Correlation of selected serum protein levels with the degree of disability and NEDA-3 status in multiple sclerosis phenotypes.

OBJECTIVE: Multiple sclerosis (MS) is a multifactorial disease that begins in 80-85% of patients as a relapsing-remitting form (RRMS), and about 50% of patients gradually develop a secondary progressive form (SPMS). Approximately 10-20% of patients are affected primarily by the progressive form (PPMS) of this disease, which is characterised by a progressive course. This work focuses on the detection of potential protein biomarkers (CHI3L1, sNfL, CXCL13, MCP-1, MMP-2, and MMP-9) in the serum of patients with multiple sclerosis, divided according to phenotype. PATIENTS AND METHODS: We detected serum (RRMS: n=40, SPMS: n=25, PPMS: n=15) concentrations of selected markers of demyelination and inflammation using ELISA and zymographic determination for accurate and reproducible recognition of individual forms of MS, as well as a comparison of their levels with a worsening of no evidence of disease activity (NEDA-3) status and patients' disability. RESULTS: We detected that concentrations of sNfL in the blood of patients with PMS were higher than in those with RRMS (about 46%, p<0.001). The association with a worsening of NEDA-3 status was confirmed in the RRMS group by positive correlation of sNfL and the expanded disability status scale (EDSS) score (r=0.579, p<0.01). The levels of MCP-1 protein were not significantly different in patients with the RRMS to the progressive form of MS (r=0.58, p=0.02), while the levels of CHI3L1 in both the RRMS and PMS groups were significantly increased in groups with evidence of disease activity (RRMS about 76%, p<0.001 and PMS about 62%, p<0.001). CONCLUSIONS: Earlier and non-invasive detection of serum biomarkers and their correlations with neurological disability can help to recognise the transition from RRMS to progressive forms of MS and complement the results of clinical and radiological follow-up of the patient and potentially help in monitoring the patient's response to the treatment.

Expression of selected inflammatory proteins and metalloproteinases in periodontitis.

OBJECTIVE: Periodontitis is a chronic inflammatory disease caused by microbial dental plaque which leads to the destruction and loss of supporting tissues of the tooth. Microbial plaque alone, however, is not enough to cause the disease. The body's response plays an important role, in which an imbalance between the pro-inflammatory and anti-inflammatory effects of cytokines leads to an inflammatory reaction. PATIENTS AND METHODS: We detected changes in mRNA expression and protein levels of MIP-1α, and metalloproteinases (MMP-2, MMP-9) contributing to cascades in the initiation and progression of inflammatory bone resorption and destruction of periodontal soft tissues in patients with aggressive (AP) or chronic (CP) forms of periodontitis in comparison with healthy individuals (control). RESULTS: MIP-1α mRNA levels were highest in AP (280 ± 23% higher than the control) also in comparison with CP. The difference in protein level was less pronounced. MMP-2 mRNA expression values were similar (300 ± 12% higher in comparison with control), but protein levels were lower, also when compared to CP. Only in CP MMP-9 mRNA levels were significantly higher than the control (30 ± 8%), while protein levels were again higher in AP. Both AP and CP showed a positive correlation between the level of MIP-1a and MMP-2 (0.879, and 0.954 respectively). However, a strong positive correlation was only found between the levels of MMP-2 and MMP-9 in CP (0.812). CONCLUSIONS: MIP-1α, MMP-2 and MMP-9 mRNA expression, along with the concentration of proteins in saliva in patients with periodontal disease, is higher than in healthy individuals and correlates with the severity of the disease.

Hypoxia factors suppression effect on the energy metabolism of a malignant melanoma cell SK-MEL-30.

OBJECTIVE: Malignant melanoma (MM), as well as other cancers, is a disorder in the cell life cycle at many levels. In terms of energy, the sync of cytosolic and mitochondrial metabolism is required for each cell. Mismatches also caused by hypoxic factors accumulate defects leading to the formation, development and invasiveness of malignant melanoma. Our aim was to compare the effect of HIF-1α and miR-210 on the metabolism of malignant melanoma cells in normoxia and pseudohypoxia. Further, we also investigated how gene silencing affects the viability in order to evaluate the potential of gene therapy in the treatment of MM. MATERIALS AND METHODS: We targeted oxidative phosphorylation by genetically suppressing HIF-1α and miR-210. We have examined mitochondrial activity, cytosolic glycolysis and cell viability. RESULTS: The ratio of NADH/NAD+ in the cytoplasm under normal conditions is stable and can thus serve as a specific cellular metabolic marker. Therefore, the study was aimed at finding the cause of the reduction in NADH levels in increasing hypoxia under ideal in vitro conditions on the SK-MEL-30 malignant melanoma cells. The relationship between HIF-1α and miR-210, their effect on transcriptional level, and the subsequent effect on metabolic process attenuation in cells was investigated. Obtained results indicate that the NADH which is accumulated by cells in hypoxia was significantly decreased upon gene silencing. CONCLUSIONS: Our studies have shown that small regulatory molecules with organelle-specific effect (such as miRs) need to be targeted, and that the resultant effect is comparable to silencing of "general" hypoxic transcription factors.

Molecular recognition of aortic valve stenosis and regurgitation.

OBJECTIVE: Aortic valve stenosis (AS) presents a disease during which there are changes of the aortic valve structure that modify the blood structure of patients. The aim of this study was to improve the effectiveness of differential diagnostics of aortic stenosis and aortic regurgitation using molecular techniques on both mRNA (RT-PCR) and protein (biochip protein). PATIENTS AND METHODS: An experimental group (n = 58) consisting of patients with aortic valve stenosis (n = 26) and aortic regurgitation (AR, n = 32) was compared with a control group (n = 35). Both blood serum and valve tissue samples were used for the determination of gene expression specific genes related to inflammatory response (CRP, IL6, IL2R, IL6R, TNFR1, and 2) as well as genes and proteins involved in remodeling of the extracellular matrix (MMP9, TIMP, Emilin-1). RESULTS: We found that hsCRP and IL6 plasma levels of patients with AS were higher than both controls and patients with AR (mean 5.6 ng/ml). The differences between AS and AR were detected only in mRNA levels of MMP9 and TIMP where increased levels characteristic for AS were found (about 74%, p < 0.01 and 87%, p < 0.001 higher than AR). CONCLUSIONS: The achieved results could contribute to the improvement of early diagnosis of selected cardiovascular disease in the future and improve the quality of patient's life.

Evaluation of molecular changes of distal organs after small bowel transplantation.

The ischemia and reperfusion of a jejunal graft during transplantation triggers the stress of endoplasmic reticulum thus inducing the synthesis of pro-inflammatory cytokines. Spreading of these signals stimulate immunological reactions in distal tissues, i.e. lung, liver and spleen. The aim of this study was to detect the molecular changes in liver and spleen induced by transplanted jejunal graft with one or six hours of reperfusion (group Tx1 and Tx6). Analysis of gene expression changes of inflammatory mediators (TNF-alpha, IL-10) and specific chaperones (Gadd153, Grp78) derived from endoplasmic reticulum (ER) was done and compared to control group. The qRT-PCR method was used for amplification of the specific genes. The levels of corresponding proteins were detected by Western blot with immunodetection. Protein TNF-alpha was in liver tissue significantly overexpressed in the experimental group Tx1 by 48 % (p<0.001). In the group Tx6 we found decreased levels of the same protein to the level of controls. However, the protein concentrations of TNF-alpha in spleen showed increased levels in group Tx1 by 31 % (p<0.001) but even higher levels in the group Tx6 by 115 % (p<0.001) in comparing to controls. Our data demonstrated that the spleen is more sensitive to post-transplantation inflammation than liver, with consequent stress of ER potentially inducing apoptosis and failure of basic functions of lymphoid tissue.

Analysis of transcriptional activities of angiogenic biomarkers during intrauterine complications leading to preterm birth.

OBJECTIVE: Pre-eclampsia, growth retardation and preterm delivery are the most common reasons leading to increased maternal and perinatal mortality. The increased expression of hypoxia induced factors, such as HIF-1, triggers the overexpression of anti-angiogenic genes. The aim of this study was to determine the transcriptional activity of individual pro- and anti-angiogenic markers (VEGF, HIF-1, sEng, Flt-1, PlGF-1) in maternal blood samples from patients with spontaneous preterm labor, preterm labor in combination with pre-eclampsia and fetal growth restriction in comparison with physiologically terminated pregnancies. PATIENTS AND METHODS: The transcriptional activity of specific genes was detected from the blood of patients using the chromatin immunoprecipitation capture method coupled with quantitative real-time PCR. RESULTS: The maximum differences in mRNA levels of PlGF-1 and VEGF-A were detected in two groups: the group of normal-term birth with complications and the group of preterm labor with complications (both significantly lower than the control, p < 0.001). In contrast, a marked increase of mRNA levels was found in the same groups of patients for the HIF-1, endoglin and Flt-1 genes (p < 0.001). CONCLUSIONS: According to our results, we can conclude that increased oxidative stress, increasing the expression levels of anti-angiogenic genes and reduction of the transcriptional activity of pro-angiogenic genes can provide additional information during diagnostics of pathological complications of labor.

Current knowledge on the active form of Vitamin D synthesized in the skin and its effects on malignant melanoma.

The link between sunlight and skin cancer is a frequently discussed topic. However, ultraviolet radiation also induces the production of Vitamin D in the body. Keratinocytes and their ability to synthesize the active form of Vitamin D, which is consumed at the place of its origin in the skin, have a unique place in this discussion. We observe a remarkable sunshine-related paradox when we monitor the relationship between the dose of solar radiation and one type of skin cancer - malignant melanoma. Recent knowledge of the non-calcemic effects of Vitamin D, which include growth regulation, DNA repair, differentiation, apoptosis, membrane transport, metabolism, cell adhesion and oxidative stress, could help to further clarify this relationship. In this context, adjuvant Vitamin D therapy is currently being considered in patients with malignant melanoma, and this is expected to reduce tumor invasiveness and micrometastases and thus improve patient prognosis and reduce the risk of relapse.

[The Importance of MITF Signaling Pathway in the Regulation of Proliferation and Invasiveness of Malignant Melanoma]. / Význam signálnej dráhy MITF pri regulácii proliferácie a invazivity malígneho melanómu.

BACKGROUND: Malignant melanoma is one of the most aggressive types of cancers. Melanoma is derived from pigment-producing cells, melanocytes, which are characterized by a specific survival mechanism. Microphthalmia-associated transcription factor (MITF-M) plays a role in the metabolism of melanoma and is involved in the regulation of the expression of multiple genes mediating processes such as melanogenesis, proliferation, differentiation, and melanocyte survival. The expression of this transcription factor in melanocytes is activated by several signaling pathways, and reduced expression or function of MITF-M can cause the dysregulation of anti-apoptotic mechanisms. MITF-M is also involved in matrix metalloproteinase 14 (MMP14) activity, which is responsible for shape changes in melanocytes and increases in their motility and invasiveness. Very low levels of expression of MITF-M are found in human melanocytes with an invasive phenotype, indicating that this transcription factor acts as a suppressor of the metastatic process. Cancer cells with low expression of cytosolic/nuclear ß-catenin have a small amount of MITF-M 14 that is insufficient to inhibit MMP transcription. The enzyme catalyzes the degradation of laminin and fibronectin, thereby changing the shape of melanocytes, which leads to their increased mobility and invasiveness. AIMS: This review describes the regulatory pathway of MITF-M activation, its involvement in the proliferation of transformed melanocytes, and its role in increasing the invasiveness of malignant melanoma. A detailed understanding of the MITF-M signaling pathway is highly topical and could help to develop new diagnostic and therapeutic applications for patients with malignant melanoma.Key words: neoplastic cell transformation - melanoma - MITF transcription factorThis work was supported by grant projects VEGA 1/0115/14 and VEGA 1/0873/16.The authors declare they have no potential confl icts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 4. 12. 2015Accepted: 14. 6. 2016.

Incidental tubal endometrioid adenocarcinoma - case report.

The fallopian tubes represent a highly important structure for ovarian carcinogenesis. They provide the passage of eutopic endometrium from the uterus, as well as metastatic cells from the ovaries. A direct source of tumor cells for ovarian cancer was also recently confirmed in the fallopian tubes. The authors present a case report of an incidental tubal endometrioid carcinoma with the coexistence of adenomyosis.

[Effect of vitamin d receptor polymorphisms on the development and progression of malignant melanoma]. / Prehlad vplyvu polymorfizmov receptora vitamínu D na vznik a progresiu malígneho melanómu.

BACKGROUND: Malignant melanoma is one of the most aggressive cutaneous tumors in men and women. The risk of developing a malignant melanoma depends on several external factors along with deregulation of mutual interaction of genotype and phenotype. Nowadays, growing attention is focused on the study of the interactions of the active form of vitamin D3 with its receptor and inhibitory effect of vitamin D3 receptor polymorphisms on multiple signaling pathways involved in proliferative and metastatic processes. OBJECTIVES: This review article addresses the relationship between factors involved in the development of malignant melanoma through Hedgehog signaling pathway (HH). It summarizes current knowledge of malignant melanoma in regard to the role of the active form of vitamin D3 binding to vitamin D3 receptor (VDR), as well as it describes the influence of polymorphisms of VDR on the inhibition of HH. Understanding of these mechanisms and critical assessment of available data is beneficial to both primary and secondary prevention of malignant melanoma particularly by means of chemo -preventive substances.

Borderline ovarian tumor - a case report with genetic testing.

Despite an accurate classification, the borderline category of ovarian tumors is one of the most controversial topics in gynecologic oncology and is confusing to both clinicians and patients. The treatment is often confronted with the necessity of a fertility-sparing approach, although under-treatment increases the risk of disease recurrence. The clear definition of a group of patients with low or high risk of relapse requiring more or less extensive treatment is lacking. Currently, the main criteria affecting the treatment extent include histopathological features, particularly the presence of microinvasion, and invasive implants. Expansion of knowledge about genetic nature of the tumor characteristics may more closely specify the scope of therapeutic approach for individual patients. The authors report a case report of serous borderline ovarian tumor patient with tumor cell dispersion into the gastrointestinal tract. The genes of tumor vascular markers GPM6B and DR6 were also studied and compared to a group of healthy patients.

[New and clinically used oncomarkers of bladder cancer]. / Nové a klinicky vyuzívané onkomarkery karcinómu mocového mechúra.

Bladder cancer is the fourth most common cancer in men and the eighth most common cancer in women. Oncomarkers play a crucial role in early detection of bladder cancer, as well as in treatment response monitoring and prognosis. Search for a new marker by molecular analysis is in progress because any diagnostic sensitivity and specificity enhancement is a great benefit for clinical practice.

Progression of apoptic signaling from mesenteric ischemia-reperfusion injury to lungs: correlation in the level of ER chaperones expression.

Multiple organ dysfunction syndrome (MODS) is characterized by the development of probably reversible, progressive dysfunction of vital systems in two or more organs, directly undamaged by surgery or other trauma. The organs which have the most common potential dysfunction are lungs, liver, kidneys, heart and gastrointestinal tract. The small intestine is the source of production of proinflammatory mediators leading and contributing to multiorgan failure. The endoplasmic reticulum (ER), after ischemia and post-ischemic reperfusion, is significantly involved in the activation of enterocyte apoptosis. The purpose of this study was to determine the stage of apoptosis in the lungs, initiated through inflammatory response from the small intestine. We analyzed changes in mRNA levels of pro-apoptotic genes Gadd153 (Chop) and anti-apoptotic genes Grp78 (Bip) in the small intestine wall and lung parenchyma. During experimental procedure the rats underwent 60 min of ischemia, caused by complete occlusion of the mesenteric arteria cranialis, with subsequent reperfusion and evaluation after 1 h, 24 h and 30 days (from R1, R24 to R30, respectively, each group n = 8). The gene expression levels were measured using RT-PCR followed by electrophoresis and visualization under UV. In the lungs we detected significantly lower level of expression Grp78 by 45 ± 6.9%. This suggests that ischemic attack and subsequent reperfusion did not promote ER stress in the lungs through induction of Gadd153 expression in the small intestine. There is still no effective approach to the treatment of affected ischemic intestine tissue, to stop the processes with could eventually lead to MODS. Therefore it is necessary to study changes in the damaged tissue at the molecular level and try to suggest possible therapeutic defined routes to the protection of tissue.

[Overview of potential oncomarkers for detection of early stages of ovarian cancer]. / Prehl'ad potenciálnych onkomarkerov detekcie skorých fáz rakoviny vajecníkov.

The causes of ovarian cancer have not been fully elucidated yet but genetic predisposition is found in approximately 10% of patients. When the disease is detected at an early stage, up to 90% of patients have a hope of recovery. However, no preventive measures or precise screening tests to detect early stages of this disease are known yet. Standard tumor markers (CA125) are usually investigated in women with an increased risk. Nevertheless, due to low sensitivity and specificity during the first stage of the cancer, CA125 determination showed a very low efficacy (less than 26%). There has been a considerable progress over the recent years in understanding the molecular mechanisms leading to tumor formation and metastasis. Gradually, 46 genes were identified, initially named tumor endothelial markers (TEM), the expression of which is increased in tumors compared to normal endothelial cells. Death receptor 6 (DR6) and glycoprotein M6B (GPM6B), both detectable from patients serum, are among the most promising candidates for a marker of an early stage of ovarian cancer. This review aims to clearly describe potential as well as clinically used tumor markers useful in an early detection of ovarian cancer. Search for new markers, characterized by increased expression in patients'blood is a highly topical issue.

Analysis of changes in pro (Gadd153) and anti apoptotic (Grp78) gene expression after ischemic-reperfusion injury of the small intestine.

Analysis of changes after ischemia-reperfusion (IR) attack to the small intestine leads to multiple organ dysfunction (multiple organ dysfunction syndrome, MODS) and the subsequent death of patients is a topic for discussion. IR stress affects the endoplasmic reticulum (ER). ER dysfunction induces responses through kinases activation that stimulate anti-apoptotic mechanism, for example Grp78 (Bip) (Yeung et al., 2008) and pro-apoptotic mechanism, for example, activation Gadd153 (Chop) (Allyson et al., 2007). We analyzed the impact of IR damage of epithelium of the small intestine of rats after 1 h ischemia and subsequent 1 h, 24 h and 30 days of reperfusion on the level of apoptotic genes expression (Gadd153) and (Bip). In this study we used RT-PCR for detection of changes in gene expression. Significantly increased levels of mRNA for Gadd153 gene were detected after 1 h ischemia and 1 h reperfusion. The mRNA level of Grp78 gene was increased 24 h after ischemia comparing with the control groups. After 30 days of reperfusion Grp78 was at the level of control groups. Still, it is necessary to analyze the changes in the damaged tissue at the molecular level to define possible pathways leading to the tissue protection.