Genes Co-Expressed with ESR2 Influence Clinical Outcomes in Cancer Patients: TCGA Data Analysis.

ERß has been assigned a tumor suppressor role in many cancer types. However, as conflicting findings emerge, ERß's tissue-specific expression and functional role have remained elusive. There remains a notable gap in compact and comprehensive analyses of ESR2 mRNA expression levels across diverse tumor types coupled with an exploration of its potential gene network. In this study, we aim to address these gaps by presenting a comprehensive analysis of ESR2 transcriptomic data. We distinguished cancer types with significant changes in ESR2 expression levels compared to corresponding healthy tissue and concluded that ESR2 influences patient survival. Gene Set Enrichment Analysis (GSEA) distinguished molecular pathways affected by ESR2, including oxidative phosphorylation and epithelial-mesenchymal transition. Finally, we investigated genes displaying similar expression patterns as ESR2 in tumor tissues, identifying potential co-expressed genes that may exert a synergistic effect on clinical outcomes, with significant results, including the expression of ACIN1, SYNE2, TNFRSF13C, and MDM4. Collectively, our results highlight the significant influence of ESR2 mRNA expression on the transcriptomic landscape and the overall metabolism of cancerous cells across various tumor types.

Effect of cellular senescence on the response of human peritoneal mesothelial cells to TGF-ß.

Transforming growth factor ß (TGF-ß) is implicated in both mesothelial-to-mesenchymal transition (MMT) and cellular senescence of human peritoneal mesothelial cells (HPMCs). We previously showed that senescent HPMCs could spontaneously acquire some phenotypic features of MMT, which in young HPMCs were induced by TGF-ß. Here, we used electron microscopy, as well as global gene and protein profiling to assess in detail how exposure to TGF-ß impacts on young and senescent HPMCs in vitro. We found that TGF-ß induced structural changes consistent with MMT in young, but not in senescent HPMCs. Of all genes and proteins identified reliably in HPMCs across all treatments and states, 4,656 targets represented overlapping genes and proteins. Following exposure to TGF-ß, 137 proteins and 46 transcripts were significantly changed in young cells, compared to 225 proteins and only 2 transcripts in senescent cells. Identified differences between young and senescent HPMCs were related predominantly to wound healing, integrin-mediated signalling, production of proteases and extracellular matrix components, and cytoskeleton structure. Thus, the response of senescent HPMCs to TGF-ß differs or is less pronounced compared to young cells. As a result, the character and magnitude of the postulated contribution of HPMCs to TGF-ß-induced peritoneal remodelling may change with cell senescence.

Effect of Flaxseed (Linum usitatissimum L.) Supplementation on Vascular Endothelial Cell Morphology and Function in Patients with Dyslipidaemia-A Preliminary Observation.

CONTEXT: Flaxseed has a characteristic fatty acids composition and unique phytonutrient profile that may have health-promoting properties. OBJECTIVE: This study aimed to determine the effects of 10 weeks of supplementation with the flaxseed (28 g/day) on endothelial cells (EC) function, serum lipids and proinflammatory mediators in patients with mild and severe dyslipidaemia. MATERIALS AND METHODS: Eleven lean patients with severe dyslipidaemia treated with apheresis (group 1; 10 weeks treated in four phases: (i) ordinary diet, (ii) ordinary diet + flaxseed, (iii) ordinary diet (wash out), (iv) ordinary diet + placebo) and eleven obese patients with mild dyslipidaemia-not treated with apheresis (group 2; 10 weeks treated in two phases: (i) ordinary diet, (ii) low fat diet + flaxseed). Flaxseed was given blindly. Serum was collected at the end of each phase of the study. ECs were exposed in vitro to the medium supplemented with pooled serum taken from patients from both groups to detect their morphological changes using light and electron microscopy. ECs proliferation was also measured at the end of each study phase. RESULTS: Serum vascular endothelial growth factor was decreased after flaxseed supplementation but only in group 1. ECs proliferation was increased after flaxseed supplementation only in obese patients. ECs exposed to medium supplemented with obese patients' serum revealed the following cellular abnormalities: accumulation of lipid droplets, changes of rough endoplasmic reticulum and mitochondria, and flaxseed did not reverse observed changes. At the same time, flaxseed supplementation decreases total cholesterol in both tested groups, low-density lipoprotein cholesterol in group 1 and triglycerides in group 2. CONCLUSIONS: Our findings support the potential role of flaxseed in treating dyslipidaemia but indicate only a slight impact on endothelial cell function.

Biological response of adrenal carcinoma and melanoma cells to mitotane treatment.

A previous case report described an adrenal incidentaloma initially misdiagnosed as adrenocortical carcinoma (ACC), which was treated with mitotane. The final diagnosis was metastatic melanoma of unknown primary origin. However, the patient developed rapid disease progression after mitotane withdrawal, suggesting a protective role for mitotane in a non-adrenal-derived tumor. The aim of the present study was to determine the biological response of primary melanoma cells obtained from that patient, and that of other established melanoma and ACC cell lines, to mitotane treatment using a proliferation assay, flow cytometry, quantitative PCR and microarrays. Although mitotane inhibited the proliferation of both ACC and melanoma cells, its role in melanoma treatment appears to be limited. Flow cytometry analysis and transcriptomic studies indicated that the ACC cell line was highly responsive to mitotane treatment, while the primary melanoma cells showed a moderate response in vitro. Mitotane modified the activity of several key biological processes, including 'mitotic nuclear division', 'DNA repair', 'angiogenesis' and 'negative regulation of ERK1 and ERK2 cascade'. Mitotane administration led to elevated levels of DNA double-strand breaks, necrosis and apoptosis. The present study provides a comprehensive insight into the biological response of mitotane-treated cells at the molecular level. Notably, the present findings offer new knowledge on the effects of mitotane on ACC and melanoma cells.

Immunohistochemical analysis of ghrelin expression in various types of adrenal tumors.

INTRODUCTION: Ghrelin, originally isolated from the endocrine cells of the gastric mucosa, is also expressed in many peripheral tissues, including normal adrenals and adrenocortical tumors. It was shown that ghrelin stimulates proliferation and inhibits apoptosis of adrenocortical cells. In the current study, we compared ghrelin expression at the protein level in various adrenal tumors. We analyzed whether immunoreactive ghrelin could be considered as a potential marker for different types of adrenal tumors. MATERIAL AND METHODS: Study was carried out on 200 adrenal specimens arranged on microscope slide in tissue microarray format. We performed standardized immunohistochemical reactions with semiquantitative reaction intensity measurements. RESULTS: At the protein level, the expression of ghrelin was significantly reduced in adrenocortical adenocarcinoma in relation to the control group and pheochromocytoma as well as cancer-adjacent normal adrenal tissue. In contrast, a relatively high ghrelin expression was found in pheochromocytoma compared to all analyzed groups, with the exception of cancer-adjacent normal adrenal tissue. CONCLUSIONS: The ghrelin expression profile at the protein level may be associated with the type of adrenal tumor. In this context, our results suggest that adrenal immunoreactive ghrelin may be considered as a sensitive and specific marker for differentiating adrenocortical carcinoma from adrenocortical adenoma and pheochromocytoma.

Infiltration of CD68+ cells correlates positively with matrix metalloproteinase 2 expression in the arteries used as aortocoronary bypass grafts. Possible clinical implications.

BACKGROUND: Late failure of arterial aortocoronary conduits may result from abnormal activity of cells found in the vessel wall, including macrophages. The purpose of this study was to assess if there are any associations between the number of macrophages and overexpression of matrix metalloproteinases (MMPs) in the wall of arterial grafts, as well as their clinical significance. METHODS: This study involved 128 consecutive patients with a mean age of 64.9 ± 9.7 years who underwent elective surgery for coronary artery disease (CAD). The surplus segments of internal thoracic artery (ITA) and radial arteries (RA) were taken for immunohistochemical analysis of macrophage numbers and MMPs expression. The participants who reached the clinical primary end-point (cardiacrelated death, acute coronary syndrome or progression of CAD) had a follow-up angiography. RESULTS: The mean numbers of macrophages were higher on RA (70 [24; 112]) than ITA cross-sections (44 [24; 59]; p < 0.001). Median expression of both MMP2 and MMP9 were stronger in the ITA than RA cross-sections (p < 0.001). A significant positive correlation of MMP2 expression and a number of macrophages infiltrating the tunica media of arterial segments were noted on both ITA and RA cross-sections. In addition, the arterial segments of the 6 patients who reached clinical end-point had higher numbers of macrophages and stronger MMP2 expression when compared to the rest of the participants. CONCLUSIONS: Macrophage infiltration of arterial wall grafts prior to harvesting may be associated with higher risk of late occlusion and MMP2 might be facilitating this process.

Analysis of Transcriptome, Selected Intracellular Signaling Pathways, Proliferation and Apoptosis of LNCaP Cells Exposed to High Leptin Concentrations.

Leptin, the first discovered adipokine, has been connected to various physiological and pathophysiological processes, including cancerogenesis. Increasing evidence confirms its influence on prostate cancer cells. However, studies on the effects of leptin on the proliferation and apoptosis of the androgen-sensitive LNCaP line of prostate cancer cells brought conflicting results. Therefore, we performed studies on the effects of high LEP concentration (1 × 10-6 M) on gene expression profile, change of selected signaling pathways, proliferation and apoptosis of LNCaP cells. RTCA (real-time cell analyzer) revealed inhibitory effect of LEP on cell proliferation, but lower LEP concentrations (10-8 and 10-10 M) did not affect cell division. Moreover, flow cytometry with a specific antibody for Cleaved PARP-1, an apoptosis marker, confirmed the activation of apoptosis in leptin-exposed LNCaP line of prostate cancer cells. Within 24 h LEP (10-6 M) increases expression of 297 genes and decreases expression of 119 genes. Differentially expressed genes (DEGs) were subjected to functional annotation and clusterization using the DAVID bioinformatics tools. Most ontological groups are associated with proliferation and apoptosis (seven groups), immune response (six) and extracellular matrix (two). These results were confirmed by the Gene Set Enrichment Analysis (GSEA). The leptin's effect on apoptosis stimulation was also confirmed using Pathview library. These results were also confirmed by qPCR method. The results of Western Blot analysis (exposure to LEP 10 min, 1, 2, 4 and 24 h) suggest (after 24 h) decrease of p38 MAPK, p44-42 mitogen-activated protein kinase and Bcl-2 phosphorylated at threonine 56. Moreover, exposure of LNCaP cells to LEP significantly stimulates the secretion of matrix metallopeptidase 7 (MMP7). Obtained results suggest activation of apoptotic processes in LNCaP cells cultured at high LEP concentration. At the same time, this activation is accompanied by inhibition of proliferation of the tested cells.

Differences in Expression of Genes Involved in Bone Development and Morphogenesis in the Walls of Internal Thoracic Artery and Saphenous Vein Conduits May Provide Markers Useful for Evaluation Graft Patency.

Coronary artery bypass grafting (CABG) is one of the most efficient procedures for patients with advanced coronary artery disease. From all the blood vessels with the potential to be used in this procedure, the internal thoracic artery (ITA) and the saphenous vein (SV) are the most commonly applied as aortocoronary conduits. Nevertheless, in order to evaluate the graft patency and efficiency effectively, basic knowledge should be constantly expanding at the molecular level as well, as the understanding of predictive factors is still limited. In this study, we have employed the expressive microarray approach, validated with Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR), to analyze the transcriptome of both venous and arterial grafts. Searching for potential molecular factors, we analyzed differentially expressed gene ontologies involved in bone development and morphogenesis, for the possibility of discovery of new markers for the evaluation of ITA and SV segment quality. Among three ontological groups of interest-"endochondral bone morphogenesis", "ossification", and "skeletal system development"-we found six genes common to all of them. BMP6, SHOX2, COL13A1, CSGALNACT1, RUNX2, and STC1 showed differential expression patterns in both analyzed vessels. STC1 and COL13A1 were upregulated in ITA samples, whereas others were upregulated in SV. With regard to the Runx2 protein function in osteogenic phenotype regulation, the RUNX2 gene seems to be of paramount importance in assessing the potential of ITA, SV, and other vessels used in the CABG procedure. Overall, the presented study provided valuable insight into the molecular background of conduit characterization, and thus indicated genes that may be the target of subsequent studies, also at the protein level. Moreover, it has been suggested that RUNX2 may be recognized as a molecular marker of osteogenic changes in human blood vessels.

An increased skin microvessel density is associated with neurovascular complications in type 1 diabetes mellitus.

The aim of this study was to assess the blood vessel density and maturity in the skin of adults with type 1 diabetes in relation to the presence of late neurovascular complications. We included 148 patients (87 men) with a median (interquartile range) age of 41 (31-49) and median diabetes duration of 21 (17-30) years. Microvessel (CD133, CD34, CD31 and von Willebrand factor) markers were evaluated by indirect immunohistochemistry assay in material from a skin biopsy. Diabetic retinopathy was diagnosed using direct ophthalmoscopy, and diabetic kidney disease was estimated in people with increased albuminuria and a 10-year duration of diabetes or evidence of diabetic retinopathy . Diabetic peripheral neuropathy diagnosis was based on Toronto definition, cardiac autonomic neuropathy on validated ProSciCard III program. Microvessel density, assessed by CD34 and CD133, was significantly higher in patients with cardiac autonomic neuropathy [160 (125-175) vs 121 (100-154)/1 mm2, p = 0.001 and 92 (83-104) vs 79 (63-92)/1 mm2, p = 0.007, respectively] and CD34 in patients with diabetic peripheral neuropathy [135 (106-168) vs 121 (95-145)/1 mm2, p = 0.018], as compared with subjects without complications. In multivariate logistic regression, density of CD34 and CD133 positive vessels was associated with presence of cardiac autonomic neuropathy [odds ratio 1.016 (95% confidence interval: 1.002-1.029), p = 0.019 and odds ratio 1.037 (95% confidence interval: 1.008-1.067), p = 0.011, respectively]. It was independent from age, sex, diabetes duration, smoking status, body mass index and HbA1c value. Density of CD34 positive vessels was also associated with diabetic peripheral neuropathy, independently from sex and diabetes duration [odds ratio 1.009 (95% confidence interval: 1.001-1.020), p = 0.037]. Skin microvessel density is increased in adults with clinical evidence of neurovascular complications of type 1 diabetes. This is associated with predominance of the vessels of low maturity.

Variability in gelatinase expression in the walls of vessels used as aortocoronary conduits may impact long-term graft patency.

BACKGROUND: An imbalance between the activity of matrix metalloproteinases (MMPs), particularly gelatinases, and tissue inhibitors of metalloproteinases (TIMPs) is considered as one of the mechanisms leading to aortocoronary graft failure. AIM: We aimed to assess the variability in gelatinase expression in the walls of aortocoronary conduits and to evaluate its impact on coronary artery bypass grafting (CABG) outcomes. METHODS: The study included 101 consecutive patients (61 men and 40 women) who underwent CABG. An immunohisto-chemical analysis of MMP-2, MMP-9, TIMP-1, and TIMP-2 expression was performed on the cross-sections of the internal thoracic artery (ITA), radial artery (RA), and saphenous vein (SV). The histological findings were compared between patients with SV graft disease (SVGD[+] group) and those without occlusions in the SV (SVGD[-] group). RESULTS: The median MMP and TIMP expression was the weakest in the ITA wall. MMP expression was comparable between the RA and SV cross-sections, whereas TIMP expression was stronger in the RA than in the SV wall (p < 0.05). In most SV segments, but not in the arteries, immunostaining intensity for MMP was comparable to or stronger than for TIMPs. In the veins harvested from the SVGD(+) group, MMP-2 and MMP-9 tissue expression was more pronounced than in the SVGD(-) group. TIMP levels were comparable between groups. CONCLUSIONS: Imbalance in the metalloproteinase-to-inhibitor tissue expression in the vessel wall might predispose to graft failure. A stronger expression of TIMPs than MMPs in the arterial grafts might explain favourable long-term outcomes.

Dermal microvessel density and maturity is closely associated with atherogenic dyslipidemia and accumulation of advanced glycation end products in adult patients with type 1 diabetes.

In patients with diabetes, functional changes in microcirculation and subclinical vascular pathology precede clinical manifestation of microangiopathic complications. The objective of this study was to evaluate the association between established vascular risk factors and density, maturity, and reactivity of dermal blood vessels in adults with type 1 diabetes (DM1). We included 148 DM1 patients (87 men) with a median (IQR) age of 40.5 (30.5-49) years and a median diabetes duration of 21 (17-29.5) years. The control group consisted of 13 healthy volunteers (6 men) with a median (IQR) age of 36 (31-43). Accumulation of advanced glycation end products (AGEs) was assessed using the AGE-Reader device. In the immunohistochemical (IHC) analyses, anti-CD133, anti-CD34, anti-CD31, and anti-vWF autoantibodies were used. Microvessel density (MVD) in the skin was calculated using the "hot spots technique". Microvascular function was examined by single-point laser-Doppler flowmetry (LDF). Median MVD, calculated for both papillary and reticular dermis, for CD31 antigen expression was 38 (19-56) per 1 mm2. The median CD34+ blood vessel density was 121 (100-155) per 1 mm2, CD133+ was 79 (63-92) per 1 mm2, and vWF+ was 50 (40-69) per 1 mm2. The average CD34/CD31 index was 2.78, the vWF/CD31 ratio was 1.32 and the CD133/CD31 ratio was 1.75. The CD34/CD31 index was positively associated with serum triglyceride concentration (Beta: 0.26, p = 0.012) and negatively associated with serum HDL cholesterol concentration (Beta: -0.22, p = 0.027), both independently from age, sex, diabetes duration, BMI, HbA1c value, presence of hypertension, and eGFR. We found a negative correlation between MVD assessed by CD31 and skin AF (r = -0.21, p = 0.016). In LDF, the area under the blood flow/time curve (AUC) correlated positively with CD31+ MVD (r = 0.21, p = 0.011) and negatively with CD34+ MVD (r = -0.20, p = 0.017). The MVD did not differ between participants with diabetes and healthy controls, and it did not differ according to the presence of retinopathy among the participants with diabetes. Atherogenic dyslipidemia is associated with increased formation of new blood vessels, characterized by high expression of CD34 and low reactivity in LDF. Conversely, chronic hyperglycemia and excessive formation of AGEs may result in decreased vascularity.

Vascular restenosis in coronary artery bypass grafting might be associated with VEGF-C/VEGFR-3 signaling pathway.

The vascular endothelial growth factor (VEGF) family of peptides and caveolins (CAVs) are reported to contribute, in early graft failure in patients, a coronary artery bypass grafting (CABG). To investigate the possible association of ultimate luminal occlusion to VEGFs and CAVs expression, a functional analysis (based on the molecular biology, bioinformatics, histology, and clinical studies) was performed. Twenty-four hundred and sixty-eight CABG patients diagnosed with multivessel stable coronary artery disease (CAD) were enrolled into prospective study and assigned to two subgroups: double- and triple-vessel CAD subjects. Distal parts of all the harvested saphenous vein (SV) and internal thoracic artery (ITA) segments were used for further tests. ITA graft failure did not differ between double-vessel and triple-vessel CAD patients. The number of SV occlusions was significantly higher in triple-vessel CAD subjects. The microarray analysis performed on SV and ITA samples obtained exclusively from triple-vessel CAD patients who developed early graft occlusion revealed 383 genes with increased and 301 genes with decreased expression in ITA samples as compared to SV grafts. This was followed by functional analysis of 'blood vessel development' group of genes. Average VEGF-C expression in ITA grafts was higher than in corresponding SV grafts; FLT4 expression was significantly higher in SV than in ITA transplants. VEGFR-3 and CAV3 expression demonstrated immunohistochemically in SMCs of the tunica media of SV grafts predicted their early restenosis in triple-vessel CAD patients. CAV2 protein expression in SMCs of ITA grafts indicated the risk of early graft failure both in double-vessel and triple-vessel CAD subjects.

Late subclinical hemolysis and long-term outcomes after aortic valve replacement with On-X mechanical prostheses - a preliminary single-center report.

INTRODUCTION: Aortic valve replacement (AVR) with a mechanical prosthesis is not free from late complications. AIM: To evaluate the prevalence of subclinical hemolysis after AVR with On-X prostheses and assess its impact on long-term outcomes. MATERIAL AND METHODS: The prospective study included 84 consecutive patients aged 58.3 ±10.3 years who underwent AVR. They were retrospectively split into group H (n = 12; 14.3%) with prosthesis-related subclinical hemolysis and a control group (C; n = 72; 85.7%). All operations were performed via median sternotomy using cardio-pulmonary bypass. At the end of follow-up, echocardiography was carried out and blood samples for morphology and biochemistry (lactate dehydrogenase (LDH), bilirubin, haptoglobin) were taken. RESULTS: The rate of subclinical hemolysis in patients with properly working prostheses was 14.3% and it was the highest (33.3%) for the smallest valves. Although an improvement in functional status was noted in both groups, it was less evident in group H than in group C (p = 0.007). At the end of follow-up, 97.2% in group C and 75.0% in group H were found in NYHA classes I and II. Patients in group H had significantly lower hemoglobin, hematocrit, and haptoglobin and higher LDH activity than group C subjects. In group H, systolic gradients of On-X valves were higher whereas effective orifice area was smaller than in group C. CONCLUSIONS: Our study proved that prosthesis-induced subclinical hemolysis is seen even after implantation of the latest generation mechanical prostheses, particularly of small diameter, and its degree may impact late outcome.

Preoperative factors predicting saphenous vein graft occlusion in coronary artery bypass grafting: a multivariate analysis.

Saphenous vein segments are frequently used as aortocoronary bypass grafts, particularly in patients over 65 years of age. In the majority of patients, venous grafts maintain their patency for 5-6 years; however, some become occluded within 12 months after surgery. There are some defined predictive biological factors used to assess saphenous vein graft long-term patency rates, but little is known about molecular parameters for estimating the risk of early vein occlusion. The pathogenesis of this process involves the proliferation of stem cells, as well as progenitor cells, in the graft wall. Histologically, this is reflected by CD34 and CD133 expression in endothelial and smooth muscle cells. Thus, the aim of present work was to perform a multivariate analysis of stem cell and progenitor cell markers in saphenous vein graft walls before transplantation to arterial circulation and correlate these results with early graft occlusion. A total of 718 patients, who underwent coronary artery bypass grafting using a saphenous vein graft, were enrolled in this prospective study. CD34, CD133 and von Willebrand factor expression was evaluated via immunohistochemistry. A multivariate analysis revealed that strong CD133 expression in smooth muscle cells can be considered a risk factor for early graft failure. Our findings suggest that CD133 expression in smooth muscle cells of the tunica media in saphenous vein grafts obtained from coronary artery bypass graft patients before graft transplantation to coronary circulation might predict the possibility of early graft occlusion.

Caveolin 2: a facultative marker of unfavourable prognosis in long-term patency rate of internal thoracic artery grafts used in coronary artery bypass grafting. Preliminary report.

OBJECTIVES: Intimal hyperplasia leading to graft failure in patients undergoing coronary artery bypass grafting (CABG) is related to vascular smooth muscle cells (SMCs) proliferation. SMCs respond to a variety of mediators, the most important of which is platelet-derived growth factor (PDGF). The platelet-derived growth factor-induced cellular response has been shown to be mediated by caveolins. The aim of this study was to analyze CAV1-3 expression in internal thoracic artery (ITA) grafts used in CABG and correlate their expression with graft occlusion. METHODS: Six hundred patients undergoing CABG with the use of ITA grafts between 2008 and 2014 were enrolled into this prospective study. CAV1-3 expression in the ITA grafts was analyzed prior to graft transplantation into the coronary circulation via immunohistochemistry. Estimated caveolins expression pattern was then correlated with the occurrence of ITA graft failure observed within 24-months of surgery. RESULTS: Thirty-four patients developed ITA graft failure (subgroup A) and 566 study participants presented no adverse events (subgroup B). CAV1 and CAV3 expression levels in SMCs of the tunica media of the ITA grafts did not differ between the study subgroups and were not associated with the risk of graft failure. CAV2 was expressed within SMCs of the ITA grafts in 94.1% of the patients from subgroup A and 2.5% from subgroup B, and its expression was associated with ITA graft occlusion observed within 24-months after CABG. CONCLUSIONS: CAV2 expression in SMCs of the tunica media in autologous ITA transplants might indicate the risk of graft failure.

Association between small fiber neuropathy and higher skin accumulation of advanced glycation end products in patients with type 1 diabetes.

INTRODUCTION Advanced glycation end products (AGEs) play a crucial role in the pathogenesis of diabetic peripheral neuropathy (DPN). OBJECTIVES The aim of the study was to assess the skin accumulation of AGEs in patients with long­lasting type 1 diabetes in relation to the presence of DPN. PATIENTS AND METHODS We evaluated 178 patients with type 1 diabetes (99 men; age, 43 years [interquartile range [IQR], 34-54 years]; disease duration, 25 years [IQR, 18-31 years]). DPN was diagnosed if 2 or more of the following 5 abnormalities were present: symptoms of neuropathy, lack of ankle reflexes, and impaired sensation of touch, temperature, and/or vibration. PGP 9.5­immunoreactive nerve fibers were counted to assess intraepidermal nerve fiber density (IENFD) in skin biopsy. The accumulation of AGEs in the skin was assessed on the basis of skin autofluorescence (AF).  RESULTS Patients with DPN (45%), compared with those without neuropathy, had higher skin AF (2.6 AU [IQR, 2.3-3.1 AU] vs 2.1 AU [IQR, 1.8-2.5 AU]; P <0.001) and lower IENFD (10 fibers/mm [IQR, 7-14 fibers/mm] vs 12 fibers/mm [IQR, 8-16 fibers/mm]; P = 0.005). We found a positive correlation between skin AF and patients' age (Rs = 0.44; P <0.001), diabetes duration (Rs = 0.32; P <0.001), and a negative correlation between skin AF and the estimated glomerular filtration rate (Rs = -0.26, P <0.001) and IENFD (Rs = -0.22; P = 0.004). In a multiple linear regression analysis, skin AF was independently associated with age (ß = 0.45; P <0.001), glycated hemoglobin level (ß = 0.19; P = 0.007), and IENFD (ß = - 0.14; P = 0.04) (R2 = 0.27; P <0.001). In multivariate logistic regression, the presence of DPN was independently associated with skin AF (odds ratio, 4.16; 95% confidence interval, 1.88-9.20; P <0.001). CONCLUSIONS The presence of DPN, and particularly small fiber neuropathy, is associated with a higher accumulation of AGEs in the skin of patients with type 1 diabetes.

Potentially positive ageing-related variations of medial smooth muscle cells in the saphenous veins used as aortocoronary bypass grafts.

INTRODUCTION: Currently, elderly people constitute a large proportion of patients undergoing coronary artery bypass grafting (CABG). Activated smooth muscle cells in the tunica media of saphenous vein (SV) grafts are thought to play a key role in the formation of neointima and development of occluding atherosclerotic plaques. The aim of this study was to identify ageing-related variations in the expression of the smooth muscle cells pro-teins that may impact on patency rate of the grafts and the CABG outcomes. MATERIAL AND METHODS: The study involved 216 consecutive patients with the mean of 62.7 ± 8.4 years who underwent isolated CABG with at least one SV aortocoronary bypass graft. Expression of a-smooth muscle actin (a-SM actin), smooth muscle-myosin heavy chain (SM-MHC), calponin (CALP), cytokeratin 8 (CK-8), metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinases-2 and -3 (TIMP-2, TIMP-3) in the SV wall was assessed by immunohistochemistry and correlated with the age of patients. RESULTS: Calponin and a-SM actin were expressed in all studied SV transplants. SM-MHC immunoreactivity was observed in SV segments in 68.5% of patients, whereas MMP-2a and TIMPs expression was found in 75% of cases. In more than 50% of analyzed SV transplants, no expression of cytokeratin-8 was found. Moderate correlations between preexisting expressions of either cytoskeletal or hemostatic proteins in the tunica media of the SV grafts and the age of CABG patients were demonstrated. They were positive for SM-MHC (r = 0.494), CALP (r = 0.548), TIMP-2 (r = 0.413) and TIMP-3 (r = 0.406) whereas negative for CK-8 (r = -0.528) and MMP-2 (r = -0.417). CONCLUSIONS: Age-dependent decreases in the expression of MMP-2 and CK-8 accompanied by increases in expression of SM-MHC, TIMP-2 and TIMP-3 may promote SV graft patency and, thus, suggest a rationale for common use of SV grafts in the elderly.

Peritoneal mesothelium promotes the progression of ovarian cancer cells in vitro and in a mice xenograft model in vivo.

The role of mesothelial cells in the intraperitoneal spread of ovarian cancer is still elusive. In particular, it is unclear whether these cells constitute a passive barrier preventing cancer cell progression or perhaps act as an active promoter of this process. In this report we show that omental human peritoneal mesothelial cells (HPMCs) stimulate adhesion and proliferation of ovarian cancer cells (A2780, OVCAR-3, SKOV-3). The latter was associated with the paracrine activity of GRO-1, IL-6, and IL-8 released to the environment by HPMCs. Furthermore, the growth dynamics of ovarian cancer xenografts produced in response to i.p. injection of ovarian cancer cells together with HPMCs was remarkably greater than for implantation of cancer cells alone. A layer of peritoneal mesothelium was consistently present in close proximity to the tumor mass in every xenograft model. In conclusion, our results indicate that HPMCs play a supporting role in the intraperitoneal invasiveness of ovarian malignancy, whose effect may be attributed to their ability to stimulate adhesion and proliferation of cancer cells.

Silane-modified surfaces in specific antibody-mediated cell recognition.

The immobilization of antibodies on various surfaces has been the subject of advanced research in various immunoassay-based diagnostic devices. The physical and chemical stabilities of the immobilized antibodies on a solid surface still cause many problems because upon immobilizing antibody molecules, the antigen-binding ability usually decreases. The silanization of surfaces with organosilanes carrying chemically active groups such as (3-aminopropyl) triethoxysilane (APTES) can accommodate these antigen-binding molecules in an appropriate orientation so that their functionality and binding activity are essentially retained. In this study, n-butyltrimethoxysilane (BMS) and 3-(octafluoropentyloxy)-propyltriethoxysilane (OFPOS) were used as "blocking silanes". The aims of this study were to compare the effectiveness of specific antibody binding of APTES, APTES + BMS and APTES + OFPOS and to characterize the modified surfaces by contact angle measurements and immunofluorescence measurements prior to and after immobilizing proteins. Additionally, we have evaluated the functionality of the immobilized antibodies by their abilities to bind EpCAM-positive human colon adenocarcinoma cell line (LoVo) and EpCAM-negative mouse embryonic fibroblast cell line (3T3). Cell enumeration was conducted on the basis of DAPI-positive signals and recorded using a confocal laser scanning biological microscope. The results of our study showed that the immobilization capability and reactivity of APTES, APTES + BMS and APTES + OFPOS differ. The modification of APTES with unreactive silanes (BMS,OFPOS) is recommended to improve the antibody binding efficiency. However, using OFPOS resulted in more effective antibody and cell binding, and it appears to be the most useful compound in specific antibody-mediated cell recognition.

Cytokeratin 8 in venous grafts: a factor of unfavorable long-term prognosis in coronary artery bypass grafting patients.

BACKGROUND: Smooth muscle cells, present in the saphenous vein (SV) tunica media, may contribute to late occlusion of venous aortocoronary grafts. The aim of present study was to evaluate expression of selected cytoskeletal proteins in tunica media of SV grafts obtained from patients undergoing coronary artery bypass grafting (CABG) and correlate procured results to late venous graft failure observed in these patients. METHODS: The study involved 218 patients (mean age of 62.5 ± 8.7 years) who underwent primary isolated CABG with the use of at least one venous aortocoronary bypass graft. Expressions of alpha-smooth muscle actin, smooth muscle-myosin heavy chain, calponin and cytokeratin 8 in SV wall were estimated by means of immunohistochemistry. The primary clinical endpoint was defined as the presence of any coronary artery disease (CAD) progression symptom while angiographic one as significant stenosis in the venous graft. RESULTS: Thirty-eight (18.1%) patients have reached the primary clinical endpoint. Freedom from clinical CAD deterioration was 0.95 ± 0.02, 0.87 ± 0.03 and 0.83 ± 0.04, for 12-, 24-,36-month follow-up, respectively. Forty-four study participants have reached the angiographic endpoint. Multivariate logistic regression analysis revealed an increased expression of cytokeratin 8 accompanied by calponin under expression in SV tunica media were independent risk factors for venous graft failure. CONCLUSIONS: An increased expression of cytokeratin 8 and weak of calponin in tunica media of SV grafts might be useful markers of unfavorable long-term prognosis in CABG patients. In the future, assessment of their expression may enable to select the most appropriate candidates for SV grafts.