AT2R deficiency mediated podocyte loss via activation of ectopic hedgehog interacting protein (Hhip) gene expression

Angiotensin II type 2 receptor (AT₂R) deficiency in AT₂R knockout (KO) mice has been linked to congenital abnormalities of the kidney and urinary tract; however, the mechanisms by which this occurs are poorly understood. In this study, we examined whether AT₂R deficiency impaired glomerulogenesis and mediated podocyte loss/dysfunction in vivo and in vitro. Nephrin‐cyan fluorescent protein (CFP)‐transgenic (Tg) and Nephrin/AT₂RKO mice were used to assess glomerulogenesis, while wild‐type and AT₂RKO mice were used to evaluate maturation of podocyte morphology/function. Immortalized mouse podocytes (mPODs) were employed for in vitro studies. AT₂R deficiency resulted in diminished glomerulogenesis in E15 embryos, but had no impact on actual nephron number in neonates. Pups lacking AT₂R displayed features of renal dysplasia with lower glomerular tuft volume and podocyte numbers. In vivo and in vitro studies demonstrated that loss of AT₂R was associated with elevated NADPH oxidase 4 levels, which in turn stimulated ectopic hedgehog interacting protein (Hhip) gene expression in podocytes. Consequently, ectopic Hhip expression activation either triggers caspase‐3 and p53‐related apoptotic processes resulting in podocyte loss, or activates TGFβ1–Smad2/3 cascades and α‐SMA expression to transform differentiated podocytes to undifferentiated podocyte‐derived fibrotic cells. We analyzed HHIP expression in the kidney disease database (Nephroseq) and then validated this using HHIP immunohistochemistry staining of human kidney biopsies (controls versus focal segmental glomerulosclerosis). In conclusion, loss of AT₂R is associated with podocyte loss/dysfunction and is mediated, at least in part, via augmented ectopic Hhip expression in podocytes. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

New Poly[(R)‐3‐hydroxybutyrate‐co‐4‐hydroxybutyrate] (P3HB4HB)‐Based Thermogels

Poly[(R )‐3‐hydroxybutyrate] (P3HB) is a potential candidate for biomaterials due to its biocompatibility and biodegradability. However, P3HB needs to have tunable hydrophobicity, modification through chemical functionalization and the right hydrolytic stability to increase their potential for water‐based biomedical applications such as using them as in situ forming gels for drug delivery. This work focuses on using a copolymer, poly[(R )‐3‐hydroxybutyrate‐co‐4‐hydroxybutyrate] (P3HB4HB) in a thermogelling multiblock system with polypropylene glycol and polyethylene glycol to study the effect of the hydrophobic P3HB4HB on gelation properties, degradation, and drug release rates with reference to P3HB. Thermogels containing P3HB4HB segments show lower critical micellization concentration values in a range from 3 × 10⁻⁴ to 1.08 × 10⁻³ g mL⁻¹ and lower critical gelation concentration values ranging from 2 to 6 wt% than that of gels containing P3HB. Furthermore, gels containing P3HB4HB degrade at a slower rate than the gels containing P3HB. Drug release studies of 5 µg mL⁻¹ of doxorubicin show that gels containing P3HB4HB exhibit sustained release although the release rates are faster than gels containing P3HB. However, this can be modified by varying the concentration of the gels used. Process optimization of purifying the starting material is one important factor before the synthesis of these biomaterials.

     

TarSeqQC: Quality control on targeted sequencing experiments in R

Targeted sequencing (TS) is growing as a screening methodology used in research and medical genetics to identify genomic alterations causing human diseases. In general, a list of possible genomic variants is derived from mapped reads through a variant calling step. This processing step is usually based on variant coverage, although it may be affected by several factors. Therefore, undercovered relevant clinical variants may not be reported, affecting pathology diagnosis or treatment. Thus, a prior quality control of the experiment is critical to determine variant detection accuracy and to avoid erroneous medical conclusions. There are several quality control tools, but they are focused on issues related to whole‐genome sequencing. However, in TS, quality control should assess experiment, gene, and genomic region performances based on achieved coverages. Here, we propose TarSeqQC R package for quality control in TS experiments. The tool is freely available at Bioconductor repository. TarSeqQC was used to analyze two datasets; low‐performance primer pools and features were detected, enhancing the quality of experiment results. Read count profiles were also explored, showing TarSeqQC's effectiveness as an exploration tool. Our proposal may be a valuable bioinformatic tool for routinely TS experiments in both research and medical genetics.     

IFN‐γR1 defects: Mutation update and description of the IFNGR1 variation database

IFN‐γ signaling is essential for the innate immune defense against mycobacterial infections. IFN‐γ signals through the IFN‐γ receptor, which consists of a tetramer of two IFN‐γR1 chains in complex with two IFN‐γR2 chains, where IFN‐γR1 is the ligand‐binding chain of the interferon‐γ receptor and IFN‐γR2 is the signal‐transducing chain of the IFN‐γ receptor. Germline mutations in the gene IFNGR1 encoding the IFN‐γR1 cause a primary immunodeficiency that mainly leads to mycobacterial infections. Here, we review the molecular basis of this immunodeficiency in the 130 individuals described to date, and report mutations in five new individuals, bringing the total number to 135 individuals from 98 kindreds. Forty unique IFNGR1 mutations have been reported and they exert either an autosomal dominant or an autosomal recessive effect. Mutations resulting in premature stopcodons represent the majority of IFNGR1 mutations (60%; 24 out of 40), followed by amino acid substitutions (28%, 11 out of 40). All known mutations, as well as 287 other variations, have been deposited in the online IFNGR1 variation database ( www.LOVD.nl/IFNGR1 ). In this article, we review the function of IFN‐γR1 and molecular genetics of human IFNGR1.     

Tumor associated macrophages provide the survival resistance of tumor cells to hypoxic microenvironmental condition through IL-6 receptor-mediated signals

Hypoxia and infiltration of tumor-associated macrophages (TAM) are intrinsic features of the tumor microenvironment. Tumor cells that remain viable in hypoxic conditions often possess an increased survival potential and tend to grow aggressively. TAM also respond to a variety of signals in the hypoxic tumor microenvironment and express a more M2-like phenotype. In this study, the established mouse tumor tissues showed a dense infiltration of CD206⁺ macrophages at the junctions between the normoxic and hypoxic regions and an increased IL-6 receptor (IL-6R) expression of tumor cells in the areas of CD206⁺ TAM accumulation, which indicates a role of M2 phenotype TAM in survival adaptation of tumor cells preparing for an impending hypoxic injury before changes in oxygen availability. Cocultured mouse FM3A or human MCF-7 tumor cells with tumor infiltrating macrophages isolated from mouse tumor tissues and M2-polarized macrophages generated from human THP-1 cells, respectively, showed significantly decreased rate of cell death in cultures exposed to hypoxia. The acquisition of survival resistance was attributed to increased IL-6 production by M2 TAM and increased expression of IL-6R in tumor cells in the coculture system. MCF-7 cells cocultured with M2 TAM showed activated JAK1/STAT3 and Raf/MEK/JNK pathways contributing to tyrosine and serine phophorylation of STAT3, respectively. However, only tyrosine phosphorylated STAT3 was detected in the nucleus, which induced upregulation of Bcl-2 and downregulation of Bax and Bak. Finally, knockdown of IL-6R by small interfering RNA significantly counteracted coculture-induced signals and completely abolished the survival resistance to hypoxic injury. Thus, we present evidence for the role of M2 phenotype TAM in IL-6 receptor-mediated signals, particularly tyrosine phosphorylation of STAT3, responsible for the prosurvival adaptation of tumor cells to hypoxia.     

Nocturnal autonomic nervous system activity and morning proinflammatory cytokines in young adult African Americans

Compromised sleep and increased sympathetic nervous system (SNS) activity are implicated in the pathogenesis of, and disparities in, cardiovascular disease. Parasympathetic dominance during sleep may be important for cardiovascular health. Sleep and autonomic balance influence immune activity, which impacts atherogenesis. We evaluated relationships between autonomic balance during sleep and morning levels of the immune activating cytokines, C‐reactive protein (CRP) and interleukin (IL)‐6. Ninety‐four (59 female) young adult African Americans without medical conditions and substance use disorders spent 2 consecutive nights in a clinical research unit for sleep recordings and blood drawing on awakening. Cardiac tracings from the second sleep recording were analysed for heart rate variability (HRV). Body mass index was the only non‐HRV measure correlated with cytokine levels. Indicators of SNS activity for the presleep, and first non‐rapid eye movement (REM) and REM sleep periods were correlated independently with morning IL‐6 levels. Altered autonomic balance during sleep may be a modifiable factor that influences immune activation.     

IL-36α induces maturation of Th1-inducing human MDDC and synergises with IFN-γ to induce high surface expression of CD14 and CD11c

We show that IL-36α induced maturation of human MDDCs and stimulated differentiation of IFN-γ producing (Type 1) CD3+ lymphocytes but was not as effective as IL-36β in doing so. For the first time, we also show that IL-36α induced expression of CD14 by MDDCs and this was highly potentiated by co-cultured with IFN-γ. In contrast, lipopolysaccharide (LPS) did not increase CD14 expression by MDDCs, suggesting that if MDDCs represent a physiologically relevant population in vivo, they need to be stimulated by relevant inflammatory cytokines prior to CD14 expression and detection of LPS, expressed by Gram negative bacteria. IFN-γ synergised with IL-36α to restore the high levels of CD11c expression by MDDCs, which was reduced by culture with these cytokines in isolation. IL-36α/IFN-γ synergy also correlated with increased binding of the opsonic complement protein (iC3b) to MDDCs. However although IL-36α increased the phagocytic capacity of MDDCs for Salmonella Typhimurium 4/74 this was not synergistically increased by IFN-γ (P > 0.05).In conclusion we report the hitherto unknown effects of IL-36α on the innate cell function of human MDDCs.