It is well-established that long-term consumption of processed and unprocessed red meat (beef, pork, lamb, and veal) increases the risk of several chronic diseases currently burdening the U.S. healthcare system. One of the strongest links between red meat consumption and disease is to cancer, particularly of the colon and rectum (i.e. colorectal cancer, CRC). While myriad factors contribute to CRC development and progression in vivo, one theory involves incorporation of a non-human sugar enriched in red meat called N-glycolylneuraminic acid (Neu5Gc). Neu5Gc, once consumed, is metabolically incorporated into endogenous sialoglycoproteins. These N-glycolyl-containing glycoconjugates are recognized by the immune system as foreign entities, and antibodies (Abs) are produced against a plethora of glycans terminating in Neu5Gc, resulting in a form of chronic inflammation known as xenosialitis. Xenosialitis is consistent with all known facts and is supported by much experimental evidence. It is the only theory that can explain the human-specific nature of CRC risk. This study will take advantage of recent discoveries at the biochemical level to propose methods and approaches to address some of the present limitations for obtaining definitive proof of the mechanism in human populations, as well as exploring ways to measure individual risk. Our solution is to assess the stable and long-lived metabolite of Neu5Gc, whereby the N-glycolyl group is transferred to chondroitin sulfate (Gc-CS), an abundant glycosaminoglycan (GAG) found in the extracellular matrix and in the serum of both omnivorous humans and “human-like” (Cmah–/–) mice fed a Neu5Gc-rich diet. A non-sulfated chondroitin metabolite (Gc-CN) can also be detected and analyzed. We hypothesize that Gc-CS and Gc-CN may serve as new biomarkers for red meat consumption and CRC risk. Moreover, we believe Abs against Gc-CS/CN exist, which, if established, will be the second known class of xeno-autoantigens and the first-known class of xeno- autoantigenic metabolites. To validate this, we will (1) generate and characterize Abs specific for Gc-CS/CN by immunizing Neu5Gc-deficient, Cmah–/– mice with Gc-CN or Gc-CS coupled to a carrier protein that recruits T cell help, screen for serum Ab response, and generate monoclonal antibodies (MAbs) not cross-reacting with N-acetylated-CS/CN (Ac-CS/CN). Using these MAbs, we will (2) determine if either Gc-CS or Gc-CN can be detected in human tumors and normal tissues. Lastly, we will (3) evaluate Gc-CS/CN and anti-Gc-CS/CN Abs in plasma from participants of three large prospective cohorts, including the Nurses' Health Study (NHS) to examine correlations, risk, and prognosis of inflammation-driven progression of tumor growth. With these archived samples, we will be able to correlate levels with red meat intake and CRC risk and prognosis during follow-up of more than 30 years.

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