Czynniki charakterystyczne dla tętniaka aorty brzusznej i jego potencjalne biomarkery

Abdominal aortic aneurysm (AAA) is a multifactorial and asymptomatic disorder with high mortality. Numerous factors inducing AAA have been postulated, but so far no key factor with a molecular, genetic or environmental basis has been identified that would contribute to the formation of an aneurysm. Damage to the structure of the extracellular matrix, apoptosis of the vascular smooth muscle cells and endothelial cells through degenerative factors induces the process of aneurysm formation in the vessel wall. This process is accompanied by a progressive inflammatory process. The test that allows the detection of AAA is abdominal ultrasonography. However, this is not a routine screening test performed for all individuals, and therefore AAA is usually diagnosed at a very advanced stage of the disease that threatens the patient’s life. The only medical procedure in AAA is surgical treatment. Therefore, it is necessary to identify molecular processes, AAA biomarkers and genes responsible for the mechanism of aneurysm formation. This would enable faster and more effective initiation of the treatment process.Tętniak aorty brzusznej (AAA) jest schorzeniem wieloczynnikowym i bezobjawowym, charakteryzującym się wysoką śmiertelnością pacjentów. Istnieje wiele indukujących go czynników, ale do tej pory nie zidentyfikowano jednego kluczowego, o podłożu molekularnym, genetycznym czy środowiskowym, który przyczynia się do powstania tętniaka. Uszkodzenie struktury macierzy zewnątrzkomórkowej, apoptoza komórek mięśni gładkich ściany naczynia i komórek śródbłonkowych poprzez czynniki degeneracyjne indukuje proces tworzenia tętniaka w ścianie naczynia. Towarzyszy temu postępujący proces zapalny. Badaniem pozwalającym na wykrycie AAA jest ultrasonografia jamy brzusznej. Nie jest to jednak rutynowe badanie przesiewowe wykonywane u wszystkich osób, dlatego do zdiagnozowania AAA dochodzi w bardzo zaawansowanym stadium choroby, co zagraża życiu pacjenta. Jedynym medycznym postępowaniem w tym przypadku pozostaje zabieg chirurgiczny, dlatego koniecznym wydaje się poznanie procesów molekularnych, biomarkerów AAA oraz genów odpowiedzialnych za mechanizm powstawania tętniaków. Umożliwiłoby to szybszą i efektywniejszą diagnostykę, a tym samym zapoczątkowałoby proces leczenia

Cytochrome P450 mRNA expressions along with in vitro differentiation of hepatocyte precursor cells from fetal, young and old rats.

Non-differentiated cells are attractive targets for cell therapy. During liver regeneration oval cells intensively proliferate and differentiate extending their metabolic activity. Hepatic cytochromes P450 (CYPs) can be linked either with metabolic activation of toxic compounds or drug metabolism. We investigated the differentiation and biotransformative potential of non-differentiated cells in primary cell cultures isolated from livers of fetuses (16-days-old), young (4-months-old) and old (20-months-old) rats. Under the conditions of experimental hepatocarcinogenesis, adult rats were fed for three weeks with CDE diet. Liver cells were cultured and precursor cells were differentiated to hepatocytes following induction with sodium butyrate (SB) or dimethyl sulphoxide (DMSO) in culture on MesenCult medium. We identified a number of cells expressing Thy-1, CD34, alpha-fetoprotein, cytokeratines--CK18 or CK19 and glutathione transferases--GSTpi or GSTalpha. In vitro differentiation of these cells, isolated from CDE-treated rats begun earlier as compared to non-treated ones. Age-dependent changes in the cell differentiation sequence, as well as CYPmRNA expression sequence accompanying precursor cells differentiation, were also observed. mRNA expression of CYP1A2, CYP2B1/2 and CYP3A1 was higher in the cells of young rats, but in the case of CYP2E1--in the cells of old rats. It was concluded that both proliferation and differentiation potential of oval cells, decreased with age

Zmiany mRNA wariantów alternatywnego składania C-endopeptydazy prokolagenu w mięśniakach macicy w zależności od fazy cyklu miesięcznego i u kobiet w okresie klimakterium

Summary Leiomyoma uteri is a monoclonal tumour of the uterus muscle layer. It is characterized by excessive, abnormal growth of extracellular matrix. The collagen types I and III are the major components of extracellular matrix. Removal of the C-propeptides in procollagens type I, II, and III by procollagen C-endopeptidase leads to spontaneous selfassembly of collagen fibrils. Thus, the procollagen C-endopeptidase is a key regulator of extracellular matrix production, its quality, and other developmental processes including angiogenesis. Objective: The objective of this study was to analyze the three alternatively spliced variants of the BMP1 gene product including the procollagen C-endopeptidase, in leiomyoma uteri tumors in comparison to normal myometrium in women being in first or second menstrual phase or in time of climacterium. Material and Methods: In the study we analyzed samples from the control and cancerous tissues from 52 women. Expression of the three alternatively spliced variants of BMP1 gene transcript were assayed by RT-PCR, following densytometric analysis. Statistical significance (p≤0,05) of the observed differences was assessed with the use of Anova test, Least Significance Different Test, and Student’s T-test. Results: Analysis of RT-PCR products revealed the presence of all alternatively spliced variants of BMP-1 mRNA and the changes in their alternative splicing intensity, depending on the phase of menstrual cycle or postmenopausal state. In the second phase of the cycle, in control tissue, the expression level of BMP-1 variant decreased when compared to women in I phase of the cycle or postmenopausal women. In the tumour, postmenopausal women showed increased expression of BMP-1at, when compared with women in the second phase of the cycle. The presence of mTLD in the tumour tissue at II phase of the cycle and in postmenopausal state was less strong when compared to women at I phase of the cycle. In control tissue this type of change was not observed. The BMP-1/HIS was present at higher level in control tissue, during II phase of the menstrual cycle and in postmenopausal state, whereas in the tumour tissue its lowest level was at II phase of the cycle. Conclusions: Regulation of alternative splicing of mRNA for procollagen C-endopeptidase in leiomyomas and myometrium depends mainly on the hormonal status of women.Streszczenie Wstęp: Mięśniak gładkokomórkowy macicy (Leiomyoma uteri) jest niezłośliwym nowotworem monoklonalnym błony mięśniowej macicy. Rozwój guza charakteryzuje nadmierny i nieprawidłowy rozrost macierzy pozakomórkowej. Główne składniki macierzy pozakomórkowej to kolageny typu I i III. Odcięcie C-propeptydow w prokolagenach typu I, II i III przez C-endopeptydazę prokolagenu wzbudza samoistne składanie włókien kolagenowych. Endopeptydaza jest kluczowym regulatorem wytwarzania macierzy pozakomórkowej, kontroli jej jakości oraz procesów rozwojowych, takich jak rozwój kości czy naczyń krwionośnych. Cel pracy: Celem pracy jest analiza syntezy trzech wariantów mRNA powstających w wyniku alternatywnego składania transkryptów genu BMP1, kodującego C-endopeptydazę prokolagenu, w mięśniakach macicy, w porównaniu do prawidłowej błony mięśniowej macicy u kobiet w I i II fazie cyklu miesiączkowego oraz w klimakterium. Materiał i Metody: W badaniach analizowano materiał pochodzący z leiomyoma uteri i tkanek kontrolnych uzyskanych od 52 pacjentek. Proces alternatywnego składania wariantów mRNA genu BMP1 był oznaczany metodą densytometryczną produktów otrzymanych z reakcji RT-PCR. Znamienność statystyczną wykrytych różnic sprawdzano testami ANOVA i Najmniejszych Istotnych Różnic (LSD) w programie mSTATC oraz testem t-Studenta dla p≤0,05. Wyniki: Analiza produktów RT-PCR ujawniła obecność mRNA trzech wariantów alternatywnego składania transkryptu genu BMP1 oraz zmiany w natężeniu alternatywnego składania wariantów zależnie od fazy cyklu menstruacyjnego lub klimakterium. W drugiej fazie cyklu, w tkance kontrolnej spadało nasilenie ekspresji wariantu BMP-1 w porównaniu do fazy pierwszej i klimakterium. Natomiast w guzie, u kobiet w okresie klimakterium, w porównaniu z kobietami w drugiej fazie cyklu zaobserwowano nasilenie ekspresji wariantu BMP-1. Poszukiwanie wariantu mTLD w tkance guzowej u kobiet w drugiej fazie cyklu i w klimakterium, z kobietami w pierwszej fazie cyklu ujawniło jego zanikanie, podczas gdy w tkance kontrolnej nie obserwowano takiej zmiany. Obecność wariantu BMP-1/HIS była najsilniej zaznaczona, w tkance kontrolnej w drugiej fazie cyklu i klimakterium, a w tkance guzowej słabiej zaznaczyła się jego obecność w drugiej fazie cyklu. Wnioski: Regulacja alternatywnego składania wariantów C-endopeptydazy prokolagenu w guzach leiomyoma uteri i błonie mięśniowej macicy zależy, przede wszystkim od fazy cyklu miesięcznego

Expression and co-expression of surface markers of pluripotency on human amniotic cells cultured in different growth media

Objectives: Despite constant advances in the field of biology and medical application of human embryonic stem cells, the molecular mechanism of pluripotency remains largely unknown. So far, definitions of pluripotent stem cells (SC) have been based on a limited number of antigenic markers and have not allowed for unambiguous determination of the homogeneity of each subpopulation. Moreover, the use of some crucial pluripotency markers such as SSEA-3 and SSEA-4 has recently been questioned due to the possibility that the pattern of surface glycans may be changed depending on the content of the cell culture medium. Aim: Quantitative analysis of amniotic SC subpopulations cultured in different media, based on the following pluripotency surface markers: SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 expression and co-expression. Material and methods: Immunofluorescence and fluorescence microscopy were used to identify and localize S.C. within a normal human placenta at term. The number of SSEA-4+, SSEA-3+, TRA-1-60+ and TRA-1-81+ cells and cells with co-expression of the above mentioned markers, cultured in media containing different protein supplements of animal origin, was counted by flow cytometry. Results and conclusions: Cells with characteristics of embryonic SC were identified in the amniotic epithelium and the chorion, but not in the decidua basalis. Amniotic epithelium contained various types of SC, with SSEA-4+ as the most numerous. Disproportion in the number of SSEA-4+, SSEA-3+, TRA-1-60+ and TRA-1-81+ cells and cells characterized by co-expression of these antigens, as well as lack of quantitative differences between S.C. subpopulations cultured in different media, was observed. In conclusion, the amniotic epithelium is composed of SC at different stages of the development but human amnion might become an alternative source of SSEA-4+ embryonic-like SC. The composition of the evaluated media, characterized by different content of animal-derived proteins, does not influence the number of cells identified within the SC subpopulations

Serologic Investigations in Children with Inflammatory Bowel Disease and Food Allergy

The aim of the study was the evaluation of frequency and titre of IgA ASCA and IgG ASCA and p-ANCA, c-ANCA in children with IBD and occurrence of ASCA antibodies in relation to coexistence of FA. Patients and methods. The study comprised 95 children at the ages of 2 to 18 years. The diagnosis of IBD was established on the basis of Porto criteria. Tests of blood serum were performed in all children: IgA and IgG ASCA, p-ANCA, c-ANCA using ELISA method. Results. IgE-dependent FA was found in 32.5% children with UC and in 21% with CD. We did not observe any relation between the occurrence of FA and the frequency and ASCA titre. p-ANCA were significantly more frequent in the group of children with UC. The occurrence of ASCA antibodies was observed in 73.7% of children with CD, 17.5% with UC and almost 30% with allergic colitis. Conclusions. Patients with CD and the presence of ASCA revealed a significantly more frequent localization of lesions within the small bowel and a tendency towards older age. We observed a connection between the occurrence of antibodies and the examined mutations of gene NOD2/CARD15

Polymorphisms in DNA repair genes – assessment of frequencies and effect on the level of DNA oxidative damage caused by lead

Introduction. The aim of this study was to evaluate the effect of polymorphisms in DNA repair genes: APE1, hOGG1, XRCC1, XPA on the level of oxidative damage to DNA as well as an assessment of the frequencies of genetic polymorphisms in the adult population of Caucasians from southern Poland. Material and methods. We examined a group of 115 men occupationally exposed to lead and 58 men with no history of occupational exposure to lead. The concentrations of lead in blood, zinc protoporphyrin in blood and 8-hydroxy-2-deoxyguanosine in urine were measured. The identification of SNP polymorphisms in genes encoding enzymes involved in DNA repair (APEX1, hOGG1, XPA, XRCC1) was performed using real-time PCR with TaqMan probes. We analyzed polymorphisms: APEX1 (rs1130409, Asp148Glu), hOGG1 (rs1052133, Ser326Cys), XPA (rs1800975, -4A/G) and XRCC1 (rs25487, Gln399Arg). Results. The mean blood lead level in the exposed group was 33,48 μg/dl and was significantly higher compared to 5.35 μg/dl (p=0.000). in the control group. The frequencies of studied polymorphisms were comparable in both groups, with the exception of -4A/G (rs1800975) in the gene XPA (p<0.0001). Conclusions. Differences were observed between genotypes in -4A/G (rs1800975) polymorphism in relations to the level of lead in blood (p=0.006) and Ser326Cys (rs1052133) polymorphism in relations to 8-hydroxy-2- deoxyguanosine in urine (p=0.01).Wstęp. Celem pracy była ocena wpływu polimorfizmów w genach naprawy DNA: APE1, hOGG1, XRCC1, XPA na poziom uszkodzeń oksydacyjnych w DNA oraz ocena częstości ich występowania w populacji dorosłych osób rasy kaukaskiej pochodzących z południowej Polski. Materiał i metody. Przebadano grupę 115 mężczyzn narażonych zawodowo na ołów oraz 58 mężczyzn bez narażenia na ołów w wywiadzie, którzy stanowili grupę kontrolną. U wszystkich osób oznaczono poziom ołowiu we krwi, cynkoprotoporfiryny, oraz stężenie 8-hydroksy- 2-deoksyguanozyny w moczu. Identyfikację polimorfizmów typu SNP w genach kodujących enzymy biorące udział w naprawie DNA (APEX1, hOGG1, XPA, XRCC1) przeprowadzono z wykorzystaniem metody real-time PCR z sondami TaqMan. Analizie poddano polimorfizmy w genach APEX1 (rs1130409, Asp148Glu), hOGG1 (rs1052133, Ser326Cys), XPA (rs1800975, -4A/G) oraz XRCC1 (rs25487, Gln399Arg). Wyniki. Średni poziom ołowiu we krwi w grupie badanej wynosił 33,48 μg/dl i był istotnie statystycznie wyższy niż w grupie kontrolnej, gdzie wynosił 5,35 μg/dl (p=0,000). Częstość występowania poszczególnych genotypów badanych polimorfizmów była porównywalna w obu grupach, z wyjątkiem polimorfizmu -4A/G (rs1800975) w genie XPA (p<0,0001). Wnioski. Zaobserwowano różnice pomiędzy poszczególnymi genotypami w polimorfizmie -4A/G (rs1800975) w odniesieniu do poziomu ołowiu we krwi (p=0,006) oraz w polimorfizmie Ser326Cys (rs1052133) w odniesieniu do poziomu 8-hydroksy-2-deoksyguanozyny w moczu (p=0,01)

Synthesis, Characterization and Cytotoxicity of Novel Thermoresponsive Star Copolymers of <i>N</i>,<i>N</i>′-Dimethylaminoethyl Methacrylate and Hydroxyl-Bearing Oligo(Ethylene Glycol) Methacrylate

Novel, nontoxic star copolymers of N,N-dimethylaminoethyl methacrylate (DMAEMA) and hydroxyl-bearing oligo(ethylene glycol) methacrylate (OEGMA-OH) were synthesized via atom transfer radical polymerization (ATRP) using hyperbranched poly(arylene oxindole) as the macroinitiator. Stars with molar masses from 100,000 g/mol to 257,000 g/mol and with various amounts of OEGMA-OH in the arms were prepared. As these polymers can find applications, e.g., as carriers of nucleic acids, drugs or antibacterial or antifouling agents, in this work, much attention has been devoted to exploring their solution behavior and their stimuli-responsive properties. The behavior of the stars was studied in aqueous solutions under various pH and temperature conditions, as well as in PBS buffer, in Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) and in organic solvents for comparison. The results indicated that increasing the content of hydrophilic OEGMA-OH units in the arms up to 10 mol% increased the cloud point temperature. For the stars with an OEGMA-OH content of 10 mol%, the thermo- and pH-responsivity was switched off. Since cytotoxicity experiments have shown that the obtained stars are less toxic than homopolymer DMAEMA stars, the presented studies confirmed that the prepared polymers are great candidates for the design of various nanosystems for biomedical applications