Queer(ing) medical spaces: queer theory as a framework for transformative social change in anesthesiology and critical care medicine.

Queer theory is a disruptive lens that can be adopted by researchers, educators, clinicians, and administrators to effect transformative social change. It offers opportunities for anesthesiologists, critical care physicians, and medical practitioners to more broadly understand what it means to think queerly and how queering anesthesiology and critical care medicine spaces improves workplace culture and patient outcomes. This article grapples with the cis-heteronormative medical gaze and queer people's apprehensions of violence in medical settings to offer new ways of thinking about structural changes needed in medicine, medical language, and the dehumanizing application of medical modes of care. Using a series of clinical vignettes, this article outlines the historical context underlying queer peoples' distrust of medicine, a primer in queer theory, and an understanding of how to begin to "queer" medical spaces using this critical framework.

RéSUMé: La théorie queer est une lentille perturbatrice qui peut être adoptée par la communauté de la recherche et de l'éducation, les personnes en clinique et les directions d'établissement pour apporter des changements sociaux transformateurs. Elle offre aux anesthésiologistes, aux intensivistes et aux médecins l'occasion de comprendre plus globalement ce que signifie le fait de penser de manière queer et comment la 'queer-icisation' des espaces d'anesthésiologie et de médecine de soins intensifs améliore la culture du milieu de travail et les devenirs des patient·es. Cet article s'attaque au regard médical cis- et hétéronormatif et aux appréhensions des personnes queer face à la violence dans les milieux médicaux afin de proposer de nouvelles façons de penser les changements structurels nécessaires en médecine, le langage médical et l'application déshumanisante des modes de soins médicaux. À l'aide d'une série de vignettes cliniques, cet article décrit le contexte historique sous-jacent à la méfiance des personnes queer à l'égard du monde médical. Il propose également une introduction à la théorie queer et une interprétation de la façon de commencer à rendre plus queer les espaces médicaux en utilisant ce cadre critique.

Identification of lamprey variable lymphocyte receptors that target the brain vasculature.

The blood-brain barrier (BBB) represents a significant bottleneck for the delivery of therapeutics to the central nervous system. In recent years, the promise of coopting BBB receptor-mediated transport systems for brain drug delivery has increased in large part due to the discovery and engineering of BBB-targeting antibodies. Here we describe an innovative screening platform for identification of new BBB targeting molecules from a class of lamprey antigen recognition proteins known as variable lymphocyte receptors (VLRs). Lamprey were immunized with murine brain microvessel plasma membranes, and the resultant repertoire cloned into the yeast surface display system. The library was screened via a unique workflow that identified 16 VLR clones that target extracellular epitopes of in vivo-relevant BBB membrane proteins. Of these, three lead VLR candidates, VLR-Fc-11, VLR-Fc-30, and VLR-Fc-46 selectively target the brain vasculature and traffic within brain microvascular endothelial cells after intravenous administration in mice, with VLR-Fc-30 being confirmed as trafficking into the brain parenchyma. Epitope characterization indicates that the VLRs, in part, recognize sialylated glycostructures. These promising new targeting molecules have the potential for brain targeting and drug delivery with improved brain vascular specificity.

Canadian Doctors for Protection from Guns: how physicians contributed to policy change.

Physicians played a key role in advancing Canada's recent assault weapons ban. Indeed, after announcing the ban in May 2020, the Trudeau government thanked Dr. Najma Ahmed and the group Canadian Doctors for Protection from Guns (CDPG) for their support of responsible gun control measures. In this piece, we explore the context in which CDPG was formed, the strategies used by the group in building nationwide support for gun control measures, and the public health framing of their messaging that proved critical in engendering political change. The work of CDPG holds valuable lessons for physicians seeking to engage in political advocacy by bearing witness to the harms experienced by their patients.

BTN3A1 governs antitumor responses by coordinating αß and γδ T cells.

Gamma delta (γδ) T cells infiltrate most human tumors, but current immunotherapies fail to exploit their in situ major histocompatibility complex-independent tumoricidal potential. Activation of γδ T cells can be elicited by butyrophilin and butyrophilin-like molecules that are structurally similar to the immunosuppressive B7 family members, yet how they regulate and coordinate αß and γδ T cell responses remains unknown. Here, we report that the butyrophilin BTN3A1 inhibits tumor-reactive αß T cell receptor activation by preventing segregation of N-glycosylated CD45 from the immune synapse. Notably, CD277-specific antibodies elicit coordinated restoration of αß T cell effector activity and BTN2A1-dependent γδ lymphocyte cytotoxicity against BTN3A1+ cancer cells, abrogating malignant progression. Targeting BTN3A1 therefore orchestrates cooperative killing of established tumors by αß and γδ T cells and may present a treatment strategy for tumors resistant to existing immunotherapies.

Differentiated agonistic antibody targeting CD137 eradicates large tumors without hepatotoxicity.

CD137 (4-1BB) is a member of the TNFR superfamily that represents a promising target for cancer immunotherapy. Recent insights into the function of TNFR agonist antibodies implicate epitope, affinity, and IgG subclass as critical features, and these observations help explain the limited activity and toxicity seen with clinically tested CD137 agonists. Here, we describe the preclinical characterization of CTX-471, a fully human IgG4 agonist of CD137 that engages a unique epitope that is shared by human, cynomolgus monkey, and mouse and is associated with a differentiated pharmacology and toxicology profile. In vitro, CTX-471 increased IFN-γ production by human T cells in an Fcγ receptor-dependent (FcγR-dependent) manner, displaying an intermediate level of activity between 2 clinical-stage anti-CD137 antibodies. In mice, CTX-471 exhibited curative monotherapy activity in various syngeneic tumor models and showed a unique ability to cure mice of very large (~500 mm3) tumors compared with validated antibodies against checkpoints and TNFR superfamily members. Extremely high doses of CTX-471 were well tolerated, with no signs of hepatic toxicity. Collectively, these data demonstrate that CTX-471 is a unique CD137 agonist that displays an excellent safety profile and an unprecedented level of monotherapy efficacy against very large tumors.

A yeast display immunoprecipitation screen for targeted discovery of antibodies against membrane protein complexes.

Yeast display immunoprecipitation is a combinatorial library screening platform for the discovery and engineering of antibodies against membrane proteins using detergent-solubilized membrane fractions or cell lysates as antigen sources. Here, we present the extension of this method for the screening of antibodies that bind to membrane protein complexes, enabling discovery of antibodies that target antigens involved in a functional protein-protein interaction of interest. For this proof-of-concept study, we focused on the receptor-mediated endocytosis machinery at the blood-brain barrier, and adaptin 2 (AP-2) was chosen as the functional interaction hub. The goal of this study was to identify antibodies that bound to blood-brain barrier (BBB) membrane protein complexes containing AP-2. Screening of a nonimmune yeast display antibody library was carried out using detergent-solubilized BBB plasma membranes as an antigen pool, and antibodies that could interact with protein complexes containing AP-2 were identified. Downstream characterization of isolated antibodies confirmed targeting of proteins known to play important roles in membrane trafficking. This functional yeast display immunoprecipitation screen may be applied to other systems where antibodies against other functional classes of protein complexes are sought.

Identification of variable lymphocyte receptors that can target therapeutics to pathologically exposed brain extracellular matrix.

Diseases that lead to blood-brain barrier (BBB) disruption will pathologically expose normally inaccessible brain extracellular matrix (ECM) to circulating blood components. Therefore, we hypothesized that brain ECM-targeting moieties could specifically target the disrupted BBB and potentially deliver therapies. Variable lymphocyte receptors (VLRs) that preferentially associate with brain ECM were identified from an immune VLR library via yeast surface display biopanning coupled with a moderate throughput ECM screen. Brain ECM binding of VLR clones to murine and human brain tissue sections was confirmed. After systemic administration, P1C10, the lead brain ECM-targeting VLR candidate, specifically accumulated in brains with mannitol-disrupted BBB and at disrupted BBB regions in two different intracranial glioblastoma models. We also demonstrate P1C10's ability to deliver doxorubicin-loaded liposomes, leading to significantly improved survival in glioblastoma-bearing mice. Thus, VLRs can be used to selectively target pathologically exposed brain ECM and deliver drug payloads.

Yeast Display-Based Antibody Affinity Maturation Using Detergent-Solubilized Cell Lysates.

It is often desired to identify or engineer antibodies that target membrane proteins (MPs). However, due to their inherent insolubility in aqueous solutions, MPs are often incompatible with in vitro antibody discovery and optimization platforms. Recently, we adapted yeast display technology to accommodate detergent-solubilized cell lysates as sources of MP antigens. The following protocol details the incorporation of cell lysates into a kinetic screen designed to obtain antibodies with improved affinity via slowed dissociation from an MP antigen.

Targeting receptor-mediated transport for delivery of biologics across the blood-brain barrier.

Biologics are an emerging class of medicines with substantial promise to treat neurological disorders such as Alzheimer's disease, stroke, and multiple sclerosis. However, the blood-brain barrier (BBB) presents a formidable obstacle that appreciably limits brain uptake and hence the therapeutic potential of biologics following intravenous administration. One promising strategy for overcoming the BBB to deliver biologics is the targeting of endogenous receptor-mediated transport (RMT) systems that employ vesicular trafficking to transport ligands across the BBB endothelium. If a biologic is modified with an appropriate targeting ligand, it can gain improved access to the brain via RMT. Various RMT-targeting strategies have been developed over the past 20 years, and this review explores exciting recent advances, emphasizing studies that show brain targeting in vivo.

Introducing glycophage arrays: facile production, purification and patterning of glycophages.

Glycosylation is a widespread post-translational modification that plays important roles in health and disease. As glycan sequence and structure are not directly coded into the genome, our understanding of glycans and their functions in biological systems is much more primitive than that of DNA and proteins.Recently, printed glycan microarrays (glycoarrays) have emerged as powerful, high-throughput tools for screening glycan-protein interactions[1,2], and have been applied in disease detection [3], drug discovery [4], the study of immunity [5], and host-pathogen interactions [1, 2], among others.Unfortunately, glycoarray applications are currently limited by the expensive and complex methods available to synthesize glycans or alternatively, by the challenges in identifying and tagging glycans from natural sources [6, 7]. In this issue of Biotechnology Journal, Çelik et al. [8] introduce a potentially powerful new method for facile, scalable production, and purification of glycans compatible with microarray patterning. Çelik et al.'s [8] approach is based on innovative deployment of filamentous phage display so that the displayed proteins can be tagged with specific glycans of interest (glycophages) and subsequently patterned in array format.

Altering the substrate specificity of RhlI by directed evolution.

REDUCING VIRULENCE: RhlI catalyzes the synthesis of N-butanoyl homoserine lactone (BHL), with a minor product N-hexanoyl homoserine lactone (HHL). By using directed evolution and a genetic screen, RhlI has been engineered for enhanced production of both BHL and HHL at a similar level. Quorum sensing regulates biofilm formation and virulence factor production in the human opportunistic pathogen Pseudomonas aeruginosa. We used directed evolution to engineer RhlI, an enzyme in the RhlI-RhlR quorum-sensing system of P. aeruginosa, to alter its substrate specificity and gain insight into the molecular mechanisms of quorum sensing. By using a genetic screen, we identified a mutant with improved production of RhlI's two signaling molecules, N-butanoyl- and N-hexanoyl-homoserine lactone (BHL and HHL). In particular, production of BHL has been enhanced by more than two-fold, and the synthesis of HHL has been improved from an undetectable level to a level similar to BHL; this change indicates a significant change in substrate specificity. No significant change in the gene expression level was observed. Sequence alignments suggest that the mutations are most likely to facilitate interactions between the enzyme and the two acylated ACP substrates. This work also demonstrates that the genetic screen/selection should be useful in engineering additional quorum-sensing components.