Recombinant protein therapeutics often suffer from short circulating half-life and poor stability, necessitating multiple injections and resulting in limited shelf-life. Conjugation to polyethylene glycol chains (PEG) extends the circulatory half-life of many proteins, but the methods for attachment often lack specificity, resulting in loss of biological activity. Using four-helix bundle cytokines as an example, we present a general platform that uses sortasemediated transpeptidation to facilitate site-specific attachment of PEG to extend cytokine half-life with full retention of biological activity. Covalently joining the N and C termini of proteins to obtain circular polypeptides, again executed using sortase, increases thermal stability. We combined both PEGylation and circularization by exploiting two distinct sortase enzymes and the use of a molecular suture that allows both site-specific PEGylation and covalent closure. The method developed is general, uses a set of easily accessible reagents, and should be applicable to a wide variety of proteins, provided that their termini are not involved in receptor binding or function.M any clinically relevant cytokines share a four-helix bundle structure, typified by IFNα2, Granulocyte colony-stimulating factor 3 (GCSF-3), Erythropoietin (EPO), IL-2, IL-4, IL-7, IL-9, and IL-15. Cocrystal structures of cytokines with receptor fragments and biochemical studies that map residues critical for interaction of a cytokine with its receptor show that the receptor contacts the sides of the helical bundles (1). This mode of interaction positions the N and C termini of the cytokine away from the receptor.Polyethylene glycol chains attached to therapeutically important proteins increase circulatory half-life, reduce clearance by kidney filtration, reduce proteolysis, and reduce the generation of neutralizing antibodies (2-4). The attachment of PEG commonly employs standard chemistries that target reactive amino acid side chains (e.g., cysteine and lysine). This strategy often generates a heterogeneous mixture in which multiple amino acids in the target are modified with a PEG chain, necessitating cumbersome separations and characterization (5). Such PEGylated molecules often show decreased biological activity, likely due to attachment of a PEG chain to a residue important for interaction with the receptor (6)-this problem may be overcome by sitespecific PEGylation. Although engineering of a carefully placed unpaired cysteine residue allows site-specific PEGylation (7, 8), this method must be tailored to the specific protein target.Sortase A from Staphylcoccus aureus (SrtA Staph ) is a thiolcontaining transpeptidase that recognizes an LPXTG motif in multiple structurally unrelated substrates (9). SrtA Staph cleaves the peptide bond between the threonine and glycine residues with concomitant formation of a thioacyl enzyme intermediate that involves the catalytic cysteine and the substrate threonine. This acyl-enzyme is resolved by nucleophilic attack by the N terminus of an oligo...
Multiple sclerosis is a devastating neurological disorder characterized by the autoimmune destruction of the central nervous system myelin. While T cells are known orchestrators of the immune response leading to MS pathology, the precise contribution of CNS resident and peripheral infiltrating myeloid cells is less well described. Here, we explore the myeloid cell function of Low-density lipoprotein receptor-related protein-1 (LRP1), a scavenger receptor involved in myelin clearance and the inflammatory response, in the context of Multiple sclerosis. Supporting its central role in Multiple sclerosis pathology, we find that LRP1 expression is increased in Multiple sclerosis lesions in comparison to the surrounding healthy tissue. Using two genetic mouse models, we show that deletion of LRP1 in microglia, but not in peripheral macrophages, negatively impacts the progression of experimental autoimmune encephalomyelitis, an animal model of Multiple sclerosis. We further show that the increased disease severity in experimental autoimmune encephalomyelitis is not due to haplodeficiency of the Cx3cr1 locus. At the cellular level, microglia lacking LRP1 adopt a pro-inflammatory phenotype characterized by amoeboid morphology and increased production of the inflammatory mediator TNF-α. We also show that LRP1 functions as a robust inhibitor of NF-kB activation in myeloid cells via a MyD88 dependent pathway, potentially explaining the increase in disease severity observed in mice lacking LRP1 expression in microglia. Taken together, our data suggest that the function of LRP1 in microglia is to keep these cells in an anti-inflammatory and neuroprotective status during inflammatory insult, including experimental autoimmune encephalomyelitis and potentially in Multiple sclerosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0343-2) contains supplementary material, which is available to authorized users.
Rheumatoid arthritis is characterized by progressive joint inflammation and affects ~1% of the human population. We noted single nucleotide polymorphisms (SNPs) in the apoptotic cell engulfment genes ELMO1, DOCK2 , and RAC1 linked to rheumatoid arthritis. As ELMO1 promotes cytoskeletal reorganization during engulfment, we hypothesized that ELMO1 loss would worsen inflammatory arthritis. Surprisingly, Elmo1 -deficient mice showed reduced joint inflammation in acute and chronic arthritis models. Genetic and cell biological studies revealed that ELMO1 associates with receptors linked to neutrophil function in arthritis and regulates activation and early neutrophil recruitment to the joints, without general inhibition of inflammatory responses. Further, neutrophils from peripheral blood of human donors that carry the SNP in ELMO1 associated with arthritis display increased migratory capacity, whereas ELMO1 knockdown reduces human neutrophil migration to chemokines linked to arthritis. These data identify ‘non-canonical’ roles for ELMO1 as an important cytoplasmic regulator of specific neutrophil receptors and promoter of arthritis.
Coronavirus disease 2019 (COVID-19) infection is associated with a plethora of neurological complications. Newly developed vaccinations targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral spike protein represent a great epidemiological promise with respect to the resolution of the pandemic. However, vaccinations are not without side effects and, in rare cases, have even been implicated in various autoimmune phenomena. In this report, we describe a case of Tolosa-Hunt syndrome (THS), a granulomatous inflammatory process of the cavernous sinus, occurring in a patient one week after getting COVID-19 vaccination. This rare diagnosis of exclusion must be considered in patients presenting with painful ophthalmoplegia.
BackgroundThe Occupational Safety and Health Administration (OSHA) of the Department of Labor requires that healthcare employers perform annual respiratory fit testing (RFT) for respiratory protection of employees with patient exposure. The annual cost of RFT in the United States is greater than $8 million and each fit test requires approximately 20 minutes. Due to the high resource expenditure for RFT, we sought to identify factors associated with changing respirators.MethodsDuring annual RFT at the University of Virginia, employees complete a questionnaire about interval clinical changes since the last RFT. Questions are based on publications indicating that certain characteristics are associated with respirator change, including: have you had dental surgery, surgery on your face, or trauma; has your weight changed by >10%; have you been or are you currently pregnant; do you recall your mask type; do you want to change masks. Answers to these questions from May 2016 through March of 2018 were compiled and analyzed by Chi-square test using Excel and R. P-value of <0.05 was considered significant.ResultsA total of 4,278 employees completed questions at least once during the time period, with 29 requiring respirator change after RFT. Requesting a mask change, and 10% weight change were significantly associated with respirator change. Pregnancy and facial trauma were not significantly associated with respirator change. Of those who changed respirator, nine reported no change in weight, no facial trauma, and no pregnancy.ConclusionThe infrequency of respirator change suggests that limiting RFT to those most likely to change their respirator may hold more value than screening all employees annually; however, questions included in this evaluation did not identify all employees who would require respirator change. We are continuing evaluation of predictors of respirator changes and association with tuberculin skin test conversion to improve efficiency of RFT.Disclosures All authors: No reported disclosures.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.