Outcomes of Cardiopulmonary Resuscitation of Oncologic Patients in Emergency Department.

OBJECTIVE: To evaluate the outcomes of cardiopulmonary resuscitation (CPR) of oncological cases versus non-oncological admitted to the emergency department as out-of-hospital cardiac arrest (OHCA) or in-hospital cardiac arrest (IHCA). STUDY DESIGN: Descriptive study. PLACE AND DURATION OF STUDY: Emergency Department (ED) of Eskisehir Osmangazi University Hospital, between January 2014 to January 2020. METHODOLOGY: Victims over the age of 18 years who had OHCA and IHCA were inducted. The outcomes of 109 patients with an oncological diagnosis and 109 controls without cancer underwent CPR and were compared. RESULTS: The median age of the participants was 65 (58-76) years. Patients with an oncological diagnosis were more likely to have an IHCA [OR: 2.98 (95% CI: 1.68-5.30), p <0.001]. The IHCA and OHCA rates of patients without an oncological diagnosis were similar. Solid-organ malignancies were observed in 102 patients (93.6%). The initial rhythm of 88 patients (80.7%) in the oncological arrest group was asystole versus 77 patients (70.6%) in the control group. Pulseless electrical activity was observed in 17 patients (15.6%) in the study group and in 24 patients (22.0%) in the control group. Although the non-oncological group was found to have a longer stay. No statistically significant difference was found between the study and control groups regarding duration of stay in the intensive care unit. Only one patient (2.0%) with cancer was discharged in stable state as against 10 (21.3%) of non-oncological arrests [OR: 12.97 (95% CI: 1.59-105.93), p = 0.008]. CONCLUSION: The presence of cancer is not a favourable prognostic factor for the success of CPR. KEY WORDS: Oncology, Cardiac arrest, IHCA, OHCA, Emergency department.

PD-L1+ neutrophils contribute to injury-induced infection susceptibility.

The underlying mechanisms contributing to injury-induced infection susceptibility remain poorly understood. Here, we describe a rapid increase in neutrophil cell numbers in the lungs following induction of thermal injury. These neutrophils expressed elevated levels of programmed death ligand 1 (PD-L1) and exhibited altered gene expression profiles indicative of a reparative population. Upon injury, neutrophils migrate from the bone marrow to the skin but transiently arrest in the lung vasculature. Arrested neutrophils interact with programmed cell death protein 1 (PD-1) on lung endothelial cells. A period of susceptibility to infection is linked to PD-L1+ neutrophil accumulation in the lung. Systemic treatment of injured animals with an anti-PD-L1 antibody prevented neutrophil accumulation in the lung and reduced susceptibility to infection but augmented skin healing, resulting in increased epidermal growth. This work provides evidence that injury promotes changes to neutrophils that are important for wound healing but contribute to infection susceptibility.

Long-acting antibody ligand mimetics for HER4-selective agonism.

Neuregulin protein 1 (NRG1) is a large (> 60-amino-acid) natural peptide ligand for the ErbB protein family members HER3 and HER4. We developed an agonistic antibody modality, termed antibody ligand mimetics (ALM), by incorporating complex ligand agonists such as NRG1 into an antibody scaffold. We optimized the linker and ligand length to achieve native ligand activity in HEK293 cells and cardiomyocytes derived from induced pluripotent stem cells (iPSCs) and used a monomeric Fc-ligand fusion platform to steer the ligand specificity toward HER4-dominant agonism. With the help of selectivity engineering, these enhanced ALM molecules can provide an antibody scaffold with increased receptor specificity and the potential to greatly improve the pharmacokinetics, stability, and downstream developability profiles from the natural ligand approach. This ligand mimetic design and optimization approach can be expanded to apply to other cardiovascular disease targets and emerging therapeutic areas, providing differentiated drug molecules with increased specificity and extended half-life.

Functional mimetic of the G-protein coupled receptor CXCR4 on a soluble antibody scaffold.

G-protein coupled receptors (GPCRs) constitute major drug targets due to their involvement in critical biological functions and pathophysiological disorders. The leading challenge in their structural and functional characterization has been the need for a lipid environment to accommodate their hydrophobic cores. Here, we report an antibody scaffold mimetic (ASM) platform where we have recapitulated the extracellular functional domains of the GPCR, C-X-C chemokine receptor 4 (CXCR4) on a soluble antibody framework. The engineered ASM molecule can accommodate the N-terminal loop and all three extracellular loops of CXCR4. These extracellular features are important players in ligand recruitment and interaction for allostery and signal transduction. Our study shows that ASMCXCR4 can be recognized by the anti-CXCR4 antibodies, MEDI3185, 2B11, and 12G5, and that ASMCXCR4 can bind the HIV-1 glycoprotein ligand gp120, and the natural chemokine ligand SDF-1α. Further, we show that ASMCXCR4 can competitively inhibit the SDF-1α signaling pathway, and be used as an immunogen to generate CXCR4-specific antibodies. This platform will be useful in the study of GPCR biology in a soluble receptor context for evaluating its extracellular ligand interactions.

Ligand- and cation-induced structural alterations of the leukocyte integrin LFA-1.

In αI integrins, including leukocyte function-associated antigen 1 (LFA-1), ligand-binding function is delegated to the αI domain, requiring extra steps in the relay of signals that activate ligand binding and coordinate it with cytoplasmic signals. Crystal structures reveal great variation in orientation between the αI domain and the remainder of the integrin head. Here, we investigated the mechanisms involved in signal relay to the αI domain, including whether binding of the ligand intercellular adhesion molecule-1 (ICAM-1) to the αI domain is linked to headpiece opening and engenders a preferred αI domain orientation. Using small-angle X-ray scattering and negative-stain EM, we define structures of ICAM-1, LFA-1, and their complex, and the effect of activation by Mn2+ Headpiece opening was substantially stabilized by substitution of Mg2+ with Mn2+ and became complete upon ICAM-1 addition. These agents stabilized αI-headpiece orientation, resulting in a well-defined orientation of ICAM-1 such that its tandem Ig-like domains pointed in the opposite direction from the ß-subunit leg of LFA-1. Mutations in the integrin ßI domain α1/α1' helix stabilizing either the open or the closed ßI-domain conformation indicated that α1/α1' helix movements are linked to ICAM-1 binding by the αI domain and to the extended-open conformation of the ectodomain. The LFA-1-ICAM-1 orientation described here with ICAM-1 pointing anti-parallel to the LFA-1 ß-subunit leg is the same orientation that would be stabilized by tensile force transmitted between the ligand and the actin cytoskeleton and is consistent with the cytoskeletal force model of integrin activation.

Enhancement of antibody functions through Fc multiplications.

Antibodies carry out a plethora of functions through their crystallizable fragment (Fc) regions, which can be naturally tuned by the adoption of several isotypes and post-translational modifications. Protein engineering enables further Fc function modulations through modifications of the interactions between the Fc and its functional partners, including FcγR, FcRn, complement complex, and additions of auxiliary functional units. Due to the many functions embedded within the confinement of an Fc, a suitable balance must be maintained for a therapeutic antibody to be effective and safe. The outcome of any Fc engineering depends on the interplay among all the effector molecules involved. In this report, we assessed the effects of Fc multiplication (or tandem Fc) on antibody functions. Using IgG1 as a test case, we found that, depending on the specifically designed linker, Fc multiplication led to differentially folded, stable molecules with unique pharmacokinetic profiles. Interestingly, the variants with 3 copies of Fc improved in vitro opsonophagocytic killing activity and displayed significantly improved protective efficacies in a Klebsiella pneumoniae mouse therapeutic model despite faster clearance compared with its IgG1 counterpart. There was no adverse effect observed or pro-inflammatory cytokine release when the Fc variants were administered to animals. We further elucidated that enhanced binding to various effector molecules by IgG-3Fc created a "sink" leading to the rapid clearance of the 3Fc variants, and identified the increased FcRn binding as one strategy to facilitate "sink" escape. These findings reveal new opportunities for novel Fc engineering to further expand our abilities to manipulate and improve antibody therapeutics.

Anti-MrkA Monoclonal Antibodies Reveal Distinct Structural and Antigenic Features of MrkA.

Antibody therapy against antibiotics resistant Klebsiella pneumoniae infections represents a promising strategy, the success of which depends critically on the ability to identify appropriate antibody targets. Using a target-agnostic strategy, we recently discovered MrkA as a potential antibody target and vaccine antigen. Interestingly, the anti-MrkA monoclonal antibodies isolated through phage display and hybridoma platforms all recognize an overlapping epitope, which opens up important questions including whether monoclonal antibodies targeting different MrkA epitopes can be generated and if they possess different protective profiles. In this study we generated four anti-MrkA antibodies targeting different epitopes through phage library panning against recombinant MrkA protein. These anti-MrkA antibodies elicited strong in vitro and in vivo protections against a multi-drug resistant Klebsiella pneumoniae strain. Furthermore, mutational and epitope analysis suggest that the two cysteine residues may play essential roles in maintaining a MrkA structure that is highly compacted and exposes limited antibody binding/neutralizing epitopes. These results suggest the need for further in-depth understandings of the structure of MrkA, the role of MrkA in the pathogenesis of Klebsiella pneumoniae and the protective mechanism adopted by anti-MrkA antibodies to fully explore the potential of MrkA as an efficient therapeutic target and vaccine antigen.

Force interacts with macromolecular structure in activation of TGF-ß.

Integrins are adhesion receptors that transmit force across the plasma membrane between extracellular ligands and the actin cytoskeleton. In activation of the transforming growth factor-ß1 precursor (pro-TGF-ß1), integrins bind to the prodomain, apply force, and release the TGF-ß growth factor. However, we know little about how integrins bind macromolecular ligands in the extracellular matrix or transmit force to them. Here we show how integrin αVß6 binds pro-TGF-ß1 in an orientation biologically relevant for force-dependent release of TGF-ß from latency. The conformation of the prodomain integrin-binding motif differs in the presence and absence of integrin binding; differences extend well outside the interface and illustrate how integrins can remodel extracellular matrix. Remodelled residues outside the interface stabilize the integrin-bound conformation, adopt a conformation similar to earlier-evolving family members, and show how macromolecular components outside the binding motif contribute to integrin recognition. Regions in and outside the highly interdigitated interface stabilize a specific integrin/pro-TGF-ß orientation that defines the pathway through these macromolecules which actin-cytoskeleton-generated tensile force takes when applied through the integrin ß-subunit. Simulations of force-dependent activation of TGF-ß demonstrate evolutionary specializations for force application through the TGF-ß prodomain and through the ß- and not α-subunit of the integrin.

A novel antibody discovery platform identifies anti-influenza A broadly neutralizing antibodies from human memory B cells.

Monoclonal antibody isolation directly from circulating human B cells is a powerful tool to delineate humoral responses to pathological conditions and discover antibody therapeutics. We have developed a platform aimed at improving the efficiencies of B cell selection and V gene recovery. Here, memory B cells are activated and amplified using Epstein-Barr virus infection, co-cultured with CHO-muCD40L cells, and then assessed by functional screenings. An in vitro transcription and translation (IVTT) approach was used to analyze variable (V) genes recovered from each B cell sample and identify the relevant heavy/light chain pair(s). We achieved efficient amplification and activation of memory B cells, and eliminated the need to: 1) seed B cells at clonal level (≤1 cell/well) or perform limited dilution cloning; 2) immortalize B cells; or 3) assemble V genes into an IgG expression vector to confirm the relevant heavy/light chain pairing. Cross-reactive antibodies targeting a conserved epitope on influenza A hemagglutinin were successfully isolated from a healthy donor. In-depth analysis of the isolated antibodies suggested their potential uses as anti-influenza A antibody therapeutics and uncovered a distinct affinity maturation pathway. Importantly, our results showed that cognate heavy/light chain pairings contributed to both the expression level and binding abilities of our newly isolated VH1-69 family, influenza A neutralizing antibodies, contrasting with previous observations that light chains do not significantly contribute to the function of this group of antibodies. Our results further suggest the potential use of the IVTT as a powerful antibody developability assessment tool.

Dimeric quaternary structure of human laforin.

The phosphatase laforin removes phosphate groups from glycogen during biosynthetic activity. Loss-of-function mutations in the gene encoding laforin is the predominant cause of Lafora disease, a fatal form of progressive myoclonic epilepsy. Here, we used hybrid structural methods to determine the molecular architecture of human laforin. We found that laforin adopts a dimeric quaternary structure, topologically similar to the prototypical dual specificity phosphatase VH1. The interface between the laforin carbohydrate-binding module and the dual specificity phosphatase domain generates an intimate substrate-binding crevice that allows for recognition and dephosphorylation of phosphomonoesters of glucose. We identify novel molecular determinants in the laforin active site that help decipher the mechanism of glucan phosphatase activity.

Shape change in the receptor for gliding motility in Plasmodium sporozoites.

Sporozoite gliding motility and invasion of mosquito and vertebrate host cells in malaria is mediated by thrombospondin repeat anonymous protein (TRAP). Tandem von Willebrand factor A (VWA) and thrombospondin type I repeat (TSR) domains in TRAP connect through proline-rich stalk, transmembrane, and cytoplasmic domains to the parasite actin-dependent motility apparatus. We crystallized fragments containing the VWA and TSR domains from Plasmodium vivax and Plasmodium falciparum in different crystal lattices. TRAP VWA domains adopt closed and open conformations, and bind a Mg(2+) ion at a metal ion-dependent adhesion site implicated in ligand binding. Metal ion coordination in the open state is identical to that seen in the open high-affinity state of integrin I domains. The closed VWA conformation associates with a disordered TSR domain. In contrast, the open VWA conformation crystallizes with an extensible ß ribbon and ordered TSR domain. The extensible ß ribbon is composed of disulfide-bonded segments N- and C-terminal to the VWA domain that are largely drawn out of the closed VWA domain in a 15 Å movement to the open conformation. The extensible ß ribbon and TSR domain overlap at a conserved interface. The VWA, extensible ß ribbon, and TSR domains adopt a highly elongated overall orientation that would be stabilized by tensile force exerted across a ligand-receptor complex by the actin motility apparatus of the sporozoite. Our results provide insights into regulation of "stick-and-slip" parasite motility and for development of sporozoite subunit vaccines.

Unexpected fold in the circumsporozoite protein target of malaria vaccines.

Circumsporozoite (CS) protein is the major surface component of Plasmodium falciparum sporozoites and is essential for host cell invasion. A vaccine containing tandem repeats, region III, and thrombospondin type-I repeat (TSR) of CS is efficacious in phase III trials but gives only a 35% reduction in severe malaria in the first year postimmunization. We solved crystal structures showing that region III and TSR fold into a single unit, an "αTSR" domain. The αTSR domain possesses a hydrophobic pocket and core, missing in TSR domains. CS binds heparin, but αTSR does not. Interestingly, polymorphic T-cell epitopes map to specialized αTSR regions. The N and C termini are unexpectedly close, providing clues for sporozoite sheath organization. Elucidation of a unique structure of a domain within CS enables rational design of next-generation subunit vaccines and functional and medicinal chemical investigation of the conserved hydrophobic pocket.

Dimerization of Vaccinia virus VH1 is essential for dephosphorylation of STAT1 at tyrosine 701.

The gene product of Vaccinia virus gene H1, VH1, is the first identified dual specificity phosphatase (DSP). The human genome encodes 38 different VH1-like DSPs, which include major regulators of signaling pathways, highly dysregulated in disease states. VH1 down-regulates cellular antiviral response by dephosphorylating activated STAT1 in the IFN-γ/STAT1 signaling pathway. In this report, we have investigated the molecular basis for VH1 catalytic activity. Using small-angle x-ray scattering (SAXS), we determined that VH1 exists in solution as a boomerang-shaped dimer. Targeted alanine mutations in the dimerization domain (aa 1-27) decrease phosphatase activity while leaving the dimer intact. Deletion of the N-terminal dimer swapped helix (aa 1-20) completely abolishes dimerization and severely reduces phosphatase activity. An engineered chimera of VH1 that contains only one active site retains wild-type levels of catalytic activity. Thus, a dimeric quaternary structure, as opposed to two cooperative active sites within the same dimer is essential for VH1 catalytic activity. Together with laforin, VH1 is the second DSP reported in literature for which dimerization via an N-terminal dimerization domain is necessary for optimal catalytic activity. We propose that dimerization may represent a common mechanism to regulate the activity and substrate recognition of DSPs, often assumed to function as monomers.

Dimeric quaternary structure of the prototypical dual specificity phosphatase VH1.

The Vaccinia virus H1 gene product, VH1, is a dual specificity phosphatase that down-regulates the cellular antiviral response by dephosphorylating STAT1. The crystal structure of VH1, determined at 1.32 A resolution, reveals a novel dimeric quaternary structure, which exposes two active sites spaced approximately 39 A away from each other. VH1 forms a stable dimer via an extensive domain swap of the N-terminal helix (residues 1-20). In vitro, VH1 can dephosphorylate activated STAT1, in a reaction that is competed by the nuclear transport adapter importin alpha5. Interestingly, VH1 is inactive with respect to STAT1 bound to DNA, suggesting that the viral phosphatase acts predominantly on the cytoplasmic pool of activated STAT1. We propose that the dimeric quaternary structure of VH1 is essential for specific recognition of activated STAT1, which prevents its nuclear translocation, thus blocking interferon-gamma signal transduction and antiviral response.