Sensitive and Specific Detection of the Non-Human Sialic Acid N-Glycolylneuraminic Acid In Human Tissues and Biotherapeutic Products

Humans are genetically defective in synthesizing the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc), but can metabolically incorporate it from dietary sources (particularly red meat and milk) into glycoproteins and glycolipids of human tumors, fetuses and some normal tissues. Metabolic incorporation of Neu5Gc from animal-derived cells and medium components also results in variable contamination of molecules and cells intended for human therapies. These Neu5Gc-incorporation phenomena are practically significant, because normal humans can have high levels of circulating anti-Neu5Gc antibodies. Thus, there is need for the sensitive and specific detection of Neu5Gc in human tissues and biotherapeutic products. Unlike monoclonal antibodies that recognize Neu5Gc only in the context of underlying structures, chicken immunoglobulin Y (IgY) polyclonal antibodies can recognize Neu5Gc in broader contexts. However, prior preparations of such antibodies (including our own) suffered from some non-specificity, as well as some cross-reactivity with the human sialic acid N-acetylneuraminic acid (Neu5Ac).We have developed a novel affinity method utilizing sequential columns of immobilized human and chimpanzee serum sialoglycoproteins, followed by specific elution from the latter column by free Neu5Gc. The resulting mono-specific antibody shows no staining in tissues or cells from mice with a human-like defect in Neu5Gc production. It allows sensitive and specific detection of Neu5Gc in all underlying glycan structural contexts studied, and is applicable to immunohistochemical, enzyme-linked immunosorbent assay (ELISA), Western blot and flow cytometry analyses. Non-immune chicken IgY is used as a reliable negative control. We show that these approaches allow sensitive detection of Neu5Gc in human tissue samples and in some biotherapeutic products, and finally show an example of how Neu5Gc might be eliminated from such products, by using a human cell line grown under defined conditions.We report a reliable antibody-based method for highly sensitive and specific detection of the non-human sialic acid Neu5Gc in human tissues and biotherapeutic products that has not been previously described

Inducing host protection in pneumococcal sepsis by preactivation of the Ashwell-Morell receptor

The endocytic Ashwell-Morell receptor (AMR) of hepatocytes detects pathogen remodeling of host glycoproteins by neuraminidase in the bloodstream and mitigates the lethal coagulopathy of sepsis. We have investigated the mechanism of host protection by the AMR during the onset of sepsis and in response to the desialylation of blood glycoproteins by the NanA neuraminidase of Streptococcus pneumoniae. We find that the AMR selects among potential glycoprotein ligands unmasked by microbial neuraminidase activity in pneumococcal sepsis to eliminate from blood circulation host factors that contribute to coagulation and thrombosis. This protection is attributable in large part to the rapid induction of a moderate thrombocytopenia by the AMR. We further show that neuraminidase activity in the blood can be manipulated to induce the clearance of AMR ligands including platelets, thereby preactivating a protective response in pneumococcal sepsis that moderates the severity of disseminated intravascular coagulation and enables host survival.Fil: Grewal, Prabhjit K.. University of California; Estados Unidos. Sanford-burnham Medical Research Institute; Estados UnidosFil: Aziz, Peter V.. University of California; Estados Unidos. Sanford-burnham Medical Research Institute; Estados UnidosFil: Uchiyama, Satoshi. University of California at San Diego; Estados UnidosFil: Rubio, Gabriel R.. University of California; Estados Unidos. Sanford-burnham Medical Research Institute; Estados UnidosFil: Lardone, Ricardo Dante. University of California; Estados Unidos. Sanford-burnham Medical Research Institute; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Le, Dzung. University of California at San Diego; Estados UnidosFil: Varki, Nissi M.. University of California at San Diego; Estados UnidosFil: Nizet, Victor. University of California at San Diego; Estados UnidosFil: Marth, Jamey D.. University of California; Estados Unidos. Sanford-burnham Medical Research Institute; Estados Unido

Inhibition of nuclear PTEN tyrosine phosphorylation enhances glioma radiation sensitivity through attenuated DNA repair

Ionizing radiation (IR) and chemotherapy are standard of care treatments for glioblastoma (GBM) patients and both result in DNA damage, however, the clinical efficacy is limited due to therapeutic resistance. We identified a mechanism of such resistance mediated by phosphorylation of PTEN on tyrosine 240 (pY240-PTEN) by FGFR2. pY240-PTEN is rapidly elevated and bound to chromatin through interaction with Ki-67 in response to IR treatment and facilitates the recruitment of RAD51 to promote DNA repair. Blocking Y240 phosphorylation confers radiation sensitivity to tumors and extends survival in GBM preclinical models. Y240FPten knock-in mice showed radiation sensitivity. These results suggest that FGFR-mediated pY240-PTEN is a key mechanism of radiation resistance and is an actionable target for improving radiotherapy efficacy

Interleukin 1 receptor signaling regulates DUBA expression and facilitates Toll-like receptor 9–driven antiinflammatory cytokine production

By dampening DUBA expression, IL-1 receptor signals facilitate TLR9-driven TRAF3 ubiquitination, antiinflammatory cytokine production, and resistance to DSS-induced colitis

MST1R kinase accelerates pancreatic cancer progression via effects on both epithelial cells and macrophages

The MST1R (RON) kinase is overexpressed in >80% of human pancreatic cancers, but its role in pancreatic carcinogenesis is unknown. In this study, we examined the relevance of Mst1r kinase to Kras driven pancreatic carcinogenesis using genetically engineered mouse models. In the setting of mutant Kras, Mst1r overexpression increased acinar-ductal metaplasia (ADM), accelerated the progression of pancreatic intraepithelial neoplasia (PanIN), and resulted in the accumulation of (mannose receptor C type 1) MRC1+, (arginase 1) Arg+ macrophages in the tumor microenvironment. Conversely, absence of a functional Mst1r kinase slowed PanIN initiation, resulted in smaller tumors, prolonged survival and a reduced tumor-associated macrophage content. Mst1r expression was associated with increased production of its ligand Mst1, and in orthotopic models, suppression of Mst1 expression resulted in reduced tumor size, changes in macrophage polarization and enhanced T cell infiltration. This study demonstrates the functional significance of Mst1r during pancreatic cancer initiation and progression. Further, it provides proof of concept that targeting Mst1r can modulate pancreatic cancer growth and the microenvironment. This study provides further rationale for targeting Mst1r as a therapeutic strategy