Reconstituted System
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Práce je dílčím výsledkem projektu, jehož cílem byl vývoj a zavedení metodiky získávání a dlouhodobého skladování erytrocytů pro strategické, speciální a léčebné potřeby Armády České republiky a státu. Jako základní zkoumaná metoda bylo zvoleno mražení erytrocytů, získaných metodou dvojité erytrocytaferézy, ve vysoké koncentraci glycerolu při -85 °C a teplotě skladování -65 °C a nižší. Glycerolizace a deglycerolizace erytrocytů, včetně jejich závěrečné resuspenze v Nutricelu (AS-3), byla prováděna uzavřeným a automatizovaným systémem na zařízení Haemonetics ACP 215. Stabilita EAK rekonstituovaných v AS-3 byla ověřena jak klinickým hodnocením u zdravých dobrovolníků pomocí tzv. indexu terapeutické účinnosti, tak biochemickými a hematologickými parametry. Rekonstituované erytrocytární jednotky byly hodnoceny ve dnech 0, 7, 14 a 21 po rozmražení 51Cr značenými erytrocyty ve dvou skupinách: mražené a skladované v primárních vacích (skupina A) a mražené a skladované po přepuštění do speciálních vaků pro hluboké zmražení (skupina B). Bylo měřeno 24hodinové přežívání autologních erytrocytů podávaných zdravým dobrovolníkům a stanoven index terapeutické účinnosti (ITE) za pomoci tzv. freeze-thaw-wash-recovery (FTW%) závislého na množství hemoglobinu v odpadu po promytí rozmražených erytrocytů. Celkem bylo zmraženo, rekonstituováno a takto vyšetřeno 104 TU erytrocytů. Hodnocené erytrocyty v obou skupinách vykazují téměř shodné hodnoty ITE: 74,01 (±CI95 %: 71,97–76,04) a 74,02 (±CI95 %: 71,18–76,87) bez ohledu na délku sledované doby uchovávání po rekonstituci (21 dní). U skupiny B bylo zjištěno 4% snížení hodnoty přežití po 24 hod. a 4% zvýšení hodnoty FTW%. Hodnoty hemolýzy na konci doby skladování byly u všech hodnocených skupin nižší než 1 %. Z hematologických a biochemických parametrů byly sledovány změny hemoglobinu, hematokritu, leukocytů, hemolýzy, osmolality, pH, K, P, ATP, NH3 a 2,3-DPG. Také výsledky měření těchto laboratorních parametrů prokázaly dobrou použitelnost zmražených erytrocytů ještě 3 týdny po rozmražení, rekonstituci v Nutricelu a uchovávání při teplotě 2–6 °C.
The article summarised partial results the project of evaluation and implementation method for collection and longterm storage packed red cells (RBC) for strategic, special and therapeutic findings Army of Czech Republic as well as the country. The essential surveyed method was freezing of RBC collected by double erythrocytapheresis in high glycerol at -80 °C and stored at -65 °C and lower. Glycerolisation, deglycerolisation and resuspension in Nutricel (AS- 3) was performed with the Haemonetics ACP 215 machine by closed system. Stability of RBC in AS-3 was evaluated by clinical study with health volunteers by index of therapeutic effectiveness (ITE) as well as by biochemical and haematological parameters. Reconstituted RBC units were evaluated in days 0,7,47 and 21 after thawing by 51Cr labelled red cells in two groups: freezing and storage in primary collection bags (group A) and freeing and storage in special bags for deep freezing (group B). 24-hour survival autologous red cell at health volunteers were observed and set ITE and via “freeze-thaw-wash-recovery” (FTW%) dependent on amount of haemoglobin in waste after washing deglycerolised RBCs. Totally 104 RBC units were frozen, reconstituted and measured. Evaluated RBCs both groups were almost statistically identical ITE: 74.01 (±CI95%: 71.97–76.04), 74.02 (±CI95%: 71.18–76.87) and 74.5 (±CI95%: 71.62–77.38), respectively, regardless of storage time after RBCs deglycerolization. The 4-percent decrease of 24-hour posttransfusion survival (%) means as well as 4-percent increase of FTW% means were statistically significant between RBCs group B and A. Haemolysis in the end of storage was below 1% at both group. From haematological and biochemical parameters haemoglobin, haematocrit, leucocytes, haemolysis, osmolality, pH, potassium, phosphorus, ATP, ammoniac and 2,3-DPG were observed. Results of these tests demonstrate good applicability frozen RBC 3 week after thawing and reconstitution in Nutricel and following storage at 2–6 °C.
- Klíčová slova
- Nutricel,
- MeSH
- finanční podpora výzkumu jako téma MeSH
- konzervace krve metody MeSH
- kryoprezervace metody využití MeSH
- lidé MeSH
- separace krevních složek metody MeSH
- transfuze erytrocytů metody MeSH
- vojenské lékařství MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- hodnotící studie MeSH
OBJECTIVES: To study the contribution of individual purified porcine CYP1A2, 2E1 and 2A19 enzymes to the biotransformation of skatole. METHODS: Individual porcine and human enzymes (CYP1A2, 2E1 or 2A6/19) were used to study their potential involvement in skatole metabolism. Furthermore, the inhibition experiments using specific inhibitors of CYP1A2, 2E1 or 2A6/19, were performed. For determination of skatole biotransformation by individual CYP forms in reconstituted systems, HPLC method with UV detection was used. RESULTS: The data presented in this paper show that porcine and human CYPs are responsible for the formation of indole-3-carbinol and 3-methyloxindole. Whereas in pig CYP2A19 and CYP1A2 seem to be the most important for metabolism of skatole, in man CYP1A2 and CYP2E1 forms are mainly responsible for the production of the metabolites mentioned above. CONCLUSIONS: The porcine and human CYP1A2, 2E1, 2A6/19 forms contribute to formation of 3-methyloxindole and indole-3-carbinol.
- MeSH
- cytochrom P-450 CYP1A2 metabolismus MeSH
- cytochrom P-450 CYP2E1 metabolismus MeSH
- indoly metabolismus MeSH
- inhibitory cytochromu P450 CYP1A2 MeSH
- inhibitory cytochromu P450 CYP2E1 MeSH
- inhibitory cytochromu P450 MeSH
- lidé MeSH
- prasata MeSH
- skatol metabolismus MeSH
- systém (enzymů) cytochromů P-450 metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- AIDS dementia complex imunologie patofyziologie MeSH
- antiretrovirové látky terapeutické užití MeSH
- centrální nervový systém patofyziologie patologie MeSH
- HIV infekce farmakoterapie imunologie komplikace MeSH
- imunitní systém fyziologie MeSH
- imunorestituční zánětlivý syndrom etiologie farmakoterapie imunologie patofyziologie MeSH
- lidé MeSH
- progresivní multifokální leukoencefalopatie komplikace MeSH
- rizikové faktory MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
- Research Support, N.I.H., Extramural MeSH
- MeSH
- demografie MeSH
- informační systémy MeSH
- lidé MeSH
- počítače MeSH
- zobrazování dat MeSH
- Check Tag
- lidé MeSH
- Geografické názvy
- Československo MeSH
Práca podáva rozbor pacientov s infekciou nervového systému hospitalizovaných na Klinike pre infekčné choroby v Košiciach v období rokov 1992 až 1996. Súbor tvorí 103 pacientov, z toho 64 mužov a 39 žien. V 37 prípadoch bola potvrdená purulentná meningitída, 5krát bazilártíia meningitída, 26krát serózna meningitída, v ďalších 29 prípadoch šlo o meningoencefalitídu a u 6 pacientov bolo ochorenie hodnotené ako neuritída alebo polyradikuloneuritída. Etiológia ochorení bola objasnená spolu v 70,9 % prípadov. V skupine purulentných meningitíd prevažovah infekcie Streptococcus pneumoniae (hlavne u starších vekových kategórií) a Neisseria meningitidis (u mladších do 20 rokov). V jednom prípade sme pozorovali infekciu kmeňom Str. pneumoniae rezistentného na penicihn a cotrimoxazol a v jednom prípade N. meningitidis s intermediámou citlivosťou na penicilín. Z nebakteriálnych vyvolávatelov neuroinfekcií bol najčastejší vírus kliešťovej encefalitídy. Celková mortalita dosiahla 4,9 %, v skupine purulentných meningitíd 10,5 %. Trvalý následok (vnútroušná nahluchlosť) bol zaznamenaný len u jedného pacienta.
An analysis is given of patients with infections of the nervous system hospitalized at the Košice Faculty Hospital Department of Infectious Diseases in the period of 1992 - 1996. The cohort of 103 patients comprises 64 males and 39 females. The diagnosis of purulent meningitis was confirmed in 37 cases, of basilary meningitis in 5 patients, serous meningitis 26 times, 29 cases of meningoencephalitis, and in 6 patients the illness was diagnosed as neuritis or polyradiculoneuritis. The etiology of the disorders was determined in 70.9 % of the patients. In cases of purulent meningitis predominated Streptococcus pneumoniae infections, chiefly in elderly patients, and Neisseria meningitidis in younger patients under 20 years of age. In one case we observed an infection caused by a strain of S. pneumoniae resistant to penicillin and cotrimoxazole, and in one case N. meningitidis with an intermediate sensitivity to penicillin. Nonbacterial neuroinfections were mostly caused by tick-borne encephalitis virus. The overall mortality was 4.9 %, in purulent meningitis it was 10.5 %. Permanent injury (inner ear deafness) was observed in only one patient.
- MeSH
- demografie MeSH
- dospělí MeSH
- infekce centrálního nervového systému epidemiologie etiologie patofyziologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- mozkomíšní mok MeSH
- prognóza MeSH
- věkové faktory MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- srovnávací studie MeSH
In bone marrow transplantation (BMT), hematopoiesis-reconstituting cells are introduced following myeloablative treatment, which eradicates existing hematopoietic cells and disrupts stroma within the hematopoietic tissue. Both hematopoietic cells and stroma then undergo regeneration. Our study compares the outcomes of a second BMT administered to mice shortly after myeloablative treatment and the first BMT, with those of a second BMT administered to mice experiencing robust hematopoietic regeneration after the initial transplant. We evaluated the efficacy of the second BMT in terms of engraftment efficiency, types of generated blood cells, and longevity of function. Our findings show that regenerating hematopoiesis readily accommodates newly transplanted stem cells, including those endowed with a robust capacity for generating B and T cells. Importantly, our investigation uncovered a window for preferential engraftment of transplanted stem cells coinciding with the resumption of blood cell production. Repeated BMT could intensify hematopoiesis reconstitution and enable therapeutic administration of genetically modified autologous stem cells.
FGF2 is secreted from cells by an unconventional secretory pathway. This process is mediated by direct translocation across the plasma membrane. Here, we define the minimal molecular machinery required for FGF2 membrane translocation in a fully reconstituted inside-out vesicle system. FGF2 membrane translocation is thermodynamically driven by PI(4,5)P2-induced membrane insertion of FGF2 oligomers. The latter serve as dynamic translocation intermediates of FGF2 with a subunit number in the range of 8-12 FGF2 molecules. Vectorial translocation of FGF2 across the membrane is governed by sequential and mutually exclusive interactions with PI(4,5)P2 and heparan sulfates on opposing sides of the membrane. Based on atomistic molecular dynamics simulations, we propose a mechanism that drives PI(4,5)P2 dependent oligomerization of FGF2. Our combined findings establish a novel type of self-sustained protein translocation across membranes revealing the molecular basis of the unconventional secretory pathway of FGF2.
- MeSH
- fibroblastový růstový faktor 2 sekrece MeSH
- fosfatidylinositol-4,5-difosfát metabolismus MeSH
- heparitinsulfát metabolismus MeSH
- membránové transportní proteiny metabolismus MeSH
- multimerizace proteinu * MeSH
- sekreční vezikuly metabolismus MeSH
- simulace molekulární dynamiky MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Proteoliposomes carrying reconstituted yeast plasma membrane H(+)-ATPase in their lipid membrane or plasma membrane vesicles are model systems convenient for studying basic electrochemical processes involved in formation of the proton electrochemical gradient (Deltamicro(H) (+)) across the microbial or plant cell membrane. Deltapsi- and pH-sensitive fluorescent probes were used to monitor the gradients formed between inner and outer volume of the reconstituted vesicles. The Deltapsi-sensitive fluorescent ratiometric probe oxonol VI is suitable for quantitative measurements of inside-positive Deltapsi generated by the reconstituted H(+)-ATPase. Its Deltapsi response can be calibrated by the K(+)/valinomycin method and ratiometric mode of fluorescence measurements reduces undesirable artefacts. In situ pH-sensitive fluorescent probe pyranine was used for quantitative measurements of pH inside the proteoliposomes. Calibration of pH-sensitive fluorescence response of pyranine entrapped inside proteoliposomes was performed with several ionophores combined in order to deplete the gradients passively formed across the membrane. Presented model system offers a suitable tool for simultaneous monitoring of both components of the proton electrochemical gradient, Deltapsi and DeltapH. This approach should help in further understanding how their formation is interconnected on biomembranes and even how transport of other ions is combined to it.
- MeSH
- arylsulfonany chemie MeSH
- biologické modely MeSH
- buněčná membrána fyziologie chemie MeSH
- elektrochemie MeSH
- financování organizované MeSH
- fluorescenční barviva analýza MeSH
- fluorescenční spektrometrie MeSH
- isoxazoly chemie MeSH
- koncentrace vodíkových iontů MeSH
- membránové potenciály MeSH
- proteolipidy chemie MeSH
- protonové ATPasy chemie MeSH
- protony MeSH
Metabolism of drugs and other foreign substances is mostly mediated by cytochromes P450 (P450, abbreviated also CYP for a particular enzyme). To find which P450 is involved in metabolism of a drug, liver microsomal monooxygenase system of P450 is reconstituted with its components: selected P450 enzyme, cytochrome b5, NADPH:P450 reductase and phospholipid. Used NADPH:P450 reductases were human recombinant and minipig or rat liver microsomal ones isolated by chromatographic separations. Chosen P450 enzyme was the CYP2E1 which is known to be of very similar primary structure among species; in this study, the minipig enzyme has been taken, as the minipigs seem to be suitable model animals for drug metabolism studies. Results obtained show that the reductase enzymes of rat and human origin can be used in reconstituted systems with a CYP2E1 as the activity of this enzyme varies in systems with reductases of different origin less than ten times. The results also indicate that the reductases from different species share a functional similarity, if not identity.
