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Although human clinical studies have suggested probiotic effects on blood glucose levels, knowledge about molecular mechanisms is still scarce. To test the hypothesis that selected Lactobacillus probiotic bacteria could regulate the activity of enterocyte glucose transporters, we aimed to measure in vitro effects of selected Lactobacillus probiotic bacteria on transcription and translation of intestinal glucose transporters sodium-dependent glucose cotransporter 1 (SGLT1) and glucose transporter 2 (GLUT2) as well as transepithelial glucose transport. Lactobacillus plantarum strains (PCS20 and PCS26), Lactobacillus rhamnosus GG (LGG) (ATCC 53103) and Lactobacillus acidophilus (L acidophilus) (ATCC 4356) were co-cultivated with noncarcinogenic porcine enterocytes (CLAB) and human epithelial colorectal adenocarcinoma cells (Caco-2) (ATCC HTB-37). Changes in transcription and expression of SGLT1 and GLUT2 were strain and cell line-specific. In CLAB, LGG was the most potent SGLT1 up-regulator, and PCS26 the most potent down-regulator of GLUT2 transcription, which was also reflected on the protein level. In Caco-2, all tested strains tended to downregulate GLUT2 gene expression, while L acidophilus most effectively reduced GLUT2 protein levels. Statistically significant effect of PCS26 and L acidophilus on GLUT2 molecular and protein levels in CLAB and Caco-2 cell lines, respectively, was also followed by a decreased rate of transepithelial glucose transport. Careful selection of specific Lactobacillus probiotic strains could be used to downregulate glucose absorption in intestinal epithelial cells and thereby could be beneficial as a support treatment of pathologies related to glucose homeostasis.

Familial Progressive Hyper‐ and Hypopigmentation is a pigmentary disorder characterized by a mix of hypo‐ and hyperpigmented lesions, café‐au‐lait spots and hypopigmented ash‐leaf macules. The disorder was previously linked to KITLG and various mutations have been reported to segregate in different families. Furthermore, association between KITLG mutations and malignancies was also suggested. Exome and SANGER sequencing were performed for identification of KITLG mutations. Functional in silico analyses were additionally performed to assess the findings. We identified a de novo mutation in exon 4 of KITLG gene causing NM_000899.4:c.[329A>T] (chr12:88912508A>T) leading to NP_000890.1:p.(Asp110Val) substitution in the 3rd alpha helix. It was predicted as pathogenic, located in a conserved region and causing an increase in hydrophobicity in the KITLG protein. Our findings clearly confirm an additional hot spot of KITLG mutations in the 3rd alpha helix, which very likely increases the risk of malignancies. To our knowledge the present study provides the strongest evidence of association of the KITLG mutation with both Familial Progressive Hyper‐ and Hypopigmentation and malignancy due to its’ location on somatic cancer mutation locus. Additionally we also address difficulties with classification of the unique phenotype and propose a subtype within broader diagnosis.

Background: The polymorphism Arg16 in β2-adrenergic receptor (ADRB2) gene has been associated with an increased risk of exacerbations in asthmatic children treated with long-acting β2-agonists (LABA). However, it remains unclear whether this increased risk is mainly attributed to this single variant or the combined effect of the haplotypes of polymorphisms at codons 16 and 27. Objective: We assessed whether the haplotype analysis could explain the association between the polymorphisms at codons 16 (Arg16Gly) and 27 (Gln27Glu) in ADRB2 and risk of asthma exacerbations in patients treated with inhaled corticosteroids (ICS) plus LABA. Methods: The study was undertaken using data from 10 independent studies (n = 5903) participating in the multi-ethnic Pharmacogenomics in Childhood Asthma (PiCA) consortium. Asthma exacerbations were defined as asthma-related use of oral corticosteroids or hospitalizations/emergency department visits in the past 6 or 12 months prior to the study visit/enrolment. The association between the haplotypes and the risk of asthma exacerbations was performed per study using haplo.stats package adjusted for age and sex. Results were meta-analysed using the inverse variance weighting method assuming random-effects. Results: In subjects treated with ICS and LABA (n = 832, age: 3–21 years), Arg16/Gln27 versus Gly16/Glu27 (OR: 1.40, 95% CI: 1.05–1.87, I2 = 0.0%) and Arg16/Gln27 versus Gly16/Gln27 (OR: 1.43, 95% CI: 1.05–1.94, I2 = 0.0%), but not Gly16/Gln27 versus Gly16/Glu27 (OR: 0.99, 95% CI: 0.71–1.39, I2 = 0.0%), were significantly associated with an increased risk of asthma exacerbations. The sensitivity analyses indicated no significant association between the ADRB2 haplotypes and asthma exacerbations in the other treatment categories, namely as-required short-acting β2-agonists (n = 973), ICS monotherapy (n = 2623), ICS plus leukotriene receptor antagonists (LTRA; n = 338), or ICS plus LABA plus LTRA (n = 686). Conclusion and clinical relevance: The ADRB2 Arg16 haplotype, presumably mainly driven by the Arg16, increased the risk of asthma exacerbations in patients treated with ICS plus LABA. This finding could be beneficial in ADRB2 genotype-guided treatment which might improve clinical outcomes in asthmatic patients.

Inhaled corticosteroids (ICS) are the most common asthma controller medication. An important contribution of genetic factors in ICS response has been evidenced. Here, we aimed to identify novel genetic markers involved in ICS response in asthma. A genome-wide association study (GWAS) of the change in lung function after 6 weeks of ICS treatment was performed in 166 asthma patients from the SLOVENIA study. Patients with an improvement in lung function ≥8% were considered as ICS responders. Suggestively associated variants (p-value ≤ 5 × 10−6) were evaluated in an independent study (n = 175). Validation of the association with asthma exacerbations despite ICS use was attempted in European (n = 2681) and admixed (n = 1347) populations. Variants previously associated with ICS response were also assessed for replication. As a result, the SNP rs1166980 from the ROBO2 gene was suggestively associated with the change in lung function (OR for G allele: 7.01, 95% CI: 3.29–14.93, p = 4.61 × 10−7), although this was not validated in CAMP. ROBO2 showed gene-level evidence of replication with asthma exacerbations despite ICS use in Europeans (minimum p-value = 1.44 × 10−5), but not in admixed individuals. The association of PDE10A-T with ICS response described by a previous study was validated. This study suggests that ROBO2 could be a potential novel locus for ICS response in Europeans.

RationaleSubstantial variability in response to asthma treatment with inhaled corticosteroids (ICS) has been described among individuals and populations, suggesting the contribution of genetic factors. Nonetheless, only a few genes have been identified to date. We aimed to identify genetic variants associated with asthma exacerbations despite ICS use in European children and young adults and to validate the findings in non-Europeans. Moreover, we explored whether a gene-set enrichment analysis could suggest potential novel asthma therapies.MethodsA genome-wide association study (GWAS) of asthma exacerbations was tested in 2681 children of European descent treated with ICS from eight studies. Suggestive association signals were followed up for replication in 538 European asthma patients. Further evaluation was performed in 1773 non-Europeans. Variants revealed by published GWAS were assessed for replication. Additionally, gene-set enrichment analysis focused on drugs was performed.Results10 independent variants were associated with asthma exacerbations despite ICS treatment in the discovery phase (p≤5×10−6). Of those, one variant at theCACNA2D3-WNT5Alocus was nominally replicated in Europeans (rs67026078; p=0.010), but this was not validated in non-European populations. Five other genes associated with ICS response in previous studies were replicated. Additionally, an enrichment of associations in genes regulated by trichostatin A treatment was found.ConclusionsThe intergenic region ofCACNA2D3andWNT5Awas revealed as a novel locus for asthma exacerbations despite ICS treatment in European populations. Genes associated were related to trichostatin A, suggesting that this drug could regulate the molecular mechanisms involved in treatment response.

Objectives Combining of genomic data of different pathologies as a single phenotype has emerged as a useful strategy to identify genetic risk loci shared among immune-mediated diseases. Our study aimed to increase our knowledge of the genetic contribution to Kawasaki disease (KD) and IgA vasculitis (IgAV) by performing the first comprehensive large-scale analysis on the genetic overlap between them. Methods A total of 1190 vasculitis patients and 11 302 healthy controls were analysed. First, in the discovery phase, genome-wide data of 405 KD patients and 6252 controls and 215 IgAV patients and 1324 controls, all of European origin, were combined using an inverse variance meta-analysis. Second, the top associated polymorphisms were selected for replication in additional independent cohorts (570 cases and 3726 controls). Polymorphisms with P-values ≤5 × 10−8 in the global IgAV–KD meta-analysis were considered as shared genetic risk loci. Results A genetic variant, rs3743841, located in an intron of the NAGPA gene, reached genome-wide significance in the cross-disease meta-analysis (P = 8.06 × 10−10). Additionally, when IgAV was individually analysed, a strong association between rs3743841 and this vasculitis was also evident [P = 1.25 × 10−7; odds ratio = 1.47 (95% CI 1.27, 1.69)]. In silico functional annotation showed that this polymorphism acts as a regulatory variant modulating the expression levels of the NAGPA and SEC14L5 genes. Conclusion We identified a new risk locus with pleiotropic effects on the two childhood vasculitides analysed. This locus represents the strongest non-HLA signal described for IgAV to date.

Background In some patients securing the peripheral intravenous cannula (PIVC) with a standard adhesive dressing can be difficult because of sweat or other body fluids. The aim of our study was to evaluate the use of tissue adhesives alone as a means to secure PIVCs inserted in the emergency department. Methods We performed a prospective interventional pilot study from November 2019 to May 2020 in a medical emergency department of an urban tertiary hospital. Patients were randomized to two groups: tissue adhesives (TA) or adhesive dressing (AD) group. After randomization we followed them until day 4. Results There were no significant differences between TA and AD groups in the rate of unplanned removal of PIVCs in the first 72 h (57.1% vs. 45.8%, p = 0.29), the rate of unplanned removal of PIVCs in the ED (0% vs. 2.1%, p = 1.00), the rate of unplanned removal of PIVC in the first 24 h (42.8% vs. 35.4%, p = 0.52), as well as in the rate of phlebitis (7.1% vs. 14.6%, p = 0.34) and the rate of any blood-stream infection (0% vs. 0%, p = 1.00). Conclusion We did not observe any significant differences when PIVCs inserted in the emergency department were secured with tissue adhesives alone, compared to standard adhesive dressings. We observed a high rate of unplanned removal of PIVCs, necessitating further research to determine more reliable ways of securing PIVCs.

IntroductionBacterial community-acquired pneumonia (CAP) in children is caused mostly by Streptococcus pneumoniae. The resistance of pneumococci to penicillin is increasing. However, most guidelines still prefer treatment with narrow-spectrum antibiotics. Therefore, we compared the effect of intravenous treatment with penicillin and broad-spectrum β-lactam antibiotics in children with CAP. The objective of our study was to assess the eligibility of treatment of bacterial CAP with intravenous penicillin.Material and methodsWe performed a prospective study and included 136 children hospitalised because of bacterial CAP. Patients were treated intravenously with either penicillin G or broad-spectrum β-lactam antibiotic monotherapy. Lung ultrasound and blood tests were performed at admission and after 2 days of treatment. The time interval from the application of antibiotics to permanent defervescence was recorded.ResultsEighty-seven (64.0%) patients were treated with penicillin G, and 49 (36.0%) were treated with broad-spectrum β-lactam antibiotics. The median time to persistent defervescence was 5 h in the penicillin group and 8 h in the broad-spectrum group (p = 0.18). There were no significant differences between the treatment regimens regarding the effect on the consolidation sizes or C-reactive protein levels. However, the decrease in the white blood cell count was marginally greater in the penicillin treatment group (p = 0.05).ConclusionsWe have shown that penicillin is as effective as broad-spectrum antibiotics in the treatment of bacterial CAP in children. Although the resistance of pneumococci to antibiotics is increasing, clinicians should still use penicillin and other narrow-spectrum β-lactam antibiotics in the treatment of bacterial CAP in children.

Multiple sclerosis is an inflammatory disorder of the central nervous system. Inflammation may create high susceptibility to subclinical atherosclerosis. The purpose of this study was to compare subclinical atherosclerosis and the role of inflammatory cytokines between the group of patients with relapsing-remitting multiple sclerosis (RRMS) and healthy controls matched for age and sex. The study group consisted of 112 non-diabetic and non-hypertensive RRMS patients treated with disease modifying drugs (DMD) and the control group was composed of 51 healthy subjects. The common carotid artery (CCA) intima media thickness (IMT) was investigated. Serum levels of risk factors for atherosclerosis and inflammatory cytokines were also determined. The mean CCA IMT (0.572 ± 0.131 mm vs. 0.571 ± 0.114 mm) did not differ (p > 0.05) between patients and controls. The RRMS patients’ CCA IMT was significantly correlated with serum interleukin 6 (IL-6) (p = 0.027), high-sensitivity C-reactive protein (hs-CRP) (p = 0.027), cystatin C (p

Objectives Previous studies have found evidence for a genetic basis for pelvic organ prolapse (POP), but no genetic studies have differentiated between types of POP. This study investigated whether genetic variants in six previously suggested candidate loci for POP modify the risk of uterine prolapse (UP). Study design One hundred patients, aged 30–55 years, who had undergone surgery due to total UP and 105 healthy controls were included in this study. After extracting the genomic DNA from peripheral blood, six single nucleotide polymorphisms (SNPs) previously identified for POP were genotyped, and association analysis was performed for contributing risk factors. RNA expression was determined from sacrouterine ligaments of patients and controls using quantitative reverse transcription polymerase chain reaction. Results The dominant genotype model for the T allele for SNP rs6051098 at the chromosome 20p13 locus was significant, and this remained significant with the risk factor regression model (p=0.046; odds ratio 1.93, 95 % confidence interval 1.01–3.66). The isocitrate dehydrogenase 3 beta (IDH3B) gene was the only potential candidate gene in the 20p13 locus that was significantly upregulated in sacrouterine biopsies in women with UP compared with controls (p = 0.034). Conclusion To the best of the authors’ knowledge, this is the first study to show that genetic risk factors contribute to UP, and suggested rs6051098 as the best candidate risk factor associated with UP. According to expression data in sacrouterine tissue, this study suggests that the IDH3B gene plays a role in the pathogenesis of UP.

The success rate of percutaneous coronary artery intervention (PCI) of chronic total occlusion (CTO) lesions have increased in the recent years. However, improvement of function is only possible when significant myocardial viability is present. One of the most important factors of maintaining myocardial viability is the opening and development of collaterals. Our hypothesis was that with a higher degree of collaterals more viable myocardium is present. In 38 patients we compared the degree of collaterals, evaluated with a conventional coronary angiogram (CCA) and graded by the Rentrop classification to transmural extent of the scar obtained in a viability study with magnetic resonance (MRI). We found a statistically significant association of the degree of collaterals determined with Rentrop method and transmural extent of the scar as measured by CMR (p = 0.001; Tau = -0.144). Additionally, associations showed an increase in the ratio between viable vs. non-viable myocardium with the degree of collaterals. Our study suggests that it may be beneficial to routinely grade the collaterals at angiography in patients with CTO as an assessment of myocardial viability.

The aetiology of community-acquired pneumonia (CAP) is not easy to establish. As lung ultrasound (LUS) has already proved to be an excellent diagnostic tool for CAP, we analysed its usefulness for discriminating between the aetiologically different types of CAP in children. We included 147 children hospitalized because of CAP. LUS was performed in all patients at admission, and follow-up LUS was performed in most patients. LUS-detected consolidations in viral CAP were significantly smaller, with a median diameter of 15 mm, compared to 20 mm in atypical bacterial CAP (p = 0.05) and 30 mm in bacterial CAP (p

Response to anti-TNF therapy is crucial for life expectancy and life quality in patients with severe Crohn's disease. We investigated if a previously reported gene expression profile predictive for infliximab response could be also applied to adalimumab response in an independent cohort. Forty-seven Slovene Crohn's disease patients indicated for adalimumab therapy were enrolled in the study. Inflamed and non-inflamed colon biopsy samples were obtained during routine colonoscopy prior to adalimumab treatment. Response to adalimumab was measured with IBDQ. Gene expression in inflamed and non-inflamed colon biopsy samples was measured with RT-qPCR. Genotypes were extracted from previously available genotype data. Statistical analysis was performed with SPSS software. The R package e1071 was used to train bootstrap aggregated support vector machines (SVM). SVM prediction model analysis was used to analyze pooled, non-inflamed, and inflamed colon tissue datasets using IBDQ response after 4, 12, 20 and 30 weeks of adalimumab treatment. The bagging approach was used in an endeavor to obtain 100 % accuracy using 10 × 100 or 100 × 100 iterations. Average adalimumab response prediction accuracy is 75.5 % for pooled samples, 90.5 % for inflamed samples, and 100 % for non-inflamed samples. Moreover, models trained on selected SNPs from analyzed genes had an average accuracy of 92.8 %, confirming the involvement of genetic regions mapping the reported genes. Finally, using combined gene expression and SNP data we observed 100 % adalimumab response prediction accuracy for pooled, inflamed, and non-inflamed datasets. Our study supports the reported genetic anti-TNF response profile and extends it for adalimumab prediction. Eine ausreichende Antwort auf Anti-TNF-Behandlung mit Infliximab oder Adalimumab ist entscheidend für Lebenserwartung und Lebensqualität bei Patienten mit schwergradigem Morbus Crohn. Wir untersuchten, ob das zuvor beschriebene Genprofil zur Prognose des Therapieerfolgs mit Infliximab auch zur Prognose des Therapieerfolgs mit Adalimumab in einer unabhängigen Kohorte anwendbar ist. 47 slowenische für Adalimumab-Behandlung indizierte Morbus-Crohn-Patienten wurden in die Studie aufgenommen. Vor der Behandlung mit Adalimumab wurden während der Kolonoskopie entzündete und nicht entzündete Gewebeproben entnommen. Die Reaktion auf Adalimumab wurde mit IBDQ gemessen. Die Genexpression in Gewebeproben wurde mit RT-qPCR gemessen. Genotypen wurden aus zuvor verfügbaren Genotypdaten extrahiert. Die statistische Analyse wurde mit SPSS-Software durchgeführt. Das R-Paket e1071 wurde zum Trainieren von Support Vector Machines benutzt. Die SVM-Vorhersagemodelle wurden verwendet, um die gepoolten, nicht entzündeten und entzündeten Darmproben-Datensätze mit Verwendung von IBDQ-Antwort nach 4, 12, 20 und 30 Wochen Adalimumab-Behandlung zu analysieren. Die Bagging-Methode wurde verwendet, um eine 100-%-Genauigkeit mit Verwendung von 10 × 100 oder 100 × 100 Iterationen zu erreichen. Die Adalimumab-Antwortvorhersage von gepoolten Datensätzen erreichte im Durchschnitt eine Genauigkeit von 75,5 %, von entzündeten Datensätzen 90,5 % und von nicht entzündeten Datensätzen 100 %. Zusätzlich haben die Modelle, die an ausgewählten SNPs in den analysierten Genen trainiert wurden, eine durchschnittliche Genauigkeit von 92,8 % erreicht. Schließlich kombinierten wir Expressions- und Genotypdaten, um den Vorhersagewert weiter zu analysieren. Mithilfe kombinierter Daten konnten wir mit Darmgewebe-Datensätzen eine Adalimumab-Antwortvorhersage mit hundertprozentiger Sicherheit feststellen. Unsere Studie unterstützt das genetische Anti-TNF-Antwortprofil, das für den Adalimumab-Therapieerfolg gleichermaßen relevant ist.

Introduction: Inhaled corticosteroids (ICS) are the most effective medication for control of asthma symptoms. However, a proportion of patients do not respond to this medication and suffer exacerbations, due to a combination of environmental and genetic factors. We performed a meta- genome-wide association study (GWAS) to identify genes associated with ICS response in European children. Methods: A GWAS meta-analysis of asthma exacerbations was performed across eight European studies from the Pharmacogenomics in Childhood Asthma (PiCA) consortium including 2,704 asthmatic children and young adults treated with ICS. Imputation of genetic variants was carried out using the Haplotype Reference Consortium as reference panel and association testing was performed by means of logistic regression models. A total of 8.1 million genetic variants with minor allele frequency ≥1% were meta-analyzed. Results: Nineteen polymorphisms were suggestively associated with asthma exacerbations despite use of ICS (p≤5x10-6). These variants were located at 10 segregating sites (minimum p=6.7x10-7). Novel associations were revealed in biologically plausible genes involved in drug metabolism (AOX1) and previously associated with lung function measurements (WNT5A and CNTNAP5). Moreover, variants located at CNTNAP5 were also nominally associated with a change in FEV1 after ICS treatment in SLOVENIA (minimum p=0.025). Conclusions: We revealed genes suggestively associated with asthma exacerbations in children and young adults despite the use of ICS. Replication will be performed in further independent studies. Study supported by Instituto de Salud Carlos III (AC15/00015) and ERACoSysMed 1st Joint Transnational Call (SysPharmPedia).

Background\ud Inhaled corticosteroids (ICS) are the most widely prescribed and effective medication to control asthma symptoms and exacerbations. However, many children still have asthma exacerbations despite treatment, particularly in admixed populations, such as Puerto Ricans and African Americans. A few genome‐wide association studies (GWAS) have been performed in European and Asian populations, and they have demonstrated the importance of the genetic component in ICS response.\ud \ud Objective\ud We aimed to identify genetic variants associated with asthma exacerbations in admixed children treated with ICS and to validate previous GWAS findings.\ud \ud Methods\ud A meta‐analysis of two GWAS of asthma exacerbations was performed in 1347 admixed children treated with ICS (Hispanics/Latinos and African Americans), analysing 8.7 million genetic variants. Those with P ≤ 5 × 10−6 were followed up for replication in 1697 asthmatic patients from six European studies. Associations of ICS response described in published GWAS were followed up for replication in the admixed populations.\ud \ud Results\ud A total of 15 independent variants were suggestively associated with asthma exacerbations in admixed populations (P ≤ 5 × 10−6). One of them, located in the intergenic region of APOBEC3B and APOBEC3C, showed evidence of replication in Europeans (rs5995653, P = 7.52 × 10−3) and was also associated with change in lung function after treatment with ICS (P = 4.91 × 10−3). Additionally, the reported association of the L3MBTL4‐ARHGAP28 genomic region was confirmed in admixed populations, although a different variant was identified.\ud \ud Conclusions and clinical relevance\ud This study revealed the novel association of APOBEC3B and APOBEC3C with asthma exacerbations in children treated with ICS and replicated previously identified genomic regions. This contributes to the current knowledge about the multiple genetic markers determining responsiveness to ICS which could lead in the future the clinical identification of those asthma patients who are not able to respond to such treatment.

Summary Background & aims Celiac disease (CD) is an immune-mediated systemic disease, caused by ingestion of gluten in genetically predisposed individuals. Gut microbiota dysbiosis might play a significant role in pathogenesis of chronic enteropathies and its modulation can be used as an intervention strategy in CD as well. In this study, we aimed to identify correlations between fecal microbiota, serum tumor necrosis factor alpha (TNF-α) and fecal short-chain fatty acids (SCFAs) in healthy children and children with CD after administration of probiotic Bifidobacterium breve BR03 and B632. Methods A double-blind placebo-controlled study enrolled 40 children with CD (CD) and 16 healthy children (HC). CD children were randomly allocated into two groups, of which 20 belonged to the placebo (PL) group and 20 to the Probiotic (PR) group. The PR group received a probiotic formulation containing a mixture of 2 strains, B. breve BR03 (DSM 16604) and B. breve B632 (DSM 24706) in 1:1 ratio for 3 months. Subsequently, for statistical analysis, blood and fecal samples from CD children (on enrolment - T0 and after 3 months, at the end of intervention with probiotic/placebo - T1) and HC children were used. The HC group was sampled only once (T0). Results Verrucomicrobia, Parcubacteria and some yet unknown phyla of Bacteria and Archaea may be involved in the disease, indicated by a strong correlation to TNF-α. Likewise, Proteobacteria strongly correlated with fecal SCFAs concentration. The effect of probiotic administration has disclosed a negative correlation between Verrucomicrobia, some unknown phyla of Bacteria, Synergistetes, Euryarchaeota and some SCFAs, turning them into an important target in microbiome restoration process. Synergistetes and Euryarchaeota may have a role in the anti-inflammatory process in healthy human gut. Conclusions Our results highlight new phyla, which may have an important relation to disease-related parameters, CD itself and health.

BackgroundCartilage tissue engineering is a fast-evolving field of biomedical engineering, in which the chondrocytes represent the most commonly used cell type. Since research in tissue engineering always consumes a lot of cells, simple and cheap isolation methods could form a powerful basis to boost such studies and enable their faster progress to the clinics. Isolated chondrocytes can be used for autologous chondrocyte implantation in cartilage repair, and are the base for valuable models to investigate cartilage phenotype preservation, as well as enable studies of molecular features, nature and scales of cellular responses to alterations in the cartilage tissue.MethodsIsolation and consequent cultivation of primary human adult articular chondrocytes from the surgical waste obtained during total knee arthroplasty (TKA) was performed. To evaluate the chondrogenic potential of the isolated cells, gene expression of collagen type 2 (COL2), collagen 1 (COL1) and aggrecan (ACAN) was evaluated. Immunocytochemical staining of all mentioned proteins was performed to evaluate chondrocyte specific production.ResultsCartilage specific gene expression of COL2 and ACAN has been shown that the proposed protocol leads to isolation of cells with a high chondrogenic potential, possibly even specific phenotype preservation up to the second passage. COL1 expression has confirmed the tendency of the isolated cells dedifferentiation into a fibroblast-like phenotype already in the second passage, which confirms previous findings that higher passages should be used with care in cartilage tissue engineering. To evaluate the effectiveness of our approach, immunocytochemical staining of the evaluated chondrocyte specific products was performed as well.DiscussionIn this study, we developed a protocol for isolation and consequent cultivation of primary human adult articular chondrocytes with the desired phenotype from the surgical waste obtained during TKA. TKA is a common and very frequently performed orthopaedic surgery during which both femoral condyles are removed. The latter present the ideal source for a simple and relatively cheap isolation of chondrocytes as was confirmed in our study.

Development of artificial materials for the facilitation of cartilage regeneration remains an important challenge in orthopedic practice. Our study investigates the potential for neocartilage formation within a synthetic polyester scaffold based on the polymerization of high internal phase emulsions. The fabrication of polyHIPE polymer (PHP) was specifically tailored to produce a highly porous (85%) structure with the primary pore size in the range of 50–170 μm for cartilage tissue engineering. The resulting PHP scaffold was proven biocompatible with human articular chondrocytes and viable cells were observed within the materials as evaluated using the Live/Dead assay and histological analysis. Chondrocytes with round nuclei were organized into multicellular layers on the PHP surface and were observed to grow approximately 300 μm into the scaffold interior. The accumulation of collagen type 2 was detected using immunohistochemistry and chondrogenic specific genes were expressed with favorable collagen type 2 to 1 ratio. In addition, PHP samples are biodegradable and their baseline mechanical properties are similar to those of native cartilage, which enhance chondrocyte cell growth and proliferation.

The early establishment of a complete microbiome has been shown to play an integral part in the development and maintenance of an intact intestine and its immune system, although much remains unknown about the specific mechanisms of immune modulation in newborns. In our study we show in a co-culture model of the undeveloped small intestine that members of Lactobacillus spp. influence STAT1 and NF-kB p65 nuclear translocation in both intestinal epithelial cells as well as underlying macrophages. Moreover, by using imaging flow cytometry we were able to monitor each individual cell and create a framework of the percentage of cells in which translocation occurred in challenged versus control cell populations. We also observed a significant difference in baseline translocation in intestinal cells when cultured alone versus those in a co-culture model, underpinning the importance of 3D models over monolayer set-ups in epithelial in vitro research. In conclusion, our work offers new insights into the potential routes by which the commensal microbiome primes the early immune system to fight pathogens, and shows how strain-specific these mechanisms really are.

World climate, environmental and other global changes would have wide-ranging and, mostly, adverse consequences for human health. These changes are a significant and emerging threat to public health, including the growing season, variability of crop yields and water demands. Above mentioned factors have been influenced in areas of food security and food safety and moreover the food industry struggle for profits. These factors control the conditions and affect the use of chemicals in the agriculture and food industry. In this chapter, the risk assessment of chemicals in food as a part of risk analysis process have been addressed. We would like to point out the importance of this process in the area of public health and consumer protection. Through food and nutrition products and most often misleading advertisements, people are exposed to different hazards that could have severe harmful consequences. Among the consumer groups, children are mostly vulnerable because of their adult dependencies, age and body weight. In this chapter, an example of risk assessment of exposure to food additives among preschool children in Slovenia is evaluated.