Search for new biologically active compounds: in vitro studies of antitumor and antimicrobial activity of dirhodium(II,II) paddlewheel complexes.

Four neutral Rh1-Rh4 complexes of the general formula [Rh2(CH3COO)4L2], where L is an N-alkylimidazole ligand, were synthesized and characterized using various spectroscopic techniques, and in the case of Rh4 the crystal structure was confirmed. Investigation of the interactions of these complexes with HSA by fluorescence spectroscopy revealed that the binding constants Kb are moderately strong (∼104 M-1), and site-marker competition experiments showed that the complexes bind to Heme site III (subdomain IB). Competitive binding studies for CT DNA using EB and HOE showed that the complexes bind to the minor groove, which was also confirmed by viscosity experiments. Molecular docking confirmed the experimental data for HSA and CT DNA. Antimicrobial tests showed that the Rh2-Rh4 complexes exerted a strong inhibitory effect on G+ bacteria B. cereus and G- bacteria V. parahaemolyticus as well as on the yeast C. tropicalis, which showed a higher sensitivity compared to fluconazole. The cytotoxic activity of Rh1-Rh4 complexes tested on three cancer cell lines (HeLa, HCT116 and MDA-MB-231) and on healthy MRC-5 cells showed that all investigated complexes elicited more efficient cytotoxicity on all tested tumor cells than on control cells. Investigation of the mechanism of action revealed that the Rh1-Rh4 complexes inhibit cell proliferation via different mechanisms of action, namely apoptosis (increase in expression of the pro-apoptotic Bax protein and caspase-3 protein in HeLa and HCT116 cells; changes in mitochondrial potential and mitochondrial damage; release of cytochrome c from the mitochondria; cell cycle arrest in G2/M phase in both HeLa and HCT116 cells together with a decrease in the expression of cyclin A and cyclin B) and autophagy (reduction in the expression of the protein p62 in HeLa and HCT116 cells).

Characterization of Oil-in-Water Emulsions Prepared with Triblock Copolymer Poloxamer 407 and Low-Molecular-Mass Surfactant Mixtures as Carriers of Grape Pomace Waste Polyphenols.

BACKGROUND: Natural antioxidants, such as grape pomace polyphenols, can be extracted by a surfactant-based green technology and incorporated into various emulsions. Therefore, this work aimed to investigate the physical stability and rheological characteristics of oil-in-water emulsions stabilized with poloxamer 407 (P407) and its mixtures with the low-molecular-mass surfactants Brij S20 (BS20) and Tween 60 (T60). Also, the influence of polyphenolic grape pomace extracts on the physical stability and rheological characteristics of the emulsions was examined. METHODS: Grape pomace polyphenols were extracted by aqueous solutions of P407 and BS20/P407 and T60/P407 mixtures. Two different types of oil-in-water emulsions were examined: emulsions prepared with pure surfactants and emulsions prepared with surfactant-based polyphenol extracts of grape pomace. Both types contained 20% sunflower oil. Characterization of the emulsions comprised droplet size evaluation, rheology characteristics and creaming stability. RESULTS: All the emulsions showed shear-thinning flow, while the rheological characteristics and creaming instability depended on the proportion of P407 in the emulsifier mixtures. Incorporation of grape pomace extracts had no effect on the investigated properties of the emulsions. CONCLUSION: The presence of extracted polyphenols in emulsifier mixtures had no significant effects on the emulsions' physico-chemical characteristics and stability. Therefore, the investigated emulsions can be considered suitable carriers for polyphenol-rich extracts.

Physicochemical properties and stability of oil-in-water emulsions stabilized by soy protein isolate and xanthan gum.

The aim of this work was to determine rheological and disperse characteristics and stability of oil-in-water emulsions stabilized by soy protein isolate (SPI) and xanthan gum (XG), as natural components. The effects of their combination on emulsion stabilization have not been investigated yet. The existence of interactions between the two macromolecules were indicated by the influence of XG on SPI surface hydrophobicity and surface tension values. Increase in SPI concentration from 1 to 3 % shift of distribution curves towards smaller particle size, while the opposite effects of further increase of SPI was obtained. The emulsions stabilized by SPI showed shear-thinning flow behavior, which changed to thixotropic at 5 % of SPI concentration. The presence of XG in emulsions at low concentrations did not affect the size distribution of the droplets, while at 0.1 % of XG Sauter mean diameter value raised and distribution curves were shifted towards a higher particle size. The presence of XG at higher concentration resulted in thixotropic flow behavior of emulsions. Also, increase in XG concentration led to the increase in consistency index and extent of non-Newtonian behavior of emulsions and enhanced the influence of the elastic modulus and creaming stability of the systems.

Assessment of Soy Protein Acid Hydrolysate-Xanthan Gum Mixtures on the Stability, Disperse and Rheological Properties of Oil-in-Water Emulsions.

There is a growing need for natural ingredients that could be utilized for the production of food, pharmaceutical, and cosmetic emulsions. Soy protein acid hydrolysate (SPAH) is a plant-based additive used in the food industry mainly as a flavor enhancer. For the purpose of this work, however, it was mixed with a well-known natural polysaccharide, xanthan gum (XG), to produce stable 30% (w/w) sunflower oil-in-water emulsions using a rotor-stator homogenizer. In order to assess the emulsifying properties of the SPAH and its mixtures with XG, the surface tension properties of their water solutions, particle size, creaming stability, and rheological properties of the emulsions were investigated. Since the emulsions prepared using only SPAH, in various concentrations, were not stable, systems containing 5% of SPAH and 0.1, 0.2, 0.3, 0.4, or 0.5% of XG were then studied. The increase in concentration of the macromolecule led to an increase in creaming stability. The emulsions with 5% SPAH and 0.5% XG were stable for at least 14 days. The increase in XG concentration led to a decrease in d4,3, while consistency index and non-Newtonian behavior increased. The systems containing SPAH, in the absence of XG, showed shear-thinning flow behavior, which was changed to thixotropic with the addition of XG. Viscoelastic properties of emulsions containing over 0.2% of XG were confirmed by oscillatory rheological tests, demonstrating the dominance of elastic (G') over viscous (G") modulus.

Myeloid p38 activation maintains macrophage-liver crosstalk and BAT thermogenesis through IL-12-FGF21 axis.

Obesity features excessive fat accumulation in several body tissues and induces a state of chronic low-grade inflammation that contributes to the development of diabetes, steatosis, and insulin resistance. Recent research has shown that this chronic inflammation is crucially dependent on p38 pathway activity in macrophages, suggesting p38 inhibition as a possible treatment for obesity comorbidities. Nevertheless, we report here that lack of p38 activation in myeloid cells worsens high-fat diet-induced obesity, diabetes, and steatosis. Deficient p38 activation increases macrophage IL-12 production, leading to inhibition of hepatic FGF21 and reduction of thermogenesis in the brown fat. The implication of FGF21 in the phenotype was confirmed by its specific deletion in hepatocytes. We also found that IL-12 correlates with liver damage in human biopsies, indicating the translational potential of our results. Our findings suggest that myeloid p38 has a dual role in inflammation and that drugs targeting IL-12 might improve the homeostatic regulation of energy balance in response to metabolic stress.

Cocoa Spread with Grape Seed Oil and Encapsulated Grape Seed Extract: Impact on Physical Properties, Sensory Characteristics and Polyphenol Content.

The aim of this study was to utilize grape pomace, as a polyphenol-rich by-product of wine production, in the manufacture of enriched cocoa spread. The formulation of the cocoa spread has been modified by substitution of refined sunflower oil with cold-pressed grape seed oil. The spread with grape seed oil (Cg) was further enriched with grape seed extract encapsulated on maltodextrins (E), where 10% and 15% of E was added to Cg obtaining the samples Cg10 and Cg15. The results showed an increase in volume-weighted mean in spread samples, from 19.17 µm in Cg to 19.71 µm in Cg10 and 21.04 µm in Cg15. Casson yield stress and Casson viscosity significantly (p ˂ 0.05) increased from 16.41 Pa and 1.58 Pa·s in Cg to 29.45 Pa and 5.70 Pa·s in Cg15 due to the reduction of the fat-phase content in enriched spreads. The addition of E had no significant effect on the melting temperature (Tpeak) of the enriched spreads, while increasing the amount of E significantly (p ˂ 0.05) increased their hardness. The incorporation of grape seed oil in the cocoa spread formulation contributed to an increase in total polyphenols and flavonoids. Moreover, the addition of 10% and 15% of E to Cg resulted in approximately 1.5× and 2× higher content of phenolic compounds in Cg10 and Cg15 compared to control spread with sunflower oil (Cs). Flavonoids increased from 0.43 mg CE/g in Cs to 0.74 mg CE/g in Cg 10 and 1.24 mg CE/g in Cg15. Encapsulates positively affected sensory characteristics of enriched spread samples by reducing their grape seed oil aroma and sweetness.

MKK6 deficiency promotes cardiac dysfunction through MKK3-p38γ/δ-mTOR hyperactivation.

Stress-activated p38 kinases control a plethora of functions, and their dysregulation has been linked to the development of steatosis, obesity, immune disorders, and cancer. Therefore, they have been identified as potential targets for novel therapeutic strategies. There are four p38 family members (p38α, p38ß, p38γ, and p38δ) that are activated by MKK3 and MKK6. Here, we demonstrate that lack of MKK6 reduces the lifespan in mice. Longitudinal study of cardiac function in MKK6 KO mice showed that young mice develop cardiac hypertrophy which progresses to cardiac dilatation and fibrosis with age. Mechanistically, lack of MKK6 blunts p38α activation while causing MKK3-p38γ/δ hyperphosphorylation and increased mammalian target of rapamycin (mTOR) signaling, resulting in cardiac hypertrophy. Cardiac hypertrophy in MKK6 KO mice is reverted by knocking out either p38γ or p38δ or by inhibiting the mTOR pathway with rapamycin. In conclusion, we have identified a key role for the MKK3/6-p38γ/δ pathway in the development of cardiac hypertrophy, which has important implications for the clinical use of p38α inhibitors in the long-term treatment since they might result in cardiotoxicity.

The human heart can increase its size to supply more blood to the body's organs. This process, called hypertrophy, can happen during exercise or be caused by medical conditions, such as high blood pressure or inherited genetic diseases. If hypertrophy is continually driven by illness, this can cause the heart to fail and no longer be able to properly pump blood around the body. For hypertrophy to happen, several molecular changes occur in the cells responsible for contracting the heart, including activation of the p38 pathway. Within this pathway is a p38 enzyme as well as a series of other proteins which are sequentially turned on in response to stress, such as inflammatory molecules or mechanical forces that alter the cell's shape. There are different types of p38 enzyme which have been linked to other diseases, making them a promising target for drug development. However, clinical trials blocking individual members of the p38 family have had disappointing results. An alternative approach is to target other proteins involved in the p38 pathway, such as MKK6, but it is not known what effect this might have. To investigate, Romero-Becerra et al. genetically modified mice to not have any MKK6 protein. As a result, these mice had a shorter lifespan, with hypertrophy developing at a young age that led to heart problems. Romero-Becerra et al. used different mice models to understand why this happened, showing that a lack of MKK6 reduces the activity of a specific member of the p38 family called p38α. However, this blockage boosted a different branch of the pathway which involved two other p38 proteins, p38γ and p38δ. This, in turn, triggered another key pathway called mTOR which also promotes hypertrophy of the heart. These results suggest that drugs blocking MKK6 and p38α could lead to side effects that cause further harm to the heart. A more promising approach for treating hypertrophic heart conditions could be to inhibit p38γ and/or p38δ. However, before this can be fully explored, further work is needed to generate compounds that specifically target these proteins.

Newly synthesized palladium(II) complexes with aminothiazole derivatives: in vitro study of antimicrobial activity and antitumor activity on the human prostate cancer cell line.

Five new complexes of the palladium(II) ion (C1-C5) having the general formula [(PdL2)]Cl2 with some 2-aminothiazoles (L1-L5), where L1 = 2-amino-4-(3,4-difluorophenyl)thiazole, L2 = 2-amino-5-methyl-4-phenylthiazole, L3 = 2-amino-4-phenylthiazole, L4 = 2-amino-4-(4-chlorophenyl)thiazole, and L5 = 2-amino-4-(2,4-difluorophenyl)thiazole, have been synthesized and characterized by elemental microanalysis and infrared, 1H NMR and 13C NMR spectroscopy. The in vitro antimicrobial activity of the five ligands and the corresponding Pd(II) complexes is investigated. Testing is performed by the microdilution method and the minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) have been determined. Testing is conducted against 11 microorganisms (nine strains of pathogenic bacteria and two yeast species). The tested ligands and palladium(II) complexes show selective, high and moderate activity. There is a difference in antimicrobial activity between the ligands and the corresponding palladium(II) complexes. The complexes have significant anti-staphylococcal activity and activity on Pseudomonas aeruginosa which is better than the positive control. The interactions of newly synthesized palladium(II) complexes with calf thymus DNA (CT-DNA) were investigated using UV-Vis absorption and fluorescence spectroscopy. Analysis of UV-absorption and fluorescence spectra indicates the formation of a complex between the palladium(II) complexes and DNA. The high values of intrinsic binding constants, Kb, of the order 104 M-1 and Stern-Volmer quenching constants, KSV, of the order 105 M-1 indicated very good binding of all complexes to CT-DNA. Also, the new Pd(II) complexes show high cytotoxic activity towards the human prostate cancer cell line and insignificant activity towards non-cancerous human fibroblasts. Future research could additionally explore the biological activity of Pd(II) complexes presented in this paper and investigate the possibility of their implementation in clinical practice.

The role of stress kinases in metabolic disease.

Obesity is a health condition that has reached pandemic levels and is implicated in the development and progression of type 2 diabetes mellitus, cancer and heart failure. A key characteristic of obesity is the activation of stress-activated protein kinases (SAPKs), such as the p38 and JNK stress kinases, in several organs, including adipose tissue, liver, skeletal muscle, immune organs and the central nervous system. The correct timing, intensity and duration of SAPK activation contributes to cellular metabolic adaptation. By contrast, uncontrolled SAPK activation has been proposed to contribute to the complications of obesity. The stress kinase signalling pathways have therefore been identified as potential targets for the development of novel therapeutic approaches for metabolic syndrome. The past few decades have seen intense research efforts to determine how these kinases are regulated in a cell-specific manner and to define their contribution to the development of obesity and insulin resistance. Several studies have uncovered new and unexpected functions of the non-classical members of both pathways. Here, we provide an overview of the role of SAPKs in metabolic control and highlight important discoveries in the field.

UV irradiation induces apoptosis in the human endometrial stromal cell line (ThESC).

PURPOSE: The treatment options of endometrial hyperplasia consist of surgical, interventional and medical therapies including apoptosis-inducing agents. The purpose of the study was to evaluate the effects of ultraviolet (UV) radiation on the viability and the type of cell death on the human endometrial stromal cells (ThESC) line. METHODS: We investigated the effect of UV exposure on human endometrial stromal cell line (ThESC) on cell viability using MTT assay as well as changes in cell morphology using phase microscopy and acridine orange (AO)/ethidium bromide (EB) cell staining. RESULTS: UV treatment significantly decreased the percentage of the viable ThESC cells compared to the viability of untreated control cells using MTT assay (p<0.05). In addition, UV treatment of ThESC cells for 60 and 90 min induced high level of cell morphology disruption, followed with loss of both the cell shape and the presence of defragmented debris and stained with intense red color. CONCLUSIONS: The obtained results suggest the potential role of UV light application as additional treatment option of benign endometrium hyperplasia alone or in combination with other treatment modalities.

Enhanced cytotoxicity and apoptosis by raloxifene in combination with estrogen and methotrexate in human endometrial stromal cells.

Endometrial hyperplasia is a condition that may lead to the development of endometrial carcinoma. Initially, changes of the endometrium are caused by the estrogen's hyperstimulation that may lead to the development of an irregular bleeding and the infertility problems. Therapy of endometrial hyperplasia is limited to medical and surgical approaches. During the past decade, the new types of drugs were developed for the treatment of the endometrial hyperplasia. Here, for the first time, we investigated the cytotoxic effects of the various combinations of estrogen, raloxifene, and methotrexate in human ThESC cell line as a possible potential treatment of the endometrial hyperplasia. Our aim was to investigate and to determine the most efficient combination of investigated drugs in ThESC cells during 24-hr period using MTT assay, FACS analysis, and immunofluorescence staining. Our results demonstrated that the combination of raloxifene with methotrexate efficiently induced both the cytotoxicity and apoptosis in ThESC cells when compared to their single effect, as well as to the effect of combined treatment of raloxifene with estrogen. The application of the low doses of methotrexate combined with raloxifene offers all advantages of a potential beneficial antitumor match in cancer chemoprevention and therapy.

Epigenome alterations in aortic valve stenosis and its related left ventricular hypertrophy.

Aortic valve stenosis is the most common cardiac valve disease, and with current trends in the population demographics, its prevalence is likely to rise, thus posing a major health and economic burden facing the worldwide societies. Over the past decade, it has become more than clear that our traditional genetic views do not sufficiently explain the well-known link between AS, proatherogenic risk factors, flow-induced mechanical forces, and disease-prone environmental influences. Recent breakthroughs in the field of epigenetics offer us a new perspective on gene regulation, which has broadened our perspective on etiology of aortic stenosis and other aortic valve diseases. Since all known epigenetic marks are potentially reversible this perspective is especially exciting given the potential for development of successful and non-invasive therapeutic intervention and reprogramming of cells at the epigenetic level even in the early stages of disease progression. This review will examine the known relationships between four major epigenetic mechanisms: DNA methylation, posttranslational histone modification, ATP-dependent chromatin remodeling, and non-coding regulatory RNAs, and initiation and progression of AS. Numerous profiling and functional studies indicate that they could contribute to endothelial dysfunctions, disease-prone activation of monocyte-macrophage and circulatory osteoprogenitor cells and activation and osteogenic transdifferentiation of aortic valve interstitial cells, thus leading to valvular inflammation, fibrosis, and calcification, and to pressure overload-induced maladaptive myocardial remodeling and left ventricular hypertrophy. This is especcialy the case for small non-coding microRNAs but was also, although in a smaller scale, convincingly demonstrated for other members of cellular epigenome landscape. Equally important, and clinically most relevant, the reported data indicate that epigenetic marks, particularly certain microRNA signatures, could represent useful non-invasive biomarkers that reflect the disease progression and patients prognosis for recovery after the valve replacement surgery.

Cytokine profile in chronic hepatitis C: An observation.

The data addressing cytokine profile in chronically infected HCV patients are conflicting, ranging from Th1 or Th2 cytokine prevalence to the expression of both types of cytokines. Therefore, the aim of this study was to evaluate cytokine profile in these patients. Cytokine sera levels in HCV patients and healthy controls were evaluated using 13plex FlowCytomix Multiplex. Median values of both proinflammatory and anti-inflammatory cytokines were lower in HCV patients then in controls. In addition, the number of subjects producing detectable quantities of cytokines was significantly lower in the group of HCV patients. Yet, cytokine levels in those patients were remarkably heterogeneous ranging from low to extremely high, much higher than the maximal values in control group. Similarly, grouping data according to HCV genotype, HCV RNA load, ALT/AST ratio and the stage of fibrosis showed marked standard deviations, reflecting high intragroup diversity. No correlation was found between each disease-related factor and cytokine levels. Patients investigated in our and similar studies were disparate pursuant to characteristics of the hosts, pathogen and course of the disease. Therefore, the inconsistency of the literature data regarding cytokine pattern in chronic HCV patients may be a consequence of the disregarded/overlooked heterogeneity of these patients.

Induction of mitochondrial apoptotic pathway by raloxifene and estrogen in human endometrial stromal ThESC cell line.

INTRODUCTION: Endometrial hyperplasia is a condition that occurs as a result of hormonal imbalance between estrogen and progesterone. Morphological disturbance of endometrial cells occurs consequently leading towards endometrial cancer. In therapy of endometrial hyperplasia SERMs are used to supress effects of locally high estrogen level in uterus. There is strong evidence suggesting that estrogen could be involved in cell death - apoptosis. There are no experimental data demstrating the direct apoptotic effect of both raloxifene and estrogen on the ThESC cell line. The aim of our study wa sto investigate both cytotoxic and apototic mechanism of raloxifene and estrogen - induced death in the ThESC cell line. MATERIAL AND METHODS: In order to determine their cytotoxic and apoptotic effects, various doses of raloxifene and estrogen were applied to the ThESC cell line for 24 h. After the treatment MTT assay, FACS analysis and immunofluoroscence method were conducted. RESULTS: The results of this study for the first time demonstrated the cytotoxic and apoptotic effects of raloxifene and estrogen on human endometrial stromal cell line suggesting the involvement of the inner, mitochondrial apoptotic pathway. CONCLUSIONS: Our results demonstrated apoptotic effects of investigated drugs in the ThESC cell line through increasing the Bax/Bcl-2 ratio and activation of caspase 3.

Outcome of Juvenile Idiopathic Arthritis Associated Uveitis in Two Disease Subtypes.

OBJECTIVES: This study aims to evaluate the efficacy of adalimumab as a first line biologic agent in specific subtypes of juvenile idiopathic arthritis (JIA) patients with associated uveitis. PATIENTS AND METHODS: We retrospectively analyzed the data of 11 JIA patients (8 males, 3 females; mean age 14.5 years; range 9 to 18 years) with associated uveitis treated with biologic therapy. All patients were diagnosed as oligoarticular/extended oligoarticular or enthesitis-related JIA subtypes, treated with methotrexate, and had active or previous history of uveitis for which adalimumab was prescribed. We tested all patients for anti-nuclear antibody presence and human leukocyte antigen genotype. We assessed disease activity and therapy efficacy by American College of Rheumatology 50%, 70%, and 100% improvement criteria. We evaluated uveitis activity by slit-lamp biomicroscopy and recorded adverse events. RESULTS: Of the JIA patients, three (27.27%) had oligoarticular/extended oligoarticular JIA and eight (72.73%) had enthesitis-related arthritis. Anti-nuclear antibody positivity was present in 27.27% (all females) while human leukocyte antigen-B51 was determined in 62.5% and human leukocyte antigen-B27 in 12.5% of patients. Mean uveitis duration before adalimumab introduction was 12.3 months. After two years of follow-up, there were no relapses of uveitis and visual acuity was stable while on adalimumab and methotrexate treatment. All patient were gradually tapered and discontinued treatment with topical steroids. Disease activity improved and seven patients (63.64%) achieved American College of Rheumatology 100% response rate (attained remission), while four patients (36.36%) achieved American College of Rheumatology 70% response rate. CONCLUSION: Anti-nuclear antibody positivity with oligoarticular/extended oligoarticular and enthesitis-related arthritis JIA subtypes, which are known for their high risk to develop uveitis, may benefit from adalimumab as a first line anti-tumor necrosis factor agent.

Ethyl Acetate Extract of Origanum vulgare L. ssp. hirtum Prevents Streptozotocin-Induced Diabetes in C57BL/6 Mice.

Type 1 diabetes (T1D) is an autoimmune disease that develops as a consequence of pancreatic ß-cell death induced by proinflammatory mediators. Because Origanum vulgare L. ssp. hirtum (Greek oregano) contains antiinflammatory molecules, we hypothesized that it might be beneficial for the treatment of T1D. An ethyl acetate extract of oregano (EAO) was prepared from the leaves by a polar extraction method. Phytochemical composition was determined by liquid chromatography-UV diode array coupled to ion-trap mass spectrometry with electrospray ionization interface (LC/DAD/ESI-MS(n) ). In vitro immunomodulatory effect of EAO was estimated by measuring proliferation (MTT) or cytokine secretion (ELISA) from immune cells. Diabetes was induced by multiple low doses of streptozotocin (MLDS) in male C57BL/6 mice and EAO was administered intraperitoneally for 10 d. Determination of cellular composition (flow cytometry) and cytokine production (ELISA) was performed on 12th d after diabetes induction. EAO suppressed the function of both macrophages and lymphocytes in vitro. In vivo, EAO treatment significantly preserved pancreatic islets and reduced diabetes incidence in MLDS-challenged mice. Besides down-modulatory effect on macrophages, EAO reduced the number of total CD4(+) and activated CD4(+) CD25(+) T cells. Furthermore, EAO affected the number of T helper 1 (Th1) and T helper 17 (Th17) cells through downregulation of their key transcription factors T-bet and RORγT. Because EAO treatment protects mice from development of hyperglycemia by reducing proinflammatory macrophage/Th1/Th17 response, this plant extract could represent a basis for future diabetes therapy.

Effects of cardiovascular drugs on TSH serum levels in patients on replacement therapy after thyroidectomy.

OBJECTIVE: Hypothyroidism is one of most common endocrine disorders resulting from deficiency of thyroid hormones. The aim of our study was to investigate whether cardiovascular drugs as well as gender, age, body-mass index, and habits, like smoking or drinking coffee affect thyroid-stimulating hormone (TSH) level in hypothyroid patients with thyroxine replacement therapy who suffer from cardiovascular disease. MATERIALS: The study was conducted on 150 hypothyroid patients who underwent total thyroidectomy for benign reasons; they were divided into five treatment groups: levothyroxine only group and, according to the drugs they had in therapy alongside levothyroxine, the angiotensinconverting enzyme inhibitors group, the selective ß-blockers group, the calcium antagonists group, as well as the nitrates group. A retrospective cohort study was conducted in the Clinical Center Kragujevac, Serbia, during the period of January 2012 to October 2014. All patients' data were collected both from participants' health records and questionnaires that patients completed, including data about habits, like smoking or drinking coffee. RESULTS: TSH values were significantly higher in the group of patients with selective ß-blockers in therapy alongside levothyroxine, compared to all the other study groups. The values of TSH level did not significantly differ among the other therapy groups. On the other hand, cigarette smoking was a risk factor that decreased TSH levels in patients on thyroid replacement therapy. CONCLUSIONS: Our study shows that selective ß-1 blockers can increase, while cigarette smoking can decrease TSH serum levels in hypothyroid patients on thyroid-replacement therapy.

METHOTREXATE AND MYOTREXATE INDUCE APOPTOSIS IN HUMAN MYOMA FIBROBLASTS (T hES CELL LINE) VIA MITOCHONDRIAL PATHWAY.

Uterine leiomyomas (fibroids) are the most common benign tumors in women of reproductive age. Although the local application of low doses of methotrexate (MTX) is used as an effective treatment of the myomas, myotrexate could be a promising new drug. This study investigated the cytotoxic and apoptotic effects of both MTX and myotrexate in human fibroblasts derived from the uterine fibroids (T hES cell line). The myotrexate adduct is an aqueous solution of MTX and L-arginine. Cells were treated with a graded concentrations of both MTX and myothrexate (0.1-16 µM) for 24 h. The cytotoxicity was assayed by MTT test, apoptosis was evaluated by Annexin V-FITC assay and their possible role in apoptosis was determined by immnu- flourescence. Both MTX and myotrexate induced apoptosis in T hES cells in a dose dependent manner (p < 0.001). Myotrexate significantly increased the percentage of AnnexinV positive cells, BAX/Bcl-2 ratio and subsequent caspase-3 activation compared to the MTX treated cells (p < 0.05). Both MTX or myotrexate treatment showed a diffuse staining of cytochrome c indicating its release from mitochondria to the cytosol, suggesting that their mechanisms of action most likely involves the mitochondrial apoptotic pathway.