Úvod: Radioimunoterapie patří mezi rychle se rozvíjející klinické metody. Často je nejvhodnější možností terapie u pacientů s relabujícím lymfomem, případně u starších pacientů. Vývoj nových typů imunokonjugátů patří proto v posledních letech mezi klíčová témata ve vývoji radiofarmak. Metody: Imunoglobulin G M75 byl značen metodou elektrofilní jodace radionuklidem 125I, který je optimální pro vývoj metody značení a také pro stanovení vlastností značeného preparátu (vazebná aktivita, imunospecifita). Výhody 125I jsou zejména jeho dlouhý poločas (59,408 dne) a nízká radiační zátěž. Zachování vazby aktivního místa na antigen bylo testováno metodou ELISA, koncentrace byla stanovena modifikovanou metodou podle Bradfordové. Fyziologická distribuce byla testována na myších CD1-Foxn1nu. Výsledky: Stanovená kumulace značené protilátky v nádorové tkáni byla > 7 % podané radioaktivity, vazebná aktivita protilátky byla první den po značení vyšší než 90 % ve srovnání s vazebnou aktivitou neznačené protilátky. Závěr: Připravený radioimunokonjugát IgG M75 s 125I si zachovává dostatečnou schopnost vazby antigenu, a má proto potenciál v terapii tumorů exprimujících karbonátdehydratasu. Pro použití v radioterapii je třeba otestovat analogický radioimunokonjugát značený radionuklidem 131I., x, Lenka Marešová, Ondřej Lebeda, Daniel Seifert, Irena Sieglová, Vlastimil Král, and Literatura
Seven- to eight-week-old rabbits were infected with Eimeria intestinalis Cheissin, 1948, a highly immunogenic coccidium, or Eimeria flavescens Marotel et Guilhon, 1941, which is weakly immunogenic. Immune response was investigated at 7, 14 and 21 days post inoculation (DPI). The level of serum immunoglobulins, lymphocyte proliferation stimulated by parasite antigens and weight of mesenteric lymph nodes (MLN) showed similar dynamics in rabbits inoculated with both coccidia species. The amount of serum IgG and IgM, but not IgA, was increased from 14 DPI. The lymphocytes from MLN of infected animals significantly reacted to stimulation with parasite antigen 14 and 21 DPI and MLN were enlarged at 14 DPI. Thus, both parasite species elicited immune response characterized by these parameters in a similar manner despite of their different immunogenicity. The only apparent difference in the responses was in the percentage of CD8+ lymphocytes in the specific site of parasite development (the last third of the small intestine in E. intestinalis, caecum in E. flavescens), which increased in rabbits infected with E. intestinalis but not with E. flavescens. This parameter reflects the status of local immunity and hence the results suggest that the local reaction plays an important role in induction of protective immunity to coccidia in rabbits.
In the present study the hypothesis that there is a feedback between juvenile hormone and adipokinetic hormones (AKHs) was investigated by topical application of the juvenoid methoprene on 9-day-old adult males of the firebug Pyrrhocoris apterus. This juvenoid (2 µg) induced a significant reduction of haemolymph lipids 24 h after treatment; however, it did not significantly reduce the ability of Pyrap-AKH (10 pmol/bug) to mobilize fat body lipids 6-72 h after the methoprene application. The same methoprene treatment elicited a significant increase of AKH content in the CNS (central nervous system: brain + corpora cardiaca + corpora allata) of experimental males 24 and 48 h after the juvenoid application. A significant decrease in the AKH level in the haemolymph was recorded at the same times and under the same experimental conditions. Similar results were observed when production of the AKHs from the CNS of topically treated males was measured under in vitro conditions. It is suggested that methoprene may reduce AKH release from the CNS resulting in an increase in the AKH content of the CNS due to accumulation rather than stimulation of AKH synthesis. Possible consequences of this phenomenon on the physiology of P. apterus are discussed.
To investigate into the relationship between two Rubisco activase (RCA) isoforms and photosynthetic rate, a set of enzyme-linked immunosorbent assay (ELISA) were developed for accurate quantification of two RCA polypeptides based on two specific monoclonal antibodies against different RCA isoforms. The results showed that content of RCA small isoform (RCAS) was 5-fold more than that of RCA large isoform (RCAL) content in all leaves and the RCAL/RCAS ratio reached maximum in the leaf with the highest photosynthetic rate. Although the difference in two RCA polypeptides accumulation in leaves was caused by different transcript level of two isoforms, the decrease of RCAL/RCAS ratio during leaf aging was not attributed to transcriptional regulation. The leaves with higher photosynthetic capacity exhibited higher RCAL/RCAS ratio and the decrease in photosynthetic rate and Rubisco activation state highly correlated with the decline of RCAL/RCAS ratio during leaf aging. Our results suggest that there is a posttranscriptional mechanism regulating the RCAL/RCAS ratio, which may play as a regulator modulating photosynthetic capacity during leaf aging in rice plant. and D. Wang ... [et al.].
Intact chloroplasts were isolated from dark-senescing primary barley (Hordeum vulgare L.) leaves in order to study selective ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) degradation by the stromal and membrane fractions. RuBPCO specific degradation was estimated and characterised applying sensitive avidin-biotin ELISA method with non-modified or oxidatively modified biotinylated RuBPCO (BR) as substrates. Distinct proteolytic activities were detected. They differed in ATP and divalent metal ion dependence, protease inhibitory profile, and dynamics in the time-course of dark-induced senescence. The results supported involvement of ATP- and metal ion-dependent serine type proteolytic activity against non-modified BR early in induced senescence and appearance of ATP-independent activity at later stage. Active oxygen-modified BR was degraded by ATP-independent serine-type protease probably containing essential SH-groups and requiring divalent metal ions. and L. Simova-Stoilova, K. Demirevska-Kepova, Z. Stoyanova.
An IgM murine monoclonal antibody (MAb) was obtained against the excretory-secretory antigen (ES-Ag) of in vitro reared protoscoleces of Echinococcus granulosus (Batsch, 1786). Western blotting revealed that the MAb recognised a 20.6 kDa protein of this ES-Ag. The MAb was used in sandwich enzyme-linked immunosorbent assay (s-ELISA) for selective sensitisation of the solid phase with the protoscolex-specific protein from its ES-Ag and somatic antigen (S-Ag) to examine serum samples of 108 cows from a cystic echinococcosis (CE) endemic area for specific antibodies and to compare the results with those from necropsies and an indirect ELISA (i-ELISA). The sensitivity of s-ELISA/ES-Ag, s-ELISA/S-Ag and i-ELISA/S-Ag was 48%, 52% and 62%, respectively. The low sensitivity of the ELISA was probably caused by the fact that 13 cows (62%) were infected with sterile cysts (acephalocysts and/or calcified foci) only. A relatively high specificity (80%) of s-ELISA/ES-Ag was observed in cows with fertile cysts. It also detected antibodies in the serum of two cows that had recovered from the disease according to the necropsy. The i-ELISA/S-Ag gave false results in testing sera from a healthy animal and from a cow with tubercular foci. Further analysis will be necessary to define more precisely the value of this study, because the duration of antibody elimination from the bloodstream of recovered cattle remains unknown. The solution of this problem will increase the specificity of the proposed test in monitoring herbivorous animals for CE., Aitbay K. Bulashev, Zhanbolat A. Suranshiev, Orken S. Akibekov, Zhanara Zh. Akanova, Gulmira A. Abulgazimova., and Obsahuje bibliografii
Cíl: Ověření základních vlastností analýzy volných lehkých řetězců v séru. Metody: Imunoturbidimetrické stanovení s použitím diagnostické soupravy FreeLite firmy The Binding Site, adaptované na analyzátor Modular Roche. Metoda ELISA, použitá v soupravě firmy BioVendor. Výsledky: Hodnoty stanovené u souboru dárců krve mužů a žen. Index kappa/lambda imunoturbidimetrií 0,53–1,92; metodou ELISA 0,24–1,17. Přesnost měření kappa imunoturbidimetrií 5,6–6,9 %, metodou ELISA 6,6–11,0 %, lambda imunoturbidimetricky 6,1 až 9,2 %, metodou ELISA 7,3–8,6 %. Studovali jsme vliv ředění vzorků na výsledky analýz a potvrdili jsme potřebnost ověřování stupně ředění u každého patologicky změněného vzorku. Regresní analýzou byla určena rovnice y = -9,84 + 1,53 x a korelační koeficient r = 0,73. U imunoturbidimetrie bylo ve sledované populaci nalezeno 58,9 % výsledků hodnocených jako patologických, metodou ELISA 45,5 %. U 4,5 % patologických výsledků byla nalezena neshoda mezi oběma metodami. U výsledků uvnitř referenčního intervalu byly nalezeny neshody u 17,8 % pozorování. Diskuse: Hlavními problémy úspěšného použití stanovení volných lehkých řetězců jsou validita referenčních intervalů, ředění vzorků individuálních pacientů, diference v diagnostické klasifikaci při použití různých metod. Závěr: Stanovení volných lehkých řetězců v séru nelze prozatím považovat za úplně rutinní záležitost, vyžaduje pečlivou přípravu vzorků a analýzu výsledků podmíněnou nadstandardní úrovní odbornosti laboratorního personálu., Objective: Verification of measurement procedures for serum free light chains analysis. Methods: Immunoturbidimetric measurement with using the FreeLite kit (The Binding Site), adapted on the Modular P (Roche). ELISA method performed by kit BioVendor. Results: The ratio kappa/lambda of blood donors values in men and women was from 0.53 to 1.92 by use FreeLite kit and from 0.24 to 1.17 by ELISA method. We obtained measurement precision as follows: kappa (FreeLite) 5.6–6.9%, 6.6–11.0% (ELISA), lambda 6.1–9.2% by FreeLite and 7.3–8.6% by ELISA kit. We assessed the influence of sample dilution on the results. It is necessary to verify dilution in each pathologically changed sample. Regression analysis is expressed by equation y = -9.84 + 1.53 x and Pearson regression coefficient is r = 0.73. We found 58.9% results obtained with immunoturbidimetry, but only 45.5% of results obtained by ELISA method out off reference limit. Four and a half % of pathologic results showed disagreement frequency in paired results while in results inside reference interval there was a disagreement frequency even 17.8%. Conclusion: It is unsuitable to consider the measurement of free light chains in serum as a routine event. This determination requires careful work and high professional level of laboratory personal., Vávrová Jaroslava, Friedecký B., Tichý M., Holečková M., Maisnar V., Hájek R., and Lit.: 9
The variations in antibody responses (total IgG and IgGl, IgG2a, IgG2b, and IgG2c subclasses) were studied in two groups of rats infected with metacercariae of the trematode Fasciola hepatica L. Animals of group 1 were 4 weeks old, and rats of group 2 were 13 weeks old. All IgG subclasses increased during the course of infection except IgG2c, which decreased. The younger rats reached more marked responses than the older, at least during the period of this trial. IgGl and IgG2a antibodies reached the highest levels, and among these two, IgG2a response was slightly superior to IgGl,
Introduction: Ankylosing spondylitis (AS) is an inflammatory rheumatic disease characterized by the development of osteoproductive changes in the spine which could possibly result in ankylosis. Treatment with tumour necrosis factor alpha (TNFα) inhibitors has proved to be an important step forward in the treatment of this disease, but for the time being it is not clear whether it favourably influences radiographic progression of the disease. Vascular endothelial growth factor most probably plays a role in the development of osteoproductive changes and recently its predictive influence on radiographic progression has been demonstrated. Bone morphogenic protein 2 (BMP-2) participates in the regulation of bone proliferation and its increased serum level has been demonstrated in patients with advanced AS and correlated with the degree of radiographic changes. Aim: The study aims to evaluate the VEGF and BMP-2 levels in patients with ankylosing spondylitis and how these levels relate to the concurrent treatment with TNFα inhibitors. Methods: Sera were evaluated from patients at the Rheumatologic Clinic of the Hradec Králové Faculty Hospital who fulfilled the modified New York Criteria for AS (n = 55). In these patients, the parameters of the activity of the disease (BASDAI = Bath Ankylosing Spondylitis Disease Activity Index, CRP = C-reactive protein) and the concurrent therapy (TNFα inhibitors, n = 21, vs. non-anti TNFα, n = 34) were recorded. The levels of VEGF and BMP-2 were analyzed using the ELISA method. Results: In patients treated with TNFα inhibitors, a significantly lower VEGF level was found when compared to untreated patients (140.3 (109.4; 262.2) vs. 261 (172.4; 396.6) pg/ml; p = 0.02). No difference was found between BMP-2 levels in both groups (treated vs. untreated patients) (254.8 (2301; 267.3) vs. 261.1 (248.6; 273.5) pg/ml; p = 0.24). A correlation analysis did not reveal any relationship between VEG F and BMP-2 (r = 0.057; p = 0.68). Serum levels of VEGF correlated with serum levels of CRP (r = 0.56; p = 0.00001) and the BASDAI value (r = 0.33; p = 0.015). Conclusion: Significantly lower VEGF levels were found in patients treated with TNFα inhibitors versus the untreated patients. These findings are in harmony with some hitherto published analyses and may give evidence of a favourable effect of TNFα inhibitors on radiographic progression. Neither influence on the BMP-2 level by treatment with TNFα inhibitors nor correlation with VEGF levels was demonstrated. and M. Tošovský, P. Bradna, C. Andrýs, K. Andrýsová, E. Čermáková, T. Soukup