Biology subjects

Stowell, S. R.

Publications and source records attributed to Stowell, S. R..

5 recordsLinked to original sources

bioRxiv2026

The Human Contraceptive Antibody Targets A Semenogelin Galectin-3 N-Linked Glycan Complex on the Sperm Surface

The human contraception antibody (HCA) is a non-hormonal topical contraceptive entering Phase II clinical trials that functions by agglutinating and immobilizing sperm. To date, the HCA epitope has been described as an N-linked polylactosamine glycan motif on CD52, but this assignment has been inferred only by colocalization with anti-CD52 and S19 antibodies in western blots. Here, we report that HCA may instead bind semenogelin-galectin-3 complexes associated with the sperm surface. Semenogelins are gel forming, highly glycosylated proteins, with molecular weights of 50-70 kDa that are cleaved into smaller segments that adhere to sperm during semen liquefaction. In this study, we isolated the HCA antigen from seminal plasma and sperm using an HCA affinity column and identified semenogelin by mass spectrometry. Western blot analysis of seminal plasma showed that HCA binds to a large smear of proteins with a higher apparent molecular mass than CD52, and that these may correspond to semenogelin sequences. Flow cytometric analysis of enzymatically deglycosylated sperm demonstrated that HCA binding requires sperm surface N-linked glycans. However, despite this apparent glycan dependence, HCA failed to bind any glycans represented on a comprehensive glycan microarray. In addition to semenogelin, Galectin-3 was identified in the HCA purified sperm lysates, supporting a model in which HCA recognizes semenogelin-derived glycopeptides that are tethered to the sperm surface via a galectin mediated interaction with an N-linked glycan. We found that O-linked glycan modifications on the semenogelin proteins contain LacNAc motifs that can bind to galectins. Finally, we found that depletion of galectins bound to the sperm surface by competitive inhibitors reduced HCA-mediated agglutination, and this could be rescued by addition of recombinant galectin-3/semenogelin-2 complexes to sperm cultures. Collectively, our findings suggest that HCA's binding site involves a semenogelin-galectin-3 complex anchored to the sperm surface via an N-linked glycan.

immunology↗

bioRxiv2026

Transforming clinical apheresis waste into a renewable source of patient-derived CD34+ hematopoietic stem cell biobank for beta-hemoglobinopathy research and therapeutic discovery

Background aimsThe development of next-generation therapies for sickle cell disease (SCD) and beta thalassemia (beta thal), including fetal globin-inducing small molecules and gene therapy approaches, depends on patient-derived CD34+ hematopoietic stem and progenitor cells (HSPCs) for discovery and preclinical validation, but commercial vendors stock only healthy donor material and disease-specific banks hold limited inventories. Recent US Food and Drug Administration and National Institutes of Health guidance favoring human cell-based methods over animal testing underscores the value of authentic patient cells. Methods: Over 14 months we recovered, purified, and biobanked CD34+ HSPCs from clinical apheresis product waste and mobilized peripheral blood (PB) otherwise discarded after clinical procedures, using immunomagnetic selection adapted for hemoglobinopathy specimens; a microfluidic technology was evaluated separately. We quantified yield and purity for bead-selected material and cell number and viability for the microfluidic pilot; engraftment was tested in NBSGW mice. Results: Immunomagnetic selection recovered a median of 4.71 x 106 CD34+ cells from just 1 to 2 mL of apheresis product waste, comparable to the 6.0 x 106 cells from a 10 to 40 fold larger volume of PB waste, with similar purity across sources and diagnoses. Because apheresis product waste is far more concentrated, it reaches equivalent yields without the density-gradient steps required for PB waste, approximately halving processing time. Recovered cells engrafted NBSGW mice, confirming preserved repopulating capacity. The microfluidic pilot (two patients, 11 specimens) recovered 2.17 x 106 CD34+ cells per specimen at greater than 90% viability and purity. Conclusions: A center with existing apheresis infrastructure can reproducibly recover, bank, and distribute research-grade patient CD34+ HSPCs, addressing a recognized gap in the hemoglobinopathy pipeline. HighlightsO_LIClinical apheresis waste is used to generate a single-center biobank of high-quality, research-grade CD34+ HSPCs from patients with sickle cell disease and beta-thalassemia. C_LIO_LIConcentrated apheresis waste matches large-volume PB waste in CD34+ yield and purity. C_LIO_LIMicrofluidic enrichment recovers CD34+ cells at >90% viability and purity across 2 patients. C_LIO_LIRecovered CD34+ HSPCs engraft mice and form erythroid cells, preserving function. C_LI

pharmacology and toxicology↗

bioRxiv2021

Innate and adaptive immunity to transfused allogenic red blood cells in mice requires MyD88

Red blood cell (RBC) transfusion therapy is essential for the survival of patients with hematological disorders such as sickle cell anemia. A potentially fatal complication of transfusion is development of non-ABO alloantibodies to polymorphic RBC antigens, yet mechanisms of alloantibody formation remain unclear. Human and mouse RBCs acquire a "storage lesion" prior to transfusion, which in mice contributes to immunogenicity. We previously reported that mouse splenic dendritic cells (DCs) are required for RBC alloimmunization and are activated by sterile and leukoreduced mouse RBCs after storage. Yet how syngeneic RBCs activate innate immune pathways to induce DC activation is unknown. We now show that DC activation to transfused RBCs occurs regardless of alloantigen presence, suggesting that RBC damage induced during storage triggers innate immune receptors. We discovered an unexpected dependence of RBC alloimmunization on the Toll-like receptor (TLR) signaling adaptor molecule MyD88. TLRs are a class of pattern recognition receptors (PRRs) that regulate DC activation and signal through two adaptor molecules, MyD88 and TRIF. We show that the inflammatory cytokine response, DC activation, and the subsequent alloantibody response to transfused syngeneic RBCs require MyD88 but not TRIF, suggesting a restricted set of PRRs are responsible for sensing RBCs and triggering alloimmunization.

immunology↗

bioRxiv2020

Endogenous Galectin-3 is Required for Skeletal Muscle Repair

Skeletal muscle has the intrinsic ability to self-repair through a multifactorial process, but many aspects of its cellular and molecular mechanisms are not fully understood. There is increasing evidence that some members of the mammalian {beta}-galactoside-binding protein family (galectins) are involved in the muscular repair process (MRP), including galectin-3 (Gal-3). However, there are many questions about the role of this protein on muscle self-repair. Here, we demonstrate that endogenous Gal-3 is required for: i) muscle repair in vivo using a chloride-barium myolesion mouse model, and ii) mouse primary myoblasts myogenic programming. Injured muscle from Gal-3 knockout mice (GAL3KO) showed persistent inflammation associated with compromised muscle repair and the formation of fibrotic tissue on the lesion site. In GAL3KO mice, osteopontin expression remained high even after 7 and 14 days of the myolesion, while MyoD and myogenin had decreased their expression. In GAL3KO mouse primary myoblast cell culture, Pax7 detection seems to sustain even when cells are stimulated to differentiation and MyoD expression is drastically reduced. These findings suggest that the detection and temporal expression levels of these transcriptional factors appear to be altered in Gal-3-deficient myoblast cell culture compared to Wild Type (WT) cells. We observed Gal-3 expression in WT states, both in vivo and in vitro, in sarcoplasm/cytoplasm and myonuclei; as differentiation proceeds, Gal-3 expression is drastically reduced, and its location is confined to the sarcolemma/plasma cell membrane. We also observed a change in the temporal-spatial profile of Gal-3 expression and muscle transcription factors levels during the myolesion. Overall, these results demonstrate that endogenous Gal-3 is required for the skeletal muscle repair process.

cell biology↗

bioRxiv2020

Galectin-1-deficient mice are protected against Trypanosoma cruzi infection through altered neutrophil migration and production of reactive oxygen species and nitric oxide

Trypanosoma cruzi is an intracellular parasite that causes Chagas disease that affects millions of people worldwide. Many cellular and molecular aspects of this neglected disease are not fully understood. Prior studies have shown that galectin-1 (Gal-1), a {beta}-galactoside-binding protein that regulates leukocyte recruitment to the inflammatory site, and promotes T. cruzi infection, but the mechanism is unclear. Here, we report that C57BL/6 mice lacking Gal-1 (Lgals1-/-) exhibited lower parasitemia and higher survival rates than their wildtype (WT) counterparts when infected with T. cruzi Y strain. Two weeks after infection, Lgals1-/- mice displayed greater neutrophil accumulation in infection site and heart tissue than WT mice. In T. cruzi-infected Lgals1-/- mice, infiltrated neutrophils produced increased levels of reactive oxygen species (ROS), while macrophages and neutrophils produced increased levels of nitric oxide (NO), which reduced replication and viability of parasites in vitro and downregulated IL-1{beta} production. Pharmacological inhibition of NADPH oxidase and NO synthase during early in vivo infection reversed the protective effect of Gal-1 deficiency in Lgals1-/- mice. Together, our findings demonstrate that lacking Gal-1 favors neutrophil migration to the infection site and increases production of ROS and NO, thereby controlling the early steps of T. cruzi infection by reducing parasitemia and prolonging survival of infected mice.

immunology↗