Lymphocytes sensitivity to Fas stimulation in healthy and asthmatic children.

The T cell hypothesis of asthma is based on the concept that the disease is driven and maintained by the persistence of a specialized subset of chronically activated T memory cells sensitized against an array of allergenic, occupational or viral antigens. Overreaction of CD4+ T cells in the peripheral blood and airway tissues is an invariant feature of asthma; therefore a potent mechanism for augmenting the number of activated T cells in this disease would be the resistance to the normally programmed pathway for cell death. The aim of the study was to evaluate the presence of apoptotic markers on peripheral blood lymphocytes from healthy and asthmatic children before and after stimulation with antiCD95 antibodies. The blood was collected from 21 children with atopic asthma suffering from allergic rhinitis because of house dust mite and/or grass pollen allergens and 8 healthy children matched for their age and sex. Blood was mixed with purified monoclonal antibody antiCD95 (Beckman Coulter), incubated for 24 hours and than stained with Annexin V andPI (Becton Dickinson). Prepared suspensions were analyzed with Cytomics FC 500 (Beckman Coulter) flow cytometer. Annexin V(+)/PI(-) cells were characterized as early apoptotic, Annexin V(+)/PI(+) as late apoptotic and Annexin V(-)/PI(+) as dead. In unstimulated sample from asthmatic children 21.09+/-11.20% cells were characterized as Annexin V positive/PI negative. After stimulation with antiCD95 Annexin V positive/PI negative cells constituted 18.72+/-9.42% of cells, p=0.1. In unstimulated sample from healthy children 11.69+/-6.70% cells were characterized as Annexin V positive/PI negative. In the sample stimulated with antiCD95 16.54+/-2.98% of cells were Annexin V positive/PI negative, p=0.02. There were no differences between results of late apoptotic and necrotic lymphocytes from healthy and asthmatic children. Performed research indicates that lymphocytes from asthmatic children are resistant to Fas mediated apoptosis

Melatonin Protects Cholangiocytes from Oxidative Stress-Induced Proapoptotic and Proinflammatory Stimuli via miR-132 and miR-34

Biosynthesis of melatonin by cholangiocytes is essential for maintaining the function of biliary epithelium. However, this cytoprotective mechanism appears to be impaired in primary biliary cholangitis (PBC). MiR-132 has emerged as a mediator of inflammation in chronic liver diseases. The effect of melatonin on oxidative stress and bile acid-induced apoptosis was also examined in cholangiocyes overexpressing miR506, as a PBC-like cellular model. In PBC patients the serum levels of melatonin were found increased in comparison to healthy controls. Whereas, in cholangiocytes within cirrhotic PBC livers the melatonin biosynthetic pathway was substantially suppressed even though the expressions of melatonin rate-limiting enzyme aralkylamine N-acetyltransferase (AANAT), and CK-19 (marker of cholangiocytes) were enhanced. In cholangiocytes exposed to mitochondrial oxidative stress melatonin decreased the expression of proapoptotic stimuli (PTEN, Bax, miR-34), which was accompanied by the inhibition of a pivotal mediator of inflammatory response Nf-κB-p65 and the activation of antiapoptotic signaling (miR-132, Bcl2). Similarly, melatonin reduced bile acid-induced proapoptotic caspase 3 and Bim levels. In summary, the insufficient hepatic expression of melatonin in PBC patients may predispose cholangiocytes to oxidative stress-related damage. Melatonin, via epigenetic modulation, was able to suppress NF-κB signaling activation and protect against biliary cells apoptotic signaling.This work was supported by grant no. 2015/17/B/NZ5/02541 from National Science Centre in Poland

Lymphocytes sensitivity to Fas stimulation in healthy and asthmatic children.

The T cell hypothesis of asthma is based on the concept that the disease is driven and maintained by the persistence of a specialized subset of chronically activated T memory cells sensitized against an array of allergenic, occupational or viral antigens. Overreaction of CD4+ T cells in the peripheral blood and airway tissues is an invariant feature of asthma; therefore a potent mechanism for augmenting the number of activated T cells in this disease would be the resistance to the normally programmed pathway for cell death. The aim of the study was to evaluate the presence of apoptotic markers on peripheral blood lymphocytes from healthy and asthmatic children before and after stimulation with antiCD95 antibodies. The blood was collected from 21 children with atopic asthma suffering from allergic rhinitis because of house dust mite and/or grass pollen allergens and 8 healthy children matched for their age and sex. Blood was mixed with purified monoclonal antibody antiCD95 (Beckman Coulter), incubated for 24 hours and than stained with Annexin V andPI (Becton Dickinson). Prepared suspensions were analyzed with Cytomics FC 500 (Beckman Coulter) flow cytometer. Annexin V(+)/PI(-) cells were characterized as early apoptotic, Annexin V(+)/PI(+) as late apoptotic and Annexin V(-)/PI(+) as dead. In unstimulated sample from asthmatic children 21.09+/-11.20% cells were characterized as Annexin V positive/PI negative. After stimulation with antiCD95 Annexin V positive/PI negative cells constituted 18.72+/-9.42% of cells, p=0.1. In unstimulated sample from healthy children 11.69+/-6.70% cells were characterized as Annexin V positive/PI negative. In the sample stimulated with antiCD95 16.54+/-2.98% of cells were Annexin V positive/PI negative, p=0.02. There were no differences between results of late apoptotic and necrotic lymphocytes from healthy and asthmatic children. Performed research indicates that lymphocytes from asthmatic children are resistant to Fas mediated apoptosis

Analysis of the visual guidance in Ghost of Tsushima

Guiding players without using quest markers and arrows can prove a difficult endeavor. Therefore, this paper provides a deeper analysis of the visual guidance techniques used in Ghost of Tsushima. A game that had the design goal to “keep the screen as clean as possible as you're going throughout the world” according to Creative Director Jason Connellin in an interview with androidcentral (2020). Furthermore, this paper explores the idea of how art principles and visual interaction cues can work together. To do so this paper uses two different frameworks, one with art principles by Brommer (2011) and one with visual interaction cues by Dillman et al. (2018). The findings of this paper highlight the usage of subtle cues in combination with art principles as the primary means of guiding the player when quest markers and arrows are not a desirable choice.

Comparison of the utility of five commercial kits for extraction of DNA from Aspergillus fumigatus spores

The aim of this study was to compare the efficiency of DNA extraction from water as well as from blood samples spiked with A. fumigatus spores, using selected commercial kits. Extraction of DNA according to manufacturer's protocols was preceded by blood cells lysis and disruption of fungal cells by enzymatic digestion or bead beating. The efficiency of DNA extraction was measured by PCR using Aspergillus-specific primers and SYBR Green I dye or TaqMan probes targeting 28S rRNA gene. All methods allowed the detection of Aspergillus at the lowest tested density of water suspensions of spores (101 cells/ml). The highest DNA yield was obtained using the ZR Fungal/Bacterial DNA kit, YeastStar Genomic DNA kit, and QIAamp DNA Mini kit with mechanical cell disruption. The ZR Fungal/Bacterial DNA and YeastStar kits showed the highest sensitivity in examination of blood samples spiked with Aspergillus (100 % for the detection of 102 spores and 75 % for 101 spores). Recently, the enzymatic method ceased to be recommended for examination of blood samples for Aspergillus, thus ZR Fungal/Bacterial DNA kit and QIAamp DNA Mini kit with mechanical cell disruption could be used for extraction of Aspergillus DNA from clinical samples

Tau Protein Modifications and Interactions: Their Role in Function and Dysfunction

Tau protein is abundant in the central nervous system and involved in microtubule assembly and stabilization. It is predominantly associated with axonal microtubules and present at lower level in dendrites where it is engaged in signaling functions. Post-translational modifications of tau and its interaction with several proteins play an important regulatory role in the physiology of tau. As a consequence of abnormal modifications and expression, tau is redistributed from neuronal processes to the soma and forms toxic oligomers or aggregated deposits. The accumulation of tau protein is increasingly recognized as the neuropathological hallmark of a number of dementia disorders known as tauopathies. Dysfunction of tau protein may contribute to collapse of cytoskeleton, thereby causing improper anterograde and retrograde movement of motor proteins and their cargos on microtubules. These disturbances in intraneuronal signaling may compromise synaptic transmission as well as trophic support mechanisms in neurons

Decreased Expression of Vitamin D Receptor Affects an Immune Response in Primary Biliary Cholangitis via the VDR-miRNA155-SOCS1 Pathway

Primary biliary cholangitis (PBC) is an immune-mediated cholestatic disease. Vitamin D receptor (VDR)-dependent signaling constrains an inflammatory response by targeting the miRNA155-SOCS1 (suppressor of cytokine signaling 1) axis. The VDR-miRNA155-SOCS1 pathway was investigated in the context of the autoimmune response associated with PBC. Human liver tissues from non-cirrhotic PBC (n = 22), cirrhotic PBC (n = 22), cirrhotic primary sclerosing cholangitis (PSC, n=13), controls (n = 23), and peripheral blood mononuclear cells (PBMC) obtained from PBC (n = 16) and PSC (n = 10) patients and healthy subjects (n = 11) were used for molecular analyses. VDR mRNA and protein expressions were substantially reduced in PBC livers (51% and 59%, respectively). Correspondingly, the decrease of SOCS1 protein expression in PBC livers, after normalization to a marker of lymphocytes and forkhead family transcriptional regulator box P3 (FOXP3, marker of Treg), was observed, and this phenomenon was accompanied by enhanced miRNA155 expression. In PSC livers, protein expressions of VDR and SOCS1 were comparable to the controls. However, in PBM cells, protein expressions of VDR and SOCS1 were considerably decreased in both PBC and PSC. We demonstrated that VDR/miRNA155-modulated SOCS1 expression is decreased in PBC which may lead to insufficient negative regulation of cytokine signaling. These findings suggest that the decreased VDR signaling in PBC could be of importance in the pathogenesis of PBC

Polymorphisms of IL12RB2 May Affect the Natural History of Primary Biliary Cholangitis: A Single Centre Study

Background. Recent GWAS in primary biliary cholangitis (PBC) showed strong associations with SNPs located within interleukin-12 receptor (IL12R) beta-2 (IL12RB2) gene. Aims. We assessed whether genetic variation of IL12RB2 is associated with laboratory and clinical features of PBC. Methods. Genomic DNA was isolated from 306 patients with PBC and 258 age/gender-matched controls. PBC-specific anti-mitochondrial antibodies (AMA) were tested in all subjects by ELISA. Two SNPs, rs3790567 and rs6679356, of IL12RB2 were genotyped using the MGB-TaqMan SNP assay. Results. Despite comparable age at diagnosis of cirrhotic and noncirrhotic PBC patients, allele A of rs3790567 and allele C of rs6679356 were overrepresented in the former rather than the latter group (p=0.0009 and p=0.002, resp.). The risk of cirrhosis at presentation increased when allele A and allele C coexisted. AMA-M2 titres were significantly higher in AA homozygotes of rs3790567 compared to GG homozygotes (132±54 versus 103±62, p=0.02) and in rs6679356 when C allele was present (p=0.038). There were no other significant associations between IL12RB2 polymorphisms and laboratory or clinical features. Conclusion. In this first study analyzing phenotypic features of PBC carriers of the IL12RB2 polymorphisms, we found that carriers are more frequently cirrhotic at diagnosis and have significantly higher titres of AMA