Clinical,biochemical, and genetic features of four patients with short‐chain enoyl‐CoA hydratase (ECHS1) deficiency

Short‐chain enoyl‐CoA hydratase (SCEH or ECHS1) deficiency is a rare inborn error of metabolism caused by biallelic mutations in the gene ECHS1 (OMIM 602292). Clinical presentation includes infantile‐onset severe developmental delay, regression, seizures, elevated lactate, and brain MRI abnormalities consistent with Leigh syndrome (LS). Characteristic abnormal biochemical findings are secondary to dysfunction of valine metabolism. We describe four patients from two consanguineous families (one Pakistani and one Irish Traveler), who presented in infancy with LS. Urine organic acid analysis by GC/MS showed increased levels of erythro‐2,3‐dihydroxy‐2‐methylbutyrate and 3‐methylglutaconate (3‐MGC). Increased urine excretion of methacrylyl‐CoA and acryloyl‐CoA related metabolites analyzed by LC‐MS/MS, were suggestive of SCEH deficiency; this was confirmed in patient fibroblasts. Both families were shown to harbor homozygous pathogenic variants in the ECHS1 gene; a c.476A > G (p.Gln159Arg) ECHS1variant in the Pakistani family and a c.538A > G, p.(Thr180Ala) ECHS1 variant in the Irish Traveler family. The c.538A > G, p.(Thr180Ala) ECHS1 variant was postulated to represent a Canadian founder mutation, but we present SNP genotyping data to support Irish ancestry of this variant with a haplotype common to the previously reported Canadian patients and our Irish Traveler family. The presence of detectable erythro‐2,3‐dihydroxy‐2‐methylbutyrate is a nonspecific marker on urine organic acid analysis but this finding, together with increased excretion of 3‐MGC, elevated plasma lactate, and normal acylcarnitine profile in patients with a Leigh‐like presentation should prompt consideration of a diagnosis of SCEH deficiency and genetic analysis of ECHS1. ECHS1 deficiency can be added to the list of conditions with 3‐MGA.     

From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium

Progress in retinal-cell therapy derived from human pluripotent stem cells currently faces technical challenges that require the development of easy and standardized protocols. Here, we developed a simple retinal differentiation method, based on confluent human induced pluripotent stem cells (hiPSC), bypassing embryoid body formation and the use of exogenous molecules, coating, or Matrigel. In 2 wk, we generated both retinal pigmented epithelial cells and self-forming neural retina (NR)-like structures containing retinal progenitor cells (RPCs). We report sequential differentiation from RPCs to the seven neuroretinal cell types in maturated NR-like structures as floating cultures, thereby revealing the multipotency of RPCs generated from integration-free hiPSCs. Furthermore, Notch pathway inhibition boosted the generation of photoreceptor precursor cells, crucial in establishing cell therapy strategies. This innovative process proposed here provides a readily efficient and scalable approach to produce retinal cells for regenerative medicine and for drug-screening purposes, as well as an in vitro model of human retinal development and disease.Irreversible blindness caused by retinal diseases, such as inherited retinopathies, age-related macular degeneration (AMD), or glaucoma, is mainly due to the impairment or loss of function of photoreceptor cells, supporting retinal pigmented epithelium (RPE) or retinal ganglion cells (RGCs). Rescuing the degenerated retina is a major challenge for which specific cell replacement is one of the most promising approaches (1, 2). Pluripotent stem cells, like human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs), have the ability to be expanded indefinitely in culture and could be used as an unlimited source of retinal cells for the treatment of retinal degenerative diseases (3, 4). Several publications have indicated that hESCs and hiPSCs can be differentiated into RPE cells spontaneously after fibroblast growth factor (FGF) 2 removal (5–7) or by different floating aggregate methods (8–11). Concerning neural retinal cells, a growing body of convergent data has demonstrated the ability of hESCs or hiPSCs to be committed into the neural retinal lineage and further differentiated into cells expressing photoreceptor markers (12–15). Recent innovative approaches using 3D cultures from embryoid bodies (EBs) of hESCs or hiPSCs allowed the self-formation of optic cup (OC) structures (16) or the generation of optic vesicle (OV)-like structures (17), depending on the addition of exogenous molecules and different substrates used. These protocols require multiple steps and trained handling, which are not always compatible with the manufacturing process for therapeutic approach or drug screening that need a large-scale production of cells of interest. Therefore, very simple and reliable approaches minimizing the use of exogenous molecules should be developed to generate hESCs or hiPSC-derived retinal cells.In the present study, we report a new retinal differentiation process using confluent hiPSCs, without cell clumps or EB formation and in the absence of Matrigel or serum. We demonstrate that integration-free hiPSCs derived from adult human dermal fibroblasts (AHDFs) cultured in proneural medium can simultaneously generate RPE cells and self-forming neural retinal (NR)-like structures within 2 wk and that, when switched to floating cultures, structures containing retinal progenitor cells (RPCs) can differentiate into all retinal cell types, including RGCs and precursors of photoreceptors, needed for therapeutic applications.     

Immune profile of pediatric renal transplant recipients following alemtuzumab induction

The incidence of developing circulating anti-human leukocyte antigen antibodies and the kinetics of T cell depletion and recovery among pediatric renal transplant recipients who receive alemtuzumab induction therapy are unknown. In a collaborative endeavor to minimize maintenance immunosuppression in pediatric renal transplant recipients, we enrolled 35 participants from four centers and treated them with alemtuzumab induction therapy and a steroid-free, calcineurin-inhibitor–withdrawal maintenance regimen. At 3 months after transplant, there was greater depletion of CD4⁺ than CD8⁺ T cells within the total, naive, memory, and effector memory subsets, although depletion of the central memory subset was similar for CD4⁺ and CD8⁺ cells. Although CD8⁺ T cells recovered faster than CD4⁺ subsets overall, they failed to return to pretransplant levels by 24 months after transplant. There was no evidence for greater recovery of either CD4⁺ or CD8⁺ memory cells than naïve cells. Alemtuzumab relatively spared CD4⁺CD25⁺FoxP3⁺ regulatory T cells, resulting in a rise in their numbers relative to total CD4⁺ cells and a ratio that remained at least at pretransplant levels throughout the study period. Seven participants (20%) developed anti-human leukocyte antigen antibodies without adversely affecting allograft function or histology on 2-year biopsies. Long-term follow-up is underway to assess the potential benefits of this regimen in children.The effects of alemtuzumab on T cell subsets have been extensively studied in adults since its introduction in the 1990s. It has been associated with profound depletion of total T cells and differential recovery among T cell subsets, with early and near-complete recovery of CD8⁺ T cells, but late, partial recovery of CD4⁺ T cells.^1–3 CD4⁺ memory T cells were relatively spared compared with other CD4⁺ subsets; some investigators reported preferential sparing of central memory (T_CM) cells, whereas others observed preferential sparing of the effector memory (T_EM) subset. Emergence of the T_EM subset, whether identified peripherally or in the allograft, has been associated with acute rejection, raising concerns about the tolerogenic potential of alemtuzumab.^1–4 Although the use of alemtuzumab was not associated with an increase in either FoxP3 expression or regulatory T cell counts in vitro, both transient and sustained expansion of regulatory T cells were observed when alemtuzumab was used in association with sirolimus in vivo.^2,5,6 Notwithstanding this, however, the combination of alemtuzumab and sirolimus in protocols free of calcineurin inhibitor (CNI) was associated with rates of acute rejection exceeding 20%, with a humoral rejection rate as high as 62.5%.^7,8 In the absence of long-term CNI treatment, alemtuzumab-treated adults may therefore have a propensity to develop alloantibodies and antibody-mediated rejection.^9,10In contrast, there is a paucity of data regarding the use of alemtuzumab in pediatric solid organ transplantation.¹¹ From a clinical perspective, two small series investigating the outcomes of selected high-risk recipients of renal, liver, and intestinal transplants treated with alemtuzumab yielded conflicting results, whereas a recent series of 42 renal transplant recipients of living donor grafts reported few cases of acute rejection and excellent graft function up to 4 years after transplant.^12–14 From a mechanistic perspective, only one study reported T cell counts in a single pediatric patient, demonstrating profound and prolonged depletion of CD3⁺, CD4⁺, CD8⁺, and CD20⁺ cells, with counts only reaching 50% of their baseline levels 12 months after transplant.¹²In this study, we investigated the longitudinal immune profiles of pediatric renal transplant recipients treated with alemtuzumab induction therapy, followed by a CNI-withdrawal regimen. Specific aims were to characterize the depletion and recovery patterns of various T cell subsets and to screen for anti-human leukocyte antigen (anti-HLA) antibody development.