related to ADD, as amongst the lowest globally reported, even within the nation, thus enhancing the theory of ethnicity and latitude contribution
Efstratiou F, Tzortzis AS, Salakos C, Kroupis C, Dimopoulou A, Vrigkou H, Papakonstantinou D, Zavras N. Clinical significance of interleukin-18 in the course of burn injured children. Minerva Pediatr.(Torino) [Internet]. 2022. WebsiteAbstract
BACKGROUND: Burn injury (BI) is one of the most serious causes of morbidity and mortality in the pediatric population. BI triggers an initial stage of hyperinflammation, followed by hypersecretion of both pro- and anti-inflammatory cytokines. IL- 18 is a vital pro-inflammatory cytokine, the effect of which has been investigated not only in animal models but also in adult patients. No study has yet examined the association of serum IL- 18 levels and the clinical significance in the course of pediatric BI. METHODS: We conducted a prospective study including all children with burn injuries who were hospitalized from December 2015 to December 2018 in a tertiary Children's Hospital. RESULTS: A total of 55 children with BI were included. In the present study, we found a strong positive correlation between total body surface area (TBSA) and the levels of IL-18 at admission and on the third day postburn, respectively. The WBC count, the number of lymphocytes and the CRP levels at admission revealed a strong, positive correlation with IL-18 levels. The correlation between IL-18 levels at admission and the length of stay (LOS) was moderate. CONCLUSIONS: This study has shown that the levels of IL-18 collected at admission correlate positively with the extent of TBSA and inflammatory indices in pediatric patients. Moreover, IL-18 levels at admission may not be the most accurate prognostic factor regarding the LOS. However, further research is needed in order to establish more accurate predictive factors for the outcome of BIs in pediatric patients
Kazantzis D, Machairoudia G, Kroupis C, Theodossiadis G, Theodossiadis P, Chatziralli I. Complete Blood Count-Derived Inflammation Indices and Retinal Vein Occlusion: A Case-Control Study. Ophthalmol.Ther. [Internet]. 2022;11(3):1241 - 1249. WebsiteAbstract
INTRODUCTION: This study evaluated complete blood count-derived inflammation indices in patients with retinal vein occlusion (RVO). METHODS: Participants in this case-control study were 54 patients with RVO and 54 age- and sex-matched control subjects. All participants underwent a thorough ophthalmic examination, as well as blood sample testing for complete blood count. Comparison of all parameters derived from complete blood count as well as calculation of specific indices was performed between patients with RVO and controls. RESULTS: Patients with RVO presented significantly higher white blood cell count (p = 0.033), neutrophil count (p = 0.003), neutrophil-to-lymphocyte ratio (NLR, p = 0.002), red cell distribution width (RDW, p = 0.009), mean platelet volume (MPV, p = 0.023), and systemic immune-inflammatory index (SII, p = 0.007) compared to controls. Receiver operator characteristic curve (ROC) analysis showed that NLR was superior to other inflammatory indices, having the greatest area under the curve. The optimal cutoff value for NLR to predict RVO was 2.29 with 46.2% sensitivity and 77.8% specificity. CONCLUSION: Patients with RVO presented increased NLR, RDW, MPV, and SII, providing evidence that inflammation plays an important role in the pathogenesis of RVO. Complete blood cell count-derived indices can be easily calculated and may serve as an easy, simple, and cost-effective tool to evaluate the degree of systemic inflammation in patients with RVO, so as to potentially guide treatment
Kazantzis D, Theodossiadis P, Kroupis C, Theodossiadis G, Chatziralli I. Vitamin B12 and Folate as Risk Factors for Retinal Vein Occlusion: A Meta-Analysis. Klin.Monbl.Augenheilkd. [Internet]. 2022;239(5):709 - 716. WebsiteAbstract
PURPOSE: To evaluate the association between serum vitamin B12/folate and retinal vein occlusion (RVO). METHODS: A comprehensive search of the PubMed database was performed, which identified 271 abstracts to be screened. Ten studies met our inclusion criteria and a meta-analysis of these comparative case-control studies was performed on the mean +/- standard deviation serum vitamin B12 and folate levels, without language restrictions. Nine studies with 720 patients with RVO and 613 controls were included in the meta-analysis for vitamin B12, and 10 studies with 784 patients with RVO and 677 controls in the meta-analysis for folate. RESULTS: There was no statistically significant difference between patients with RVO and controls in serum vitamin B12 levels (mean difference: - 40.25 pg/mL, p = 0.28), either central RVO (mean difference: - 18.24 pg/mL, p = 0.71) or branch RVO (mean difference: - 23.56 pg/mL, p = 0.48). On the contrary, the plasma folate level was significantly lower in RVO patients than in controls (mean difference: - 1.34 ng/mL, p = 0.001), as well as in patients with CRVO compared to controls (mean difference: - 1.48 ng/mL, p = 0.006), but not in BRVO patients (mean difference: - 0.72 ng/mL, p = 0.11). CONCLUSIONS: RVO is associated with low serum folate levels, but not with serum vitamin B12 levels
Papadopoulou A, Fragkou PC, Maratou E, Dimopoulou D, Kominakis A, Kokkinopoulou I, Kroupis C, Nikolaidou A, Antonakos G, Papaevangelou V, et al. Angiotensin-converting-enzyme insertion/deletion polymorphism, ACE activity, and COVID-19: A rather controversial hypothesis. A case-control study. J.Med.Virol. [Internet]. 2022;94(3):1050 - 1059. WebsiteAbstract
Accumulating data has shown a contribution of the renin-angiotensin system in COVID-19 pathogenesis. The role of angiotensin-converting enzyme (ACE) insertion (I)/deletion (D) polymorphism as a risk factor in developing COVID-19 disease comes from epidemiological data and is controversially discussed. We conducted a retrospective case-control study and assessed the impact of ACE I/D genotype in COVID-19 disease prevalence and severity. In 81 COVID-19 patients explicitly characterized and 316 controls, recruited during the first wave of COVID-19 pandemic, ACE I/D genotype, and ACE activity were determined. A generalized linear model was used and Poisson regression analysis estimated the risk ratios (RRs) of alleles and genotypes for disease severity. DD patients had almost 2.0-fold increased risk (RR: 1.886, confidence limit [CL] 95%: 1.266-2.810, p = 0.0018) of developing a more severe disease when contrasted to ID and II individuals, as did D allele carriers compared to I carriers (RR: 1.372; CL 95%: 1.051-1.791; p = 0.0201). ACE activity (expressed as arbitrary units, AU/L) was lower in patients (3.62 +/- 0.26) than in controls (4.65 +/- 0.13) (p < 0.0001), and this reduction was observed mainly among DD patients compared to DD controls (3.97 +/- 0.29 vs. 5.38 +/- 0.21; p = 0.0014). Our results demonstrate that ACE DD genotype may predispose to COVID-19 increased disease severity via a mechanism associated, at least in part, with the significant fall in their ACE activity. Our findings suggest a more complex pattern of synergy between this polymorphism and ACE activity in COVID-19 patients compared to healthy individuals and set the grounds for large-scale studies assessing ACE genotype-based optimized therapies with ACE inhibitors and angiotensin receptor blockers
Chatziralli I, Kazantzis D, Kroupis C, Machairoudia G, Dimitriou E, Theodossiadis G, Theodossiadis P, Sergentanis TN. The impact of laboratory findings and optical coherence tomography biomarkers on response to intravitreal anti-VEGF treatment in patients with retinal vein occlusion. Int.Ophthalmol. [Internet]. 2022;42(11):3449 - 3457. WebsiteAbstract
AIMS: To investigate potential laboratory and imaging biomarkers as treatment response predictors to intravitreal anti-vascular endothelial growth factor (anti-VEGF) agents in patients with retinal vein occlusion (RVO). METHODS: Participants in this prospective study were 53 patients with treatment naive macular edema secondary to RVO, treated with intravitreal anti-VEGF agents and followed-up for 12 months. At baseline, all participants underwent best-corrected visual acuity measurement, dilated fundoscopy, optical coherence tomography and fluorescein angiography (FFA), while full blood count and biochemical analysis of various parameters was also performed. At month 12, treatment response was examined and classified as "favorable" or "non-response". Potential associations between laboratory/imaging biomarkers and treatment response were assessed. RESULTS: Univariate analysis showed that "favorable" response at month 12 after initiation of anti-VEGF treatment was correlated with baseline central subfield thickness (CST) < 464 mum (p < 0.001), absence of subretinal fluid (p = 0.004), absence of hyperreflective foci (HF) (p = 0.004), intact ellipsoid zone (EZ) and external limiting membrane (ELM) (p < 0.001 and p = 0.001, respectively), absence of epiretinal membrane (ERM) (p = 0.020) and absence of macular ischemia on FFA (p < 0.001), while increased monocytes-to-lymphocytes ratio was also associated with "favorable" treatment response (p = 0.010). All other laboratory parameters did not reach statistical significance. However, at the multivariate analysis, EZ and ELM status, HF, macular ischemia and monocytes-to-lymphocytes ratio were found to be independent predictors of treatment response. CONCLUSIONS: Intact EZ and ELM, absence of HF, absence of macular ischemia and increased monocytes-to-lymphocytes ratio at baseline can predict "favorable" treatment response in patients with treatment naive macular edema secondary to RVO
Dimitrakis E, Katsarou MS, Lagiou M, Papastefanopoulou V, Spandidos DA, Tsatsakis A, Papageorgiou S, Moutsatsou P, Antoniou K, Kroupis C, et al. Association of vitamin D receptor gene haplotypes with late-onset Alzheimer's disease in a Southeastern European Caucasian population. Exp.Ther.Med. [Internet]. 2022;24(3):584. WebsiteAbstract
Vitamin D receptor (VDR) gene single nucleotide polymorphisms (SNPs) have been investigated over the past years with the aim of identifying any association with the development of Alzheimer's disease (AD). However, information regarding the potential association of VDR SNP haplotypes with AD is limited. The aim of the present study was to provide additional knowledge on the effects of VDR haplotypes on the development of late-onset AD in a cohort of Southeastern European Caucasians (SECs). The study sample included 78 patients with late-onset AD and 103 healthy subjects as the control group. VDR SNPs that were analyzed were TaqI (rs731236), BsmI (rs1544410) and FokI (rs2228570). The CAC (TaqI, BsmI and FokI) haplotype was found to be associated with a 53% lower risk of developing the disease (OR, 0.47; 95% CI, 0.23-0.96; P=0.04) and the TAC (TaqI, BsmI and FokI) haplotype was associated with an ~6-fold greater risk of developing AD (OR, 6.19; 95% CI, 1.91-20.13; P=0.0028). Female subjects carrying the TAC haplotype had a ~9-fold greater risk of developing AD in comparison to female control subjects (OR, 9.27; 95% CI, 1.86-46.28; P<0.05). The TaqI and BsmI polymorphisms were in high linkage disequilibrium (D'=0.9717, r=0.8467) and produced a haplotype with a statistically significant different frequency between the control and AD group. The TA (TaqI and BsmI) haplotype was associated with an ~8-fold greater risk of developing AD (OR, 8.27; 95% CI, 2.70-25.28; P<0.05). Female TA carriers had an ~14-fold greater risk of developing the disease in comparison to female control subjects (OR, 13.93; 95% CI, 2.95-65.87; P<0.05). On the whole, the present study demonstrates that in the SEC population, TAC and TA are risk haplotypes for AD, while the CAC haplotype may act protectively. SEC women carrying the TAC or TA haplotype are at a greater risk of developing AD, thus suggesting that women are markedly affected by the poor utilization of vitamin D induced by the VDR haplotype
Dimitrakis E, Katsarou MS, Lagiou M, Papastefanopoulou V, Stanitsa E, Spandidos DA, Tsatsakis A, Papageorgiou S, Moutsatsou P, Antoniou K, et al. Association of vitamin D receptor gene TaqI polymorphism with Alzheimer's disease in a Southeastern European Caucasian population. Exp.Ther.Med. [Internet]. 2022;23(5):341. WebsiteAbstract
The role of vitamin D in Alzheimer's Disease (AD) has been studied over the past years. The results from numerous studies have indicated that the molecular pathways involved in the development of AD are closely related to the molecular pathways of the mechanisms of action of vitamin D. However, only a limited number of studies have described the key role of vitamin D receptor (VDR) in the regulation of the functions of vitamin D and the potential effect of single nucleotide polymorphisms (SNPs) of the VDR gene. Thus, the aim of the present study was to investigate the VDR TaqI polymorphism in relation to AD in a Southeastern European Caucasian (SEC) cohort. Further, the present study aimed to compare the results obtained with those of other AD populations. For this purpose, blood samples from 90 confirmed patients with AD [median age, 74 years; median mini-mental state examination (MMSE) score of 21; median frontal assessment battery (FAB) score of 10] and 103 healthy controls (median age, 57 years) were analyzed to determine the genotypes of TaqI (rs731236) using quantitative PCR. The frequencies (%) of the TaqI TT, TC and CC genotypes in the controls/patients were 34/48.9, 47.6/41.1 and 18.4/10.0, respectively. Statistically significant differences were observed for the TaqI C allele [odds ratio (OR). 0.54; 95% confidence interval (CI), 0.30-0.96; P=0.035], the TaqI TT genotype (OR, 1.86; 95% CI, 1.04-3.32; P=0.035) and the TaqI CC genotype (OR, 0.119; 95% CI, 0.014-0.995; P=0.032,) in relation to the MMSE score <21 in the patient's group. The TaqI TT allele was found to increase the risk of developing AD by 1.86-fold in the SEC population, while the TaqI C allele may act protectively, with a 46% lower risk of developing the disease. Patients with the TaqI CC genotype were found to have an 88% less likelihood of developing severe cognitive impairment based on the MMSE score. On the whole, the present study did not confirm the results of previous studies on the VDR TaqI C allele in patients with AD
Ananikas K, Stavrakas P, Kroupis C, Christou EE, Brouzas D, Petrou P, Papakonstantinou D. Molecular Biologic Milieu in Rhegmatogenous Retinal Detachment and Proliferative Vitreoretinopathy: A Literature Review. Ophthalmic Res. [Internet]. 2022;65(6):637 - 646. WebsiteAbstract
Multiple lines of evidence support an immunologic response along with inflammation to be implicated in the pathophysiology of primary rhegmatogenous retinal detachment (RRD) and the development of proliferative vitreoretinopathy (PVR). The purpose of this review is to provide an update on the signaling molecules in the vitreous and subretinal fluid (SRF) involved in these processes. A detailed literature search was performed in PubMed database until November 2021. We identified all papers referring to inflammatory and immunological mediators in the context of primary RRD and in cases complicated by PVR. We analyzed prospective and retrospective cohort studies and reference lists of the retrieved articles. A comprehensive investigation of immunological and inflammatory responses provides significant evidence for the implication of varying signaling molecules in the pathophysiology of RRD and the development of PVR. The reviewed series has revealed that disruption of the normal equilibrium during these processes may be present in the vitreous and SRF of these eyes. The precise role of cytokines, chemokines, and growth factors in the pathophysiology of these disorders remains to be clearly elucidated. Overall, immunological and inflammatory signaling molecules are widely implicated in both primary RRD and PVR. The reviewed literature indicates that precise knowledge concerning the pathological milieu sheds light on the underlying pathophysiology and potential therapeutic targets and highlights unmet needs to be addressed by future research
2021
Wieringa G, Queralto J, Homsak E, Jassam N, Cavalier E, Svinarov D, Krleza JL, Christou S, Pikner R, Larsen TR, et al. A proposed Common Training Framework for Specialists in Laboratory Medicine under EU Directive 2013/55/EC (The Recognition of Professional Qualifications). Clin.Chem.Lab Med. [Internet]. 2021;59(3):505 - 512. WebsiteAbstract
European Union (EU) Directive 2013/55/EC (The Recognition of Professional Qualifications) allows Member States to decide on a common set of minimum knowledge, skills and competences that are needed to pursue a given profession through a Common Training Framework. To be adopted the framework must combine the knowledge, skills and competences of at least one third of the Member States. Professionals who have gained their qualifications under a Common Training Framework will be able to have these recognised automatically within the Union. The backbone of the European Federation of Clinical Chemistry and Laboratory Medicine's (EFLM) proposed Common Training Framework for non-medical Specialists in Laboratory Medicine is outlined here. It is based on an Equivalence of Standards in education, training, qualifications, knowledge, skills, competences and the professional conduct associated with specialist practice. In proposing the recognition of specialist practice EFLM has identified 15 EU Member States able to meet Equivalence and in whom the profession and/or its training is regulated (an additional EU Commission requirement). The framework supports and contributes to the Directive's enabling goals for increasing professional mobility, safeguarding consumers and ensuring a more equitable distribution of skills and expertise across the Member States. It represents EFLM's position statement and provides a template for professional societies and/or competent authorities to engage with the EU Commission
Stanitsa E, Economou A, Beratis I, Kontaxopoulou D, Fragkiadaki S, Papastefanopoulou V, Pavlou D, Papantoniou P, Kroupis C, Papatriantafyllou J, et al. Effect of Apolipoprotein E4 on the Driving Behavior of Patients with Amnestic Mild Cognitive Impairment or Mild Alzheimer's Disease Dementia. J.Alzheimers.Dis. [Internet]. 2021;84(3):1005 - 1014. WebsiteAbstract
BACKGROUND: The driving behavior of patients with mild Alzheimer's disease dementia (ADD) and patients with mild cognitive impairment (MCI) is frequently characterized by errors. A genetic factor affecting cognition is apolipoprotein E4 (APOE4), with carriers of APOE4 showing greater episodic memory impairment than non-carriers. However, differences in the driving performance of the two groups have not been investigated. OBJECTIVE: To compare driving performance in APOE4 carriers and matched non-carriers. METHODS: Fourteen APOE4 carriers and 14 non-carriers with amnestic MCI or mild ADD underwent detailed medical and neuropsychological assessment and participated in a driving simulation experiment, involving driving in moderate and high traffic volume in a rural environment. Driving measures were speed, lateral position, headway distance and their SDs, and reaction time. APOE was genotyped through plasma samples. RESULTS: Mixed two-way ANOVAs examining traffic volume and APOE4 status showed a significant effect of traffic volume on all driving variables, but a significant effect of APOE4 on speed variability only. APOE4 carriers were less variable in their speed than non-carriers; this remained significant after a Bonferroni correction. To further examine variability in the driving performance, coefficients of variation (COV) were computed. Larger headway distance COV and smaller lateral position COV were observed in high compared to moderate traffic. APOE4 carriers had smaller speed COV compared to non-carriers. CONCLUSION: The lower speed variability of APOE4 carriers in the absence of neuropsychological test differences indicates reduced speed adaptations, possibly as a compensatory strategy. Simulated driving may be a sensitive method for detecting performance differences in the absence of cognitive differences
Papadopoulou MK, Chatziralli I, Tzika K, Chiras D, Kitsos G, Kroupis C. Correlation of the intronic LOXL1 polymorphism rs11638944 with pseudoexfoliation syndrome and glaucoma in a Greek population. Ophthalmic Genet. [Internet]. 2021;42(4):405 - 411. WebsiteAbstract
BACKGROUND: The purpose of this study is the development and validation of a novel and robust genotyping method for a new lysyl oxidase-like 1 (LOXL1) intronic polymorphism (rs11638944, C > G) and the investigation of its potential association with pseudoexfoliation syndrome (PXS) and pseudoexfoliation glaucoma (PXG) in a Greek population. MATERIAL AND METHODS: 242 DNA samples from 49 PXS, 64 PXG, 50 primary open-angle glaucoma (POAG) patients and 79 healthy age-matched controls were analyzed. Novel methodologies were developed and optimized, in order to genotype the intronic LOXL1 polymorphism: a) a real-time qPCR and melting curve analysis in the Light Cycler platform for rapid and cost-effective analysis and, b) a conventional PCR-RFLP method for analysis of a small number of samples. In selected samples, validity was checked with the reference DNA Sequencing method. RESULTS: The real-time qPCR methodology was reliable, demonstrating good efficiency, reproducibility, accuracy in genotyping (100% concordance with the PCR-RFLP method and DNA Sequencing), with good allele discrimination (Tm = 53.26 degrees C for C allele, Tm = 61.83 degrees C for G allele, DeltaTm = 8.57 degrees C). The results were characterized by Hardy-Weinberg equilibrium in all groups. An increase from 18% in healthy controls to 61% in PXS patients was detected for the G/G homozygote thus, the C allele is protective for PXS with OR = 0.22 (95%CI: 0.11-0.42, p < .0001). Moreover, an increase from 18% in healthy controls to 70% in PXG patients was detected for the G/G homozygote thus, the C allele is protective for PXG with OR = 0.13 (95%CI: 0.06-0.25, p < .0001). CONCLUSIONS: A statistically significant association was verified for the intronic LOXL1 polymorphism rs11638944 and PXS/PXG in a Greek population
Can CH, Vanstapel F, Thelen M, Bernabeu-Andreu FA, van Schrojenstein LM, Brugnoni D, Mesko BP, Milinkovic N, Linko S, Vaubourdolle M, et al. Improving the laboratory result release process in the light of ISO 15189:2012 standard. Clin.Chim.Acta [Internet]. 2021;522:167 - 173. WebsiteAbstract
The ISO 15189:2012 standard section 5.9.1 requires laboratories to review results before release, considering quality control, previous results, and clinical information, if any, and to issue documented procedures about it. While laboratory result reporting is generally regarded as part of the post-analytical phase, the result release process requires a general view of the total examination process. Reviewing test results may follow with troubleshooting and test repetition, including reanalyzing an individual sample or resampling. A systematic understanding of the result release may help laboratory professionals carry out appropriate test repetition and ensure the plausibility of laboratory results. In this paper, we addressed the crucial steps in the result release process, including evaluation of sample quality, critical result notification, result reporting, and recommendations for the management of the result release, considering quality control alerts, instrument flags, warning messages, and interference indexes. Error detection tools and plausibility checks mentioned in the present paper can support the daily practice of results release
Bourbouli M, Paraskevas GP, Rentzos M, Mathioudakis L, Zouvelou V, Bougea A, Tychalas A, Kimiskidis VK, Constantinides V, Zafeiris S, et al. Genotyping and Plasma/Cerebrospinal Fluid Profiling of a Cohort of Frontotemporal Dementia-Amyotrophic Lateral Sclerosis Patients. Brain Sci. [Internet]. 2021;11(9). WebsiteAbstract
Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are part of the same pathophysiological spectrum and have common genetic and cerebrospinal fluid (CSF) biomarkers. Our aim here was to identify causative gene variants in a cohort of Greek patients with FTD, ALS and FTD-ALS, to measure levels of CSF biomarkers and to investigate genotype-phenotype/CSF biomarker associations. In this cohort of 130 patients (56 FTD, 58 ALS and 16 FTD-ALS), we performed C9orf72 hexanucleotide repeat expansion analysis, whole exome sequencing and measurement of "classical" (Abeta(42), total tau and phospho-tau) and novel (TDP-43) CSF biomarkers and plasma progranulin. Through these analyses, we identified 14 patients with C9orf72 repeat expansion and 11 patients with causative variants in other genes (three in TARDBP, three in GRN, three in VCP, one in FUS, one in SOD1). In ALS patients, we found that levels of phospho-tau were lower in C9orf72 repeat expansion and MAPT c.855C>T (p.Asp285Asp) carriers compared to non-carriers. Additionally, carriers of rare C9orf72 and APP variants had lower levels of total tau and Abeta(42), respectively. Plasma progranulin levels were decreased in patients carrying GRN pathogenic variants. This study expands the genotypic and phenotypic spectrum of FTD/ALS and offers insights in possible genotypic/CSF biomarker associations
Charoniti E, Papastefanopoulou V, Florou-Hatziyiannidou C, Koros C, Stanitsa E, Papatriantafyllou JD, Papageorgiou SG, Kroupis C. TARDBP p.I383V, a recurrent alteration in Greek FTD patients. J.Neurol.Sci. [Internet]. 2021;428:117566. WebsiteAbstract
BACKGROUND: A significant proportion of FTD (Frontotemporal Degeneration) cases can be attributed to mutations in major genes such as GRN, MAPT and C9orf72. Our previous report on a Greek FTD cohort revealed the presence of the single nucleotide polymorphism (SNP) p.I383V (rs80356740) in the TARDBP gene in three unrelated patients. Our objective was to develop a novel, fast and accurate method for the detection of this particular SNP and evaluate the assay in a larger cohort. METHODS AND RESULTS: A real-time qPCR-melting curve analysis method was developed, validated and tested in 142 FTD patients and 111 healthy control subjects. The SNP was detected in another two patients raising its yield in FTD patients to 3.5% (5 out of 142 patients) while one in 111 healthy controls was found to be a carrier. However, its frequency in the general population has been reported extremely low in international SNP databases (0.002%). CONCLUSION: This fact along with the indicated pathogenicity of this SNP in some bioinformatics tools, suggest that TARDBP p.I383V is recurrent and likely pathogenic for the Greek FTD population. Our high-throughput method could be used for genotyping in other larger patient cohorts and in other populations. Additionally, functional in vitro studies are required for the final adjudication of this TARDBP alteration as a pathogenic alteration
2020
Roelofsen-de BR, Wielders J, Boursier G, Vodnik T, Vanstapel F, Huisman W, Vukasovic I, Vaubourdolle M, Sonmez C, Linko S, et al. Validation and verification of examination procedures in medical laboratories: opinion of the EFLM Working Group Accreditation and ISO/CEN standards (WG-A/ISO) on dealing with ISO 15189:2012 demands for method verification and validation. Clin.Chem.Lab Med. [Internet]. 2020;58(3):361 - 367. WebsiteAbstract
This paper reflects the opinion of the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group Accreditation and ISO/CEN standards (WG-A/ISO). It aims to provide guidance for drawing up local/national documents about validation and verification of laboratory methods. We demonstrate how risk evaluation can be used to optimize laboratory policies to meet intended use requirements as well as requirements of standards. This is translated in a number of recommendations on how to introduce risk evaluation in various stages of the implementation of new methods ultimately covering the whole process cycle
Frogoudaki AA, Pantelakis I, Bistola V, Kroupis C, Birba D, Ikonomidis I, Alexopoulos D, Filippatos G, Parissis J. Global Longitudinal Strain of the Systemic Ventricle Is Correlated with Plasma Galectin-3 and Predicts Major Cardiovascular Events in Adult Patients with Congenital Heart Disease. Medicina (Kaunas.) [Internet]. 2020;56(6). WebsiteAbstract
Backround and Objective: We sought to assess in adult congenital heart disease (ACHD) patients the prognostic value of plasma galectin-3 (Gal-3) levels and systemic ventricular global longitudinal strain (SV GLS) as well as their association with NTproBNP and arrhythmogenesis. Materials and Methods: We studied 58 patients (26 men, mean age 37 +/- 16.8 years) with various congenital heart diseases. Patients underwent echocardiogram, 24 h ambulatory ECG monitoring, while NTproBNP and Gal-3 were measured. They were followed up (median of 790.5 days -IQR 350.3 days) and major cardiovascular events (MACE) were recorded. Results. Mean Gal-3 levels were 17.07 +/- 6.38 ng/m. Plasma Gal-3 was correlated with LogNTproBNP (r = 0.456, p = 0.001).Gal-3 levels associated with supraventricular tachycardia (SVT) (p < 0.001) and ventricular tachycardia (VT) (p < 0.001), but was not associated with MACE (HR 1.018, 95% CI 0.944-1.098, p = 0.641).Mean SVGLS in patients with systemic left ventricle was -15.91% +/- 4.09%, which was significantly lower compared to patients with systemic right ventricle and patients with single ventricle (-11.42% +/- 3.37% and -11.9% +/- 5.06%, respectively, p = 0.021).SV GLS correlated with plasma Gal-3 (r = 0.313, p = 0.027) and logNTproBNP (r = 0.479, p < 0.001). SVGLS correlated with VT arrhythmias (p = 0.004). NTproBNP predicted MACE (AUC 0.750, p = 0.03). SVGLS also predicted MACE (AUC 0.745, p = 0.03. In multivariate analysis, SVGLS and logNTproBNP maintained their predictive value (p = 0.004 and p = 0.009, respectively) Conclusion: In ACHD patients, SV GLS was found to predict MACE independently from NTproBNP and correlated with VT. Gal-3 correlated with NTproBNP and SVGLS as well as SVT and VT, but has not been shown to bear significant prognostic potential
2019
Thelen M, Vanstapel F, Brguljan PM, Gouget B, Boursier G, Barrett E, Kroupis C, Lohmander M, Sprongl L, Vodnik T, et al. Documenting metrological traceability as intended by ISO 15189:2012: A consensus statement about the practice of the implementation and auditing of this norm element. Clin.Chem.Lab Med. [Internet]. 2019;57(4):459 - 464. WebsiteAbstract
ISO15189:2012 requires medical laboratories to document metrological traceability of their results. While the ISO17511:2003 standard on metrological traceability in laboratory medicine requires the use of the highest available level in the traceability chain, it recognizes that for many measurands there is no reference above the manufacturer's selected measurement procedure and the manufacturer's working calibrator. Some immunoassays, although they intend to measure the same quantity and may even refer to the same reference material, unfortunately produce different results because of differences in analytical selectivity as manufacturers select different epitopes and antibodies for the same analyte. In other cases, the cause is the use of reference materials, which are not commutable. The uncertainty associated with the result is another important aspect in metrological traceability implementation. As the measurement uncertainty on the clinical samples is influenced by the uncertainty of all steps higher in the traceability chain, laboratories should be provided with adequate and appropriate information on the uncertainty of the value assignment to the commercial calibrators that they use. Although the between-lot variation in value assignment will manifest itself as part of the long-term imprecision as estimated by the end-user, information on worst-case to be expected lot-lot variation has to be communicated to the end-user by the IVD provider. When laboratories use ancillary equipment that potentially could have a critical contribution to the reported results, such equipment needs verification of its proper calibration and criticality to the result uncertainty could be assessed by an approach based on risk analysis, which is a key element of ISO15189:2012 anyway. This paper discusses how the requirement for metrological traceability as stated in ISO15189 should be met by the medical laboratory and how this should be assessed by accreditation bodies
Ramos EM, Koros C, Dokuru DR, Van, Berlo V, Kroupis C, Wojta K, Wang Q, Andronas N, Matsi S, Beratis IN, et al. Frontotemporal dementia spectrum: first genetic screen in a Greek cohort. Neurobiol.Aging [Internet]. 2019;75:224 - 224. WebsiteAbstract
Frontotemporal dementia (FTD) is a heterogeneous group of neurodegenerative syndromes associated with several causative and susceptibility genes. Herein, we aimed to determine the incidence of the most common causative dementia genes in a cohort of 118 unrelated Greek FTD spectrum patients. We also screened for novel possible disease-associated variants in additional 21 genes associated with FTD or amyotrophic lateral sclerosis. Pathogenic or likely pathogenic variants were identified in 16 cases (13.6%). These included repeat expansions in C9orf72 and loss-of-function GRN variants, and likely pathogenic variants in TARDBP, MAPT, and PSEN1. We also identified 14 variants of unknown significance in other rarer FTD or amyotrophic lateral sclerosis genes that require further segregation and functional analysis. Our genetic screen revealed a high genetic burden in familial Greek FTD cases (30.4%), whereas only two of the sporadic cases (3.5%) carried a likely pathogenic variant. A substantial number of familial cases still remain without an obvious causal variant, suggesting the existence of other FTD genetic causes besides those currently screened in clinical routine
Moschos MM, Diamantopoulou A, Gouliopoulos N, Droutsas K, Bagli E, Chatzistefanou K, Kitsos G, Kroupis C. TCF4 and COL8A2 Gene Polymorphism Screening in a Greek Population of Late-onset Fuchs Endothelial Corneal Dystrophy. In Vivo [Internet]. 2019;33(3):963 - 971. WebsiteAbstract
BACKGROUND/AIM: Fuchs' endothelial corneal dystrophy (FECD) is a hereditary, progressive, bilateral, and irreversible disorder of the corneal endothelium. The purpose of this study was to develop a novel, accurate and high-throughput real-time polymerase chain reaction (PCR) method and melting-curve analysis in order to genotype the rs613872 polymorphism in the transcription factor 4 (TCF4) gene and to implement it on a well-ascertained sample of 22 Greek FECD patients and 58 healthy individuals, age- and sex-matched. PATIENTS AND METHODS: DNA was extracted from blood samples, which were screened with the DNA sequencing method in order to detect the g.31753T>G/p.L450W (rs8035192) and g.31767C>A/p.Q455K (rs8035191) mutations in a COL8A2 genomic region. RESULTS: TCF4 risk G allele frequency increased to 48% in FECD patients compared to 17% in healthy-subjects [OR=4.82 (95% CI=1.98-11.73)]. No p.L450W and p.Q455K COL8A2 gene mutations were detected. CONCLUSION: We confirmed that rs613872 in the TCF4 gene is strongly and statistically associated with late-onset FECD in a Greek population
Antalis E, Spathis A, Kottaridi C, Kossyvakis A, Pastellas K, Tsakalos K, Mentis A, Kroupis C, Tsiodras S. Th17 serum cytokines in relation to laboratory-confirmed respiratory viral infection: A pilot study. J.Med.Virol. [Internet]. 2019;91(6):963 - 971. WebsiteAbstract
BACKGROUND: Th17 cytokines are associated with modulation of inflammation and may be beneficial in clearing influenza infection in experimental models. The Th17 cytokine profile was evaluated in a pilot study of respiratory virus infections. METHODS: Consecutive patients with symptoms of respiratory tract infection visiting the emergency department of a tertiary care hospital during the winter influenza season of 2014 to 2015 were evaluated. CLART PneumoVir kit, (GENOMICA, Madrid, Spain) was used for viral detection of all known respiratory viruses. Th17 cytokine profile was evaluated with the MILLIPLEX MAP Human TH17 Magnetic Bead Panel (Millipore Corp., Billerica, MA). Correlation of the TH17 profile with viral detection was performed with univariate and multivariate analysis. RESULTS: Seventy-six patients were evaluated (median age 56 years, 51.3% female); a respiratory virus was identified in 60 (78.9%) patients; 45% had confirmed influenza. Influenza A (H3N2) correlated with higher levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 1beta (IL-1beta), IL-17A, IL-17E, IL-17F, IL-21, IL-22, and IL-23 (P < 0.05 by analysis of variance [ANOVA]) compared with respiratory syncytial virus (RSV). Parainfluenza virus (PIV) similarly had higher levels of GM-CSF, IL-1b, IL-17A, IL-22 compared with those detected in RSV, influenza B and any other virus infection ( P < 0.05; ANOVA). Increasing age (beta-coefficient = 1.11, 95% CI, 1.04-1.2, P < 0.01) as well as IL-17A levels (beta-coefficient = 1.03, 95% CI, 1.001-1.05, P = 0.04) predicted hospital admission. CONCLUSION: Main Th17 cell effector cytokines were upregulated in laboratory-confirmed A(H3N2) influenza and PIV. Excessive amounts of Th17 cytokines may be implicated in the pathogenesis and immune control of acute influenza and PIV infection in humans and may predict the severity of disease
Delimitsou A, Fostira F, Kalfakakou D, Apostolou P, Konstantopoulou I, Kroupis C, Papavassiliou AG, Kleibl Z, Stratikos E, Voutsinas GE, et al. Functional characterization of CHEK2 variants in a Saccharomyces cerevisiae system. Hum.Mutat. [Internet]. 2019;40(5):631 - 648. WebsiteAbstract
Genetic testing for cancer predisposition leads to the identification of a number of variants with uncertain significance. To some extent, variants of BRCA1/2 have been classified, in contrast to variants of other genes. CHEK2 is a typical example, in which a large number of variants of unknown clinical significance were identified and still remained unclassified. Herein, the CHEK2 variant assessment was performed through an in vivo, yeast-based, functional assay. In total, 120 germline CHEK2 missense variants, distributed along the protein sequence, and two large in-frame deletions were tested, originating from genetic test results in breast cancer families, or selected from the ClinVar database. Of these, 32 missense and two in-frame deletions behaved as non-functional, 73 as functional, and 15 as semi-functional, after comparing growth rates of each strain with positive and negative controls. The majority of non-functional variants were localized in the CHK2 kinase and forkhead-associated domains. In vivo results from the non-functional variants were in agreement with in silico predictions, and, where available, with strong breast cancer family history, to a great extent. The results of the largest, to date, yeast-based assay, evaluating CHEK2 variants, can complement and assist in the classification of rare CHEK2 variants with unclear clinical significance
Economopoulou P, Koutsodontis G, Avgeris M, Strati A, Kroupis C, Pateras I, Kirodimos E, Giotakis E, Kotsantis I, Maragoudakis P, et al. HPV16 E6/E7 expression in circulating tumor cells in oropharyngeal squamous cell cancers: A pilot study. PLoS.One. [Internet]. 2019;14(5):e0215984. WebsiteAbstract
OBJECTIVES: Human papillomavirus-related oropharyngeal squamous cell carcinoma (HPV+ OPSCC) is increasing in incidence. Although HPV+ OPSCC has favorable prognosis, 10 to 25% of HPV+ OPSCCs eventually recur. We sought to evaluate the feasibility of detection of HPV16 E6/E7 expression in Circulating Tumor Cells (CTCs) and its utility as a prognostic tool in HPV16-associated OPSCC. MATERIALS AND METHODS: We developed a highly sensitive RT-qPCR assay for HPV mRNA expression in EpCAM(+) CTCs. In 22 patients with early stage and locally advanced OPSCC we evaluated HPV16 E6/E7 expression in the EpCAM(+) CTC fraction at baseline and at the end of concurrent chemoradiotherapy. HPV status in pre-therapy formalin-fixed paraffin-embedded (FFPE) tumor biopsies was assessed by p16 immunohistochemistry and polymerase chain reaction (PCR) and double positives were subjected to Real-time qPCR assay for detection of HPV16, 18 and 31 types. RESULTS: Fourteen of 22 OPSCC (63.6%) were HPV DNA+/p16+. Among HPV+/p16+ patients, 10 patients (71.4%) were HPV16 DNA+. HPV16 E6/E7(+) CTCs were detected in 3 of 10 patients (30%) at baseline and 4 of 9 patients (44.4%) at the end-of-treatment, all of which were p16+/HPV16 DNA+. Survival analysis showed a significantly higher risk for disease relapse (p = 0.001) and death (p = 0.005) in patients with HPV16 E6/E7(+) baseline CTCs. CONCLUSION: Detection of HPV E6/E7(+) CTCs might be a useful noninvasive test in liquid biopsy samples for determination of a clinically relevant HPV infection in HPV+ OPSCC. Combined interpretation of HPV E6/E7(+) CTCs with UICC staging data may lead to alteration of risk definition of patient subsets, with improved risk discrimination in early-stage disease
Moore KM, Nicholas J, Grossman M, McMillan CT, Irwin DJ, Massimo L, Van Deerlin VM, Warren JD, Fox NC, Rossor MN, et al. Age at symptom onset and death and disease duration in genetic frontotemporal dementia: an international retrospective cohort study. Lancet Neurol. [Internet]. 2019. WebsiteAbstract
BACKGROUND: Frontotemporal dementia is a heterogenous neurodegenerative disorder, with about a third of cases being genetic. Most of this genetic component is accounted for by mutations in GRN, MAPT, and C9orf72. In this study, we aimed to complement previous phenotypic studies by doing an international study of age at symptom onset, age at death, and disease duration in individuals with mutations in GRN, MAPT, and C9orf72. METHODS: In this international, retrospective cohort study, we collected data on age at symptom onset, age at death, and disease duration for patients with pathogenic mutations in the GRN and MAPT genes and pathological expansions in the C9orf72 gene through the Frontotemporal Dementia Prevention Initiative and from published papers. We used mixed effects models to explore differences in age at onset, age at death, and disease duration between genetic groups and individual mutations. We also assessed correlations between the age at onset and at death of each individual and the age at onset and at death of their parents and the mean age at onset and at death of their family members. Lastly, we used mixed effects models to investigate the extent to which variability in age at onset and at death could be accounted for by family membership and the specific mutation carried. FINDINGS: Data were available from 3403 individuals from 1492 families: 1433 with C9orf72 expansions (755 families), 1179 with GRN mutations (483 families, 130 different mutations), and 791 with MAPT mutations (254 families, 67 different mutations). Mean age at symptom onset and at death was 49.5 years (SD 10.0; onset) and 58.5 years (11.3; death) in the MAPT group, 58.2 years (9.8; onset) and 65.3 years (10.9; death) in the C9orf72 group, and 61.3 years (8.8; onset) and 68.8 years (9.7; death) in the GRN group. Mean disease duration was 6.4 years (SD 4.9) in the C9orf72 group, 7.1 years (3.9) in the GRN group, and 9.3 years (6.4) in the MAPT group. Individual age at onset and at death was significantly correlated with both parental age at onset and at death and with mean family age at onset and at death in all three groups, with a stronger correlation observed in the MAPT group (r=0.45 between individual and parental age at onset, r=0.63 between individual and mean family age at onset, r=0.58 between individual and parental age at death, and r=0.69 between individual and mean family age at death) than in either the C9orf72 group (r=0.32 individual and parental age at onset, r=0.36 individual and mean family age at onset, r=0.38 individual and parental age at death, and r=0.40 individual and mean family age at death) or the GRN group (r=0.22 individual and parental age at onset, r=0.18 individual and mean family age at onset, r=0.22 individual and parental age at death, and r=0.32 individual and mean family age at death). Modelling showed that the variability in age at onset and at death in the MAPT group was explained partly by the specific mutation (48%, 95% CI 35-62, for age at onset; 61%, 47-73, for age at death), and even more by family membership (66%, 56-75, for age at onset; 74%, 65-82, for age at death). In the GRN group, only 2% (0-10) of the variability of age at onset and 9% (3-21) of that of age of death was explained by the specific mutation, whereas 14% (9-22) of the variability of age at onset and 20% (12-30) of that of age at death was explained by family membership. In the C9orf72 group, family membership explained 17% (11-26) of the variability of age at onset and 19% (12-29) of that of age at death. INTERPRETATION: Our study showed that age at symptom onset and at death of people with genetic frontotemporal dementia is influenced by genetic group and, particularly for MAPT mutations, by the specific mutation carried and by family membership. Although estimation of age at onset will be an important factor in future pre-symptomatic therapeutic trials for all three genetic groups, our study suggests that data from other members of the family will be particularly helpful only for individuals with MAPT mutations. Further work in identifying both genetic and environmental factors that modify phenotype in all groups will be important to improve such estimates. FUNDING: UK Medical Research Council, National Institute for Health Research, and Alzheimer's Society
Ntzifa A, Kroupis C, Haliassos A, Lianidou E. A pilot plasma-ctDNA ring trial for the Cobas(R) EGFR Mutation Test in clinical diagnostic laboratories. Clin.Chem.Lab Med. [Internet]. 2019;57(5):e97 - e101. Website
2018
Rizou T, Perlikos F, Lagiou M, Karaglani M, Nikolopoulos S, Toumpoulis I, Kroupis C. Development of novel real-time RT-qPCR methodologies for quantification of the COL11A1 mRNA general and C transcripts and evaluation in non-small cell lung cancer specimens. J.BUON. [Internet]. 2018;23(6):1699 - 1710. WebsiteAbstract
PURPOSE: The purpose of this study was the development of new quantitative methodologies for the general (total) COL11A1 gene and the C transcript (RT-qPCR methods for A and E transcripts have already been developed by our group previously), the quantification of all COL11A1 transcripts and the investigation for the first time of their potential association with histopathological prognostic factors in lung cancer. METHODS: Real-time RT-qPCR methodologies with dual hybridization probes were developed on the Light Cycler 1.5 platform (Roche,Germany). All COL11A1 transcripts were measured in 27 cDNA lung tissue specimens in a blinded fashion (8 control and 19 non-small cell lung cancer (NSCLC) tissues with known histopathological data). Statistical analysis was performed with the IBM SPSS program. RESULTS: The novel real-time RT-qPCR methodologies were appropriately validated. All 19 NSCLC samples were positive for the general COL11A1 transcript (range 11.2-1198.0 copies/mug total RNA, while 5 out of 8 control samples were negative: mean values were also statistically significantly different (p<0.001). In 4 tumor samples (21%), no specific COL11A1 transcript was detected. Transcript C was detected in only 3 tumor samples. Regarding transcripts A and E, 13 out of 19 tumor samples were positive for either one (68%) and 11 for both (58%). CONCLUSIONS: No other statistically significant association of the specific transcripts with histopathological data was observed, most probably due to the limited number of samples. As the number of general COL11A1 transcripts/microg exceeds the sum of A+E+C transcripts in all samples, there is opportunity for discovery and identification of other transcripts as well
Francis K, Dougali A, Sideri K, Kroupis C, Vasdekis V, Dima K, Douzenis A. Brain-derived neurotrophic factor (BDNF) in children with ASD and their parents: a 3-year follow-up. Acta Psychiatr.Scand. [Internet]. 2018;137(5):433 - 441. WebsiteAbstract
OBJECTIVE: Several lines of evidence point to a probable relationship between brain-derived neurotrophic factor (BDNF) and autism spectrum disorder (ASD), but studies have yielded inconsistent findings on the BDNF serum level in ASD. The study aimed to assess those levels in children with ASD and their families. METHOD: BDNF serum levels were measured in 45 ASD children without intellectual disability (ID) and allergies, age 30-42 months and age-matched normal controls. BDNF serum levels in the parents of the ASD subjects were compared to normal controls. BDNF serum levels in the ASD subjects were followed up for 3 years and correlated with adaptive functioning changes. RESULTS: BDNF serum levels were measured to be lower in children with ASD and independent of all the major baseline characteristics of the subjects. Having a child with ASD raises the BDNF levels in parents comparing to controls. Prospectively, no correlation between the change of BDNF variables in time and the change of the Vineland scores was found. CONCLUSIONS: Our results contradict those from recent published meta-analyses with the age, the presence of ID and allergies being possible contributing factors. The parents' data indeed point to a role of BDNF in the pathophysiology of ASD
Perlikos F, Lagiou M, Papalois A, Rizou T, Kroupis C, Toumpoulis IK. Lazaroid (U-74389G) ameliorates lung injury due to lipid peroxidation and nitric oxide synthase-dependent reactive oxygen species generation caused by remote systematic ischemia-reperfusion following thoracoabdominal aortic occlusion. Int.J.Surg. [Internet]. 2018;55:156 - 161. WebsiteAbstract
INTRODUCTION: Lung ischemia-reperfusion injury after thoracoabdominal aortic occlusion represents a major complication, which increases morbidity and mortality. In the present study we hypothesized that lazaroid U-74389G intravenous administration protects from lung ischemia-reperfusion injury through lipid peroxidation inhibition. MATERIALS AND METHODS: A total of 24 pigs were randomized in three groups. Group I (n=8) underwent sham operation, group II (n=8) underwent thoracoabdominal aortic occlusion for 45min and received placebo and group III (n=8) received 3 doses of lazaroid (3mg/kg) 60 and 30min before thoracoabdominal aortic occlusion and at 30min during thoracoabdominal aortic occlusion (duration 45min). Aortic occlusion was performed with aortic balloon-catheters under fluoroscopic guidance. All animals were sacrificed at the 7th postoperative day and lung specimens were harvested for molecular analysis. RESULTS: mRNA levels of leukotrienes LB4 (LTB4R2), LC4 (LTC4S) and nitric oxide synthase (NOS) isoforms including iNOS, nNOS and eNOS were determined with real-time RT-qPCR. Nitric oxide can either induce (iNOS) or inhibit (nNOS and eNOS) lipid peroxidation based on its specific isoform origin. Group III showed significantly reduced mRNA levels of LTB4R2 (-63.7%), LTC4S (-35.9%) and iNOS (-60.2%) when compared with group II (P < 0.05, for all). The mRNA levels of nNOS was significantly increased (+37.4%), while eNOS was slightly increased (+2.1%) in group III when compared with group II (P < 0.05 and P = 0.467 respectively). CONCLUSION: Lazaroid U-74389G may represent an effective pharmacologic intervention in reducing lung ischemia-reperfusion injury following thoracoabdominal aortic occlusion
2017
Psyrri A, Fortpied C, Koutsodontis G, Avgeris M, Kroupis C, Goutas N, Menis J, Herman L, Giurgea L, Remenar E, et al. Evaluation of the impact of tumor HPV status on outcome in patients with locally advanced unresectable head and neck squamous cell carcinoma (HNSCC) receiving cisplatin, 5-fluorouracil with or without docetaxel: a subset analysis of EORTC 24971 study. Ann.Oncol. [Internet]. 2017;28(9):2213 - 2218. WebsiteAbstract
Background: EORTC 24971 was a phase III trial demonstrating superiority of induction regimen TPF (docetaxel, cisplatin, 5-fluorouracil) over PF (cisplatin/5-fluorouracil), in terms of progression-free (PFS) and overall survival (OS) in locoregionally advanced unresectable head and neck squamous cell carcinomas. We conducted a retrospective analysis of prospectively collected data aiming to evaluate whether only HPV(-) patients (pts) benefit from adding docetaxel to PF, in which case deintensifying induction treatment in HPV(+) pts could be considered. Patients and methods: Pretherapy tumor biopsies (blocks or slides) were assessed for high-risk HPV by p16 immunohistochemistry, PCR and quantitative PCR. HPV-DNA+ and/or p16+ tumors were subjected to in situ hybridization (ISH) and HPV E6 oncogene expression qRT-PCR analysis. Primary and secondary objectives were to evaluate the value of HPV/p16 status as predictive factor of treatment benefit in terms of PFS and OS. The predictive effect was analyzed based on the model used in the primary analysis of the study with the addition of a treatment by marker interaction term and tested at two-sided 5% significance level. Results: Of 358, 119 pts had available tumor samples and 58 of them had oropharyngeal cancer. Median follow-up was 8.7 years. Sixteen of 119 (14%) evaluable samples were p16+ and 20 of 79 (25%) evaluable tumors were HPV-DNA+. 13 of 40 pts (33%) assessed with HPV-DNA ISH and 12 of 28 pts (43%) assessed for HPV E6 mRNA were positive. The preplanned analysis showed no statistical evidence of predictive value of HPV/p16 status for PFS (P = 0.287) or OS (P = 0.118). Conclusions: The incidence of HPV positivity was low in the subset of EORTC 24971 pts analyzed. In this analysis only powered to detect a large treatment by marker interaction, there was no statistical evidence that treatment effect found overall was different in magnitude in HPV(+) or HPV(-) pts. These results do not justify selection of TPF versus PF according to HPV status
Flevari P, Leftheriotis D, Kroupis C, Antonakos G, Lekakis J, Dima K. Response to the letter to editor: Copeptin and its clinical implications in the setting of vasovagal syncope. Int.J.Cardiol. [Internet]. 2017;242:29. Website
Flevari P, Leftheriotis D, Kroupis C, Antonakos G, Lekakis J, Dima K. Copeptin levels in patients with vasovagal syncope. Int.J.Cardiol. [Internet]. 2017;230:642 - 645. WebsiteAbstract
BACKGROUND AND PURPOSE: Vasovagal syncope (VVS) is linked to more than one pathophysiologic mechanisms. Copeptin, an emerging cardiovascular marker, is a surrogate for arginine-vasopressin, which increases following VVS. We aimed to assess the dynamic pattern of copeptin levels in typical VVS, categorized by the degree of vasoconstriction during orthostasis, and healthy controls. METHODS: The following groups were studied: Group A (n=21), with adequate limb vasoconstriction during the first min. of tilt, assessed by limb plethysmography (at least 30% flow reduction); Group B (n=15), showing impaired vasoconstriction during orthostasis (<10% reduction); Group C (n=18), history of VVS and negative tilt test result; Group D (n=18), healthy controls. Copeptin plasma levels were assessed before and 5min following tilt test positivity or termination. RESULTS: Baseline copeptin values were similar in all groups (8.3+/-6.4 in Group A, 5.7+/-2.3pmol/l in B, 6.0+/-1.9 in C, and 6.9+/-2.6 in D, p: 0.41). Significant increases in copeptin during tilt were observed in all Groups of VVS patients (A, B, C), including those with negative tilt (Group C: from 6.0+/-1.9 to 27.7+/-12.6pmol/l, p: 0.001), but not in controls. Following tilt termination, a greater increase was observed in copeptin values in Group B vs all other Groups A, C, and D (111.6+/-63.5 vs 29.5+/-51.3, 27.7+/-12.6, and 8.3+/-2.9, respectively). CONCLUSIONS: Copeptin increases following tilt not only in VVS with a positive response, but also in typical history patients with a negative test. Increased copeptin levels following orthostasis may be useful for diagnosing VVS
Sarli A, Skalidakis I, Velissari A, Koutsandrea C, Stefaniotou M, Petersen MB, Kroupis C, Kitsos G, Moschos MM. Investigation of associations of ARMS2, CD14, and TLR4 gene polymorphisms with wet age-related macular degeneration in a Greek population. Clin.Ophthalmol. [Internet]. 2017;11:1347 - 1358. WebsiteAbstract
BACKGROUND: Age-related macular degeneration (AMD) is a multifactorial degenerative ocular disease that leads to loss of central vision. Functional gene polymorphisms have already been associated with the disease (for example, ARMS2 A69S, rs10490924). AIM: The goal of our study was to verify the correlation of the aforementioned ARMS2 variation with the disease, to examine, for the first time, the role of the CD14 C260T variation (rs2569190), and to investigate the association of two TLR4 polymorphisms (Asp299Gly or rs4986790 and Thr399Ile or rs4986791) in a Greek population with the wet form of AMD. PATIENTS AND METHODS: Genomic DNAs were isolated from blood samples of 103 healthy controls and 120 Greek patients with wet AMD who were age- and sex-matched, and all of whom were clinically evaluated. For the genotyping of all selected polymorphisms, polymerase chain reaction-restriction fragment length polymorphism analysis was performed. RESULTS AND CONCLUSIONS: This study confirmed the association between the ARMS2 variation and AMD, detecting the T risk allele in a significantly higher frequency in the patient group, compared with the control subjects (45% vs 29.13%, P<0.001, odds ratio [OR] 1.99, confidence interval 1.34-2.95). For the CD14 polymorphism, no statistically significant correlation was observed. As for the TLR4 polymorphisms, the percentage of heterozygotes increased from 2.9% to 11.7% in the patient population for Asp299Gly and from 1.9% to 10% for the Thr399Ile polymorphism (ORs 4.40 [P=0.01] and 5.61 [P=0.0088], respectively). Although our ARMS2 and CD14 results provided definite conclusions, the role of innate immunity TLR4 gene awaits further investigation in larger AMD populations with more clinical data collected on past microbial infections
2016
Henny J, Vassault A, Boursier G, Vukasovic I, Mesko BP, Lohmander M, Ghita I, Andreu FA, Kroupis C, Sprongl L, et al. Recommendation for the review of biological reference intervals in medical laboratories. Clin.Chem.Lab Med. [Internet]. 2016;54(12):1893 - 1900. WebsiteAbstract
This document is based on the original recommendation of the Expert Panel on the Theory of Reference Values of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), updated guidelines were recently published under the auspices of the IFCC and the Clinical and Laboratory Standards Institute (CLSI). This document summarizes proposals for recommendations on: (i) The terminology, which is often confusing, noticeably concerning the terms of reference limits and decision limits. (ii) The method for the determination of reference limits according to the original procedure and the conditions, which should be used. (iii) A simple procedure allowing the medical laboratories to fulfill the requirements of the regulation and standards. The updated document proposes to verify that published reference limits are applicable to the laboratory involved. Finally, the strengths and limits of the revised recommendations (especially the selection of the reference population, the maintenance of the analytical quality, the choice of the statistical method used...) will be briefly discussed
Boursier G, Vukasovic I, Brguljan PM, Lohmander M, Ghita I, Bernabeu Andreu FA, Barrett E, Brugnoni D, Kroupis C, Sprongl L, et al. Accreditation process in European countries - an EFLM survey. Clin.Chem.Lab Med. [Internet]. 2016;54(4):545 - 551. WebsiteAbstract
BACKGROUND: Accreditation is a valuable resource for medical laboratories. The development of quality systems based on ISO 15189 has taken place in many laboratories in the European countries but data about accreditation remain scarce. The EFLM Working Group "Accreditation and ISO/CEN standards" conducted a survey that reviews the current state of the accreditation process in European countries. METHODS: An on-line questionnaire was addressed to delegates of 39 EFLM scientific societies in March 2014. One answer by country was taken into account. The survey was dealing with mandatory status, number of accredited medical laboratories in each country, possibility of flexible scope and concerned medical fields. The status of point-of-care testing (POCT) in each country was also studied. RESULTS: Twenty-nine responses (74%) were registered. All the assessed countries (100%) have begun an accreditation process in various ways. All the national accreditation bodies (NAB) offer or are working to offer an ISO 15189 accreditation. The accreditation process most often concerns all phases of the examination and various medical fields. Medical laboratories are responsible for POCT in 20 (69%) countries. The accreditation process for POCT, according to ISO 15189 and ISO 22870, is also developing. CONCLUSIONS: While there are several variations in the approaches to accreditation of medical laboratories in the European countries, the ISO 15189 accreditation project has been widely accepted. The use of a unique standard and the cooperation among countries due to scientific societies, EFLM, accreditation bodies and EA enable laboratory professionals to move toward uniform implementation of the accreditation concept
Poumpouridou N, Goutas N, Tsionou C, Dimas K, Lianidou E, Kroupis C. Development of a novel PTT assay for mutation detection in PALB2 large exons and PALB2 screening in medullary breast cancer. Fam.Cancer [Internet]. 2016;15(2):183 - 191. WebsiteAbstract
Beyond BRCA1 and BRCA2 genes, PALB2 (Partner and localizer of BRCA2) emerges as the third breast cancer susceptibility gene due to its role in the same DNA repair pathway: homologous recombination. In most populations studied so far, PALB2 mutations are detected in 1-2% of BRCA negative female patients. PALB2 gene contains 13 exons; exons 4 and 5 consist 65% of the coding area. We developed a protein truncation test (PTT) for quick screening of truncating pathogenic mutations of these two large exons. Specific primers were de novo, in silico designed and the PTT-PCR products were translated in the presence of biotinylated lysine and detected colorimetrically. The assay was initially tested in 30 patients with hereditary breast cancer, negative for BRCA mutations and then, in 17 patients with the rare medullary breast cancer subtype. Small PALB2 exons were screened with high-resolution melting curve analysis (HRMA) and the large DNA rearrangements with multiplex ligation-dependent probe amplification (MLPA). Any alterations detected were verified by Sanger DNA Sequencing. The developed PTT methodology is highly specific for clinical significant mutations; positive control samples that produce truncated PALB2 peptides were correctly identified and the method was accurate when compared to DNA sequencing. We did not detect any deleterious PALB2 mutation in both groups of patients. HRMA and MLPA were also negative for all tested samples. However, our novel, fast and cost-effective PTT method for pathogenic mutation detection of the two large PALB2 exons can be applied in screening of a large number of breast cancer patients
Poumpouridou N, Acha-Sagredo A, Goutas N, Vlachodimitropoulos D, Chatziioannidou I, Lianidou E, Liloglou T, Kroupis C. Development and validation of molecular methodologies to assess PALB2 expression in sporadic breast cancer. Clin.Biochem. [Internet]. 2016;49(3):253 - 259. WebsiteAbstract
OBJECTIVES: Recent reports have included PALB2 (Partner and localizer of BRCA2) in the growing list of hereditary cancer genes. PALB2 mutations confer a moderate breast cancer risk in heterozygotes and Fanconi anemia in biallelic mutation carriers. PALB2 protein co-localizes with BRCA2 and BRCA1 in nuclear structures and enables error-free homologous recombination repair of double-stranded DNA breaks. This important contribution could be severely diminished if affected by epigenetic mechanisms such as promoter CpG island methylation. The aim of our study was to develop molecular methodologies in order to assess accurately PALB2 expression in breast cancer tissues. DESIGN AND METHODS: DNA and RNA were extracted from 91 sporadic fresh-frozen breast tissues with known histopathological data. DNA was subjected to sodium bisulfite conversion reaction and the CpG island of the PALB2 promoter was analyzed by pyrosequencing. RNA was converted to cDNA and analyzed by a newly developed and validated RT-qPCR assay based on a hydrolysis probe (TaqMan) in the Light Cycler. RESULTS: PALB2 promoter was not methylated in any of the samples tested. 87 out of 91 (95.6%) primary tumors were positive for PALB2 expression, as checked at the mRNA level. When levels of PALB2 mRNA were compared to histopathological data (tumor size, grade, lymph node involvement, metastasis, hormone receptors and HER2 overexpression), no significant statistical correlation was found. CONCLUSIONS: DNA methylation is an unlike mechanism for PALB2 transcriptional regulation. PALB2 mRNA expression does not seem be a promising prognostic biomarker for sporadic breast cancer
Marouga A, Dalamaga M, Kastania AN, Kroupis C, Lagiou M, Saounatsou K, Dimas K, Vlahakos DV. Circulating resistin is a significant predictor of mortality independently from cardiovascular comorbidities in elderly, non-diabetic subjects with chronic kidney disease. Biomarkers [Internet]. 2016;21(1):73 - 79. WebsiteAbstract
CONTEXT: Resistin is associated with inflammation, atherosclerosis and cardiovascular (CV) disease. OBJECTIVE: To associate circulating resistin with all-cause and CV mortality in chronic kidney disease (CKD) patients. METHODS: Serum resistin was determined in a cohort of 80 elderly, non-diabetic patients with stable CKD at different stages in a follow-up period of 5 years. RESULTS: Circulating resistin was significantly elevated in deceased compared to alive patients. Resistin emerged as an independent biomarker of all-cause and CV mortality after a 5-year follow-up period. CONCLUSION: Elevated circulating resistin was a significant independent predictor of CV and all-cause mortality in elderly, non-diabetic CKD patients
2015
Makris K, Stefani D, Makri E, Panagou I, Lagiou M, Sarli A, Lelekis M, Kroupis C. Evaluation of a particle enhanced turbidimetric assay for the measurement of neutrophil gelatinase-associated lipocalin in plasma and urine on Architect-8000: Analytical performance and establishment of reference values. Clin.Biochem. [Internet]. 2015;48(18):1291 - 1297. WebsiteAbstract
OBJECTIVES: Neutrophil gelatinase-associated lipocalin (NGAL) is a promising biomarker for acute kidney injury. NGAL can be measured in both blood and urine. Apart from kidney injury, NGAL levels in both plasma and urine can be influenced by various pathological situations. Accurate evaluation and comparison of results deriving from clinical studies require robust assays, appropriate specimen handling and reference intervals that will reflect its levels in a healthy population for both biological matrices. METHODS: We report the analytical validation of a latex particle-enhanced turbidimetric immunoassay (PETIA) aimed to measure NGAL in plasma and urine on an automated biochemistry analyzer (ABBOTT-Architect-8000). Assay performance characteristics were evaluated using standard protocols. Urine and plasma specimen storage requirements were determined and reference ranges for blood and urine were determined using healthy controls. RESULTS: The assay is precise (total CV%<4.8%), and sensitive (limit of quantification: 8.4 ng/mL for plasma and 9.0 ng/mL for urine), showing no hook effect. Calibration is stable for at least 30 days. The assay showed excellent linearity over the studied interval (20-4450 ng/mL). The analyte is stable at 4 degrees C for at least 5 days, and at 20 degrees C for 4h. Gender specific reference ranges for plasma (male: 38.7-157.6 ng/mL, female: 24.4-142.5 ng/mL) and unisex for urine (<9.0-49.41 ng/mL) are proposed. CONCLUSION: Our data indicate that NGAL can be measured with adequate precision and sensitivity on automated biochemistry analyzers and its measurement could easily be added to a standard panel to screen kidney diseases
Velissari A, Skalidakis I, Oliveira SC, Koutsandrea C, Kitsos G, Petersen MB, Kroupis C. Novel association of FCGR2A polymorphism with age-related macular degeneration (AMD) and development of a novel CFH real-time genotyping method. Clin.Chem.Lab Med. [Internet]. 2015;53(10):1521 - 1529. WebsiteAbstract
BACKGROUND: Age-related macular degeneration (AMD) is a degenerative ocular disease, which may lead to loss of central vision. In Caucasian populations, a strong correlation has been established with polymorphism Y402H (rs1061170) in the complement factor H gene (CFH). The H131R polymorphism (rs1801274) in the FCGR2A gene has been associated with many inflammatory diseases, but has not been investigated in relation to AMD. The goal of our study was the development of a novel method for Y402H (g.43097C>T) genotyping, the confirmation of its association with AMD in the Greek population and the investigation of the H131R polymorphism in AMD. METHODS: DNAs were extracted from blood samples of 120 patients with the severe wet form of AMD and 103 age- and sex-matched controls, all of whom were clinically evaluated. A real-time PCR and melting curve analysis method for Y402H genotyping was developed in the LightCycler platform, after in silico design of appropriate primers and probes. Genotyping for H131R was performed using a real-time PCR method previously described by our group. RESULTS: The novel genotyping method for Y402H in the CFH gene is fast, reproducible (Efficiency=1.79, reproducibility CVCq=3.33%, Tm C allele 53.36 degrees C and T allele 61.91 degrees C, DeltaTm=8.55) and accurate as results were confirmed with the gold standard DNA Sequencing method. CONCLUSIONS: The present study confirmed the association between CFH Y402H SNP and wet AMD in the Greek population (OR=1.77, p=0.002). FCGR2A H131R polymorphism was investigated for the first time in this present study for possible correlation with wet AMD and a statistically significant association was detected (OR=1.74, p=0.006), that awaits further confirmation in a larger set of samples
Thelen MH, Vanstapel FJ, Kroupis C, Vukasovic I, Boursier G, Barrett E, Bernabeu AF, Brguljan PM, Brugnoni D, Lohmander M, et al. Flexible scope for ISO 15189 accreditation: a guidance prepared by the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group Accreditation and ISO/CEN standards (WG-A/ISO). Clin.Chem.Lab Med. [Internet]. 2015;53(8):1173 - 1180. WebsiteAbstract
The recent revision of ISO15189 has further strengthened its position as the standard for accreditation for medical laboratories. Both for laboratories and their customers it is important that the scope of such accreditation is clear. Therefore the European co-operation for accreditation (EA) demands that the national bodies responsible for accreditation describe the scope of every laboratory accreditation in a way that leaves no room for doubt about the range of competence of the particular laboratories. According to EA recommendations scopes may be fixed, mentioning every single test that is part of the accreditation, or flexible, mentioning all combinations of medical field, examination type and materials for which the laboratory is competent. Up to now national accreditation bodies perpetuate use of fixed scopes, partly by inertia, partly out of fear that a too flexible scope may lead to over-valuation of the competence of laboratories, most countries only use fixed scopes. The EA however promotes use of flexible scopes, since this allows for more readily innovation, which contributes to quality in laboratory medicine. In this position paper, the Working Group Accreditation and ISO/CEN Standards belonging to the Quality and Regulation Committee of the EFLM recommends using an approach that has led to successful introduction of the flexible scope for ISO15189 accreditation as intended in EA-4/17 in The Netherlands. The approach is risk-based, discipline and competence-based, and focuses on defining a uniform terminology transferable across the borders of scientific disciplines, laboratories and countries
Chiras D, Kitsos G, Petersen MB, Skalidakis I, Kroupis C. Oxidative stress in dry age-related macular degeneration and exfoliation syndrome. Crit Rev.Clin.Lab Sci. [Internet]. 2015;52(1):12 - 27. WebsiteAbstract
Oxidative stress refers to cellular or molecular damage caused by reactive oxygen species, which especially occurs in age-related conditions as a result of an imbalance between the production of reactive oxygen species and the antioxidant defense response. Dry age-related macular degeneration (AMD) and exfoliation syndrome (XFS) are two common and complex age-related conditions that can cause irreversible vision loss. Two subtypes of AMD, which is the leading cause of blindness in the Western world, exist: the most prevalent dry type and the most severe wet type. Early dry AMD is characterized by formation of drusen, which are sub-retinal deposits, in the macular area and may progress to geographic atrophy with more dramatic manifestation. XFS is a systemic disorder of the extracellular matrix characterized by the accumulation of elastic fibrils that leads, in most cases, to glaucoma development with progressive and irreversible vision loss. Due to the aging population, the prevalence of these already-widespread conditions is increasing and is resulting in significant economic and psychological costs for individuals and for society. The exact composition of the abnormal drusen and XFS material as well as the mechanisms responsible for their production and accumulation still remain elusive, and consequently treatment for both diseases is lacking. However, recent epidemiologic, genetic and molecular studies support a major role for oxidative stress in both dry AMD and XFS development. Understanding the early molecular events in their pathogenesis and the exact role of oxidative stress may provide novel opportunities for therapeutic intervention for the prevention of progression to advanced disease
Karaglani M, Toumpoulis I, Goutas N, Poumpouridou N, Vlachodimitropoulos D, Vasilaros S, Rizos I, Kroupis C. Development of novel real-time PCR methodology for quantification of COL11A1 mRNA variants and evaluation in breast cancer tissue specimens. BMC.Cancer [Internet]. 2015;15:694. WebsiteAbstract
BACKGROUND: Collagen XI is a key structural component of the extracellular matrix and consists of three alpha chains. One of these chains, the alpha1 (XI), is encoded by the COL11A1 gene and is transcribed to four different variants at least (A, B, C and E) that differ in the propensity to N-terminal domain proteolysis and potentially in the way the extracellular matrix is arranged. This could affect the ability of tumor cells to invade the remodeled stroma and metastasize. No study in the literature has so far investigated the expression of these four variants in breast cancer nor does a method for their accurate quantitative detection exist. METHODS: We developed a conventional PCR for the general detection of the general COL11A1 transcript and real-time qPCR methodologies with dual hybridization probes in the LightCycler platform for the quantitative determination of the variants. Data from 90 breast cancer tissues with known histopathological features were collected. RESULTS: The general COL11A1 transcript was detected in all samples. The developed methodologies for each variant were rapid as well as reproducible, sensitive and specific. Variant A was detected in 30 samples (33 %) and variant E in 62 samples (69 %). Variants B and C were not detected at all. A statistically significant correlation was observed between the presence of variant E and lymph nodes involvement (p = 0.037) and metastasis (p = 0.041). CONCLUSIONS: With the newly developed tools, the possibility of inclusion of COL11A1 variants as prognostic biomarkers in emerging multiparameter technologies examining tissue RNA expression should be further explored
2014
Pavlidou A, Kroupis C, Goutas N, Dalamaga M, Dimas K. Validation of a real-time quantitative polymerase chain reaction method for the quantification of 3 survivin transcripts and evaluation in breast cancer tissues. Clin.Breast Cancer [Internet]. 2014;14(2):122 - 131. WebsiteAbstract
BACKGROUND: Survivin is a novel antiapoptotic gene, which is a member of the inhibitor of apoptosis protein (IAP) family. Recently, 3 splice variants of this gene were cloned and characterized. This study aimed to validate a sensitive and specific method for the detection of survivin variants in breast cancer. METHODS: Real-time quantitative polymerase chain reaction (qPCR) was performed on the cDNA with a reverse primer specific for each splice variant and a pair of common hybridization probes. RESULTS: The expression of wild-type survivin was significantly correlated with survivin-2b, survivin-DeltaEx3, and the ratio of survivin-DeltaEx3 to wild-type survivin (P < .001). The ratio of survivin-2b to wild-type survivin was strongly associated with the ratio of survivin-DeltaEx3 to wild-type survivin (P < .001). There was a strong positive association between the grade of the tumor and survivin-2b mRNA, survivin-DeltaEx3 mRNA, and the ratio of survivin-DeltaEx3 to wild-type survivin mRNA (P < .05). The ratio of survivin-2b to wild-type survivin was significantly associated with the presence of estrogen receptors (P = .05). CONCLUSION: Our validated data suggest that survivin isoforms may be related to clinicopathological features and could be used as molecular prognostic tools or as new therapy targets
Pavlidou A, Kroupis C, Dimas K. Association of survivin splice variants with prognosis and treatment of breast cancer. World J.Clin.Oncol. [Internet]. 2014;5(5):883 - 894. WebsiteAbstract
The purpose of this study was the overview of current knowledge regarding the use of survivin and its isoforms in prognosis and treatment of breast cancer. An advanced search of Medline was performed using the following search strategy: "(survivin isoforms) OR (survivin transcript variants) AND (breast cancer) AND (neoplasm OR tumor OR cancer OR carcinoma)". Relevant studies were retrieved and processed thoroughly in order to analyze the related data. Besides wild-type survivin full-length transcript, another six splice variants have been identified. Overexpression of survivin and its isoforms leads to shorter overall and disease-free survival; the transcript variants are correlated with apoptosis and could assist prognosis prediction. It has been proved through numerous studies that inhibiting survivin isoforms might become a promising target of drug therapy of carcinomas. Use of small molecule YM155 could offer new therapy for triple negative breast cancer patients, while, chemotherapy with 5-fluorouracil + epirubicin + cyclophosphamide and Tax-Epi could be guided by survivin splice variants measurements. Survivin transcript variants could become prognostic biomarkers and could provide information about clinical management of patients suffering from breast cancer
2013
Chiras D, Tzika K, Kokotas H, Oliveira SC, Grigoriadou M, Kastania A, Dima K, Stefaniotou M, Aspiotis M, Petersen MB, et al. Development of novel LOXL1 genotyping method and evaluation of LOXL1, APOE and MTHFR polymorphisms in exfoliation syndrome/glaucoma in a Greek population. Mol.Vis. [Internet]. 2013;19:1006 - 1016. WebsiteAbstract
PURPOSE: In the Greek population of Epirus, exfoliation syndrome (XFS) and exfoliation glaucoma (XFG) occur at a high prevalence. In this study, we validate a novel lysyl oxidase-like 1 (LOXL1) genotyping method, investigate the previously reported association of LOXL1 with XFS/XFG, and evaluate apolipoprotein E (APOE) and methylenetetrahydrofolate reductase (MTHFR) polymorphisms as genetic risk factors for both conditions in our population. METHODS: Blood samples were collected from 82 patients with XFG, 69 patients with XFS, 52 patients with primary open-angle glaucoma (POAG), and 107 controls. APOE and MTHFR 677C>T genotyping was performed from extracted genomic DNA with established methods. A novel methodology of real-time PCR and melting curve analysis was developed and validated to accurately genotype the LOXL1 G153D and R141L polymorphisms by using two different fluorescent channels of the LightCycler instrument (Roche) examining each SNP separately. RESULTS: No significant differences were observed for the APOE and MTHFR polymorphisms between the patients with XFS, the patients with XFG, and the control subjects. The APOE epsilon2 allele appears to be associated with elevated risk of POAG in our population. Our novel LOXL1 genotyping method was easy to perform, fast, and accurate. A statistically significant association was found for the LOXL1 gene with XFS/XFG in this Greek population. The association of XFS and XFG with G153D appeared to be less powerful in this population (XFS: odds ratio [OR]=2.162, p=0.039, XFG: OR=2.794, p=0.002) compared to other populations, and for R141L, the association was proven only with XFG (OR=3.592, p<0.001). Neither of the two LOXL1 SNPs was significantly associated with POAG. CONCLUSIONS: We confirmed the association between LOXL1 and XFS/XFG, but the APOE and MTHFR polymorphisms are not significant risk factors for the development of XFS/XFG in our population of patients from Epirus (Greece)
2012
Kokotas H, Kroupis C, Chiras D, Grigoriadou M, Lamnissou K, Petersen MB, Kitsos G. Biomarkers in primary open angle glaucoma. Clin.Chem.Lab Med. [Internet]. 2012;50(12):2107 - 2119. WebsiteAbstract
Glaucoma, a leading cause of blindness worldwide, is currently defined as a disturbance of the structural or functional integrity of the optic nerve that causes characteristic atrophic changes in the optic nerve, which may lead to specific visual field defects over time. This disturbance usually can be arrested or diminished by adequate lowering of intraocular pressure (IOP). Glaucoma can be divided roughly into two main categories, ' open angle ' and ' closed angle ' glaucoma.Open angle, chronic glaucoma tends to progress at a slower rate and patients may not notice loss of vision until the disease has progressed significantly. Primary open angle glaucoma(POAG) is described distinctly as a multifactorial optic neuropathy that is chronic and progressive with a characteristic acquired loss of optic nerve fibers. Such loss develops in the presence of open anterior chamber angles, characteristic visual field abnormalities, and IOP that is too high for the healthy eye. It manifests by cupping and atrophy of the optic disc, in the absence of other known causes of glaucomatous disease. Several biological markers have been implicated with the disease. The purpose of this study was to summarize the current knowledge regarding the non-genetic molecular markers which have been predicted to have an association with POAG but have not yet been validated
Melichar B, Kroupis C. Cancer epigenomics: moving slowly, but at a steady pace from laboratory bench to clinical practice. Clin.Chem.Lab Med. [Internet]. 2012;50(10):1699 - 1701. Website
Flevari P, Theodorakis G, Leftheriotis D, Kroupis C, Kolokathis F, Dima K, Anastasiou-Nana M, Kremastinos D. Serum markers of deranged myocardial collagen turnover: their relation to malignant ventricular arrhythmias in cardioverter-defibrillator recipients with heart failure. Am.Heart J. [Internet]. 2012;164(4):530 - 537. WebsiteAbstract
BACKGROUND: Pathologic collagen remodeling has been involved in the occurrence of ventricular arrhythmias and sudden cardiac death in heart failure. The aim of the study was to investigate the relationship between malignant ventricular arrhythmias and cardiac collagen turnover indexes, expressing specific types of derangement in collagen physiology, in stable patients with an implantable cardioverter-defibrillator (ICD). METHODS: Seventy-four patients with an ICD and heart failure were studied. They had coronary artery disease (n = 42) or dilated cardiomyopathy, New York Heart Association classes I and II, and left ventricular ejection fraction 29% +/- 1%. An ICD had been implanted for secondary (n = 36) or primary prevention of sudden cardiac death. We assessed (1) markers of collagen types I and III synthesis and their ratio: procollagen type I carboxyterminal peptide (PICP), procollagen type III aminoterminal peptide (PIIINP), and PICP/PIIINP; (2) markers of collagen degradation, degradation inhibition, and their ratio: matrix metalloproteinase 9 (MMP-9), tissue inhibitor of matrix metalloproteinase (TIMP) 1 (TIMP-1), and MMP-9/TIMP-1. Patients were prospectively followed up for 1 year. The number of episodes necessitating appropriate interventions for ventricular tachyarrhythmias (>170 beat/min) was related to the assessed parameters. RESULTS: Multivariate analysis revealed a significant relation between the number of tachyarrhythmic episodes and MMP-9/TIMP-1 (P = .007), PICP/PIIINP (P = .007), and ejection fraction (P = .04). No other significant relation was observed between arrhythmias and the remaining parameters. CONCLUSION: In heart failure, biochemical markers indicative of a deranged equilirium in myocardial collagen deposition/degradation and collagen I/III synthesis are related to ventricular arrhythmogenesis. Further studies are needed to investigate their predictive ability
Poumpouridou N, Kroupis C. Hereditary breast cancer: beyond BRCA genetic analysis; PALB2 emerges. Clin.Chem.Lab Med. [Internet]. 2012;50(3):423 - 434. WebsiteAbstract
Abstract Despite the initial enthusiasm following the discovery of the association of BRCA germline mutations with hereditary breast and/or ovarian cancer, in many families affected by the syndrome no pathogenic mutations were detected in the two genes, although exhaustively searched. Many other genes have also been implicated due to their role in the same pathway of DNA repair where the BRCA1/2 genes are involved: homologous recombination (HR). Among them, PALB2 clearly emerges as the third breast cancer susceptibility gene. Its mutations have been detected in most populations investigated so far, albeit rarely: in 1%-4% of families negative for BRCA mutations, with either partial or complete penetrance. In some populations, PALB2 recurrent mutations have been identified and the estimated hazard risks are comparable to those of BRCA mutations. Since new effective targeted therapeutic options are becoming available ("synthetic lethality" with novel PARP inhibitors, etc.) that are applicable to all those patients with a defect in HR pathway, it is imperative to detect all these candidate patients. Data obtained from laboratory tests in the tumor (simple immunohistochemistry, gene expression analysis, etc.) can assist in the recognition of a specific pattern (BRCA1ness, HRless) so that even patients that look "sporadic" could benefit from these targeted therapies. Therefore, a genetic analysis algorithm is proposed, although with the advent of Next Generation Sequencing it is predicted that in the future most germline genetic alterations and also somatic or epigenetic events in the tumor of these genes will be detected
Mourtzikou A, Stamouli M, Kroupis C, Christodoulou S, Skondra M, Kastania A, Pectasides D, Athanasas G, Dimas C. Evaluation of carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule EpCAM (GA733-2), and carbohydrate antigen 19-9 (CA 19-9) levels in colorectal cancer patients and correlation with clinicopathological. Clin.Lab [Internet]. 2012;58(5-6):441 - 448. WebsiteAbstract
BACKGROUND: Colorectal cancer (CRC) is a major public health problem and one of the leading causes of death worldwide. The aim of our study was: a) to determine the CEA, CA 19-9, EGFR, and EpCAM (GA733-2) levels both in healthy volunteers and in colorectal cancer patients, b) to evaluate the ELISA method for EGFR and EpCAM (GA733-2) measurement, and c) to correlate the tumor marker levels with clinicopathological findings in the CRC patients group. METHODS: Our study was conducted on 50 blood samples obtained from CRC patients and 40 blood samples from healthy individuals. CEA and CA 19-9 measurements were performed using electrochemiluminescence immune-assay technology, while EGFR and EpCAM (GA733-2) measurements were performed by an in-house enzyme immunoassay. RESULTS: CEA, CA 19-9, and EpCAM (GA733-2) levels were higher in the CRC patients group than in the control group. EGFR levels were lower in the patients group than in the control group. The mean levels of CA 19-9 and EpCAM (GA733-2) vary at different colon cancer stages. CEA, CA19-9, and EpCAM (GA733-2) vary according to performance status. CONCLUSIONS: CEA, CA 19-9, and EpCAM (GA733-2) showed similar specificity (80%, 80% and 84%, respectively). EGFR showed the lowest sensitivity and specificity. CA 19-9 was the marker with the highest sensitivity. The need for convenient tumour marker tests with high sensitivity is of great importance for early diagnosis and monitoring of CRC
2011
Kroupis C, Vourlidis N. Human papilloma virus (HPV) molecular diagnostics. Clin.Chem.Lab Med. [Internet]. 2011;49(11):1783 - 1799. WebsiteAbstract
Human Papilloma Virus (HPV) is becoming a menace worldwide, especially to the developing world, due to its involvement in a variety of malignancies, with cervical cancer being the most important and prevalent. There are many HPV types; HPV 16/18 are the most carcinogenic but few others are also characterized as high-risk (HR). They can cause a variety of low- or high-grade cellular abnormalities, most frequently detected in a routine Pap test. Most infections clear within 2 years, however, a minority persists and potentially could progress to cervical cancer. Molecular tests detecting HPV DNA, RNA or proteins are now being available either commercially or in-house developed. DNA detection is nowadays an established tool for diagnosis and monitoring of HPV-related disease, however, there is lack of a reference method and standardization with reference materials. The various available test formats create confusion on which molecular test to choose and what are its limitations. Therefore, the need for lab accreditation and participation in proficiency testing has to be stressed. Novel HPV biomarkers (RNA, protein etc.) are now intensively examined for their inclusion as adjunct tools. Recently, developed prophylactic vaccines for HPV 16/18 have already proven safe and efficient and raise high expectations for the complete eradication of these types in the future
Pavlidou A, Dalamaga M, Kroupis C, Konstantoudakis G, Belimezi M, Athanasas G, Dimas K. Survivin isoforms and clinicopathological characteristics in colorectal adenocarcinomas using real-time qPCR. World J.Gastroenterol. [Internet]. 2011;17(12):1614 - 1621. WebsiteAbstract
AIM: To investigate three isoforms of survivin in colorectal adenocarcinomas. METHODS: We used the LightCycler Technology (Roche), along with a common forward primer and reverse primers specific for the splice variants and two common hybridization probes labeled with fluorescein and LightCycler-Red fluorophore (LC-Red 640). Real time quantitative polymerase chain reaction (PCR) was performed on cDNAs from 52 tumor specimens from colorectal cancer patients and 10 unrelated normal colorectal tissues. In the patients group, carcinoembryonic antigen (CEA) and CA19-9 tumor markers were also measured immunochemically. RESULTS: Wild type survivin mRNA isoform was expressed in 48% of the 52 tumor samples, survivin-2b in 38% and survivin-DeltaEpsilonx3 in 29%, while no expression was found in normal tissues. The mRNA expression of wild type survivin presented a significant correlation with the expression of the ratio of survivin-2b, survivin-DeltaEpsilonx3, survivin-2b/wild type survivin and survivin-DeltaEpsilonx3/wild type survivin (P < 0.001). The mRNA expression of wild-survivin and survivin-DeltaEpsilonx3 was related with tumor size and invasion (P = 0.006 and P < 0.005, respectively). A significant difference was found between survivin-2b and morphologic cancer type. Also, the ratio of survivin-DeltaEx3/wild-survivin was significantly associated with prognosis. No association was observed between the three isoforms and grade, metastasis, Dukes stage and gender. The three isoforms were not correlated with CEA and CA19-9. CONCLUSION: Survivin isoforms may play a role in cell apoptosis and their quantification could provide information about clinical management of patients suffering from colorectal cancer
Iakovis P, Anyfantakis ZA, LIMAS C, Kroupis C, Degiannis D, Cokkinos DV. Increased inflammatory response in patients with dilated cardiomyopathy is associated with dyslipidemia: Effects of statin therapy. Angiology [Internet]. 2011;62(1):55 - 61. Publisher's VersionAbstract
Dilated cardiomyopathy (DCM) is associated with increased inflammatory response reflected among other markers in high-sensitivity C-reactive protein (hsCRP) and soluble interleukin-2 receptor (sIL-2R) levels. We examined prospectively 60 consecutive patients with DCM. Of them, 30 were dyslipidemic (group I) and 30 normolipidemic (group II). Group I patients were randomized to either simvastatin therapy (20 mg/day, group Ia, n = 15) or hypolipidemic diet therapy (group Ib, n = 15). Patients were re-evaluated 6 months later. High-sensitivity C-reactive protein and sIL-2R levels were significantly higher in group I compared with group II patients (19.5 +/- 3.4 vs 3.03 +/- 3.5 mg/L, P = .01, 1137 +/- 441 vs 599 +/- 235 pg/mL, P = .001, respectively). There was a significant correlation between sIL-2R and hsCRP levels in dyslipidemic patients but not in normolipidemic patients. Significant reduction of hsCRP and sIL-2R levels was observed only in group Ia patients. Patients with DCM having dyslipidemia have increased inflammatory response, which is reduced after 6 months of statin therapy
2010
Kroupis C, Theodorou M, Chaidaroglou A, Dalamaga M, Oliveira SC, Cokkinos DV, Degiannis D, Manginas A. The association between a common FCGR2A polymorphism and C-reactive protein and coronary artery disease revisited. Genet.Test.Mol.Biomarkers [Internet]. 2010;14(6):839 - 846. WebsiteAbstract
INTRODUCTION: the FcgammaRIIa receptor is responsible for the clearance of large immune complexes and recently has been proved to be a C-reactive protein (CRP) receptor as well. A polymorphism in the corresponding FCG2RA gene resulting in an amino acid change (R131H) has been implicated, with conflicting results in the pathogenesis of various autoimmune or inflammatory disorders (e.g., atherosclerosis and coronary artery disease [CAD]). METHODS: we recently developed a real-time polymerase chain reaction and melting curve analysis method for the genotyping of the above polymorphism. We further looked at its validity with bioinformatics study and DNA sequencing. Then we genotyped 134 CAD patients and 45 angiographically normal controls and determined serum high-sensitivity CRP by nephelometry (Dade-Behring). Also, we used apparently healthy platelet donors (n = 206) as a larger control group. RESULTS: our method is accurate and devoid of problems with homologs and copy number variants. The need for reference materials is stressed. There were statistically significant differences (p < 0.05) between the CAD patients and each of the two other control groups, with the percentage of RR genotype rising from 6.5% and 11% in the control groups to an average of 19% in all CAD patients (17%, 24%, and 18.5% in stable angina, unstable angina, and myocardial infarction, respectively). In a logistic regression model that included known risk factors for CAD including CRP, the RR genotype remained a significant predictor for CAD (odds ratio: 6.3 [1.1-36.3]). Also after linear regression analysis, CRP levels were reduced in the RR carriers (vs. HH + HR), controlling for age, sex, and disease (marginal p = 0.07). CONCLUSIONS: with our accurate genotyping method, the RR genotype was correlated with atherothrombotic CAD events. The inverse correlation found between CRP levels and genotype supports the in vitro data of RR cells binding CRP stronger than HH
Vorkas PA, Poumpouridou N, Agelaki S, Kroupis C, Georgoulias V, Lianidou ES. PIK3CA hotspot mutation scanning by a novel and highly sensitive high-resolution small amplicon melting analysis method. J.Mol.Diagn. [Internet]. 2010;12(5):697 - 704. WebsiteAbstract
Somatic mutations in the PIK3CA gene have been discovered in many human cancers, and their presence correlates to therapy response. Three "hotspot" mutations within the PIK3CA gene are localized in exons 9 and 20. High-resolution melting analysis (HRMA) is a highly sensitive, robust, rapid, and cost-effective mutation analysis technique. We developed a novel methodology for the detection of hotspot mutations in exons 9 and 20 of the PIK3CA gene that is based on a combination of PCR and HRMA. The PIK3CA HRMA assay was evaluated by performing repeatability, sensitivity, and comparison with DNA sequencing studies and was further validated in 129 formalin-fixed paraffin-embedded breast tissue samples: 99 tumors, 20 noncancerous, and 10 fibroadenomas. The developed methodology was further applied in a selected group of 75 breast cancer patients who underwent Trastuzumab treatment. In sensitivity studies, the assay presented a capability to detect as low as 1% of mutated dsDNA in the presence of wtDNA for both exons. In the 99 tumor samples (validation group), 12/99 (12.1%) exon 9 mutations and 20/99 (20.2%) exon 20 mutations were found. No mutations were found in noncancerous tissues. In fibroadenomas, we report one PIK3CA mutation for the first time. In the selected group, 30/75 (40%) samples were detected as mutants. The PIK3CA HRMA assay is highly sensitive, reliable, cost-effective, and easy-to-perform, and therefore can be used as a screening test in a high-throughput pharmacodiagnostic setting
Vorkas PA, Christopoulos K, Kroupis C, Lianidou ES. Mutation scanning of exon 20 of the BRCA1 gene by high-resolution melting curve analysis. Clin.Biochem. [Internet]. 2010;43(1-2):178 - 185. WebsiteAbstract
OBJECTIVES: 5382insC frameshift mutation along with 5331G>A (G1738R) missense mutation, both found in exon 20 of the BRCA1 gene, are relatively frequent among the Greek breast and ovarian cancer population (46%). Our goal was to develop a novel, reliable and rapid genotyping/scanning method for mutation detection of the exon 20 of the BRCA1 gene, using high-resolution melting curve analysis. DESIGN AND METHODS: The developed methodology was based on real-time PCR and high-resolution melting curve analysis in the presence of LCGreen I dye. Two amplicons on the exon 20 of BRCA1 gene were designed (157 bp and 100 bp), one flanking the exon's boundaries, and one embracing the 5382insC mutation. Our methodology was first optimized and validated by using genomic DNA samples with the 5382insC and 5331G>A (G1738R) mutations and wild-type. In total, the developed methodology was applied on 90 peripheral blood and 127 formalin-fixed paraffin-embedded breast tissue samples. RESULTS: Sensitivity studies with gDNA isolated from peripheral blood showed that mutated DNA could be reliably detected in the presence of wild-type DNA at 5% and 0.5% ratio with the larger and the smaller amplicon, respectively. By using the developed methodology we successfully identified 5382insC, 5331G>A and 5370C>T (R1751X) mutations, in genomic DNA isolated from peripheral blood samples and 5382insC mutation in two breast tumors, as verified by DNA sequencing. CONCLUSIONS: The combination of real-time PCR and high-resolution melting curve analysis provides a cost-efficient, simple and rapid approach to successfully scan exon 20 of BRCA1 gene for these clinically important and frequent mutations
2009
Tsangaris I, Tsantes A, Bagos P, Nikolopoulos G, Kroupis C, Kopterides P, Dimopoulou I, Orfanos S, Kardoulaki A, Chideriotis S, et al. The effect of plasma homocysteine levels on clinical outcomes of patients with acute lung injury/acute respiratory distress syndrome. Am.J.Med.Sci. [Internet]. 2009;338(6):474 - 477. WebsiteAbstract
BACKGROUND: Several reports have shown that homocysteine promotes thrombosis by disturbing the procoagulant-anticoagulant balance, whereas alterations in coagulation and fibrinolysis have been suggested as important pathogenetic and prognostic determinants of mortality in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). The objective of the study was to evaluate the effect of plasma homocysteine levels on the outcomes of patients with ALI/ARDS. METHODS: Sixty-nine consecutive ventilated patients with ALI/ARDS were studied. Blood samples were drawn within 3 days of clinical recognition of ARDS. Measurement of plasma homocysteine, vitamin B12, folate, creatinine, protein C and plasminogen-activator inhibitor-1 antigen levels, and genotyping of the methylenetetrahydrofolate reductase gene C677T and A1298C polymorphisms were carried out. The primary outcomes were 28- and 90-day mortality, whereas secondary outcomes included nonpulmonary organ failure-free days, liberation from mechanical ventilation up to day 28, and ventilator-free days during the 28 days after enrollment. RESULTS: In the multivariable analysis, plasma homocysteine concentration adjusted for age, Acute Physiology and Chronic Health Evaluation II score, methylenetetrahydrofolate reductase C677T and A1298C polymorphisms, and levels of plasminogen-activator inhibitor-1 antigen, protein C, creatinine, vitamin B12, and folate was not found to affect significantly mortality at 28 and 90 days (P = 0.39 and P = 0.83, respectively), days without organ failure besides lungs (P = 0.38), the probability of being free from mechanical ventilation at day 28 (P = 0.63), and days without ventilation assistance (P = 0.73). CONCLUSION: Our data suggest that increased plasma homocysteine levels, either alone or in synergy with other thrombophilic risk factors, do not seem to adversely affect the prognosis in patients with ALI/ARDS
Kroupis C, Theodorou M, Kounavi M, Oliveira SC, Iliopoulou E, Mavri-Vavayanni M, Melissari EN, Degiannis D. Development of a real-time PCR detection method for a FCGR2A polymorphism in the LightCycler and application in the heparin-induced thrombocytopenia syndrome. Clin.Biochem. [Internet]. 2009;42(16-17):1685 - 1693. WebsiteAbstract
OBJECTIVE: The FcgammaRIIa receptor is responsible for the activation of platelets by antibodies in heparin-induced thrombocytopenia (HIT). The c.497G>A polymorphism in the corresponding FCG2RA gene (H131R) has been implicated in the HIT syndrome and we aimed at its rapid and reliable determination. DESIGN AND METHODS: We designed a novel asymmetric real-time PCR method in the LightCycler that uses two hybridization probes and is followed by melting curve analysis. Seventy-one post-cardiac-surgery HIT Greek patients well ascertained by clinical data, immunological and functional tests (PAT, CD62P-selectin and microparticle flow cytometric detection) were studied, along with a clinically relevant group of 49 thrombocytopenic control patients and 119 healthy subjects. RESULTS: The developed method has excellent analytical characteristics (linear and efficient amplification, precision), has wide DeltaT(m) between the two alleles H and R (11.53 degrees C), and is in 100% concordance with validated controls and another commonly used screening method. The RR percentage increased from 10% in the control populations to 24% in the HIT patient group. CONCLUSION: The described method is technically simple, robust, fast, and accurate. A statistically significant difference was found in the comparison between the groups of HIT patients and healthy subjects [RR vs. RH+ HH, chi(2) test, p=0.01, OR (95% C.I.) 2.81 (1.21-4.68)]. The RR frequency in the Greek population was found to be the lowest among Caucasians
2008
Delimaris I, Georgopoulos S, Kroupis C, Zachari A, Liberi M, Bastounis E, Dionyssiou-Asteriou A. Serum oxidizability, total antioxidant status and albumin serum levels in patients with aneurysmal or arterial occlusive disease. Clin.Biochem. [Internet]. 2008;41(9):706 - 711. WebsiteAbstract
OBJECTIVES: Oxidative stress is involved in the pathophysiology of atherosclerosis. The aim of the present study was to estimate the extent of oxidative stress in patients with aneurysmal and arterial occlusive disease (AAOD) by analyzing the magnitude of serum oxidizability, total antioxidant status and serum antioxidants and to evaluate their potential clinical significance. DESIGN AND METHODS: The study was conducted on 47 patients with AAOD and 49 healthy individuals. Oxidative stress was assessed by: a) copper-induced lipid oxidation described in terms of "lag-time" (t(LAG)) and "maximal rate of accumulation of absorbing products" (RA) and b) the measurement of serum total antioxidant status (TAS) and hydrophilic serum antioxidants (albumin, uric acid, transferrin, bilirubin). LDL-cholesterol and HDL-cholesterol were also estimated. RESULTS: A decrease of t(LAG) and albumin levels in patients as compared to controls was observed. t(LAG) was negatively correlated with RA in both patients and controls. RA and LDL-cholesterol did not differ between the two groups. HDL-cholesterol was decreased in patients in comparison to controls. There is statistically significant evidence that low albumin serum levels are associated with increased risk of AAOD. CONCLUSIONS: The results support the involvement of oxidative stress in AAOD. Significant alterations in serum oxidizability were found in patients with AAOD and low albumin serum levels were correlated with the disease. Clinical evaluation of both findings needs further investigation
Kroupis C, Christopoulos K, Devetzoglou M, Tsiagas I, Lianidou ES. Asymmetric real-time PCR detection of BRCA1 5382insC mutation by melting curve analysis in the LightCycler. Clin.Chim.Acta [Internet]. 2008;390(1-2):141 - 144. WebsiteAbstract
BACKGROUND: 5382insC BRCA1 frameshift mutation is a common founder mutation for many populations worldwide and a high-risk allele for the development of hereditary breast and/or ovarian cancer. Our goal was to develop a novel, reliable and rapid method for its detection. METHODS: We developed an asymmetric real-time PCR method with hybridization probes in the LightCycler. Genotyping was performed by melting curve analysis. RESULTS AND CONCLUSIONS: The developed method was in concordance with reference methods when tested in 85 peripheral blood and 107 tumor DNA samples from Greek breast and/or ovarian cancer patients. The described method proved to be simple, cost-effective, easy to perform and rapid enough for routine use as a screening method in high-risk families and especially in the Greek, Slavic and Jewish populations where 5382insC mutation is the most common BRCA1 mutation
2007
Kroupis C, Thomopoulou G, Papathomas TG, Vourlidis N, Lazaris AC. Population-based study of human papillomavirus infection and cervical neoplasia in Athens, Greece. Epidemiol.Infect. [Internet]. 2007;135(6):943 - 950. WebsiteAbstract
The aim of our study is to describe the prevalence of the different HPV types in women with pre-neoplastic lesions of the cervix in Greece. Cervical scrapes from 841 women were obtained for both cytological evaluation and analysis for the presence of HPV DNA. PCR was performed on specimens from these 841 women. The Pap test results were normal or showed benign cellular changes in 45.8% of the women, atypical squamous cells of undetermined significance (ASCUS) in 23.2%, low-grade squamous intra-epithelial lesion (LSIL) in 27.9% and high-grade squamous intra-epithelial lesion (HSIL) in 3.1%. HPV DNA was demonstrated in 23.6% of cytologically normal women. We detected HPV in 60% of the total samples. Of these, HPV-16 was the most common HPV DNA detected. Interestingly, HPV-58 was inversely correlated with positive cytological findings. A clear pattern of decreasing prevalence of HPV with age was also observed. Our results indicate that HPV infections, especially those with HPV-16, represent a significant public health concern in Greece
2006
Theodorakis GN, Flevari P, Kroupis C, Adamopoulos S, Livanis EG, Kostopoulou A, Kolokathis F, Paraskevaidis IA, Leftheriotis D, Kremastinos DT. Antiinflammatory effects of cardiac resynchronization therapy in patients with chronic heart failure. Pacing Clin.Electrophysiol. [Internet]. 2006;29(3):255 - 261. WebsiteAbstract
BACKGROUND: Cardiac resynchronization therapy (CRT) pacing has been proposed as an additional treatment to medical therapy to improve heart failure patients with left ventricular asynchrony. The aim of this study was to evaluate the influence of CRT treatment on proinflammatory cytokines in patients with heart failure. METHODS: Twenty patients, with a mean age 64 +/- 2 years, with severe chronic heart failure NYHA class II-IV (mean ejection fraction 25 +/- 2%), were included in the study. Patients were treated with CRT pacing, after failure of optimal therapy. Blood samples were taken at baseline, 3 months after pacing therapy, and after a subsequent 3-month period of no pacing for the assessment of proinflammatory cytokines TNF-alpha and its receptors (sTNFR-I, sTNFR-II), IL-6, adhesion molecules sICAM-1 and sVCAM-1, and the apoptotic indices sFas and sFas-Ligand. RESULTS: Levels of TNF-alpha, sTNFR-I, and sTNFR-II were reduced at the end of 3 months of CRT therapy and further reduced at the end of the no pacing period (P < 0.05, compared to baseline). Levels of IL-6 also declined after 3 months of CRT pacing (from 8.9 +/- 2.5 pg/mL to 4.7 +/- 1.3 pg/mL, P < 0.05) and this was maintained during the no pacing period (3.9 +/- 1.1 pg/mL P < 0.05 compared to baseline). The adhesion molecule sICAM-1 levels also reduced (from 265 +/- 17 ng/mL to 235 +/- 12, P < 0.05) after 3 months of CRT pacing and remained unchanged at the end of the no pacing period (219 +/- 12 ng/mL, P < 0.05 compared to baseline values). CONCLUSION: Major proinflammatory cytokines and the adhesion molecule sICAM-1 are reduced with CRT therapy and this effect is maintained for at least 3 months after discontinuation of pacing
Kroupis C, Markou A, Vourlidis N, Dionyssiou-Asteriou A, Lianidou ES. Presence of high-risk human papillomavirus sequences in breast cancer tissues and association with histopathological characteristics. Clin.Biochem. [Internet]. 2006;39(7):727 - 731. WebsiteAbstract
OBJECTIVE: To examine the presence of human papillomavirus (HPV) in breast cancer tissues. DESIGN AND METHODS: Four different PCR methods for detection and verification of genital HPVs were applied in frozen breast cancer specimens. Tumors were also evaluated for various histopathological parameters. RESULTS: Seventeen samples out of 107 tested positive (15.9%). HPV RFLP typing identified a total of 21 high-risk viruses: fourteen HPV 16 (67% of all detected HPV types), three HPV 59, two HPV 58, one HPV 73 and one HPV 82 (one sample with double infection and two samples with triple infection). Breast cancer patients harboring high-risk HPV DNA sequences in their tumor were younger than the rest of the patients. Furthermore, they were less estrogen-receptor-positive and more proliferative as observed in the corresponding indices: Ki-67 staining, S-phase/proliferative fractions and percentage of cells with DNA content over 5C. CONCLUSION: The presence of high-risk HPV DNA sequences in the breast cancer tissues studied was verified, and a possible association with acceleration of malignancy was examined
Flevari P, Theodorakis G, Paraskevaidis I, Kolokathis F, Kostopoulou A, Leftheriotis D, Kroupis C, Livanis E, Kremastinos DT. Coronary and peripheral blood flow changes following biventricular pacing and their relation to heart failure improvement. Europace. [Internet]. 2006;8(1):44 - 50. WebsiteAbstract
AIMS: To study the effect of cardiac resynchronization therapy (CRT) on coronary and peripheral arterial circulation and to assess whether their changes are related to the improvement in patients' functional capacity and prognostically important biochemical markers. METHODS AND RESULTS: Twenty-five patients were studied (New York Heart Association classes III and IV, left ventricular ejection fraction <35%, QRS>120 ms, mean age 66 +/- 2.1 years). Coronary blood flow (CBF), forearm blood flow (FBF), and their reserve were measured by transoesophageal echocardiography (in cm/s) and venous occlusion plethysmography (in mL/100 mL/min) at baseline and following 3 months of CRT. N-terminal-pro-brain natriuretic peptide (Nt-pro-BNP) and serum adhesion molecules, sICAM-1 and sVCAM-1 levels were also assessed. CRT induced a non-significant increase in resting CBF (baseline vs. CRT: 52.1 +/- 5.5 vs. 58.2 +/- 3.6, P: NS), whereas hyperaemic CBF was increased by CRT (baseline vs. CRT: 67.8 +/- 6.8 vs. 79.8 +/- 6.2, P < 0.05). Significant increases were observed in resting FBF (baseline vs. CRT: 1.6 +/- 0.2 vs. 2.6 +/- 0.2, P < 0.05) and hyperaemic FBF (baseline vs. CRT: 2.1 +/- 0.2 vs. 3.2 +/- 0.3, P < 0.05). The per cent difference in hyperaemic FBF was related to the per cent change in Nt-pro-BNP (r = -0.71, P < 0.05) and the per cent improvement in exercise duration (r = 0.80, P < 0.05). CONCLUSION: CRT induces favourable changes in coronary and peripheral arterial function. Changes in peripheral blood flow are related to patients' improvement and may be prognostically significant
2005
Zygalaki E, Stathopoulou A, Kroupis C, Kaklamanis L, KYRIAKIDES Z, Kremastinos D, Lianidou ES. Real-time reverse transcription-PCR quantification of vascular endothelial growth factor splice variants. Clin.Chem. [Internet]. 2005;51(8):1518 - 1520. Website
Kroupis C, Stathopoulou A, Zygalaki E, Ferekidou L, Talieri M, Lianidou ES. Development and applications of a real-time quantitative RT-PCR method (QRT-PCR) for BRCA1 mRNA. Clin.Biochem. [Internet]. 2005;38(1):50 - 57. WebsiteAbstract
OBJECTIVES: To develop a real-time quantitative RT-PCR method for BRCA1 mRNA and then use it for the study of BRCA1 gene expression in human MCF-7 breast cancer cells after their exposure to antineoplastic agents and gamma irradiation. DESIGN AND METHODS: The developed QRT-PCR method is based on the real-time monitoring of a fluorescein-labeled TaqMan probe, specific for BRCA1 mRNA, during PCR in the LightCycler. A BRCA1 PCR amplicon was purified, quantitated and used as a standard of known concentration for the development and analytical evaluation of the assay. The method was applied to study the alteration of BRCA1 gene expression after exposure to taxol, doxorubicin, 5-fluorouracil, etoposide or gamma irradiation in human MCF-7 breast cancer cells. RESULTS: The developed method is quantitative, highly specific for mRNA and highly sensitive (detection limit of 4 BRCA1 copies per mug of total RNA). We observed a reduction of BRCA1 expression for all antineoplastic agents used, while the gamma irradiated MCF-7 cells had an increase of expression with a peak at the 10 Gy dose. CONCLUSIONS: The developed BRCA1 QRT-PCR method is quantitative, highly sensitive and specific. The proposed method is rapid, automated, and cost effective and can be used to study BRCA1 expression in a variety of clinical samples
2004
LIMAS CJ, Iakovis P, Anyfantakis A, Kroupis C, Cokkinos DV. Familial clustering of autoimmune diseases in patients with dilated cardiomyopathy. Am.J.Cardiol. [Internet]. 2004;93(9):1189 - 1191. WebsiteAbstract
In this study, we examined the hypothesis that dilated cardiomyopathy (DCM) shares genetic risk factors with other diseases of presumed autoimmune etiology, and, therefore, the same multiple genes in combination with environmental factors lead to numerous different autoimmune diseases. In accordance with this hypothesis, we showed an increased prevalence of autoimmune diseases in first-degree relatives of patients with DCM. Also, T-cell activation, as reflected in high levels of the soluble interleukin-2 receptor, appears to identify patients with DCM with a clustering of autoimmune diseases
Kroupis C, Lianidou E, Goutas N, Vasilaros S, Yannoukakos D, Petersen MB. Genetic counseling of medullary breast cancer patients. Clin.Genet. [Internet]. 2004;65(4):343 - 344. Website
2003
Sbarouni E, Flevari P, Kroupis C, Kyriakides ZS, Koniavitou K, Kremastinos DT. The effects of raloxifene and simvastatin on plasma lipids and endothelium. Cardiovasc.Drugs Ther. [Internet]. 2003;17(4):319 - 323. WebsiteAbstract
PURPOSE: Raloxifene is a selective estrogen receptor modulator and an attractive alternative to estrogen replacement as it obviates the need for a progestin and does not increase C-reactive protein levels. We compared the effects of simvastatin and raloxifene treatments on the lipid profile, the levels of adhesion molecules and the endothelium dependent and independent vasoreactivity. SUBJECTS & METHODS: We treated 12 postmenopausal women with hypercholesterolemia and coronary artery disease with raloxifene 60 mg/day and simvastatin 20 mg/day in a randomized, double-blind, crossover study. Each treatment period was 8 weeks long with a 4-week washout interval. Plasma lipids and cellular adhesion molecules were evaluated and peripheral blood flow studies with venous occlusion plethysmography were performed. RESULTS: Both simvastatin and raloxifene significantly reduced total [33% (27-40), 12% (0-24)] and LDL [44% (36-52), 16% (0-33)] cholesterol compared to baseline values (p < 0.05) but simvastatin was more effective than raloxifene (p < 0.005). None of the treatments had any significant effect on HDL cholesterol and triglyceride levels. Only raloxifene significantly reduced Lp(a) [18% (1-36)] and ICAM-1 [17% (8-25)] and VCAM-1 [24% (15-33)] plasma levels compared to baseline (p = 0.019, p < 0.0001 and p = 0.003, respectively). Hyperemic blood flow response on raloxifene was significantly higher compared to baseline [52% (0-105)], (p < 0.05), whereas no significant change was noted on simvastatin. Endothelium independent blood flow induced by nitroglycerine was not influenced by either active treatment. CONCLUSIONS: Raloxifene administration is associated with lower ICAM-1, VCAM-1 and Lp(a) plasma levels and enhanced endothelium dependent dilation compared to simvastatin although simvastatin is more powerful in total and LDL cholesterol reduction
Kroupis C, Lianidou E, Goutas N, Ladopoulou A, Konstantopoulou I, Pantazidis A, Yannoukakos D, Efstathiou E, Vourlidis N, Tsionou C. Atypical medullary breast carcinoma in a family carrying the 5382insC BRCA-1 mutation. Breast J. [Internet]. 2003;9(3):260 - 262. Website
LIMAS CJ, Hasikidis C, Iakovou J, Kroupis C, Haidaroglou A, Cokkinos DV. Prognostic significance of soluble interleukin-2 receptor levels in patients with dilated cardiomyopathy. Eur.J.Clin.Invest [Internet]. 2003;33(6):443 - 448. Publisher's VersionAbstract
BACKGROUND: Activation of T lymphocytes is thought to mediate myocardial dysfunction in dilated cardiomyopathy (CMP), probably through cytotoxic cytokines, but its value as a prognostic factor has not been evaluated. METHODS: For 2 years we prospectively followed 76 patients (65 males, 11 females, age 49 +/- 7 years) with CMP and New York Heart Association(NYHA) Class II-III heart failure; left ventricular (LV) function was assessed echocardiographically. Thirty-three patients (28 males, five females, age 52 +/- 6 years) with ischaemic heart disease (IHD) and similar NYHA and LV function characteristics were used as controls. Serum sIL-2R levels, peripheral blood lymphocyte proliferation (basal, + concanavalin A) and HLA-DQB1 genotyping was carried out in all patients. RESULTS: The CMP patients had increased sIL-2R levels (1259 +/- 130 pg mL-1) compared with the IHD patients (703 +/- 80 pg mL-1, P < 0.01, only 3 > 800 pg mL-1). In the CMP patients, there was a significant (r = +0.45, P= 0.04) correlation between sIL-2R and the LV end-diastolic diameter but not with the LV ejection fraction or NYHA Class. During the 24-month follow up, 17 of the CMP patients had an adverse clinical course (death, need for cardiac transplantation, or worsening heart failure). Of these, 14 (75%) had elevated (>or= 800 pg mL-1) sIL-2R levels (Group I) compared with only five (6%) with a stable clinical course (Group II). Neither [3H] thymidine incorporation into the peripheral blood lymphocytes nor the excess of HLA-DQB1-30 histidine homozygotes in the Group I patients (38% vs. 17%, P < 0.05) could predict the clinical outcome. CONCLUSION: Increased sIL-2R levels in CMP patients are an independent predictor of a more aggressive clinical course
2002
Adamopoulos S, Parissis J, Karatzas D, Kroupis C, Georgiadis M, Karavolias G, Paraskevaidis J, Koniavitou K, Coats AJ, Kremastinos DT. Physical training modulates proinflammatory cytokines and the soluble Fas/soluble Fas ligand system in patients with chronic heart failure. J.Am.Coll.Cardiol. [Internet]. 2002;39(4):653 - 663. WebsiteAbstract
OBJECTIVES: We sought to investigate the effects of physical training on circulating proinflammatory cytokines and the soluble apoptosis mediators Fas (sFas) and Fas ligand (sFasL) in patients with chronic heart failure (CHF). BACKGROUND: Recent investigations have shown an overexpression of circulating proinflammatory cytokines and soluble apoptosis mediators in patients with CHF, which may be related to their exercise intolerance and clinical deterioration. METHODS: Plasma levels of tumor necrosis factor-alpha (TNF-alpha), soluble TNF receptors I and II (sTNF-RI and sTNF-RII, respectively), interleukin-6 (IL-6), soluble IL-6 receptor (sIL-6R), sFas and sFasL were measured in 24 patients with stable CHF (New York Heart Association functional class II/III; left ventricular ejection fraction 23.2 +/- 1.3%) and in 20 normal control subjects before and after a 12-week program of physical training in a randomized, crossover design. Functional status of patients with CHF was evaluated by using a cardiorespiratory exercise test to measure peak oxygen consumption (VO2max). RESULTS: Physical training produced a significant reduction in plasma levels of TNF-alpha (7.5 +/- 1.0 pg/ml vs. 4.6 +/- 0.7 pg/ml, p < 0.001), sTNF-RI (3.3 +/- 0.2 ng/ml vs. 2.7 +/- 0.2 ng/ml, p < 0.005), sTNF-RII (2.6 +/- 0.2 ng/ml vs. 2.3 +/- 0.2 ng/ml, p = 0.06), IL-6 (8.3 +/- 1.2 pg/ml vs. 5.9 +/- 0.8 pg/ml, p < 0.005), sIL-6R (34.0 +/- 3.0 ng/ml vs. 29.2 +/- 3.0 ng/ml, p < 0.01), sFas (5.5 +/- 0.7 ng/ml vs. 4.5 +/- 0.8 ng/ml, p = 0.05) and sFasL (34.9 +/- 5.0 pg/ml vs. 25.2 +/- 4.0 pg/ml, p < 0.05), as well as a significant increase in VO2max (16.3 +/- 0.7 ml/kg per min vs. 18.7 +/- 0.8 ml/kg per min, p < 0.001). Good correlations were found between a training-induced increase in VO2max and a training-induced reduction in levels of the proinflammatory cytokine TNF-alpha (r = -0.54, p < 0.01) and the apoptosis inducer sFasL (r = -0.57, p < 0.005) in patients with CHF. In contrast, no significant difference in circulating cytokines and apoptotic markers was found with physical training in normal subjects. CONCLUSIONS: Physical training reduces plasma levels of proinflammatory cytokines and the sFas/sFasL system in patients with CHF. These immunomodulatory effects may be related to the training-induced improvement in functional status of patients with CHF
Ladopoulou A, Kroupis C, Konstantopoulou I, Ioannidou-Mouzaka L, Schofield AC, Pantazidis A, Armaou S, Tsiagas I, Lianidou E, Efstathiou E, et al. Germ line BRCA1 & BRCA2 mutations in Greek breast/ovarian cancer families: 5382insC is the most frequent mutation observed. Cancer Lett. [Internet]. 2002;185(1):61 - 70. WebsiteAbstract
BRCA1 and BRCA2 genes were screened for loss-of-function mutations in a series of 85 patients having at least one first- or second-degree relative affected by breast and/or ovarian cancer. All BRCA1 exons and BRCA2 exons 10 and 11 were screened with a combination of methods including SSCP, PTT and direct sequencing. We have found disease-associated mutations in 14 families (16.5%), eleven in BRCA1 and three in BRCA2. The known founder mutation 5382insC of BRCA1 was identified in seven unrelated families. The other mutations identified include the non-sense R1751X, the splice junction variant 5586G>A of BRCA1 and three frameshifts, 2024del5, 3034del4, and 6631del5, of BRCA2. Nine out of these 14 families had a family history of three or more breast/ovarian cancer cases. A large number of polymorphic or unclassified variants is also reported. Combined with our previously published data 5382insC was found in nine out of 20 families (45%), suggesting that this mutation may represent a common founder mutation in the Greek population
Adamopoulos S, Parissis JT, Georgiadis M, Karatzas D, Paraskevaidis J, Kroupis C, Karavolias G, Koniavitou K, Kremastinos DT. Growth hormone administration reduces circulating proinflammatory cytokines and soluble Fas/soluble Fas ligand system in patients with chronic heart failure secondary to idiopathic dilated cardiomyopathy. Am.Heart J. [Internet]. 2002;144(2):359 - 364. WebsiteAbstract
BACKGROUND: Recent studies have shown that an abnormal proinflammatory cytokine expression and apoptotic process contribute to adverse left ventricular remodeling and progress of chronic heart failure. This study investigates the effects of growth hormone (GH) administration on serum levels of representative proinflammatory cytokines and soluble apoptosis mediators in patients with chronic heart failure secondary to idiopathic dilated cardiomyopathy (IDC). METHODS: Serum levels of tumor necrosis factor-alpha (TNF-alpha), its soluble receptors (sTNF-RI, sTNF-RII), interleukin-6 (IL-6), soluble IL-6 receptor (sIL-6R), soluble Fas (sFas) and soluble Fas Ligand (sFasL) were determined (enzyme-linked immunosorbent assay method) in 10 patients with IDC (New York Heart Association class III, ejection fraction 24% +/- 2%) before and after a 3-month subcutaneous administration of 4 IU GH every other day (randomized crossover design). Peak oxygen consumption (Vo(2)max) was also used to evaluate the functional status of patients with IDC. RESULTS: Treatment with GH produced a significant reduction in serum levels of TNF-alpha (8.2 +/- 1.2 vs 5.7 +/- 1.1 pg/mL, P <.05), sTNF-RI (3.9 +/- 0.4 vs 3.2 +/- 0.3 ng/mL, P <.05), sTNF-RII (2.6 +/- 0.3 vs 2.2 +/- 0.2 ng/mL, P <.05), IL-6 (5.5 +/- 0.6 vs 4.4 +/- 0.4 pg/mL, P =.05), sIL-6R (32.7 +/- 3.0 vs 28.2 +/- 3.0 ng/mL, P <.05), sFas (4.4 +/- 0.8 vs 3.1 +/- 0.6 ng/mL, P <.05), and sFasL (34.2 +/- 11.7 vs 18.8 +/- 7.3 pg/mL, P <.01). A significant improvement was also observed in VO2max after the completion of 3 months' treatment with GH (15.0 +/- 0.8 vs 17.2 +/- 1.0 mL/kg/min, P <.05). Good correlations were found between GH-induced reduction in TNF-alpha levels and increase in VO2max (r = -0.64, P <.05) as well as between GH-induced reduction in sFasL and increase in VO2max (r = -0.56, P =.08). CONCLUSIONS: GH administration reduces serum levels of proinflammatory cytokines and soluble Fas/FasL system in patients with IDC. These immunomodulatory effects may be associated with improvement in clinical performance and exercise capacity of patients with IDC
LIMAS CJ, Kroupis C, Haidaroglou A, Cokkinos DV. Hyperprolactinaemia in patients with heart failure: clinical and immunogenetic correlations. Eur.J.Clin.Invest [Internet]. 2002;32(2):74 - 78. Publisher's VersionAbstract
BACKGROUND: Prolactin represents a stimulatory link between the neuroendocrine and immune systems, but its involvement in the neurohumoral adaptations to heart failure (HF) has not been explored. METHODS: We prospectively studied 55 patients (45 males, 10 females, age 48 +/- 7 years) with NYHA Class II/III HF due either to dilated cardiomyopathy (CMP) (n = 33) or ischemic heart disease (IHD) (n = 22). Serum prolactin levels were determined by radioimmunoassay, soluble interleukin-2 receptor (sIL-2R) levels by enzyme-linked immunoassay and HLA-DQ genotyping with PCR. Left ventricular ejection fraction (LVEF) and end-diastolic diameter (LVEDd) were assessed echocardiographically. RESULTS: Hyperprolactinaemia (17.3 +/- 4 ng mL-1 [Group I] vs. 4.64 +/- 2 ng mL-1 [Group II], P < 0.01) was found in 14 patients (8 with IHD, 6 with CMP). The distribution of HLA-DQB1 alleles was compared in the two groups and showed a significant increase in the frequency of *0301 (86% in Group I vs. 45% in Group II, P < 0.05). Histidine at position 30 of the HLA-DQB1 gene was found in 22% of Group II but in none of Group I patients. Furthermore, there was an inverse correlation between the presence of histidine at position 30 and the levels of serum prolactin. Both sIL-2R levels, a marker of T-cell activation, and concanavalin A-stimulated lymphocyte proliferation were lower in Group I patients (561 +/- 106 vs. 804 +/- 109 pg mL-1 and 20.8 +/- 4 vs. 37.3 +/- 5 cpmX103 [3H] thymidine, respectively). LVEF was significantly higher (32 +/- 5%) and LVEDd smaller (62.0 +/- 6 mm) in Group I compared to Group II (25 +/- 4% and 68.0 +/- 5 mm, respectively, P < 0.01) patients. CONCLUSION: Hyperprolactinaemia presents in 25% of patients with HF and may reflect decreased activation of T-lymphocytes associated with relatively preserved LV systolic function which is under immune-genetic control at the HLA-DQ locus
2001
Adamopoulos S, Parissis J, Kroupis C, Georgiadis M, Karatzas D, Karavolias G, Koniavitou K, Coats AJ, Kremastinos DT. Physical training reduces peripheral markers of inflammation in patients with chronic heart failure. Eur.Heart J. [Internet]. 2001;22(9):791 - 797. WebsiteAbstract
AIMS: Previous studies have shown an abnormal expression of cellular adhesion molecules and cytokines in chronic heart failure, which may be related to endothelial dysfunction characterizing this syndrome. Our study investigates the effects of physical training on serum activity of some peripheral inflammatory markers associated with endothelial dysfunction, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage chemoattractant protein-1 (MCP-1), soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) in patients with chronic heart failure. METHODS AND RESULTS: Serum levels of GM-CSF, MCP-1, sICAM-1 and sVCAM-1 were determined in 12 patients with stable chronic heart failure (ischaemic heart failure: 6/12, dilated cardiomyopathy: 6/12, New York Heart Association: II-III, ejection fraction: 24+/-2%) before and after a 12-week programme of physical training in a randomized crossover design. In addition, the functional status of chronic heart failure patients was evaluated by using a cardiorespiratory exercise stress test to measure peak oxygen consumption. Physical training produced a significant reduction in serum GM-CSF (28+/-2 vs 21+/-2 pg. ml(-1), P<0.001), MCP-1 (192+/-5 vs 174+/-6 pg. ml(-1), P<0.001), sICAM-1 (367+/-31 vs 314+/-29 ng. ml(-1), P<0.01) and sVCAM-1 (1247+/-103 vs 1095+/-100 ng. ml(-1), P<0.01) as well as a significant increase in peak oxygen consumption (14.6+/-0.5 vs 16.5+/-0.5 ml. kg(-1)min(-1), P<0.005). A significant correlation was found between the training-induced improvement in peak oxygen consumption and percentage reduction in soluble adhesion molecules sICAM-1 (r=-0.72, P<0.01) and sVCAM-1 (r=-0.67, P<0.02). CONCLUSION: Physical training affects beneficially peripheral inflammatory markers reflecting monocyte/macrophage-endothelial cell interaction. Training-induced improvement in exercise tolerance is correlated with the attenuation of the inflammatory process, indicating that inflammation may contribute significantly to the impaired exercise capacity seen in chronic heart failure
2000
Konstantopoulou I, Kroupis C, Ladopoulou A, Pantazidis A, Boumba D, Lianidou ES, Petersen MB, Florentin L, Chiotellis E, Nounesis G, et al. BRCA1 mutation analysis in breast/ovarian cancer families from Greece. Hum.Mutat. [Internet]. 2000;16(3):272 - 273. WebsiteAbstract
Germline mutations in BRCA1 gene account for varying proportions of breast/ovarian cancer families, and demonstrate considerable variation in mutational spectra coincident with ethnic and geographical diversity. We have screened for mutations the entire coding sequence of BRCA1 in 30 breast/ovarian cancer women with family history of two or more cases of breast cancer under age 50 and/or ovarian cancer at any age. Genomic DNA from patient was initially analyzed for truncating mutations in exon 11 with PTT followed by DNA sequencing. In the cases where no frameshift mutation was observed in exon 11, all other exons were screened with direct sequencing. Two novel (3099delT, 3277insG) and three already described (3741insA, 1623del5-TTAAA, 5382insC-twice) truncating mutations were identified. In addition, 6 point mutations (L771L, P871L, E1038G, K1183R, S1436S, S1613G) which are already classified as polymorphisms were identified. Three unclassified intronic variants (IVS16-68 G>A, IVS16-92 G>A, IVS18+65G>A) were also detected. These results show that BRCA1 deleterious mutations are present in a fraction (20%) of Greek breast/ovarian cancer families similar to other European countries. Mutations were detected in high- (>/=3 members) as well as in moderate-risk (2 members) families. This is the first report of BRCA1 mutation analysis in Greece
Sbarouni E, Kroupis C, Kyriakides ZS, Koniavitou K, Kremastinos DT. Cell adhesion molecules in relation to simvastatin and hormone replacement therapy in coronary artery disease. Eur.Heart J. [Internet]. 2000;21(12):975 - 980. WebsiteAbstract
AIMS: To assess the effect of simvastatin, hormone replacement therapy and their combination on soluble cell adhesion molecules and plasma lipids, in hypercholesterolaemic post-menopausal women with coronary artery disease. METHODS: We studied 16 post-menopausal women with coronary artery disease and hypercholesterolaemia (total cholesterol >200mg x dl(-1) and LDL cholesterol >130 mg x dl(-1)). We compared simvastatin (20 mg daily) with hormone replacement therapy (0.625 mg conjugated oestrogen and 2.5 mg medroxyprogesterone acetate daily) and their combination, in a randomized, crossover, placebo controlled study. Each treatment period was 8 weeks long with a 4 week washout interval between treatments. Circulating cell adhesion molecules and plasma lipids were evaluated at the end of each treatment period. RESULTS: All three active treatments--simvastatin, hormone replacement therapy and the combination therapy--significantly reduced total and LDL cholesterol, compared to placebo (P<0.001). Only hormone replacement therapy, alone and in combination with simvastatin, significantly decreased lipoprotein(a) when compared to placebo (P<0.05), whereas simvastatin had no significant effect. Likewise, hormone replacement therapy and the combination therapy significantly reduced the intercellular adhesion molecule (ICAM-1) plasma levels (P=0.03 and P=0.02, respectively), while simvastatin, which was superior to hormone replacement therapy in lowering total and LDL cholesterol, did not modify ICAM-1 levels; the combination therapy was not more effective than hormone replacement therapy alone in ICAM-1 reduction. Neither the effect, on any treatment when compared to placebo, of VCAM-1 nor E-selectin levels differed significantly. CONCLUSIONS: Hormone replacement therapy may limit the inflammatory response to injury by modulating the expression of cell adhesion molecules from the endothelial cells, possibly in association with lipoprotein (a) reduction
1999
Antoniadi T, Hatzis T, Kroupis C, Economou-Petersen E, Petersen MB. Prevalence of factor V Leiden, prothrombin G20210A, and MTHFR C677T mutations in a Greek population of blood donors. Am.J.Hematol. [Internet]. 1999;61(4):265 - 267. WebsiteAbstract
The pathogenesis of venous thrombosis involves the interaction of genetic and environmental factors. In order to estimate the frequency of the factor V Leiden, the prothrombin G20210A, and the MTHFR C677T mutations in the Greek population, we analyzed 160 healthy Greek blood donors by PCR amplification and detected allele frequencies of 2.5%, 2.2%, and 35.3%, respectively. The allele frequencies were compared with reported frequencies of other populations of southern Europe. The identification of these common genetic risk factors for thrombosis should enable easy DNA diagnosis and carrier detection in a high proportion of cases and will contribute to a better understanding of the interaction of genetic and environmental risk factors
Antoniadi T, Rabionet R, Kroupis C, Aperis GA, Economides J, Petmezakis J, Economou-Petersen E, Estivill X, Petersen MB. High prevalence in the Greek population of the 35delG mutation in the connexin 26 gene causing prelingual deafness. Clin.Genet. [Internet]. 1999;55(5):381 - 382. Website
1998
Degiannis D, Kalteziotis V, Thalassinos A, Kapsalis A, Kroupis C, Koniavitou K. Effect of cyclosporine and Sirolimus on interleukin-15-driven proliferation of OKT3-preactivated human lymphocytes. Transplant.Proc. [Internet]. 1998;30(4):948 - 949. Website
1995
Condrescu M, Gardner JP, Chernaya G, Aceto JF, Kroupis C, Reeves JP. ATP-dependent regulation of sodium-calcium exchange in Chinese hamster ovary cells transfected with the bovine cardiac sodium-calcium exchanger. J.Biol.Chem. [Internet]. 1995;270(16):9137 - 9146. WebsiteAbstract
Chinese hamster ovary cells expressing the bovine cardiac Na/Ca exchanger were treated with ouabain to increase [Na+]i and stimulate Ca2+ influx by Na/Ca exchange. Depletion of cellular ATP inhibited 45Ca uptake by 40% or more and reduced the half-maximal Na+ concentration for inhibition of 45Ca uptake from 90 to 55 mM. ATP depletion also reduced the rate of rise in [Ca2+]i when [Na+]o was reduced and inhibited the decline in [Ca2+]i when high [Na+]o was restored. The effects of ATP depletion were either absent or reduced in cells expressing a mutant exchanger missing most of the cytosolic hydrophilic domain. We were unable to detect a phosphorylated form of the exchanger in immunoprecipitates from 32P-labeled cells. ATP depletion caused a breakdown in the actin cytoskeleton of the cells. Treatment of the cells with cytochalasin D mimicked the effects of ATP depletion on the [Na+] inhibition profile for 45Ca uptake. Thus, ATP depletion inhibits both the Ca2+ influx and Ca2+ efflux modes of Na/Ca exchange, and may alter the competitive interactions of extracellular Na+ and Ca2+ with the transporter. The latter effect appears to be related to changes in the actin cytoskeleton
1993
Pijuan V, Zhuang Y, Smith L, Kroupis C, Condrescu M, Aceto JF, Reeves JP, Smith JB. Stable expression of the cardiac sodium-calcium exchanger in CHO cells. Am.J.Physiol [Internet]. 1993;264(4 Pt 1):C1066 - C1074. WebsiteAbstract
A line of Chinese hamster ovary (CHO) cells called CK1.4 was produced by transfection with the gene for the bovine cardiac Na(+)-Ca2+ exchanger. CK1.4 cells stably expressed substantial exchange activity and exchanger protein as shown by immunoprecipitation. Exchange activity was quantified as 45Ca2+ influx that depended on both increasing intracellular Na+ and lowering the concentration of external Na+. Replacing external Na+ with K+ slightly increased 45Ca2+ uptake by CK1.4 cells with basal Na+ and greatly increased 45Ca2+ uptake by Na(+)-loaded cells. Neither exchange activity nor exchanger protein was detected in the nontransfected parental line. By contrast to CK1.4 cells, replacing external Na+ with K+ decreased 45Ca2+ uptake in the nontransfected cells whether or not they were Na+ loaded. Changes in cytosolic free Ca2+ determined with fura-2 were consistent with the 45Ca2+ uptake data. Analysis of poly(A)(+)-RNA by Northern blot confirmed that CK1.4 cells, but not the parental line, expressed the exchanger. Expression of the exchanger was also observed in aortic myocytes and a renal epithelial cell line (LLC-MK2) but not in other lines of renal epithelial cells (MDCK, LLC-PK1) or human dermal fibroblasts. The cardiac exchanger produced substantial 45Ca2+ efflux from CK1.4 cells in response to hormone-evoked release of stored Ca2+. CK1.4 cells are an attractive model for studies of the regulation of the cardiac exchanger because they stably express sufficient exchanger for biochemical and immunological analysis
McGinnis K, Kroupis C, Wilson DB. Dimerization of Thermomonospora fusca beta-1,4-endoglucanase E2. Biochemistry [Internet]. 1993;32(32):8146 - 8150. WebsiteAbstract
Unboiled Thermomonospora fusca endoglucanase E2 electrophoresed on SDS-polyacrylamide gels migrated in the range of 80-90 kDa, but when boiled it migrated in the 40-42-kDa range. Sedimentation equilibrium centrifugation as well as chemical cross-linking experiments confirmed that E2 is a dimer. The dimer was reversibly dissociated at low pH. The E2 dimer was stable up to 70 degrees C, but began to dissociate at this temperature after a 30-60-min incubation. A nondimerizing mutant was obtained using region-specific chemical mutagenesis. DNA sequencing of this mutant revealed a single base change that substituted Gly for Glu-263. Chemical modification of carboxylic acid residues in E2 disrupted the dimer interaction
1992
Aceto JF, Condrescu M, Kroupis C, Nelson H, Nelson N, Nicoll D, Philipson KD, Reeves JP. Cloning and expression of the bovine cardiac sodium-calcium exchanger. Arch.Biochem.Biophys. [Internet]. 1992;298(2):553 - 560. WebsiteAbstract
Two clones (p17 and p13), each containing the complete coding sequence for the bovine cardiac Na+/Ca2+ exchanger, were obtained from a lambda gt10 cDNA library by screening with cDNA probes from the canine exchanger. The coding sequence of clone p17 was 92 and 98% identical to the canine cDNA at the nucleotide and amino acid levels, respectively. Nine of the 21 amino acid differences between the two exchangers were found within the 32-amino acid signal sequence. The sequenced portions of the 3' untranslated regions of the cow and dog clones were 88% identical. Na+/Ca2+ exchange activity was expressed in Xenopus laevis oocytes injected with cRNA from clone p17, and in COS cells transfected with expression vectors containing p17. Immunoprecipitation of 35S-labeled proteins from transfected cells with an antibody against the N-terminal portion of the bovine exchanger showed the presence of a 120-kDa protein corresponding to the intact cardiac exchanger. The second bovine clone (p13) did not express exchange activity in either of the above expression systems, presumably because it contained a 300-bp insert with multiple stop codons which interrupted the coding sequence. Comparison of the 5' untranslated regions of p13 and p17 revealed a 156-bp segment in p17 that was apparently spliced out of p13. This segment contained a short open reading frame. A chimera encoding the 5' untranslated region of p13 and the coding sequence of p17 exhibited only a modest (74%) increase in expressed exchange activity in transfected cells compared to p17, suggesting that the presence of the upstream open reading frame in p17 did not greatly reduce translation efficiency. The results suggest that alternate splicing mechanisms may be involved in processing mRNA for the bovine cardiac exchanger