Human plasma cells engineered to secrete bispecifics drive effective in vivo leukemia killing.

Bispecific antibodies are an important tool for the management and treatment of acute leukemias. As a next step toward clinical translation of engineered plasma cells, we describe approaches for secretion of bispecific antibodies by human plasma cells. We show that human plasma cells expressing either fragment crystallizable domain-deficient anti-CD19 × anti-CD3 (blinatumomab) or anti-CD33 × anti-CD3 bispecific antibodies mediate T cell activation and direct T cell killing of B acute lymphoblastic leukemia or acute myeloid leukemia cell lines in vitro. We demonstrate that knockout of the self-expressed antigen, CD19, boosts anti-CD19-bispecific secretion by plasma cells and prevents self-targeting. Plasma cells secreting anti-CD19-bispecific antibodies elicited in vivo control of acute lymphoblastic leukemia patient-derived xenografts in immunodeficient mice co-engrafted with autologous T cells. In these studies, we found that leukemic control elicited by engineered plasma cells was similar to CD19-targeted chimeric antigen receptor-expressing T cells. Finally, the steady-state concentration of anti-CD19 bispecifics in serum 1 month after cell delivery and tumor eradication was comparable with that observed in patients treated with a steady-state infusion of blinatumomab. These findings support further development of ePCs for use as a durable delivery system for the treatment of acute leukemias, and potentially other cancers.

Interventional Techniques for the Management of Knee Osteoarthritis: A Literature Review.

Osteoarthritis of the knee is a prevalent condition that causes pain, discomfort, and disability that can severely impact the quality of life. This literature review aims to review the various interventional pain management techniques available to treat knee osteoarthritis. It analyzes the efficacy of various interventions such as intra-articular corticosteroids, prolotherapy, viscosupplementation, platelet-rich plasma, and genicular nerve blocks with radiofrequency ablation or cryoneurolysis. We searched databases for studies published in the past 20 years. A total of 37 articles were included. The literature supports the idea that a comprehensive treatment plan consisting of the various aforementioned techniques can provide relief for patients while delaying or avoiding joint replacement surgery.

Multimodal Pain Management for Parsonage-Turner Syndrome in the Acute Rehabilitation Setting: A Case Report.

Parsonage-Turner syndrome (PTS) is a rare neurological condition characterized by acute shoulder pain and novel neuromuscular weakness in the distribution of the brachial plexus. We present a case report highlighting the difficulties in the diagnosis and management of this condition. A multidisciplinary approach is often required to control this unique type of pain, consisting of neuropathic medications, non-steroidal anti-inflammatory drugs (NSAIDs) to control neuromuscular pain, and modalities provided by experienced physical therapists. Finally, a comprehensive, structured rehabilitation program focusing on the range of motion, neuromuscular re-education, and strengthening is imperative to regain function, reduce symptoms, and improve recovery.

Human plasma cells engineered to secrete bispecifics drive effective in vivo leukemia killing.

Bispecific antibodies are an important tool for the management and treatment of acute leukemias. Advances in genome-engineering have enabled the generation of human plasma cells that secrete therapeutic proteins and are capable of long-term in vivo engraftment in humanized mouse models. As a next step towards clinical translation of engineered plasma cells (ePCs) towards cancer therapy, here we describe approaches for the expression and secretion of bispecific antibodies by human plasma cells. We show that human ePCs expressing either fragment crystallizable domain deficient anti-CD19 × anti-CD3 (blinatumomab) or anti-CD33 × anti-CD3 bispecific antibodies mediate T cell activation and direct T cell killing of specific primary human cell subsets and B-acute lymphoblastic leukemia or acute myeloid leukemia cell lines in vitro. We demonstrate that knockout of the self-expressed antigen, CD19, boosts anti-CD19 bispecific secretion by ePCs and prevents self-targeting. Further, anti-CD19 bispecific-ePCs elicited tumor eradication in vivo following local delivery in flank-implanted Raji lymphoma cells. Finally, immunodeficient mice engrafted with anti-CD19 bispecific-ePCs and autologous T cells potently prevented in vivo growth of CD19+ acute lymphoblastic leukemia in patient-derived xenografts. Collectively, these findings support further development of ePCs for use as a durable, local delivery system for the treatment of acute leukemias, and potentially other cancers.

Pain Management Interventions in Lumbar Spinal Stenosis: A Literature Review.

Lumbar spinal stenosis (LSS) occurs due to the narrowing of the space within the vertebral canal and or intervertebral foramina. This results in the compression of the spinal cord and possibly the roots of the spinal nerves. Lower back pain and neurogenic claudication (NC) are major symptoms of spinal stenosis. This is a literature review that summarizes the important findings pertaining to pain management of spinal stenosis. Twenty-four original articles were assessed. Pain can be treated through non-invasive or surgical methods. Conservative techniques include physical exercises, epidural corticosteroid injection, local anesthetic injection therapy, and oral analgesics. Surgical intervention deals with the decompression of the affected spinal region, with or without vertebral fusion surgery. Other novel surgical techniques include implantation of specific equipment, known as interspinous spacer devices and minimally invasive lumbar decompression (MILD). Most studies offering a comparative analysis have demonstrated that surgical intervention is more efficacious than non-surgical interventions to manage pain associated with spinal stenosis.

Scoping review of telehealth use by Indigenous populations from Australia, Canada, New Zealand, and the United States.

INTRODUCTION: Telehealth has the potential to address health disparities experienced by Indigenous people, especially in remote areas. This scoping review aims to map and characterize the existing evidence on telehealth use by Indigenous people and explore the key concepts for effective use, cultural safety, and building therapeutic relationships. METHODS: A search for published and gray literature, written in English, and published between 2000 and 2022 was completed in 17 electronic databases. Two reviewers independently screened retrieved records for eligibility. For included articles, data were extracted, categorized, and analyzed. Synthesis of findings was performed narratively. RESULTS: A total of 321 studies were included. The most popular type of telehealth used was mHealth (44%), and the most common health focuses of the telehealth interventions were mental health (26%) and diabetes/diabetic retinopathy (13%). Frequently described barriers to effective telehealth use included concerns about privacy/confidentiality and limited internet availability; meanwhile, telehealth-usage facilitators included cultural relevance and community engagement. Although working in collaboration with Indigenous communities was the most frequently reported way to achieve cultural safety, 40% of the studies did not report Indigenous involvement. Finally, difficulty to establish trusting therapeutic relationships was a major concern raised about telehealth, and evidence suggests that having the first visit-in-person is a potential way to address this issue. CONCLUSION: This comprehensive review identified critical factors to guide the development of culturally-informed telehealth services to meet the needs of Indigenous people and to achieve equitable access and positive health outcomes.

Transcriptomic analysis of pathways associated with ITGAV/alpha(v) integrin-dependent autophagy in human B cells.

Macroautophagy/autophagy proteins have been linked with the development of immune-mediated diseases including lupus, but the mechanisms for this are unclear due to the complex roles of these proteins in multiple immune cell types. We have previously shown that a form of noncanonical autophagy induced by ITGAV/alpha(v) integrins regulates B cell activation by viral and self-antigens, in mice. Here, we investigate the involvement of this pathway in B cells from human tissues. Our data reveal that autophagy is specifically induced in the germinal center and memory B cell subpopulations of human tonsils and spleens. Transcriptomic analysis show that the induction of autophagy is related to unique aspects of activated B cells such as mitochondrial metabolism. To understand the function of ITGAV/alpha(v) integrin-dependent autophagy in human B cells, we used CRISPR-mediated knockdown of autophagy genes. Integrating data from primary B cells and knockout cells, we found that ITGAV/alpha(v)-dependent autophagy limits activation of specific pathways related to B cell responses, while promoting others. These data provide new mechanistic links for autophagy and B-cell-mediated immune dysregulation in diseases such as lupus.

Ex vivo engineered human plasma cells exhibit robust protein secretion and long-term engraftment in vivo.

Due to their unique longevity and capacity to secrete high levels of protein, plasma B cells have the potential to be used as a cell therapy for protein replacement. Here, we show that ex vivo engineered human plasma cells exhibit single-cell RNA profiles, scanning electron micrograph ultrastructural features, and in vivo homing capacity of long-lived plasma cells. After transferring human plasma cells to immunodeficient mice in the presence of the human cytokines BAFF and IL-6, we observe increases in retention of plasma cells in the bone marrow, with engraftment exceeding a year. The most profound in vivo effects of human IL-6 are observed within 20 days of transfer and could be explained by decreased apoptosis in newly differentiated plasma cells. Collectively, these results show that ex vivo engineered and differentiated human plasma cells have the potential for long-lived in vivo protein secretion, which can be modeled in small animals.

Activated interleukin-7 receptor signaling drives B-cell acute lymphoblastic leukemia in mice.

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk subtype of B-ALL often associated with genetic variants that alter cytokine receptor signaling, including mutations in the interleukin-7 receptor (IL7R). To investigate whether IL7R variants are leukemia-initiating, we built mouse models expressing activated Il7r (aIL7R). B-cell intrinsic aIL7R mice developed spontaneous B-ALL, demonstrating sufficiency of Il7r activating mutations in leukemogenesis. Concomitant introduction of a knock-out allele in the associated adapter protein Lnk (encoded by Sh2b3) or a dominant-negative variant of the transcription factor Ikaros (Ikzf1) increased disease penetrance. The resulting murine leukemias displayed monoclonality and recurrent somatic Kras mutations and efficiently engrafted into immunocompetent mice. Phosphoproteomic analyses of aIL7R leukemic cells revealed constitutive Stat5 signaling and B cell receptor (BCR)-like signaling despite the absence of surface pre-BCR. Finally, in vitro treatment of aIL7R leukemic B-cells with Jak, mTOR, or Syk inhibitors blocked growth, confirming that each pathway is active in this mouse model of IL7R-driven B-ALL.

Mast cell surfaceome characterization reveals CD98 heavy chain is critical for optimal cell function.

BACKGROUND: Mast cells are involved in many distinct pathologic conditions, suggesting that they recognize and respond to various stimuli and thus require a rich repertoire of cell surface proteins. However, mast cell surface proteomes have not been comprehensively characterized. OBJECTIVE: We aimed to further characterize the mast cell surface proteome to obtain a better understanding of how mast cells function in health and disease. METHODS: We enriched for glycosylated surface proteins expressed in mouse bone marrow-derived cultured mast cells (BMCMCs) and identified them using mass spectrometry analysis. The presence of novel surface proteins in mast cells was validated by real-time quantitative PCR and flow cytometry analysis in BMCMCs and peritoneal mast cells (PMCs). We developed a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing approach to disrupt genes of interest in BMCMCs. RESULTS: The glycoprotein enrichment approach resulted in the identification of 1270 proteins in BMCMCs, 378 of which were localized to the plasma membrane. The most common protein classes among plasma membrane proteins were small GTPases, receptors, and transporters. One such cell surface protein was CD98 heavy chain (CD98hc), encoded by the Slc3a2 gene. Slc3a2 gene disruption resulted in a significant reduction in CD98hc expression, adhesion, and proliferation. CONCLUSIONS: Glycoprotein enrichment coupled with mass spectrometry can be used to identify novel surface molecules in mast cells. Moreover, CD98hc plays an important role in mast cell function.

Tunable electroconductive decellularized extracellular matrix hydrogels for engineering human cardiac microphysiological systems.

Cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) offer tremendous potential when used to engineer human tissues for drug screening and disease modeling; however, phenotypic immaturity reduces assay reliability when translating in vitro results to clinical studies. To address this, we have developed hybrid hydrogels comprised of decellularized porcine myocardial extracellular matrix (dECM) and reduced graphene oxide (rGO) to provide a more instructive microenvironment for proper cell and tissue development. A tissue-specific protein profile was preserved post-decellularization, and through the modulation of rGO content and degree of reduction, the mechanical and electrical properties of the hydrogels could be tuned. Engineered heart tissues (EHTs) generated using dECM-rGO hydrogel scaffolds and hiPSC-derived cardiomyocytes exhibited significantly increased twitch forces and had increased expression of genes that regulate contractile function. Improvements in various aspects of electrophysiological function, such as calcium-handling, action potential duration, and conduction velocity, were also induced by the hybrid biomaterial. dECM-rGO hydrogels could also be used as a bioink to print cardiac tissues in a high-throughput manner, and these tissues were utilized to assess the proarrhythmic potential of cisapride. Action potential prolongation and beat interval irregularities was observed in dECM-rGO tissues at clinical doses of cisapride, indicating that the enhanced electrophysiological function of these tissues corresponded well with a capability to produce physiologically relevant drug responses.

Multiplexed Functional Assessment of Genetic Variants in CARD11.

Genetic testing has increased the number of variants identified in disease genes, but the diagnostic utility is limited by lack of understanding variant function. CARD11 encodes an adaptor protein that expresses dominant-negative and gain-of-function variants associated with distinct immunodeficiencies. Here, we used a "cloning-free" saturation genome editing approach in a diploid cell line to simultaneously score 2,542 variants for decreased or increased function in the region of CARD11 associated with immunodeficiency. We also described an exon-skipping mechanism for CARD11 dominant-negative activity. The classification of reported clinical variants was sensitive (94.6%) and specific (88.9%), which rendered the data immediately useful for interpretation of seven coding and splicing variants implicated in immunodeficiency found in our clinic. This approach is generalizable for variant interpretation in many other clinically actionable genes, in any relevant cell type.

TWIST1 Heterodimerization with E12 Requires Coordinated Protein Phosphorylation to Regulate Periostin Expression.

Diffuse invasion into adjacent brain matter by glioblastoma (GBM) is largely responsible for their dismal prognosis. Previously, we showed that the TWIST1 (TW) bHLH transcription factor and its regulated gene periostin (POSTN) promote invasive phenotypes of GBM cells. Since TW functional effects are regulated by phosphorylation and dimerization, we investigated how phosphorylation of serine 68 in TW regulates TW dimerization, POSTN expression, and invasion in glioma cells. Compared with wild-type TW, the hypophosphorylation mutant, TW(S68A), impaired TW heterodimerization with the E12 bHLH transcription factor and cell invasion in vitro but had no effect on TW homodimerization. Overexpression of TW:E12 forced dimerization constructs (FDCs) increased glioma cell invasion and upregulated pro-invasive proteins, including POSTN, in concert with cytoskeletal reorganization. By contrast, TW:TW homodimer FDCs inhibited POSTN expression and cell invasion in vitro. Further, phosphorylation of analogous PXSP phosphorylation sites in TW:E12 FDCs (TW S68 and E12 S139) coordinately regulated POSTN and PDGFRa mRNA expression. These results suggested that TW regulates pro-invasive phenotypes in part through coordinated phosphorylation events in TW and E12 that promote heterodimer formation and regulate downstream targets. This new mechanistic understanding provides potential therapeutic strategies to inhibit TW-POSTN signaling in GBM and other cancers.

Synthesizing Signaling Pathways from Temporal Phosphoproteomic Data.

We present a method for automatically discovering signaling pathways from time-resolved phosphoproteomic data. The Temporal Pathway Synthesizer (TPS) algorithm uses constraint-solving techniques first developed in the context of formal verification to explore paths in an interaction network. It systematically eliminates all candidate structures for a signaling pathway where a protein is activated or inactivated before its upstream regulators. The algorithm can model more than one hundred thousand dynamic phosphosites and can discover pathway members that are not differentially phosphorylated. By analyzing temporal data, TPS defines signaling cascades without needing to experimentally perturb individual proteins. It recovers known pathways and proposes pathway connections when applied to the human epidermal growth factor and yeast osmotic stress responses. Independent kinase mutant studies validate predicted substrates in the TPS osmotic stress pathway.

Development of Selected Reaction Monitoring Methods to Systematically Quantify Kinase Abundance and Phosphorylation Stoichiometry in Human Samples.

Protein phosphorylation, one of the most common types of post-translational modifications, is the central regulatory mechanism of cellular signaling networks. In human cells, thousands of proteins are continuously and dynamically phosphorylated and dephosphorylated at specific sites and times in response to external and internal stimuli. Reversible phosphorylation is facilitated by the action of two protein superfamilies: kinases and phosphatases. Kinases play an essential role in almost every relevant physiological process in human cells and their deregulation is linked to pathologies ranging from cancer to autoimmune diseases.Systematic identification of kinases expressed in a particular cell type, quantification of their abundance, and precise determination of their phosphorylation stoichiometry are essential to understand the cellular signaling networks and physiology of a sample. Our protocol outlines the steps to build and use a high-throughput, comprehensive, modular, and robust selected reaction monitoring (SRM) proteomics framework to facilitate quantification of the kinome state in research or clinical human samples.

Integrated systems biology analysis of KSHV latent infection reveals viral induction and reliance on peroxisome mediated lipid metabolism.

Kaposi's Sarcoma associated Herpesvirus (KSHV), an oncogenic, human gamma-herpesvirus, is the etiological agent of Kaposi's Sarcoma the most common tumor of AIDS patients world-wide. KSHV is predominantly latent in the main KS tumor cell, the spindle cell, a cell of endothelial origin. KSHV modulates numerous host cell-signaling pathways to activate endothelial cells including major metabolic pathways involved in lipid metabolism. To identify the underlying cellular mechanisms of KSHV alteration of host signaling and endothelial cell activation, we identified changes in the host proteome, phosphoproteome and transcriptome landscape following KSHV infection of endothelial cells. A Steiner forest algorithm was used to integrate the global data sets and, together with transcriptome based predicted transcription factor activity, cellular networks altered by latent KSHV were predicted. Several interesting pathways were identified, including peroxisome biogenesis. To validate the predictions, we showed that KSHV latent infection increases the number of peroxisomes per cell. Additionally, proteins involved in peroxisomal lipid metabolism of very long chain fatty acids, including ABCD3 and ACOX1, are required for the survival of latently infected cells. In summary, novel cellular pathways altered during herpesvirus latency that could not be predicted by a single systems biology platform, were identified by integrated proteomics and transcriptomics data analysis and when correlated with our metabolomics data revealed that peroxisome lipid metabolism is essential for KSHV latent infection of endothelial cells.

A computationally engineered RAS rheostat reveals RAS-ERK signaling dynamics.

Synthetic protein switches controlled with user-defined inputs are powerful tools for studying and controlling dynamic cellular processes. To date, these approaches have relied primarily on intermolecular regulation. Here we report a computationally guided framework for engineering intramolecular regulation of protein function. We utilize this framework to develop chemically inducible activator of RAS (CIAR), a single-component RAS rheostat that directly activates endogenous RAS in response to a small molecule. Using CIAR, we show that direct RAS activation elicits markedly different RAS-ERK signaling dynamics from growth factor stimulation, and that these dynamics differ among cell types. We also found that the clinically approved RAF inhibitor vemurafenib potently primes cells to respond to direct wild-type RAS activation. These results demonstrate the utility of CIAR for quantitatively interrogating RAS signaling. Finally, we demonstrate the general utility of our approach in design of intramolecularly regulated protein tools by applying it to the Rho family of guanine nucleotide exchange factors.

Endogenous N-terminal Domain Cleavage Modulates α1D-Adrenergic Receptor Pharmacodynamics.

The α1D-adrenergic receptor (ADRA1D) is a key regulator of cardiovascular, prostate, and central nervous system functions. This clinically relevant G protein-coupled receptor has proven difficult to study, as it must form an obligate modular homodimer containing the PDZ proteins scribble and syntrophin or become retained in the endoplasmic reticulum as non-functional protein. We previously determined that targeted removal of the N-terminal (NT) 79 amino acids facilitates ADRA1D plasma membrane expression and agonist-stimulated functional responses. However, whether such an event occurs in physiological contexts was unknown. Herein, we report the ADRA1D is subjected to innate NT processing in cultured human cells. SNAP near-infrared imaging and tandem-affinity purification revealed the ADRA1D is expressed as both full-length and NT truncated forms in multiple human cell lines. Serial truncation mapping identified the cleavage site as Leu(90)/Val(91) in the 95-amino acid ADRA1D NT domain, suggesting human cells express a Δ1-91 ADRA1D species. Tandem-affinity purification MS/MS and co-immunoprecipitation analysis indicate NT processing of ADRA1D is not required to form scribble-syntrophin macromolecular complexes. Yet, label-free dynamic mass redistribution signaling assays demonstrate that Δ1-91 ADRA1D agonist responses were greater than WT ADRA1D. Mutagenesis of the cleavage site nullified the processing event, resulting in ADRA1D agonist responses less than the WT receptor. Thus, we propose that processing of the ADRA1D NT domain is a physiological mechanism employed by cells to generate a functional ADRA1D isoform with optimal pharmacodynamic properties.

Dynamic mass redistribution reveals diverging importance of PDZ-ligands for G protein-coupled receptor pharmacodynamics.

G protein-coupled receptors (GPCRs) are essential membrane proteins that facilitate cell-to-cell communication and co-ordinate physiological processes. At least 30 human GPCRs contain a Type I PSD-95/DLG/Zo-1 (PDZ) ligand in their distal C-terminal domain; this four amino acid motif of X-[S/T]-X-[φ] sequence facilitates interactions with PDZ domain-containing proteins. Because PDZ protein interactions have profound effects on GPCR ligand pharmacology, cellular localization, signal-transduction effector coupling and duration of activity, we analyzed the importance of Type I PDZ ligands for the function of 23 full-length and PDZ-ligand truncated (ΔPDZ) human GPCRs in cultured human cells. SNAP-epitope tag polyacrylamide gel electrophoresis revealed most Type I PDZ GPCRs exist as both monomers and multimers; removal of the PDZ ligand played minimal role in multimer formation. Additionally, SNAP-cell surface staining indicated removal of the PDZ ligand had minimal effects on plasma membrane localization for most GPCRs examined. Label-free dynamic mass redistribution functional responses, however, revealed diverging effects of the PDZ ligand. While no clear trend was observed across all GPCRs tested or even within receptor families, a subset of GPCRs displayed diminished agonist efficacy in the absence of a PDZ ligand (i.e. HT2RB, ADRB1), whereas others demonstrated enhanced agonist efficacies (i.e. LPAR2, SSTR5). These results demonstrate the utility of label-free functional assays to tease apart the contributions of conserved protein interaction domains for GPCR signal-transduction coupling in cultured cells.

Individual protomers of a G protein-coupled receptor dimer integrate distinct functional modules.

Recent advances in proteomic technology reveal G-protein-coupled receptors (GPCRs) are organized as large, macromolecular protein complexes in cell membranes, adding a new layer of intricacy to GPCR signaling. We previously reported the α1D-adrenergic receptor (ADRA1D)-a key regulator of cardiovascular, urinary and CNS function-binds the syntrophin family of PDZ domain proteins (SNTA, SNTB1, and SNTB2) through a C-terminal PDZ ligand interaction, ensuring receptor plasma membrane localization and G-protein coupling. To assess the uniqueness of this novel GPCR complex, 23 human GPCRs containing Type I PDZ ligands were subjected to TAP/MS proteomic analysis. Syntrophins did not interact with any other GPCRs. Unexpectedly, a second PDZ domain protein, scribble (SCRIB), was detected in ADRA1D complexes. Biochemical, proteomic, and dynamic mass redistribution analyses indicate syntrophins and SCRIB compete for the PDZ ligand, simultaneously exist within an ADRA1D multimer, and impart divergent pharmacological properties to the complex. Our results reveal an unprecedented modular dimeric architecture for the ADRA1D in the cell membrane, providing unexpected opportunities for fine-tuning receptor function through novel protein interactions in vivo, and for intervening in signal transduction with small molecules that can stabilize or disrupt unique GPCR:PDZ protein interfaces.