Zmiana ekspresji mRNA dla CTLA-4, CD28, VDR i CD45 w limfocytach T u osób z chorobą Hashimoto — badanie pilotowe

Introduction: CD28/T-cell receptor (TCR)/cytotoxic T-lymphocyte antigen 4 (CTLA4) complex controls T-cell tolerance and autoimmunity in Hashimoto’s thyroiditis (HT). In addition, CD45 protein tyrosine phosphatase (PTPase) and vitamin D receptor (VDR) cooperatively interact with the TCR complex to affect autoimmune processes central to the pathogenesis of HT. Nevertheless, their role in HT aetiology has been less well established. In this study, we aimed to explore mRNA expression levels of CTLA4, CD28, CD45, and VDR in T-cells, across different outcomes of HT. Material and methods: The study included 45 HT patients and 13 euthyroid, healthy controls. T-lymphocytes were isolated from peripheral blood mononuclear cells, total mRNA was extracted from T-cells, and gene expression was studied by reverse transcription-polymerase chain reaction (RT-PCR) and ImageQuant method relative to glyceraldehyde-3-phosphate dehydrogenase RT-PCR products. Results: Nominally higher expression levels of VDR, CTLA4, CD28, and CD45RAB mRNA were found in unsorted T-lymphocytes of healthy controls when compared to the HT patients. No difference was observed between hypothyroid/untreated, spontaneously euthyroid and LT4-treated HT patients. VDR mRNA expression was linked to both T3 levels and CTLA4 gene expression, whilst CD45RB mRNA expression coincided with CTLA4 and CD28 transcript levels. Conversely, older age and lower T3 levels were associated with increased abundance of CD45R0 isoform in HT patients. Conclusions: The results suggest a cross talk between endocrine and immune functions in HT pathology: an altered peripheral T cell mRNA profile with reduced VDR, CTLA4, CD28, and CD45RAB transcript levels is accompanied by age-related shift from naive to memory/late-differentiated T cell CD45R mRNA signature and associated with thyroid hormone status in the HT patients.Wstęp: Kompleks antygenu CD28/receptora limfocytów T (TCR)/antygenu 4 związan ego z limfocytem T cytotoksycznym (CTLA4) reguluje tolerancję limfocytów T oraz autoimmunogenność w chorobie Hashimoto (HT). Ponadto białkowa fosfataza tyrozynowa (PTPase) CD45 oraz receptor witaminy D (VDR) wchodzą w interakcję z kompleksem TCR, modyfikując procesy autoimmunologiczne mające podstawowe znaczenie w patogenezie HT. Jednak rola tych cząsteczek w etiologii HT nie została dokładnie ustalona. Celem autorów badania była ocean poziomów ekspresji mRNA dla CTLA4, CD28, CD45 i VDR w limfocytach T w zależności od różnego statusu HT. Materiał i metody: Do badania włączono 45 chorych na HT i 13 zdrowych osób z prawidłową czynnością tarczycy. Limfocyty T wyizolowano spośród komórek jędnojądrzastych krwi obwodowej, wyekstrahowano z nich całkowity mRNA i określono ekspresję genów za pomocą łańcuchowej reakcji polimerazowej z odwrotną transkryptazą (RT-PCR) i metody ImageQuant związanej produktami reakcji RT-PCR dehydrogenazy aldehydu 3-fosfoglicerynowego. Wyniki: U osób zdrowych stwierdzono nominalnie wyższy poziom ekspresji mRNA dla VDR, CTLA4, CD28 i CD45RAB w niesortowanych limfocytach T niż u chorych na HT. Nie zaobserwowano żadnych różnic między chorymi na HT z niedoczynnością tarczycy/nieleczonymi, u których samoistnie nastąpiło przywrócenie eutyreozy, i stosującymi leczenie LT4. Ekspresja VDR mRNA była powiązana zarówno ze stężeniem T3, jak i ekspresją genu CTLA4, natomiast ekspresja mRNA dla CD45RB wiązała się z poziomami transkryptu CTLA4 i CD28. Z kolei starszy wiek i niższe stężenia T3 wiązały się ze zwiększoną ilością izoformy CD45R0 u chorych na HT. Wnioski: Uzyskane wyniki sugerują interferencje między czynnością wewnątrzwydzielniczą i immunologiczną w patologii HT: zmiana profilu mRNA obwodowych limfocytów T z ograniczeniem poziomu transkryptu białek VDR, CTLA4, CD28 i CD45RAB współistnieje z zależnym od wieku przesunięciem sygnatury mRNA limfocytów CD45R od komórek naiwnych do komórek pamięci immunologicznej/ zróżnicowanych i jest powiązana ze stężeniami hormonów tarczycy u chorych na HT

Effect of Different Diet on the Lung Fatty Acid Modification

The effect of dietary fat on the composition of lung fatty acids was studied in Fischer rats fed two experimental semisynthetic, isocaloric diets containing different qualities of fat (low fat diet (LFD) and medium-chain triacylglycerols diet (MCTD)). The effect of MCTs preparation on the lung phospho- and neutral lipids is manifested as a change in the degree of saturation expressed as a ratio of saturation index (RSj) MCTD vs. CD. The saturation increased; in neutral lipids by 70%, in phosphatidylcholine fraction by 152.6%, and in other phospholipids by 39.5%. The results on the fatty acid composition of lipids obtained using LFD showed the effect of desaturation expressed as a ratio of desaturation index (RD;), in phosphatidylcholine fraction -1.95. The observed effect of desaturation was a consequence of the administered LFD, which contains a lower amount of essential fatty acids, which strongly affects the conversion of palmitic acid into palmitooleic acid. The LFD can be useful as a carbohydrate diet in a special clinical situation only if enriched with a certain amount of essential fatty acids, especially linoleic acid

Medical Laboratory Diagnostics

Medicinsko-laboratorijska dijagnostika sastavni je dio moderne medicine. Danas se više od 70% odluka u kliničkom liječenju oslanja na rezultate pretraga učinjenih od strane medicinsko-dijagnostičkih laboratorija. S obzirom na brz razvoj molekularne medicine, personalizirane medicine, kao i metoda i tehnologija koje se koriste u laboratorijskoj medicini, od ključne je važnosti kontinuirano stručno i znanstveno usavršavanje stručnjaka iz područja medicinsko-laboratorijske djelatnosti. To se postiže obrazovanjem na diplomskoj i poslijediplomskoj razini. Zapošljavanjem visokoobrazovanih stručnjaka, obrazovanih za rad u sustavu zdravstva i zdravstvene zaštite iz područja medicinsko-laboratorijske dijagnostike, unaprjeđuje se sustav analize bioloških uzoraka te značajno doprinosi procesu prevencije, otkrivanja i liječenja bolesti.Medical laboratory diagnostics is an integral segment of modern medicine. Today, more than 70% of decisions in clinical treatment rely on the results of tests performed by medical-diagnostic laboratories. Considering the rapid development of molecular medicine, personalized medicine, as well as the methods and technologies used in laboratory medicine, the continuous professional and scientific training of experts in the field of medical-laboratory activities is of crucial importance. This is achieved through education at the graduate and postgraduate level. By employing highly educated experts, trained to work in the health and health care system in the field of medical-laboratory diagnostics, the system of analysis of biological samples is improved and significantly contributes to the process of prevention, detection and treatment of diseases

PAX8-PPARg Oncogene in Follicular Thyroid Tumors: RT-PCR and Immunohistochemical Analyses

US-guided fine needle aspiration cytology is currently the best diagnostic tool for thyroid nodules. However, it is not sensitive and specific enough for differentiating between benign and malignant follicular tumors. A potentially useful marker for this differentiation is the PAX8-PPARg rearrangement, identified in follicular thyroid carcinomas, but not in follicular adenomas or other types of thyroid tumors. The aim of this research was to determine the clinical significance of the PAX8-PPARg oncogene in diagnostics follicular thyroid tumors. The study included 62 patients with follicular or Hürthle cell tumors. Gene expression was determined by reverse transcription-polymerase chain reaction (RT-PCR) from paraffin embedded tissues, and PCR products were checked using the agarose gel electrophoresis. The immunohistochemical analysis was performed on archive paraffin embedded tissues with the monoclonal PPARã antibody. The statistical analysis has indicated that neither the expression of PAX8-PPARg mRNA, nor the immunohystochemical analysis with the PPARg antibody correlate with the patohystological diagnosis. The oncogene PAX8-PPARg has not met the expectations as a reliable tumor marker for differentiation between benign and malignant thyroid tumors, which makes the only reliable histological criteria – capsular and vascular invasion

9-Deazaguanine and Its Methyl Derivatives: Synthesis, Antitumor Activity in vitro and Effects on Purine Nucleoside Phosphorylase Gene Expression

9-Deazaguanine 9-DG, 1-methyl-9-deazaguanine AG-19-K1 and 1,7-dimethyl-9-deazaguanine AG-3 were synthesized and their antiproliferative activity against five leukemia and four solid tumor cell lines as well as inhibitory properties vs. calf spleen purine nucleoside phosphorylase (PNP) were tested. Synthesis of 9-DG involves reaction of 2-amino-6-methyl-5-nitropyrimidin- 4(3H)-one (2) with DMF-dimethylacetal (amount ratio, n(2) / n(DMF-dimethylacetal) = 1:2.5) and use of the benzyloxymethyl group to protect the N-3 position of 2-(N-dimethylaminomethylene) amino-6-methyl-5-nitropyrimidin-4(3H)-one (4). Reaction of 2 with DMF-dimethylacetal (amount ratio, n(2) / n(DMF-dimethylacetal) = 1:6) gave the N-3 methyl substituted intermediate 3. Dithionite reduction of this product afforded N-methyl derivatives AG-19-K1 and AG-3. AG-19-K1 and AG-3 were inactive vs. calf spleen PNP at a concentration of 75 mmol dm–3. Cytotoxic effects of 9-deazaguanine derivatives on cell growth were determined by the MTT assay. Investigated derivatives showed moderate antiproliferative activity towards examined tumor cells. At a concentration of 10–3 mol dm–3, AG-19-K1 inhibited the growth of JURKAT, K562 and AGS cells by approximately 80 %. At the same concentration, AG-3 and 9-DG inhibited cell proliferation by 40-50 % of all tested lines, except MOLT-4 and HL-60. The PNP gene expression was changed in treated leukemia cells after exposure to AG-19-K1 and 9-DG in a time-dependent manner

Effects of Hyperglycemia and Metformin on Expression of Adhesion Molecules on Human Aortic Endothelial Cells

Expression of adhesion molecules on the endothelial cell surface as a response to elevated glucose concentration is considered as a main event in the development of atherosclerosis. The influence of high glucose concentration and metformin, a wide used anti-diabetic drug, on the expression of E-selectin, vascular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) on human aortic endothelial cells (HAECs) was investigated. HAECs were cultured 4 h in a medium with 5.5, 8.0, 12.0, and 16.5 mmol dm-3 glucose with or without metformin addition. The expression of cell adhesion molecules (CAM) was measured by flow-cytometry. Compared to CAM expression in the medium with 5.5 mmol dm-3 glucose, glucose concentration of 12.0 mmol dm-3 increased expression of E-selectin (62 %), VCAM-1 (four fold) and ICAM-1 (81 %). Metformin administration in the medium with 12.0 mmol dm-3 glucose additionally enhanced E-selectin and VCAM-1 expression compared to effects of corresponding glucose concentration. ICAM-1 expression was only significantly increased in the medium with metformin and 8.0 mmol dm-3 glucose. In conclusion, metformin in condition with elevated glucose concentration additionally increased expression of CAM on HAECs which could contribute to development of macrovascular complications in diabetic patients

3D Cell Technology in Biomedical Research

Traditional two dimensional cell culture has enabled great strides in biomedicine but needs to be improved to be able to keep up with the demands of modern biomedical research. 2D monolayer culture cannot replicate tissue responses and needs to be supplemented with extensive animal research. Growing cells in three dimensional scaffolds provides a more functional model for biomedical research than traditional monolayer culture. Depending on the needs and the complexity of the model there are several ways that 3D models can be initiated. Simple spheroids can be grown in low adherence plates and in hanging drops while larger spheroids and co-cultured ones need to be grown in systems with greater support such as hydro gels. The system that offers the greatest flexibility is the magnetic levitation approach. In the paper we offer a brief resume to various 3D methods and their characteristics to ease the choice of methods for implementing 3D cell culture techniques