Introduction of continuous glucose monitoring (CGM) is a key factor in decreasing HbA1c in war refugee children with type 1 diabetes.

AIMS: Our aim was to describe the changes in therapy and diabetes control in Ukrainian war refugee children with diabetes (CwD) during the first year of their stay in Czechia. METHODS: A total of 124 CwD (62 male, 62 female) were enrolled into this observational study. Anthropometric, laboratory and diabetes management data were acquired at baseline and at 3 months intervals for 12 months. All CwD were offered a CGM device during their first visit. Generalized Estimating Equation models were fitted in order to estimate the dynamics of studied characteristics. RESULTS: Median baseline HbA1c was 58 mmol/mol (IQR [48; 73]mmol/mol) (7.5 %, IQR[6.5;8.8]%). The HbA1c decreased significantly throughout the course of the study at a pace of - 2.2 mmol/mol (-0.2 %pt.) per visit (P = 0.01, CI[-3.2;-1.1]). The pace of the decrease in the average HbA1c was significantly higher in the group of CwD who received CGM in Czechia than in those who already had it from Ukraine by 2.9 mmol/mol (0.27 %pt.) per visit (P < 0.001, CI [-4.4; -1.3]). CONCLUSIONS: The steepest decrease in HbA1c was observed in CwD with newly initiated CGM underlining its vital role in improving the glucose control of CwD regardless of their background.

A molecular-based approach to the thermodynamics of aqueous solutions: binary mixture of water and carbon dioxide.

A simple model and theory of molecular fluids is applied to a binary mixture of water and carbon dioxide. An approach based on the perturbation theory is followed using a reference system of so-called pseudo-hard bodies for water and hard triatomics for carbon dioxide. Pseudo-hard bodies bear the traits of the non-additive nature of association supplementing the common excluded volume effect. The reference term is parametrized using Monte Carlo simulation data on the compressibility factor. After adding a simple mean-field term to the reference equation, fluid phase equilibria are qualitatively reproduced.

Serum levels of matrix metalloproteinases 2 and 9 in patients with acute myocardial infarction.

The myocardial extracellular matrix plays an important role in maintaining the structural and functional integrity of the heart and is centrally involved in post-myocardial infarction repair processes. We analysed some genetic and proteomic aspects that could play an important role in the development of myocardial infarction. Matrix metalloproteinases are enzymes that contribute strongly to the degradation of extracellular matrix components. In this study the serological levels of MMP-2 and MMP-9 were investigated using immunological testing in 34 patients with myocardial infarction and 34 matched control subjects. The serum levels of MMPs were determined by ELISA. Changes in serum levels were characterized within 24 h and after 6 months post myocardial infarction. Significantly higher levels of MMP-2 (299.47 ± 117.61 ng/ml) and MMP-9 (93.56 ± 53.74 ng/ml) were determined in patients with myocardial infarction compared to the controls, in both cases P < 0.001. MMP-9 levels decreased significantly in the 6 months after cardiac event, whereas the levels of MMP-2 were almost equal to the post-infarction ones. While comparing the results from four patients that died of cardiovascular cause within 6 months we found significantly higher MMP-2 (435.00 ± 55.83 ng/ml, P = 0.003) and MMP-9 (166.25 ± 41.07 ng/ml, P = 0.018) values. Microarray analysis was used to determine the gene expression of selected genes for MMPs and their regulators from peripheral blood. The selected genes did not show satisfactory results that could have a potential implication for diagnostics of tissue degeneration.

Serum levels of matrix metalloproteinases 2 and 9 and TGFBR2 gene screening in patients with ascending aortic dilatation.

Development of ascending aortic dilatation (AAD) in about 10 % of patients operated for aortic valve disease (AVD) is probably based on intrinsic pathology of the aortic wall. This may involve an abnormality in the process of extracellular matrix remodelling. The present study evaluated the serum levels of specific metalloproteinases (MMP-2 and MMP-9) and investigated the gene for transforming growth factor receptor 2 (TGFBR2) in 28 patients with AVD associated with AAD (mean age 60.6 years), in 29 patients (68.9 years) with AVD without AAD, and in 30 healthy controls (45.3 years). The serum levels of MMPs were determined by ELISA. Further, we focused on genetic screening of the TGFBR2 gene. Plasma MMP-2 concentrations were significantly higher in the groups of patients compared to the controls: median 1315.0 (mean 1265.2 ± SD 391.3) in AVD with AAD, 1240.0 (1327.8 ± 352.5) in AVD without AAD versus 902.5 (872.3 ± 166.2) ng/ml in the healthy controls, in both cases P < 0.001. The serum levels of MMP-9 were significantly higher in AVD with AAD patients [107.0 (202.3 ± 313.0)] and in AVD without AAD patients [107.0 (185.8 ± 264.3)] compared to the healthy controls [14.5 (21.2 ± 24.8) ng/ml], in both cases P < 0.001. No significant correlation was observed between plasma MMP-2 and MMP-9 and ascending aorta diameter. Genetic screening did not reveal any variation in the TGFBR2 gene in the patients. Measurement of MMP levels is a simple and relatively rapid laboratory test that could be used as a biochemical indicator when evaluated in combination with imaging techniques.

Poly(HEMA) brushes emerging as a new platform for direct detection of food pathogen in milk samples.

Surface plasmon resonance (SPR) biosensors capable of in real time detection of Cronobacter at concentrations down to 106 cells mL⁻¹ in samples of consumer fresh-whole fat milk, powder whole-fat milk preparation, and powder infant formulation were developed for the first time. Antibodies against Cronobacter were covalently attached onto polymer brushes of poly(2-hydroxyethyl methacrylate) (poly(HEMA)) grafted from the SPR chip surface. The lowest detection limit, 104 cells mL⁻¹, was achieved in phosphate buffered saline (pH 7.4) with sensors prepared by covalent immobilization of the same antibodies onto a self assembled monolayer (SAM) of hexa(ethylene glycol) undecanethiol (EG6). However, when the EG6 based sensors were challenged with milk samples the non-specific response due to the deposition of non-targeted compounds from the milk samples was much higher than the specific response to Cronobacter hampering the detection in milk. Similar interfering fouling was observed on antifouling polymer brushes of hydroxy-capped oligoethylene glycol methacrylate and even a 10 times higher fouling was observed on the widely used SAM of mixed hydroxy- and carboxy-terminated alkanethiols. Only poly(HEMA) brushes totally suppressed the fouling from milk samples. The robust well-controlled surface initiated atom transfer radical polymerization of HEMA allowed the preparation of highly dense brushes with a minimal thickness so that the capture of antigens by the antibodies immobilized on the brush layer could take place close to the gold SPR surface to provide a stronger optical response while the fouling was still suppressed. A minimum thickness of 19 nm of poly(HEMA) brush layer was necessary to suppress completely non-specific sensor response to fouling from milk.

Antitumor activity of apoptotic nuclease TBN1 from L. esculentum.

Nuclease from tomato (TBN1) was produced by in planta biotechnology purified and tested for its anticarcinogenic properties. The nuclease was cytostatic after its intratumoral administration to nude mice bearing human melanoma or prostate carcinoma or after tumor targeting by TBN1 administration intravenously as conjugate with polyethylene glycol (PEG). Inhibitory effects of TBN1 on tumor growth were comparable to effects of bovine seminal RNase (BS-RNase), but the inhibition was reached at about ten times lower protein concentration. Simultaneously, TBN1 exhibited a lower degree of embryotoxicity compared to BS-RNAse and other nucleases. TBN1 showed significant stability in vivo, because it was readily detected after its administration intratumorally or intravenously by the fluorescence methods. Intravenous administration of TBN1-PEG caused significant inhibition of tumor proliferation without obvious degenerative changes, while direct administration of TBN1 into melanoma tumors led to rapid tumor tissue degeneration. The fact can be essential for the mode of TBN1 biological action that mature nuclease is a small (36 kDa) thermostable glycoprotein that has ability to destroy human 28S, 18S, 7S and 5.8S RNA, circular RNAs, double-stranded RNA in vitro and shows DNase and 3'nucleotidase activities.

[Long-term improvement of fasting glycaemia after switching basal insulin from NPH to determir in children with type 1 diabetes: a 1-year multicentre study]. / Zmena bazálního inzulínu z NPH na inzulínový analog detemir vede k dlouhodobému zlepsení ranní glykémie nalacno u detí s diabetes mellitus 1. typu: výsledky rocní multicentrické studie.

BACKGROUND: Paper presents an evaluation of diabetes control after switching from NPH insulin to detemir in children with type 1 diabetes. METHODS AND RESULTS: We performed a non-randomized, observational, multicentre study on the first group of children whose treatment switched from NPH to insulin detemir in four centers of paediatric diabetes. A total of 72 children (39 boys and 33 girls) were included in the analysis. The average age at intervention was 10.6 +/- 4.7 yrs, the average age at diabetes onset was 6.2 +/- 4.3 yrs. Diabetes control was assessed 3 months prior to the switch and subsequently during 3-month intervals. RESULTS: Mean HbA(1c) decreased from 6.9% at baseline to 6.4% after 3 months of detemir therapy (p = 0.0003). However, in the next months we observed a trend for increasing the HbA(1c), and no statistically significant difference in HbA(1c) was observed at the 6, 9 and 12 months visits vs. baseline. Fasting glycaemia decreased significantly after 3 months of treatment with detemir in comparison with the baseline (the mean value of the difference was 2.1 mmol/l, CI 95% 1.5-2.6, p = 1.4*10(-10)), and this effect was detectable during all the observational period (month 12 vs. baseline 2.6 mmol/l, p < 10(-8)). CONCLUSIONS: Switching basal insulin from NPH insulin to detemir resulted in a short-term improvement of HbA(1c), and a long-term decreasing of fasting glycaemia.

Characterization of rabbit antibodies for immunochemical detection of Yersinia enterocolitica.

Rabbit IgG raised against whole cells of Yersinia enterocolitica O:3, O:9 and against a group of pathogenic Y. enterocolitica strains (serotypes O:3, O:5,27, O:8. and O:9) were prepared. The antibody limiting titers were within the range of 1:9.5 x 10(4)-1:7.5 x 10(5). The immunoblotting analysis of Yersinia lipopolysacchides separated by SDS-PAGE showed that IgG against the single serotype O:3 interacted with high-molar-mass LPS of O:3 whereas other antibodies were bound to low-molar-mass LPS of serotypes O:3, O:5,27, O:9 and strain Y. enterocolitica (CNCTC Y 2/68). IgG against the group of pathogenic serotypes also weakly interacted with low-molar-mass LPS of serotypes O:5, O:6,30, and O:10. The cross-reactivity of the antibodies with Y. pseudotuberculosis Ia and/or Y. rohdei b, d, e, f, i, which was observed by means of dot-blotting procedure using the whole bacterial cells as an antigen, was shown not to be caused by LPS of these bacteria. The prepared antibodies were used in the development of indirect competitive ELISA. At the optimum concentration of the immunoreactants the detection limits were within the range of 3-7 x 10(6) colony-forming units per mL.

Mung bean sprout (Phaseolus aureus) nuclease and its biological and antitumor effects.

Bovine seminal ribonuclease (BS RNase), a dimeric homolog of bovine pancreatic ribonuclease (RNase A), is known to display special biological activities namely cytotoxicity for human tumor cells. Because some plant ribonucleases have a similar mass weight and structure as the animal ribonuclease, effects of a commercial product of Mung bean (Phaseolus aureus) nuclease (PhA) were studied on proliferation of ML-2 human tumor cells, as well as it's aspermatogenic, embryotoxic, immunogenic, and immunosuppressive activity, and therapeutic efficiency in athymic mice bearing human melanoma tumor. Concerning the antiproliferative activity, PhA nuclease was almost non-effective in vitro on ML-2 cells and also immunosuppressive activity on human lymphocyte in mixed culture was very low compared to that of BS RNase. However, significant antitumor activity was detected on human melanoma tumor after intratumoral or intraperitoneal administration into the mice. Furthermore conjugate of PhA nuclease with polyethylene glycol (PEG) injected seven times at the dose of 10 microg intraperitoneally showed identical antitumor activity as that of bovine seminal ribonuclease (BS RNase) injected by the same way at ten times higher dose. Both PhA and BS RNases exerted strong aspermatogenic effect on the width of spermatogenic layers while RNase A administration at ten times higher concentration was ineffective. PhA nuclease when compared by means of antibody cross reaction with RNase A, BS RNase and wheat leaf neutral RNase (WLN-RNase) was found to be immunologically similar to RNase A and WLN-RNase, meanwhile BS RNase showed much higher antigenicity in comparison with them.

Hypertrophic cardiomyopathy--molecular genetic analysis of exons 9 and 11 of the TNNT2 gene in Czech patients.

OBJECTIVES: Our research is a pilot study that specializes in the molecular genetic investigation of the TNNT2 gene in Czech patients with HCM/FHC disease. This study was initiated with exons 9 and 11 of TNNT2 because of their crucial role in the binding ability of cardiac troponin T to alpha-tropomyosin, and continued with analyses in other regions of the gene. METHODS: Hundred and eighty-one Czech probands with HCM/FHC were enrolled in this study. The study group consisted of 24 families with FHC and probands without FHC history but with HCM diagnosis. The clinical diagnosis was based on echocardiography. DNA was isolated from peripheral blood lymphocytes and subsequently analyzed by the polymerase chain reaction (PCR), followed by DNA sequencing analyses, which were cross-sequenced. RESULTS: The DeltaGlu160 mutation was observed in a sequence of the TNNT2 gene in a patient with the severe form of hypertrophic cardiomyopathy. No sequence alteration was found in exons 9 and 11 of the TNNT2 gene found in the rest of the DNA samples. CONCLUSION: The DeltaGlu160 mutation was observed in patients with severe forms of hypertrophic cardiomyopathy. This region is responsible for binding troponin T to alpha-tropomyosin. This mutation may lead to functional and structural effects on the troponin T protein. Mutations in this region are reported relatively rarely and therefore it was unique to observe the DeltaGlu160 mutation in our study.

Some biological actions of PEG-conjugated RNase A oligomers.

Previously we have shown that monomeric RNase A has no significant biological activity, whereas its oligomers (dimer to tetramer) prepared by lyophilizing from 50% acetic acid solutions, show remarkable aspermatogenic and antitumor activities. Furthermore, conjugates prepared by chemical binding of native RNase A to polyethylene glycol (PEG) have shown a significant aspermatogenic and antitumor activities. In this work we show that the chemical conjugation of PEG to the RNase A C-dimer, and to the two RNase A trimers (NC-trimer and C- trimer) decreases the aspermatogenic activity of the oligomers while increasing their inhibitory activity on the growth of the human UB900518 amelanotic melanoma transplanted in athymic nude mice. Moreover, the PEG-conjugated RNaseA oligomers are devoid, like the free oligomers, of any embryotoxic activity.

Development of an indirect competitive ELISA for detection of Campylobacter jejuni subsp.jejuni O:23 in foods.

An indirect enzyme immunoassay for rapid detection of Campylobacter jejuni subsp. jejuni 0:23 has been developed. Optimum concentrations of immobilized cells, polyclonal chicken IgY, and rabbit anti-IgY antibody-horseradish peroxidase conjugate were 3.1 CFU/nL, 10 microg/mL, and 8 microg/mL, respectively. Under such conditions, the detection limit reached 50 CFU/microL, limit of quantification being 480 CFU/microL. By testing 5 chromogens, viz. 1,2-benzenediamine, 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid), 3,3',5,5'-tetramethylbenzidine, bi(4,4'-anisidine) and 3-methyl-2-benzothiazolinone hydrazone, in horseradish peroxidase substrate, 1,2-benzenediamine or 3,3',5,5'-tetramethylbenzidine as H-donors in the enzyme substrate provided the highest ELISA sensitivity. The applied polyclonal antibody was specific for homogeneous antigen. The cross-reactions were observed only with one strain of C. sputorum subsp. sputorum (21.5 %) and with G+ bacterium Micrococcus luteus (6.1 %). Preliminary tests have been performed with a limited number of artificially contaminated food samples. No matrix effects on the ELISA sensitivity were observed. The results (by means of ELISA) were comparable with those given by both a standard cultivation method performed according to CSN ISO 10272 and commercially available Singlepath Campylobacter GLISA-Rapid Test.

The detection of human beta 2-microglobulin by grating coupler immunosensor with three dimensional antibody networks.

Immunosensors for the detection of human beta 2-microglobulin (B2M) were prepared by immobilisation of covalently crosslinked assemblies containing various numbers of molecular layers of monoclonal antibody against B2M (anti-B2M) on the surface of a Ta2O5 grating coupler sensor. The immobilisation procedure consisted of repeated successive adsorption of anti-B2M and dextran sulfate (DS) followed by glutaraldehyde (GA) crosslinking of anti-B2M and washing out DS. The flexibility of the resulting anti-B2M networks was evaluated from the sensor response to the reversible expansion and contraction of the networks induced by changing pH of the ambient solution. A decreased GA concentration and the use of a higher-molecular-mass DS increased the network flexibility. The sensor sensitivity to B2M increased with increasing flexibility of the antibody networks and with increasing number of anti-B2M molecular layers, indicating that B2M can penetrate inside the antibody network.

Immobilisation of multilayer bioreceptor assemblies on solid substrates.

Multilayer assemblies were prepared by alternating adsorption of monolayers of monoclonal antibody against horse radish peroxidase (anti-HRP) and dextran sulfate (DS) on solid supports at acid pH. After crosslinking with glutaraldehyde, DS was washed out of the film with buffered physiological saline, while the antibody remained immobilised on the support. Assembly was monitored in situ on germanium supports by infrared multi-internal reflection spectroscopy. The binding capacity of the immobilised antibodies for HRP was measured by ELISA and by optical waveguide light mode spectroscopy. The activity of an immobilised anti-HRP bilayer was approximately twice that of a monolayer prepared by simple physiosorption. An addition of further anti-HRP layers could increase the activity only up to 2.5 of the monolayer activity independently of a number of layers in the assembly. The non-specific adsorption of proteins from human blood plasma was three times lower on the immobilised anti-HRP multilayer film than on the surface covered only with a physiosorbed anti-HRP monolayer.

Effect of salt concentration gradient on separation of different types of specific immunoglobulins by ion-exchange chromatography on DEAE cellulose.

A three-stage process, consisting of an ammonium sulfate precipitation step, dialysis desalination with microporous anion-exchange Neosepta membranes and anion-exchange chromatography on DEAE-cellulose DE-52 was used for the isolation of mouse monoclonal antibodies specific against different antigens. The ascites fluids contained monoclonal antibodies against human IgG, against horseradish peroxidase and against the heavy chain of human IgM. The effect of the salt concentration gradient in the elution buffer was examined with the aim of optimizing chromatographic conditions. The quality of separation of protein zones was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions. The immunoreactivity of purified monoclonal antibodies was determined by enzyme-linked immunosorbent assay using a solid-phase adsorbed antigens against which each monoclonal antibody type was directed.

Separation of specific immunoglobulin. 1. Desalination using a membrane system.

Separation of the ammonium sulfate precipitated protein fraction of mouse ascitic fluid, containing the specific immunoglobulin (pI 6.7-6.8; molecular weight 180000), from ammonium sulfate was investigated by means of non-traditional dialysis, based on the difference in diffusion rates of small and large molecules through porous membranes. The experiments were carried out in spiral membrane modules equipped with a Neosepta (AM-2 or ACS-SB) anion exchange membrane and a microfiltration membrane (Synpore or Sartorius). To enhance the driving force for penetration of ammonium sulfate and low-molecular-weight components from solution of ascitic protein fraction into water, a counterpressure was imposed on the side of microfiltration membrane. The flow rate, counterpressure and the pore sizes of microfiltration membranes had a significant effect on the separation process, as expected. The type of the anion exchange membrane had only a small effect. This process makes it possible to desalt the immunoglobulin fraction with high purity and yield in a few hours instead of 5 days.

[Palpation and sonographic evaluation of the size of the thyroid gland]. / Palpacní a sonografické hodnocení velikosti stítné zlázy.

The author compared in 88 13-year-old children evaluation of the size of the thyroid gland by palpation and sonography. Evaluation by the two methods was in agreement in 64 cases (73%) and controversial in 24 cases (27%). Palpation led in 24 instances to overestimation of the size and in 7 children (8%) to underestimation, as compared with sonography. Sonography is an essential method for evaluation of the thyroid gland in children.

[Muscular exertion--"embarrassment" in the evaluation of aminotransferases and creatine kinase serum levels]. / Svalová zátez-- "rozpaky" pri hodnocení hladiny aminotransferáz a kreatinkinázy v séru.

The authors draw attention to the problem of elevated values of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and creatine kinase (CK) serum activities in adolescents which they encountered recently in two patients hospitalized in their clinic. In common practice it is important when evaluating results of ALT, AST and CK in serum to take into account the effect of physical load, encountered frequently in this age group of adolescents.