Ex vivo culture resting time impacts transplantation outcomes of genome-edited human hematopoietic stem and progenitor cells in xenograft mouse models.

Ex vivo resting culture is a standard procedure following genome editing in hematopoietic stem and progenitor cells (HSPCs). However, prolonged culture may critically affect cell viability and stem cell function. We investigated whether varying durations of culture resting times impact the engraftment efficiency of human CD34+ HSPCs edited at the BCL11A enhancer, a key regulator in the expression of fetal hemoglobin. We employed electroporation to introduce CRISPR-Cas9 components for BCL11A enhancer editing and compared outcomes with nonelectroporated (NEP) and electroporated-only (EP) control groups. Post-electroporation, we monitored cell viability, death rates, and the frequency of enriched hematopoietic stem cell (HSC) fractions (CD34+CD90+CD45RA- cells) over a 48-hour period. Our findings reveal that while the NEP group showed an increase in cell numbers 24 hours post-electroporation, both EP and BCL11A-edited groups experienced significant cell loss. Although CD34+ cell frequency remained high in all groups for up to 48 hours post-electroporation, the frequency of the HSC-enriched fraction was significantly lower in the EP and edited groups compared to the NEP group. In NBSGW xenograft mouse models, both conditioned with busulfan and nonconditioned, we found that immediate transplantation post-electroporation led to enhanced engraftment without compromising editing efficiency. Human glycophorin A+ (GPA+) red blood cells (RBCs) sorted from bone marrow of all BCL11A edited mice exhibited similar levels of γ-globin expression, regardless of infusion time. Our findings underscore the critical importance of optimizing the culture duration between genome editing and transplantation. Minimizing this interval may significantly enhance engraftment success and minimize cell loss without compromising editing efficiency. These insights offer a pathway to improve the success rates of genome editing in HSPCs, particularly for conditions like sickle cell disease.

Associations of Urine Biomarkers of Kidney Tubule Health With Incident Hypertension and Longitudinal Blood Pressure Change in Middle-Aged Adults: The CARDIA Study.

BACKGROUND: Urine biomarkers of kidney tubule injury associate with incident hypertension in older adults with comorbidities, but less is known about these associations in younger adults. METHODS: In 1170 participants of the CARDIA study (Coronary Artery Risk Development in Young Adults; mean age, 45 years; 40% Black people; 56% women) without hypertension, cardiovascular disease, or kidney disease at baseline, we examined associations of urine MCP-1 (monocyte chemoattractant protein-1), α1m (alpha-1-microglobulin), KIM-1 (kidney injury molecule-1), EGF (epidermal growth factor), IL (interleukin)-18, YKL-40 (chitinase-3-like protein 1), and UMOD (uromodulin) with incident hypertension (onset of systolic blood pressure [BP] ≥130 mm Hg or diastolic BP ≥80 mm Hg or initiation of hypertension medications) and longitudinal BP change in models adjusted for hypertension risk factors, estimated glomerular filtration rate, and albuminuria. RESULTS: After a median 9.9 (interquartile range, 5.9-10.2) years, 376 participants developed incident hypertension. In demographic-adjusted analyses, higher tertiles of EGF associated with lower risk of incident hypertension in both Black and White participants. After multivariable adjustment, the risk of incident hypertension remained lower in tertile 2 (hazard ratio, 0.70 [95% CI, 0.50-0.97]) and tertile 3 (hazard ratio, 0.58 [0.39-0.85]) of EGF versus tertile 1. In fully adjusted models, participants in EGF tertile 3 had smaller 10-year increases in systolic (-3.4 [95% CI, -6.1 to -0.7] mm Hg) and diastolic BP (-2.6 [95% CI, -4.6 to -0.6] mm Hg) than tertile 1. Other biomarkers showed inconsistent associations with incident hypertension and BP change. CONCLUSIONS: In middle-aged adults without hypertension, cardiovascular disease, or kidney disease, higher urine EGF associated with lower risk of incident hypertension and lower 10-year BP elevations.

Complete Resolution of Paraneoplastic Membranous Nephropathy Following Curative Therapy of Triple-Negative Breast Cancer.

A paraneoplastic syndrome, which includes glomerulopathy, is a manifestation of malignancy unexplained by direct tumor burden. Membranous nephropathy (MN) may be associated with malignancies that are primarily solid tumors of the lung, prostate and gastrointestinal tract. It is rarely associated with breast cancer. To our knowledge, we herein report the first case of MN associated with triple-negative carcinoma of the breast. The patient initially presented with MN as a paraneoplastic nephrotic syndrome. Treatment resulting in a complete pathological response of the breast cancer also resolved the MN. Neither has recurred after a 48-month follow-up. The patient exhibited autoantibodies against phospholipase A2 receptor and was also antinuclear antibody (ANA) and anti-Smith (anti-Sm) antibody positive. These results suggest that the neoplasm evoked an autoimmune response, which resolved with treatment. ANA and anti-SM positivity closely correlated with the neoplasm activity supporting this hypothesis.