Biology subjects

Brehm, M. A.

Publications and source records attributed to Brehm, M. A..

5 recordsLinked to original sources

bioRxiv2026

Humanized FLT3 mice display enhanced tissue engraftment and support HIV-1 persistence and rebound

Despite advances in antiretroviral therapy (ART), HIV-1 cure efforts remain hindered by viral reservoirs in long-lived myeloid cells and immune-privileged tissues that are less accessible and therefore unlikely to be assessed in human clinical trials. Consequently, there is a critical need for robust research platforms such as immune cell humanized mice to bridge preclinical and clinical HIV research. However, previously described humanized mouse models have demonstrated incomplete hematopoietic development, particularly showing low levels of NK or myeloid cells. Herein, we present the novel humanized FLT3 mouse model that develops NK cells, myeloid progenitors, monocytes, and both functional conventional (cDCs) and plasmacytoid dendritic cells (pDCs) to support HIV-1 infection. Human cord blood derived CD34+ hematopoietic stem cells (HSC) were engrafted in the FLT3 (Hu-FLT3) and NSG (Hu-NSG) mouse strains for comparison. Our data showed that while Hu-NSG and Hu-FLT3 mice have comparable human lymphocyte levels, the proportion of myeloid cells (including monocytes, pDCs and cDCs) in Hu-FLT3 mice (16.2 %) was three-fold higher than in Hu-NSG mice (5.6 %) and the proportion of NK cells was six-fold higher (12.8 % and 1.9 %, respectively). Both strains successfully supported HIV-1 infection, maintain viral replication for 17 weeks in untreated mice, and proviral DNA was detectable in peripheral blood, bone marrow and spleen. While ART effectively reduced viral load to undetectable levels in four weeks in both strains, we observed viral rebound after treatment discontinuation within 3 weeks, reaching the same levels of viral load pre-ART and mimicking what is observed in people living with HIV (PLWH).Human immune cells and HIV-1 RNA were higher in tissues of Hu-FLT3 mice compared to Hu-NSG mice, mirroring features reported in human tissue reservoirs. Our findings demonstrated that Hu-FLT3 mice support enhanced development of human innate immune cells in blood and tissues, which are associated with higher levels of HIV-1 replication compared to Hu-NSG mice. This study establishes a novel, robust and accessible in vivo platform to investigate potential HIV cure and persistence-targeting interventions with translational relevance to human therapeutic development thanks to the improved and more complete human immune repertoire in Hu-FLT3.

immunology↗

bioRxiv2023

A human immune/muscle xenograft model of FSHD muscle pathology

BackgroundFacioscapulohumeral muscular dystrophy (FSHD) disease progression is associated with muscle inflammation, although its role in FSHD muscle pathology is unknown. MethodsWe have developed a novel humanized mouse strain, NSG-SGM3-W41, that supports the co- engraftment of human hematopoietic stem cells (HSCs) and muscle myoblasts as an experimental model to investigate the role of innate immunity in FSHD muscle pathology. ResultsThe NSG-SGM3-W41 mouse supports the selective expansion of human innate immune cell lineages following engraftment of human HSCs and the co-engraftment and differentiation of patient-derived FSHD or control muscle myoblasts. Immunohistological and NanoString RNA expression assays establish that muscle xenografts from three FSHD subjects were immunogenic compared to those from unaffected first-degree relatives. FSHD muscle xenografts preferentially accumulated human macrophages and B cells and expressed early complement genes of the classical and alternative pathways including complement factor C3 protein, which is a mediator of early complement function through opsonization to mark damaged cells for macrophage engulfment. FSHD muscle xenografts also underwent immune donor dependent muscle turnover as assayed by human spectrin {beta}1 immunostaining of muscle fibers and by NanoString RNA expression assays of muscle differentiation genes. ConclusionsThe NSG-SGM3-W41 mouse provides an experimental model to investigate the role of innate immunity and complement in FSHD muscle pathology and to develop FSHD therapeutics targeting DUX4 and the innate immunity inflammatory responses.

immunology↗

bioRxiv2022

Minimization of metabolic cost of transport predicts changes in gait mechanics over a range of ankle-foot orthosis stiffnesses in individuals with bilateral plantar flexor weakness

Neuromuscular disorders often lead to ankle plantar flexor muscle weakness, which impairs ankle push-off power and forward propulsion during gait. To improve walking speed and reduce metabolic cost of transport (mCoT), patients with plantar flexor weakness are provided dorsal-leaf spring ankle-foot orthoses (AFOs). The mCoT during gait depends on the AFO stiffness where an optimal AFO stiffness exists that minimizes mCoT. The biomechanics of why and how there exists a unique optimal stiffness for individuals with plantar flexor weakness are not well understood. To help understand why, we hypothesized that gait adaptations can be predicted by mCoT minimization. To explain how, we hypothesized that the AFO would reduce the required support moment and, hence, metabolic costs from the ankle plantar flexor and knee extensor muscles during stance and reduce hip flexor metabolic cost to initiate swing. To test these hypotheses, we generated neuromusculoskeletal simulations to represent gait of an individual with bilateral plantar flexor weakness wearing an AFO with varying stiffness. Predictions were predicated on the goal of minimizing mCoT at each stiffness level, and the motor patterns were determined via dynamic optimization. The simulation results were compared to experimental data from subjects with bilateral plantar flexor weakness walking with varying AFO-stiffness. Our simulations demonstrated that minimization of mCoT predicts gait adaptations in response to varying AFO stiffness levels in individuals with bilateral plantar flexor weakness. Initial reductions in mCoT with increasing stiffness were attributed to reductions in quadriceps metabolic cost during midstance. Increases in mCoT above optimum stiffness were attributed to the increasing metabolic cost of both hip flexor and hamstrings muscles. The insights gained from our simulations could inform clinicians on the prescription of personalized AFOs. With further model individualization, simulations based on mCoT minimization may sufficiently predict adaptations to an AFO in individuals with plantar flexor weakness. Author SummaryNeuromuscular disorders like stroke, Charcot-Marie-Tooth disease, and poliomyelitis often lead to calf muscle weakness, which makes walking slower and more demanding. To improve walking speed and reduce energy demand, patients with calf muscle weakness are frequently provided ankle-foot orthoses (AFOs). The energy demand of walking is affected by the AFOs stiffness and there is a stiffness that minimizes the energy demand for an individual with calf weakness. To uncover the optimal stiffness, we generated simulations of an individual with calf muscle weakness walking with an AFO over a range of stiffnesses. Stable walking patterns were generated that minimized the energy demand for a given stiffness. We found that the initial reductions in energy demand as stiffness increased, were attributed to reductions in quadriceps muscle energy. Increases in energy demand as stiffness increased above the optimum were attributed to the increased energetic cost of both hip flexor and hamstrings muscles. With further model individualization, we believe that simulations based on minimizing the energy demand of movement can sufficiently predict adaptations to an AFO. Simulations can enable the prescription of personalized AFOs for individuals with neuromuscular disorders that help them walk with sufficient speed and efficiency to keep up with their peers.

bioengineering↗

bioRxiv2021

A Conformational Transition of Von Willebrand Factor's D'D3 Domain Primes It For Multimerization

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that is critically involved in hemostasis. Biosynthesis of long VWF concatemers in the endoplasmic reticulum and the (trans-)Golgi is still not fully understood. We use the single-molecule force spectroscopy technique magnetic tweezers to analyze a previously hypothesized conformational change in the DD3 domain crucial for VWF multimerization. We find that the interface formed by submodules C8-3, TIL3, and E3 wrapping around VWD3 can open and expose two previously buried cysteines that are known to be vital for multimerization. By characterizing the conformational change at varying levels of force, we are able to quantify the kinetics of the transition and the stability of the interface. We find a pronounced destabilization of the interface upon lowering the pH from 7.4 to 6.2 and 5.5. This is consistent with initiation of the conformational change that enables VWF multimerization at the DD3 domain by a decrease in pH in the trans-Golgi network and Weibel-Palade bodies. Furthermore, we find a stabilization of the interface in the presence of coagulation factor VIII (FVIII), providing evidence for a previously hypothesized binding site in submodule C8-3. Our findings highlight the critical role of the DD3 domain in VWF biosynthesis and function and we anticipate our methodology to be applicable to study other, similar conformational changes in VWF and beyond.

biophysics↗

bioRxiv2018

Modular, ultra-stable, and highly parallel protein force spectroscopy in magnetic tweezers using peptide linkers

Single-molecule force spectroscopy has provided unprecedented insights into protein folding, force-regulation, and function. Here, we present a modular magnetic tweezers force spectroscopy approach that uses elastin-like polypeptide linkers to provide a high yield of protein tethers. Our approach extends protein force spectroscopy into the low force (<1 pN) regime and enables ultra-stable measurements on many molecules in parallel. We present (un-)folding data for the single protein domain ddFLN4 and for the large multi-domain dimeric protein von Willebrand factor (VWF) that is critically involved in primary hemostasis. The measurements reveal exponential force-dependencies of unfolding and refolding rates, directly resolve the stabilization of the VWF A2 domain by Ca2+, and discover transitions in the VWF C-domain stem at low forces that likely constitute the first steps of VWF activation in vivo. Our modular attachment approach will enable precise and multiplexed force spectroscopy measurements for a wide range of proteins in the physiologically relevant force regime.

biophysics↗