DeltaF508 CFTR Hetero- and Homozygous Paediatric Patients with Cystic Fibrosis Do Not Differ with Regard to Nutritional Status.

The purpose of this study was to compare the nutritional status between deltaF508 CFTR hetero- and homozygous paediatric patients with cystic fibrosis. We assessed the percentage profiles of fatty acids measured in erythrocyte membranes and the serum levels of vitamins A, D3, E and K1 in the studied groups. We also measured the weights and heights and calculated the body mass indexes (BMIs). The studied groups consisted of 34 heterozygous and 30 homozygous patients. No statistically significant differences were found in the serum vitamins or erythrocyte membrane fatty acid profiles between the hetero- and homozygous patient groups, except for heptadecanoic acid (p = 0.038). The mean percentiles of height, weight and BMI did not differ significantly between the two groups. The homozygous and heterozygous paediatric patients with cystic fibrosis were similar in terms of their nutritional statuses.

Serum chemerin level, cytokine profile and nutritional status in children with cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) is characterized by malnutrition and chronic inflammation predominantly occurring in lungs. Evidence suggests a relation between inflammatory activity and nutritional status. Proinflammatory cytokines, playing crucial role in pulmonary destruction in CF, are regarded as a component of the pathogenesis of illness-related malnutrition. Chemerin - a novel marker of a crosstalk between nutrition and inflammation, has not been investigated in children with cystic fibrosis. The aim of this study was to determine serum level of chemerin, interleukin-1b (IL-1b), interleukin-6 (IL-6), tumor necrosing factor α (TNF-α) and interleukin-10 (IL-10) and to verify if they correlate with the nutritional status in children with CF. METHODS: The study included 72 pediatric patients with cystic fibrosis. The control group was comprised of 30 healthy children. Nutritional status parameters: Body Mass Index (BMI), fat mass percentage (FM %) and fat free mass percentage (FFM%) have been assessed in all the subjects basing on bioimpedance and anthropometry according to Slaughter. Serum concentrations of chemerin and cytokines were estimated with ELISA. RESULTS: No statistically significant difference in serum chemerin was found between the studied and the control group. We have documented a significantly higher level of IL-1b, IL-6, TNF-α and IL-10 in CF patients when compared to healthy controls. Neither the chemerin nor the cytokine levels correlated with parameters of nutritional status in our cohort. No statistically significant correlation was found between the serum chemerin and the inflammatory cytokines: IL-1b, IL-6, and TNFα. CONCLUSIONS: Our results show that chemerin is not associated with the nutritional status in children with cystic fibrosis. Chemerin has no impact on the levels of IL-1b, IL-6, TNFα in CF patients. IL-1b, IL6, TNFα and also IL10 are upregulated in cystic fibrosis.

Serum chemerin in children with excess body weight may be associated with ongoing metabolic complications - A pilot study.

PURPOSE: The aim of this study was to verify if serum chemerin in children correlates with body weight, fat mass, selected inflammatory markers, parameters of liver function, lipid and glucose metabolism. MATERIALS AND METHODS: The study included children aged 5-17 years with normal body weight (<85th BMI percentile, n=43) or overweight (≥85th BMI percentile, n=58). Serum concentrations of chemerin were determined with ELISA. RESULTS: Children with excess body weight presented with significantly higher serum concentrations of chemerin. Serum chemerin correlated positively with body weight, absolute BMI and its percentile, fat mass, systolic blood pressure, CRP, ALT, insulin and HOMA-IR. CONCLUSIONS: Serum level of chemerin may serve as a measure of ongoing obesity-related inflammation, early marker of subclinical liver dysfunction and metabolic syndrome in overweight pediatric patients.

The LC-MS method for the simultaneous analysis of selected fat-soluble vitamins and their metabolites in serum samples obtained from pediatric patients with cystic fibrosis.

Cystic fibrosis (CF) is one of the most common genetic diseases in children and affects mainly respiratory and digestive system functions. Despite the prolonged supplementation of vitamins, malnutrition manifested by poor growth and weight loss in children is a major complication in CF related to pancreatic insufficiency and difficulty in absorbing fat-soluble vitamins. In the present study, we have developed and validated a sensitive and accurate high-performance liquid chromatography coupled to mass spectrometry (LC-MS) method for the simultaneous quantification of three fat-soluble vitamins (A, E and K1) and two vitamin D3 active metabolites: 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 in serum samples obtained from pediatric patients with CF. In optimized conditions, the LC-MS method was highly sensitive and presented excellent linearity with a regression coefficient higher than 0.999. The accuracy was in the range of 87.55-95.58 % for all analytes. The precision of the method, expressed as% RSD, ranged from 1.36 % to 3.74 % as the intra-day variability and from 2.35 % to 7.98 % as the inter-day precision for all the studied compounds. Sample preparation included a protein precipitation step with the use of methanol followed by liquid-liquid extraction with n-hexane. The statistical analysis (t-test and principal component analysis (PCA)) of the obtained results revealed significant changes in the plasma level of the analyzed compounds, with 25-hydroxyvitamin D3, vitamin E and K1 present at extremely low concentrations in patients with cystic fibrosis in comparison to healthy controls. The elaborated method reached the expectations for the fast and reliable assessment of fat-soluble vitamin status in children with cystic fibrosis in order to diagnose the disease and monitor the treatment process.

Capillary electromigration techniques as tools for assessing the status of vitamins A, C and E in patients with cystic fibrosis.

The purpose of this work is the evaluation of the nutritional status of patients with cystic fibrosis (CF), based on the level of vitamin C in urine and vitamins A and E in serum, using the fast, selective and fully automated micellar electrokinetic capillary chromatographic (MEKC) and microemulsion electrokinetic capillary chromatographic (MEEKC) methods. The optimization of parameters affecting the electrophoretic separation provided adequate separation of the analytes of interest in the short time of 8 min (MEKC) and 20 min (MEEKC). The developed methods were practical applications to evaluate the levels of vitamins A, C and E in real samples from 28 children suffering from cystic fibrosis and from 10 healthy volunteers. Based on the mean concentration values obtained in the two groups, it can be seen that the levels of each vitamin were lower in patients with CF than in healthy volunteers. In the case of vitamin E, these differences in both groups were statistically significant, while the disproportion of concentrations of vitamins A and C in both the studied groups were not so relevant. On the other hand, a principal component analysis (PCA) confirmed that in some patients with CF the concentration of vitamin A was significantly lower than in the control group. Thus, the future evaluation of the status of fat-soluble vitamins in the longer term for the evaluation of the nutritional status of patients with CF should be continued. The presented CE methods can become useful tools for the evaluation of the nutritional status of patients with CF.

[Engineered spider silk: the intelligent biomaterial of the future. Part I]. / Inzynierowany jedwab pajeczy: inteligentny biomaterial przyszlosci. Czesc I.

The unique properties of spider silk such as strength, extensibility, toughness, biocompatibility and biodegradability are the reasons for the recent development in silk biomaterial technology. For a long time scientific progress was impeded by limited access to spider silk. However, the development of the molecular biology strategy was a breaking point in synthetic spider silk protein design. The sequences of engineered spider silk are based on the consensus motives of the corresponding natural equivalents. Moreover, the engineered silk proteins may be modified in order to gain a new function. The strategy of the hybrid proteins constructed on the DNA level combines the sequence of engineered silk, which is responsible for the biomaterial structure, with the sequence of polypeptide which allows functionalization of the silk biomaterial. The functional domains may comprise receptor binding sites, enzymes, metal or sugar binding sites and others. Currently, advanced research is being conducted, which on the one hand focuses on establishing the particular silk structure and understanding the process of silk thread formation in nature. On the other hand, there are attempts to improve methods of engineered spider silk protein production. Due to acquired knowledge and recent progress in synthetic protein technology, the engineered silk will turn into intelligent biomaterial of the future, while its industrial production scale will trigger a biotechnological revolution.

[Engineered spider silk: the intelligent biomaterial of the future. Part II]. / Inzynierowany jedwab pajeczy: inteligentny biomaterial przyszlosci. Czesc II.

The development and progress in engineered spider silk manufacturing has enabled its practical application. Recombinant spider silk can assemble in several morphological forms such as films, hydrogels, fibers, scaffolds, microcapsules, and micro- and nanospheres. The in vitro fiber formation takes place by mimicking the natural spinning process in the spider spinning gland: in the presence of phosphate ions and dragging forces. Films are obtained by evaporation of solvent from the silk solution, while the result of evaporation of the solvent in the presence of porogens is a silk scaffold. Hydrogels are formed by spontaneous polymerization of silk particles in solutions at low pH. The silk film assembled at the interface of two immiscible phases forms microcapsules. The smallest of the described forms--silk spheres--are obtained by salting out the silk protein solution after addition of the phosphate ions. Common properties of the silk biomaterials are biocompatibility and biodegradability, which make them suitable for a number of applications in medicine and pharmacy. Moreover, the strategy of hybrid proteins which provides the desired function to biomaterial will further expand their potential use.

Mouse ataxin-3 functional knock-out model.

Spinocerebellar ataxia 3 (SCA3) is a genetic disorder resulting from the expansion of the CAG repeats in the ATXN3 gene. The pathogenesis of SCA3 is based on the toxic function of the mutant ataxin-3 protein, but the exact mechanism of the disease remains elusive. Various types of transgenic mouse models explore different aspects of SCA3 pathogenesis, but a knock-in humanized mouse has not yet been created. The initial aim of this study was to generate an ataxin-3 humanized mouse model using a knock-in strategy. The human cDNA for ataxin-3 containing 69 CAG repeats was cloned from SCA3 patient and introduced into the mouse ataxin-3 locus at exon 2, deleting it along with exon 3 and intron 2. Although the human transgene was inserted correctly, the resulting mice acquired the knock-out properties and did not express ataxin-3 protein in any analyzed tissues, as confirmed by western blot and immunohistochemistry. Analyses of RNA expression revealed that the entire locus consisting of human and mouse exons was expressed and alternatively spliced. We detected mRNA isoforms composed of exon 1 spliced with mouse exon 4 or with human exon 7. After applying 37 PCR cycles, we also detected a very low level of the correct exon 1/exon 2 isoform. Additionally, we confirmed by bioinformatic analysis that the structure and power of the splicing site between mouse intron 1 and human exon 2 (the targeted locus) was not changed compared with the native mouse locus. We hypothesized that these splicing aberrations result from the deletion of further splicing sites and the presence of a strong splicing site in exon 4, which was confirmed by bioinformatic analysis. In summary, we created a functional ataxin-3 knock-out mouse model that is viable and fertile and does not present a reduced life span. Our work provides new insights into the splicing characteristics of the Atxn3 gene and provides useful information for future attempts to create knock-in SCA3 models.

In vitro multicompartmental bladder model for assessing blockage of urinary catheters: effect of hydrogel coating on dynamics of Proteus mirabilis growth.

OBJECTIVES: To investigate the effect of a hydrogel coating on the dynamics of bacterial growth in laboratory models of the catheterized bladder. Infection of the urinary tract by Proteus mirabilis can result in catheter blockage by crystalline biofilm, a common complication in patients undergoing long-term bladder catheterization. METHODS: Two series of catheters were tested in the infected bladder models: test series 1, silicone catheters impregnated with triclosan (0.5%, 1%, 4%), or silicone catheters with 0% triclosan impregnated with pure solvents and hydrogel coated (based on polyvinylpyrrolidone); and test series 2, silicone catheters, hydrogel-coated with hydrogel plus iodine (polyvinylpyrrolidone plus iodine) or hydrogel plus polyhexamethylene biguanide. Test series 1 was used to detect the influence of triclosan, solvents, impregnation time, and the presence of hydrogel coating on the interval to catheter blockage by P. mirabilis biofilm. The experiments with test series 2 focused on the dynamic interaction of the hydrogel coating and biofilm formation. The division of the catheterized bladder model into 3 sampling zones brought more information about the spatial segregation of the bacterial population. RESULTS: The bacteriostatic efficiency of the water-soluble polyhexamethylene biguanide and polyvinylpyrrolidone iodine complex was limited to the first hours after catheterization. Only catheters containing triclosan resisted encrustation for significantly longer (up to >7 days). In contrast, the uncoated and hydrogel-coated catheters were occluded by day 2. CONCLUSIONS: The hydrogel layer can increase aggregation of the planktonic cells and newly nucleated crystals, leading to even faster catheter blockage than in the case of uncoated silicone. However, the addition of active agents were able to suppress this negative effect.

Determination of urethral catheter surface lubricity.

Device for in-vitro measurement of static and kinetic friction coefficient of catheter surface was developed. Tribometer was designed and constructed to work with exchangeable counter-faces (polymers, tissue) and various types of tubes, in wet conditions in order to mimic in-vivo process. Thus seven commercially available urethral catheters, made from vinyl polymers, natural latex with silicone coating, all-silicone or hydrogel coated, and one made from polyvinylchloride with polyurethane/polyvinylpyrrolidone hydrogel coating obtained in our laboratory, were tested against three various counter faces: polymethacrylate (organic glass), inner part of porcine aorta and porcine bladder mucosa. Additionally, the hydrophility/hydrophobity of tested catheters was stated via water wetting contact angle measurement. Super-hydrophilic biomaterials revealed low friction on tissue and hydrophobic counter-face; slightly hydrophobic showed higher friction in both cases, while more hydrophobic manifested low friction on tissue but high on hydrophobic polymer. The smoothest friction characteristic was achieved in all cases on tissue counter-faces. The measured values of the static coefficient of friction of catheters on bladder mucosa counter-face were as follows: the highest (0.15) for vinyl and siliconised latex catheters and 3 folds lower (0.05) for all-silicone ones. Hydrogel coated catheters exhibited the lowest static and kinetic friction factors.