rapid and robust cd4 and cd8 t nk b cell dendritic cell and monocyte reconstitution after nicotinamide expanded cord blood transplantation

Introduction Nicotinamide-expanded cord blood (NiCord) is a potential alternative source for allogeneic hematopoietic cell transplantation (HCT) when an HLA-id donor is lacking. A phase 1/2 trial with standalone NiCord HCT showed rapid neutrophil- (11 days) and platelet engraftment (34 days). We previously reported that successful CD4+ immune reconstitution (IR) is crucial for infectious and relapse control associated with favorable survival (JACI 2017) and is a better predictor for event-free survival than neutrophil reconstitution. We performed unique in-depth immune monitoring to evaluate and compare IR after NiCord and conventional HCT. Methods In this phase1/2 international multicenter trial, we compared IR after NiCord HCT to cohorts of adolescent and young adult (AYA) patients receiving either unmanipulated cord blood transplantation (unCBT) or T-repleted-unrelated bone marrow transplantation (BMT). All patients received HCT for a hematologic malignancy with myeloablative conditioning without serotherapy. Immune monitoring was performed (harmonized sampling, handling and analyses) in a central lab. The primary endpoint was probability of achieving CD4+ IR (>50*106/L within 100 days). Secondary endpoints were IR of B-cells, CD4+ and CD8+ T-cells, natural killer (NK)-cells, monocytes, and dendritic cells (DC) 7-365 days after HCT. In addition, TREC analyses were performed on CD3+ MACs-sorted cells. Linear-mixed effects modelling in LOESS-regression curves and two-sided log-rank test for univariate comparisons in cumulative incidence plots were used. Results 27 NiCord recipients (median 41.5; 13.4-61.7yrs) were included. NiCord cell dose consisted of median 6.4*106 CD34+/kg, and 2.3*106 CD3+T-cells/kg of the co-infused negative fraction (following CD133+ selection). Of these patients, 91% achieved successful CD4+ IR, which was comparable (p=0.76, Figure 1) to the 27 unCBT (median 15.4; 12.2-22.1 yrs) and 20 BMT (median 14.3; 12.1-19.7 yrs) recipients included in this study. We observed similar reconstitution of T-cells (p=0.15), monocytes (p=0.94), conventional DCs (p=0.41), and plasmacytoid DCs (p=0.52). Interestingly, reconstitution of NK-cells (p Conclusions In-depth immune monitoring reveals fast and full IR after NiCord HCT in adult patients, which is equal or even faster to IR after unCBT or BMT, despite the younger age of the AYA cohorts (expected to reconstitute faster). This may be explained by the higher stem cell dose and higher proliferative capacity of the NiCord-expanded product. Optimal comparison of IR in NiCord vs. unCBT in a randomized phase 3 trial is underway

Exome sequencing in patient-parent trios suggests new candidate genes for early-onset primary sclerosing cholangitis

BACKGROUND & AIMS Primary sclerosing cholangitis (PSC) is a rare bile duct disease strongly associated with inflammatory bowel disease (IBD). Whole-exome sequencing (WES) has contributed to understanding the molecular basis of very early-onset IBD, but rare protein-altering genetic variants have not been identified for early-onset PSC. We performed WES in patients diagnosed with PSC METHODS In this multicentre study, WES was performed on 87 DNA samples from 29 patient-parent trios with early-onset PSC. We selected rare (minor allele frequency <2%) coding and splice-site variants that matched recessive (homozygous and compound heterozygous variants) and dominant (de novo) inheritance in the index patients. Variant pathogenicity was predicted by an in-house developed algorithm (GAVIN), and PSC-relevant variants were selected using gene expression data and gene function. RESULTS In 22 of 29 trios we identified at least 1 possibly pathogenic variant. We prioritized 36 genes, harbouring a total of 54 variants with predicted pathogenic effects. In 18 genes, we identified 36 compound heterozygous variants, whereas in the other 18 genes we identified 18 de novo variants. Twelve of 36 candidate risk genes are known to play a role in transmembrane transport, adaptive and innate immunity, and epithelial barrier function. CONCLUSIONS The 36 candidate genes for early-onset PSC need further verification in other patient cohorts and evaluation of gene function before a causal role can be attributed to its variants.Peer reviewe

Can hysterosalpingo-foam sonography replace hysterosalpingography as first-choice tubal patency test? A randomized non-inferiority trial

Funding Information: The FOAM study was an investigator-initiated study funded by ZonMw, The Netherlands organization for Health Research and Development (project number 837001504). ZonMw funded the whole project. IQ Medical Ventures provided the ExEm-foamVR kits free of charge. The funders had no role in study design, collection, analysis and interpretation of the data. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.Peer reviewedPublisher PD

The FOAM study : Is Hysterosalpingo foam sonography (HyFoSy) a cost-effective alternative for hysterosalpingography (HSG) in assessing tubal patency in subfertile women? Study protocol for a randomized controlled trial

This is an investigator initiated trial, VU medical center Amsterdam is the sponsor, contact information: prof. CJM de Groot, Department of Obstetrics and Gynaecology, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands, Tel: + 31-204444444. This study is funded by ZonMw, a Dutch organization for Health Research and Development, project number 837001504. ZonMW gives financial support for the whole project. IQ Medical Ventures provides the ExEm FOAM® kits. The funding bodies have no role in the design of the study; collection, analysis, and interpretation of data; and in writing the manuscript.Peer reviewedPublisher PD

Cost-effective sequence analysis of 113 genes in 1,192 probands with retinitis pigmentosa and Leber congenital amaurosis

Introduction: Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are two groups of inherited retinal diseases (IRDs) where the rod photoreceptors degenerate followed by the cone photoreceptors of the retina. A genetic diagnosis for IRDs is challenging since >280 genes are associated with these conditions. While whole exome sequencing (WES) is commonly used by diagnostic facilities, the costs and required infrastructure prevent its global applicability. Previous studies have shown the cost-effectiveness of sequence analysis using single molecule Molecular Inversion Probes (smMIPs) in a cohort of patients diagnosed with Stargardt disease and other maculopathies. Methods: Here, we introduce a smMIPs panel that targets the exons and splice sites of all currently known genes associated with RP and LCA, the entire RPE65 gene, known causative deep-intronic variants leading to pseudo-exons, and part of the RP17 region associated with autosomal dominant RP, by using a total of 16,812 smMIPs. The RP-LCA smMIPs panel was used to screen 1,192 probands from an international cohort of predominantly RP and LCA cases. Results and discussion: After genetic analysis, a diagnostic yield of 56% was obtained which is on par with results from WES analysis. The effectiveness and the reduced costs compared to WES renders the RP-LCA smMIPs panel a competitive approach to provide IRD patients with a genetic diagnosis, especially in countries with restricted access to genetic testing.This study received funding from Novartis. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. This work was supported by grants from Foundation Fighting Blindness Career Development Award CDGE-0621-0809-RAD (SR), Foundation Fighting Blindness project program award PPA-0123-0841-UCL (SR and SdB), Retinitis Pigmentosa Fighting Blindness, Fight for Sight UK (RP Genome Project GR586), Ghent University Special Research Fund (BOF20/GOA/023) (EDB and BL); EJP RD Solve-RET EJPRD19-234 (EDB, BL, SB, CR, FC, and SR). EDB (1802220N) and BL (1803816N) are FWO Senior Clinical Investigators of the Research Foundation Flanders (FWO). EDB, BL, SB, FC, and SR are members of ERN-EYE (Framework Partnership Agreement No. 739534)

The Development of Metabolomic Sampling Procedures for Pichia pastoris, and Baseline Metabolome Data

Metabolic profiling is increasingly being used to investigate a diverse range of biological questions. Due to the rapid turnover of intracellular metabolites it is important to have reliable, reproducible techniques for sampling and sample treatment. Through the use of non-targeted analytical techniques such as NMR and GC-MS we have performed a comprehensive quantitative investigation of sampling techniques for Pichia pastoris. It was clear that quenching metabolism using solutions based on the standard cold methanol protocol caused some metabolite losses from P. pastoris cells. However, these were at a low level, with the NMR results indicating metabolite increases in the quenching solution below 5% of their intracellular level for 75% of metabolites identified; while the GC-MS results suggest a slightly higher level with increases below 15% of their intracellular values. There were subtle differences between the four quenching solutions investigated but broadly, they all gave similar results. Total culture extraction of cells + broth using high cell density cultures typical of P. pastoris fermentations, was an efficient sampling technique for NMR analysis and provided a gold standard of intracellular metabolite levels; however, salts in the media affected the GC-MS analysis. Furthermore, there was no benefit in including an additional washing step in the quenching process, as the results were essentially identical to those obtained just by a single centrifugation step. We have identified the major high-concentration metabolites found in both the extra- and intracellular locations of P. pastoris cultures by NMR spectroscopy and GC-MS. This has provided us with a baseline metabolome for P. pastoris for future studies. The P. pastoris metabolome is significantly different from that of Saccharomyces cerevisiae, with the most notable difference being the production of high concentrations of arabitol by P. pastoris

The GC-Rich Mitochondrial and Plastid Genomes of the Green Alga Coccomyxa Give Insight into the Evolution of Organelle DNA Nucleotide Landscape

Most of the available mitochondrial and plastid genome sequences are biased towards adenine and thymine (AT) over guanine and cytosine (GC). Examples of GC-rich organelle DNAs are limited to a small but eclectic list of species, including certain green algae. Here, to gain insight in the evolution of organelle nucleotide landscape, we present the GC-rich mitochondrial and plastid DNAs from the trebouxiophyte green alga Coccomyxa sp. C-169. We compare these sequences with other GC-rich organelle DNAs and argue that the forces biasing them towards G and C are nonadaptive and linked to the metabolic and/or life history features of this species. The Coccomyxa organelle genomes are also used for phylogenetic analyses, which highlight the complexities in trying to resolve the interrelationships among the core chlorophyte green algae, but ultimately favour a sister relationship between the Ulvophyceae and Chlorophyceae, with the Trebouxiophyceae branching at the base of the chlorophyte crown