Do Human iPSC-Derived Cardiomyocytes Cultured on PLA Scaffolds Induce Expression of CD28/CTLA-4 by T Lymphocytes?

Many research groups have developed various types of tissue-engineered cardiac constructs. However, the immunological properties of such artificial tissues are not yet fully understood. Previously, we developed microfiber scaffolds carrying human iPSC-derived cardiomyocytes (hiPSC-CM). In this work, we evaluated the ability of these tissue-engineered constructs to activate the expression of CD28 and CTLA-4 proteins on T lymphocytes, which are early markers of the immune response. For this purpose, electrospun PLA microfiber scaffolds were seeded with hiPSC-CM and cultured for 2 weeks. Allogeneic mononuclear cells were then co-cultured for 48 h with three groups of samples: bare scaffolds, pure cardiomyocyte culture and tissue-engineered constructs, followed by analysis of CD28/CTLA-4 expression on T lymphocytes using flow cytometry. PLA scaffolds and concanavalin A stimulation (positive control) statistically significantly increased CD28 expression on CD4+ T cells (up to 61.3% and 66.3%) CD8+ T cells (up to 17.8% and 21.7%). CD28/CTLA-4 expression was not increased when T lymphocytes were co-cultured with cardiac tissue-engineered constructs and iPSC-CM monolayers. Thus, iPSC-CM in monolayers and on PLA microfiber scaffolds did not induce T cell activation, which suggests that such cardiac constructs would not be a cause of rejection after implantation.

Early Markers of Cardiovascular Disease Associated With Clinical Data and Autosomal Ancestry in Patients With Type 1 Diabetes: A Cross-sectional Study in an Admixed Brazilian Population.

BackgroundPatients with type 1 diabetes (T1D) have a higher risk of developing cardiovascular disease (CVD), which is a major cause of death in this population. The objective of this study was to investigate early markers of CVD associated with clinical data and autosomal ancestry in T1D patients from an admixed Brazilian population. MethodsA cross-sectional study was conducted with 99 T1D patients. The early markers of CVD included the ankle-brachial index (ABI), coronary artery calcium score (CACS), and carotid Doppler sonography. Demographic, clinical, and serum data were collected. A panel of autosomal informational insertion/deletion ancestry markers was used to estimate the individual proportions of European, African, and Amerindian ancestry.ResultsThe study sample had a mean age of 27.6 years and 14.4 years of duration of T1D. The prevalence of alterations in early CVD markers was: ABI (< 0,9) 19.6%, CACS (> 0 +) 4.1%, and carotid Doppler 5.0%. There was significant agreement between CACS and carotid Doppler, and these were correlated with traditional risk factors for CVD. There was a predominance of European ancestry (47.3%), followed by African (28%) and Ameridian (24.7%). There were no association between early CVD markers and autosomal ancestry proportions.ConclusionThe ABI was useful in the early identification of CVD in asymptomatic young patients with T1D and with a short duration of disease, and showed agreement with the carotid Doppler. Although CACS and carotid Doppler are non-invasive tests, carotid Doppler is more cost-effective, and both have limitations in screening for CVD in young patients with a short duration of T1D. We did not find a statistically significant relationship between autosomal ancestry proportions and early CVD markers in an admixed Brazilian population.

Do Human iPSC Derived Cardiomyocytes Cultured on PLA Scaffolds Induce Expression of CD28/CTLA 4 by T Lymphocytes?

Different types of engineered cardiac constructs are being developed nowadays by many research groups. However, the immunological properties of such artificial tissues are not yet clearly understood. Previously, we have studied microfiber scaffolds carrying iPSC-derived cardiomyocytes. In this work, we evaluated the ability of these tissue-engineered constructs to activate the expression of CD28 and CTLA-4 proteins in T-lymphocytes which are early markers of the immune response. For this purpose electrospun PLA nanofibrous scaffolds were seeded with human iPSCs-CM and cultivated for 2 weeks. After, allogeneic mononuclear cells were co-cultured during 48 hours with 3 groups of samples that were tissue-engineered constructs, pure culture of cardiomyocytes and bare scaffolds followed by analysis of CD28/CTLA-4 expression on T-lymphocytes via flow cytometry. PLA scaffolds and concanavalin A (positive control) stimulation statistically significantly increased CD28 expression on CD4+ cells (up to 61.3% and 66.3%) and on CD8+ cells (up to 17.8% and 21.7%). CD28/CTLA-4 expression didn&rsquo;t increase during co-cultivation of T-lymphocytes with cardiac engineered constructs and iPSC-CM monolayers. Thus, iPSCs-CM in monolayers and on PLA nanofibrous scaffolds didn&rsquo;t cause T-cell activation, which allows us to expect that such cardiac constructs are not a cause of rejection after implantation.

Revisiting The Role of Notch in Nephron Segmentation: Notch is required for proximal, not distal fate selection during mouse and human nephrogenesis

Notch signaling promotes maturation of nephron epithelia, but its proposed contribution to nephron segmentation into proximal and distal domains has been called into doubt. We leveraged single cell and bulk RNA-seq, quantitative immunofluorescent lineage/fate tracing, and genetically modified human iPSC to revisit this question in developing mouse kidneys and human kidney organoids. We confirmed that Notch signaling is needed for maturation of all nephron lineages, and thus mature lineage markers fail to detect a fate bias. By contrast, early markers identified a distal fate bias in cells lacking Notch2, and a concomitant increase in early proximal and podocyte fates in cells expressing hyperactive Notch1 was observed. Orthogonal support for a conserved role for Notch signaling in the distal/proximal axis segmentation is provided by the ability of Nicastrin-deficient hiPSCs-derived organoids to differentiate into TFA2B+ distal tubule and CDH1 connecting segment progenitors, but not into HNF4A+ or LTL+ proximal progenitors.

Green-Synthesized Magnesium Hydroxide Nanoparticles Induced Osteoblastic Differentiation in Bone Co-Cultured Cells

In this work, magnesium hydroxide NPs were synthesized using water (Mg(OH)2 NPs) or a rose hip (RH) extract (Mg(OH)2RH NPs) and tested for the bone cells’ effects in co-cultured osteoblastic and osteoclastic cells, using a Transwell® insert system, allowing reciprocal cell paracrine interactions. Behavior of each cell population was characterized for typical phenotype markers, at days 1 and 6. Cell cultures treated with osteogenic/osteoclastogenic inducers were used as positive control of cell differentiation. The NPs presented a round shape morphology with an average diameter ~90 nm (Mg(OH)2 NPs) and below 10 nm (Mg(OH)2RH NPs. Both NPs induced osteoblastic and osteoclastic behavior similarly to that observed in induced osteoblastic and osteoclastic cultures (positive controls). Differences between the two types of particles were evident at the gene expression level. Compared to Mg(OH)2 NPs, the green-synthesized NPs greatly increased the expression of osteoblastic genes coding for the early markers ALP and collagen type 1 and the later transcription factor osterix, while decreasing the expression of osteoclastogenic genes, namely the essential transcription factor NFATC1, TRAP and the genes coding for the functional markers CA2 and CTSK. Overall, a positive added effect could be hypothesized for Mg(OH)2RH NPs with potential usefulness to promote bone formation in regenerative applications.

Highly sensitive HBsAg, anti-HBc and anti HBsAg titres in Early Diagnosis of HBV Reactivation in Anti-HBc-Positive onco-haematological Patients.

The role of novel HBV markers in predicting Hepatitis B virus reactivation (HBV-R) in HBsAg-negative/anti-HBc-positive oncohaematological patients was examined. One hundred and seven HBsAg-negative/anti-HBc-positive oncohaematological patients, receiving anti-HBV prophylaxis for > 18 months were included. At baseline, all patients had undetectable HBV DNA, and 67.3% were anti-HBs positive. HBV-R occurred in 17 (15.9%) patients: 6 during and 11 after the prophylaxis period. At HBV-R, the median (IQR) HBV-DNA was 44 (27–40509) IU/ml, and the alanine aminotransferase upper limit of normal (ULN) was 44% (median [IQR]: 81[49–541] U/L). An anti-HBc>3 cut-off index (COI) plus anti-HBs persistently/declining to <50 mIU/ml was predictive for HBV-R (OR [95% CI]: 9.1 [2.7–30.2]; 63% of patients with vs. 15% without this combination experienced HBV-R, P<0.001). The detection of highly sensitive (HS) HBsAg and/or HBV-DNA confirmed at > 2 time points, also predicts HBV-R (OR [95% CI ]: 13.8 [3.6–52.6]; 50% of positive vs. 7% of negative patients to these markers experienced HBV-R, P=0.001).HS-HBs and anti-HBc titration proved useful early markers of HBV-R.The use of these markers demonstrated that HBV-R frequently occurs in oncohaematological patients with signs of resolved HBV infection, raising issues of proper HBV-R monitoring.

Mid-gestation cytokine profiles in mothers of children affected by autism spectrum disorder: a case–control study

Autism Spectrum disorder is one of the commonest and most important neurodevelopmental conditions affecting children today. With an increasing prevalence and an unclear aetiology, it is imperative we find early markers of autism, which may facilitate early identification and intervention. Alterations of gestational cytokine profiles have been reported in mothers of autistic children. Increasing evidence suggests that the intrauterine environment is an important determinant of autism risk. This study aims to examine the mid-gestational serum cytokine profiles of the mothers of autistic children from a well-characterised birth cohort. A nested sub-cohort within a large mother–child birth cohort were identified based on a confirmed multi-disciplinary diagnosis of autism before the age 10 years and neuro-typical matched controls in a 2:1 ratio. IFN-γ, IL-1β, IL-4, IL-6, IL-8, IL-17A, GMCSF and TNFα were measured in archived maternal 20-week serum using MesoScale Diagnostics multiplex technology and validation of our IL-17A measurements was performed using an ultrasensitive assay. From a cohort of 2137 children, 25 had confirmed autism before 10 years and stored maternal serum from mid-gestation. We examined the sera of these 25 cases and 50 matched controls. The sex ratio was 4:1 males to females in each group, and the mean age at diagnosis was 5.09 years (SD 2.13). We found that concentrations of IL-4 were significantly altered between groups. The other analytes did not differ significantly using either multiplex or ultra-sensitive assays. In our well-characterised prospective cohort of autistic children, we confirmed mid-gestational alterations in maternal IL-4 concentrations in autism affected pregnancies versus matched controls. These findings add to promising evidence from animal models and retrospective screening programmes and adds to the knowledge in this field.