Mutations of the splicing factor–encoding gene U2AF1 are frequent in the myelodysplastic syndromes (MDS), a myeloid malignancy, and other cancers. Patients with MDS suffer from peripheral blood cytopenias, including anemia, and an increasing percentage of bone marrow myeloblasts. We studied the impact of the common U2AF1S34F mutation on cellular function and mRNA splicing in the main cell lineages affected in MDS. We demonstrated that U2AF1S34F expression in human hematopoietic progenitors impairs erythroid differentiation and skews granulomonocytic differentiation toward granulocytes. RNA sequencing of erythroid and granulomonocytic colonies revealed that U2AF1S34F induced a higher number of cassette exon splicing events in granulomonocytic cells than in erythroid cells. U2AF1S34F altered mRNA splicing of many transcripts that were expressed in both cell types in a lineage-specific manner. In hematopoietic progenitors, the introduction of isoform changes identified in the U2AF1S34F target genes H2AFY, encoding an H2A histone variant, and STRAP, encoding serine/threonine kinase receptor–associated protein, recapitulated phenotypes associated with U2AF1S34F expression in erythroid and granulomonocytic cells, suggesting a causal link. Furthermore, we showed that isoform modulation of H2AFY and STRAP rescues the erythroid differentiation defect in U2AF1S34F MDS cells, suggesting that splicing modulators could be [...] Research Article Hematology

Introduction: Cytotoxic T lymphocytes (CTL) that encounter antigen proliferate from a single naïve T-cell to form a population with diverse effector function and differentiation states, that control and prevent infection and cancer. Antigen-specific CTL are extremely heterogeneous in terms of clonotype, differentiation state and efficacy. Factors that define CTL of superior efficacy are well explored. TCR signal strength is known to modulate the breadth and magnitude of CTL effector functions, and epigenetic marks modulate intrinsic states such as exhaustion that limit activation. It is not clear if or how TCR signal strength modulates contact-dependent killing, and TCR affinity, TCR avidity, or factors downstream of the TCR could all contribute. There is a critical period of contact when a TCR engages cognate peptide- MHC class I molecules on a target cell that determines if the cell will be killed or survive. This is important to explore as not all T-cell therapy products to treat cancer are equally efficacious despite lentiviral transduction with the same chimeric-antigen-receptor. Lack of CTL efficacy is also encountered in chronic viral infections. Indeed, antigen-specific CTL from HIV controllers suppress HIV replication in vitro unlike antigen-specific CTL from chronic progressors. Viral suppression correlates with target cell elimination suggesting a critical role for contact-dependent killing.

The aim of this study was to evaluate the performance of the overall antioxidant of Lactobacillus fermentum LF31 bacterium with prebiotic supplement in human colon cultured cells.The antioxidant capability of L. fermentum LF31 has been assayed in vitro on human colon adenocarcinoma HT-29 cell line using the oxygen radical absorbance capacity method.The analysis revealed that the interaction of probiotic strain cells supplemented with a prebiotic exerts a remarkable antioxidant capacity.The L. fermentum used in the present study exhibited significant in vitro antioxidant capacity, increasing the total antioxidant potential.

The splicing factor SF3B1 is the most frequently mutated gene in myelodysplastic syndromes (MDS), and is strongly associated with the presence of ring sideroblasts (RS). We have performed a systematic analysis of cryptic splicing abnormalities from RNA sequencing data on hematopoietic stem cells (HSCs) of SF3B1 -mutant MDS cases with RS. Aberrant splicing events in many downstream target genes were identified and cryptic 3′ splice site usage was a frequent event in SF3B1- mutant MDS. The iron transporter ABCB7 is a well-recognized candidate gene showing marked downregulation in MDS with RS. Our analysis unveiled aberrant ABCB7 splicing, due to usage of an alternative 3′ splice site in MDS patient samples, giving rise to a premature termination codon in the ABCB7 mRNA. Treatment of cultured SF3B1 -mutant MDS erythroblasts and a CRISPR/Cas9-generated SF3B1 -mutant cell line with the nonsense-mediated decay (NMD) inhibitor cycloheximide showed that the aberrantly spliced ABCB7 transcript is targeted by NMD. We describe cryptic splicing events in the HSCs of SF3B1 -mutant MDS, and our data support a model in which NMD-induced downregulation of the iron exporter ABCB7 mRNA transcript resulting from aberrant splicing caused by mutant SF3B1 underlies the increased mitochondrial iron accumulation found in MDS patients with RS.

Background A major gap in the management of sarcoidosis is the lack of accessible and objective methods to measure disease activity. Since 90% of patients have pulmonary involvement, we explored if a disease activity score based on thoracic CT scans could address this clinical issue. Methods High-resolution CT scans from 100 consecutive patients with sarcoidosis at a regional sarcoidosis service were scored for extent of CT abnormalities known to relate to granuloma or lymphocytic infiltration from published CT-pathological studies. These individual abnormality scores were then correlated against serum ACE, sIL-2R and change in FVC to identify CT abnormalities that reflect contemporaneous disease activity. The sum of these scores, or CT Activity Score (CTAS), was then validated against FVC response to treatment. Findings CT extent scores for nodularity, ground-glass opacification, interlobular septal thickening and consolidation correlated significantly with at least one of the disease activity parameters and were used to form CTAS. CTAS was found to predict FVC response to treatment at 1 year and was highly reproducible between radiologists. An abbreviated CTAS (aCTAS), constructed from presence or absence of the four CT abnormalities, also showed significant correlation with FVC response to treatment. CTAS and aCTAS also correlated with response to treatment in the fibrotic subgroup. Interpretation CTAS provides a concept for an objective and reproducible CT scoring method to quantify disease activity in sarcoidosis. The score can potentially be used to stratify patients according to disease activity, determine response to treatment and establish if fibrotic sarcoidosis is active.

Introduction Monocytes are potential key cellular players inthe early pathogenesis of sarcoidosis. Having previously identified altered monocyte activity in sarcoidosis,1 we now examine monocyte whole genome expression profile in order to determine potential processes and pathways involved in the perturbed immune activity of these cells. Methods Patients with tissue-confirmed sarcoidosis (three high-activity, three low-activity, identified by a predefined clinical activity score)2 were recruited: gene expression profiles compared with age and gender matched healthy controls. All donors were Caucasian, corticosteroid (and other immunemodulatory treatment) naïvenon-smokers. Monocytes were isolated by CD14-negative magnetic selection within 3 h of venesection, RNA extracted using a proprietary kit and stored at -80°C prior to single batch hybridisation with Illumina HumanHT-12 v4 chips. Results A total of 3437 genes were differentially expressed in sarcoidosis monocytes compared with controls (adjusted p value of <0.05). Filtering by Log2 fold change of at least1.5 identified 151 differentially expressed genes among these. Principal component analysis demonstrated clear segregation between sarcoidosis and controls, and between those with high and low activity, with low activity clustering closer to healthy controls. IL-6 was the most significantly upregulated gene (Log2 FC 4.723 adjusted p < 0.001). Other significantly upregulated genes included the pro-inflammatory cytokines IL1A (2.987, 0.001) and IL1B (2.952, 0.001); and the monocyte chemotactic factors CCL20 (4.212, 0.002), CXCL2 (4.057, <0.001) and CCL3 (3.470, 0.003). Gene set enrichment analysis identified gene ontology (GO) gene sets relating to inflammatory andimmune system responses to be amongst the most positively enriched genes in the monocytes from patients with active disease (p < 0.001, normalised enrichment score [NES] 2.22 and 2.20 respectively). Conclusions Sarcoidosis monocytes sarcoidosis have a distinct gene expression profile exhibiting a pro-inflammatory, chemotactic phenotype. IL6 may be implicated in the initiation and maintenance of alveolitis and hypergammaglobulinemia in sarcoidosis and the recent interest in the use of humanised anti-IL-6R antibodies in the treatment of rheumatoid arthritis and other immune mediated disease makes functional characterisation of the role of IL-6 in the pathogenesis of sarcoidosis an exciting prospect. References Crawshaw A et al. Eur J Immunol 2014 Kendrick YR et al. Am J Respir Crit Care Med 2013;187:A1391