Stem Cell Res

Primary hyperoxaluria type 1 (PH1) is a rare autosomal recessive disorder of the liver metabolism due to functional deficiency of the peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT). AGT deficiency results in overproduction of oxalate which complexes with calcium to form insoluble calcium-oxalate salts in urinary tracts, ultimately leading to end-stage renal disease. Currently, the only curative treatment for PH1 is combined liver-kidney transplantation, which is limited by donor organ shortage and lifelong requirement for immunosuppression. Transplantation of genetically modified autologous hepatocytes is an attractive therapeutic option for PH1. However, the use of fresh primary hepatocytes suffers from limitations such as organ availability, insufficient cell proliferation, loss of function, and the risk of immune rejection. We developed patient-specific induced pluripotent stem cells (PH1-iPSCs) free of reprogramming factors as a source of renewable and genetically defined autologous PH1-hepatocytes. We then investigated additive gene therapy using a lentiviral vector encoding wild-type AGT under the control of the liver-specific transthyretin promoter. Genetically modified PH1-iPSCs successfully provided hepatocyte-like cells (HLCs) that exhibited significant AGT expression at both RNA and protein levels after liver-specific differentiation process. These results pave the way for cell-based therapy of PH1 by transplantation of genetically modified autologous HLCs derived from patient-specific iPSCs

Mapping of transrectal ultrasonographic prostate biopsies: quality control and learning curve assessment by image processing

Objective: Mapping of transrectal ultrasonographic (TRUS) prostate biopsies is of fundamental importance for either diagnostic purposes or the management and treatment of prostate cancer, but the localization of the cores seems inaccurate. Our objective was to evaluate the capacities of an operator to plan transrectal prostate biopsies under 2-dimensional TRUS guidance using a registration algorithm to represent the localization of biopsies in a reference 3-dimensional ultrasonographic volume. Methods: Thirty-two patients underwent a series of 12 prostate biopsies under local anesthesia performed by 1 operator using a TRUS probe combined with specific third-party software to verify that the biopsies were indeed conducted within the planned targets. RESULTS: The operator reached 71% of the planned targets with substantial variability that depended on their localization (100% success rate for targets in the middle and right parasagittal parts versus 53% for targets in the left lateral base). Feedback from this system after each series of biopsies enabled the operator to significantly improve his dexterity over the course of time (first 16 patients: median score, 7 of 10 and cumulated median biopsy length in targets of 90 mm; last 16 patients, median score, 9 of 10 and a cumulated median length of 121 mm; P = .046). Conclusions: In addition to being a useful tool to improve the distribution of prostate biopsies, the potential of this system is above all the preparation of a detailed "map" of each patient showing biopsy zones without substantial changes in routine clinical practices

An Orthotopic Model of Glioblastoma Is Resistant to Radiodynamic Therapy with 5-AminoLevulinic Acid

Radiosensitization of glioblastoma is a major ambition to increase the survival of this incurable cancer. The 5-aminolevulinic acid (5-ALA) is metabolized by the heme biosynthesis pathway. 5-ALA overload leads to the accumulation of the intermediate fluorescent metabolite protoporphyrin IX (PpIX) with a radiosensitization potential, never tested in a relevant model of glioblastoma. We used a patient-derived tumor cell line grafted orthotopically to create a brain tumor model. We evaluated tumor growth and tumor burden after different regimens of encephalic multifractionated radiation therapy with or without 5-ALA. A fractionation scheme of 5 × 2 Gy three times a week resulted in intermediate survival [48-62 days] compared to 0 Gy (15-24 days), 3 × 2 Gy (41-47 days) and, 5 × 3 Gy (73-83 days). Survival was correlated to tumor growth. Tumor growth and survival were similar after 5 × 2 Gy irradiations, regardless of 5-ALA treatment (RT group (53-67 days), RT+5-ALA group (40-74 days), HR = 1.57, p = 0.24). Spheroid growth and survival were diminished by radiotherapy in vitro, unchanged by 5-ALA pre-treatment, confirming the in vivo results. The analysis of two additional stem-like patient-derived cell lines confirmed the absence of radiosensitization by 5-ALA. Our study shows for the first time that in a preclinical tumor model relevant to human glioblastoma, treated as in clinical routine, 5-ALA administration, although leading to important accumulation of PpIX, does not potentiate radiotherapy

CD63-GPC1-Positive Exosomes Coupled with CA19-9 Offer Good Diagnostic Potential for Resectable Pancreatic Ductal Adenocarcinoma

Tumor-released extracellular vesicles (EVs) contain tumor-specific cargo distinguishing them from healthy EVs, and making them eligible as circulating biomarkers. Glypican 1 (GPC1)-positive exosome relevance as liquid biopsy elements is still debated. We carried out a prospective study to quantify GPC1-positive exosomes in sera from pancreatic ductal adenocarcinoma (PDAC) patients undergoing up-front surgery, as compared to controls including patients without cancer history and patients displaying pancreatic preneoplasic lesions. Sera were enriched in EVs, and exosomes were pulled down with anti-CD63 coupled magnetic beads. GPC1-positive bead percentages determined by flow cytometry were significantly higher in PDAC than in the control group. Diagnosis accuracy reached 78% (sensitivity 64% and specificity 90%), when results from peripheral and portal blood were combined. In association with echo-guided-ultrasound-fine-needle-aspiration (EUS-FNA) negative predictive value was 80% as compared to 33% for EUS-FNA only. This approach is clinically relevant as a companion test to the already available diagnostic tools, since patients with GPC1-positive exosomes in peripheral blood showed decreased tumor free survival

Covichem: A biochemical severity risk score of COVID-19 upon hospital admission

Clinical and laboratory predictors of COVID-19 severity are now well described and combined to propose mortality or severity scores. However, they all necessitate saturable equipment such as scanners, or procedures difficult to implement such as blood gas measures. To provide an easy and fast COVID-19 severity risk score upon hospital admission, and keeping in mind the above limits, we sought for a scoring system needing limited invasive data such as a simple blood test and co-morbidity assessment by anamnesis. A retrospective study of 303 patients (203 from Bordeaux University hospital and an external independent cohort of 100 patients from Paris Pitié-Salpêtrière hospital) collected clinical and biochemical parameters at admission. Using stepwise model selection by Akaike Information Criterion (AIC), we built the severity score Covichem. Among 26 tested variables, 7: obesity, cardiovascular conditions, plasma sodium, albumin, ferritin, LDH and CK were the independent predictors of severity used in Covichem (accuracy 0.87, AUROC 0.91). Accuracy was 0.92 in the external validation cohort (89% sensitivity and 95% specificity). Covichem score could be useful as a rapid, costless and easy to implement severity assessment tool during acute COVID-19 pandemic waves

Biologie Végétale - Nutrition et Métabolisme

Biologie Végétale - Nutrition et Métabolism

Mapping of transrectal ultrasonographic prostate biopsies: quality control and learning curve assessment by image processing.

International audienceOBJECTIVE: Mapping of transrectal ultrasonographic (TRUS) prostate biopsies is of fundamental importance for either diagnostic purposes or the management and treatment of prostate cancer, but the localization of the cores seems inaccurate. Our objective was to evaluate the capacities of an operator to plan transrectal prostate biopsies under 2-dimensional TRUS guidance using a registration algorithm to represent the localization of biopsies in a reference 3-dimensional ultrasonographic volume. METHODS: Thirty-two patients underwent a series of 12 prostate biopsies under local anesthesia performed by 1 operator using a TRUS probe combined with specific third-party software to verify that the biopsies were indeed conducted within the planned targets. RESULTS: The operator reached 71% of the planned targets with substantial variability that depended on their localization (100% success rate for targets in the middle and right parasagittal parts versus 53% for targets in the left lateral base). Feedback from this system after each series of biopsies enabled the operator to significantly improve his dexterity over the course of time (first 16 patients: median score, 7 of 10 and cumulated median biopsy length in targets of 90 mm; last 16 patients, median score, 9 of 10 and a cumulated median length of 121 mm; P = .046). CONCLUSIONS: In addition to being a useful tool to improve the distribution of prostate biopsies, the potential of this system is above all the preparation of a detailed "map" of each patient showing biopsy zones without substantial changes in routine clinical practices

CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells

International audienceCRISPR-Cas9 is a promising technology for gene therapy. However, the ON-target genotoxicity of CRISPR-Cas9 nuclease due to DNA double-strand breaks has received little attention and is probably underestimated. Here we report that genome editing targeting globin genes induces megabase-scale losses of heterozygosity (LOH) from the globin CRISPR-Cas9 cut-site to the telomere (5.2 Mb). In established lines, CRISPR-Cas9 nuclease induces frequent terminal chromosome 11p truncations and rare copy-neutral LOH. In primary hematopoietic progenitor/stem cells, we detect 1.1% of clones (7/648) with acquired megabase LOH induced by CRISPR-Cas9. In-depth analysis by SNP-array reveals the presence of copy-neutral LOH. This leads to 11p15.5 partial uniparental disomy, comprising two Chr11p15.5 imprinting centers (H19/IGF2:IG-DMR/IC1 and KCNQ1OT1:TSS-DMR/IC2) and impacting H19 and IGF2 expression. While this genotoxicity is a safety concern for CRISPR clinical trials, it is also an opportunity to model copy-neutral-LOH for genetic diseases and cancers