David S Newburg, PhD

My initial studies on nutrition in brain development led to a pioneering research program in 1984 on bioactive components of human milk. Human milk oligosaccharides (HMOS) are the third largest component of milk, representing 10% of the maternal caloric input, but are essentially indigestible by the infant, suggesting other essential functions. First, specific HMOS contain moieties that mimic intestinal glycans that are used by enteric pathogens as receptors; competitive inhibition of binding by specific HMOS protects breastfeeding infants from infection. Second, although (and because) HMOS are essentially indigestible by the mammalian intestinal mucosa, they promote growth in distal gut of specific mutualist symbionts of the microbiota. We found a unique transcellular signaling pathway by which healthy microbiota communicate with intestinal mucosal epithelial cells through fut2 expression, promoting mucosal homeostasis, and resilience to damage by pathogen infection, physical, or immunologic insult. This prebiotic effect indirectly reduces gut inflammation. Third, we find that individual HMOS directly inhibit specific pro-inflammatory signaling pathways. These data strongly support our original hypothesis that the glycans of human milk constitute an innate immune system whereby the mother confers potent clinically significant protection to her nursing infant. 

BS: University of Massachusetts (Amherst) Amherst, MA, 1970 (Chemistry)

PhD: Boston University Boston, MA, 1976 (Biochemistry (Nutrition and Neuroscience))

My research focuses on glycobiology of human milk, with emphasis on functional interactions with intestinal mucosa, including control of glycan ontogeny in gut development and function. Developing and validating instrumental methods of analysis and biological models of mucosal signaling have allowed the following five significant research contributions:
Human milk glycans inhibit pathogens.  An essential step for most enteropathogens is to bind to their target receptor in the gut mucosa, through adhesin (bacteria) or capsid (virus) ligation. The uniquely rich array of glycans in human milk include moieties that enteric pathogens use for docking to intestinal glycans, allowing competitive inhibition of pathogen binding, thereby protecting breastfeeding infants from infection. We pioneered identification and characterization of these human milk glycans. For example, a human milk fucosylated oligosaccharide inhibits stable toxin of enterotoxigenic E. coli in vivo. Campylobacter binds to fucosylated H-2 host cell receptors , and the binding is inhibited by 2¢-fucosyllactose (2’-FL) in milk. Norovirus binding is inhibited by lacto-N-difucohexaose-I. Pathogens that use non-fucosylated glycan moieties for binding to their mucosal target are inhibited by milk glycans that contain these other relevant epitopes: Enterohemorrhagic E. coli binding is inhibited by a mannosylated glycopeptide. Rotavirus infection is inhibited by the glycoprotein lactadherin. Binding of gp120 of HIV to CD4 of its target T-4 lymphocytes is inhibited by glycosaminoglycans and sulfatides. Sulfatides also inhibit recruitment of polymorphonucleocytes by salmonella. Strains of noroviruses bind to distinct carbohydrate epitopes and are inhibited by the corresponding milk glycans. Thus, this type of protection is through inhibition of pathogen binding. 
Interkingdom signaling in the gut underlies strong mutualism. Signaling between gut microbes and the mucosa heretofore has always involved inflammatory pathways, but we found that pioneering microbes can induce adaptive gene expression without activating inflammation. Post-weaning murine gut is heavily fucosylated, whereas suckling gut has sparse fucosylation. The possible proximate signal for this transition includes 1) change in diet; 2) innate timing by developmentally sensitive genes; 3) modified hormonal milieu; or 4) a shift in microbiota. In germ-free mice the shift in fucosylation did not occur at weaning, eliminating all candidates except 4) microbiota. Furthermore, colonization of adult germ-free mice induces fucosylation of their mucosa. Moreover, when standard mice are treated with antibiotics, the gut microbes are depleted and dysbiotic, and the mucosa reverts to the immature, minimally fucosylated state. This can be reversed by recolonization by fecal slurry or just a single fucose-utilizing isolate of normal microbiota, such as Bacteriodes fragilis. Such recolonization rapidly induces fut2 (secretor) gene expression through up-regulation of the ERK and JNK signaling pathways, their nuclear transcription factors ATF-2 and jun, which activate AP-1 control elements found in the fut2 gene. The NF-kB pro-inflammatory signaling usually associated with bacterial recognition is not activated. The induced fut2 gene product, FucT 2, adds a1,2 linked L-fucose to glycans targeted to the extracellular glycocalyx of the intestinal mucosa, providing a fucosylated niche favored by mutualists of the microbiota. This ostensible mutualism implies a host benefit. Mice whose bacteria have been disrupted by antibiotics with limited intestinal fucosylation more fragile to mucosal injury. Restoration of the microbiota or recolonization with only B. fragilis (9343) fully reinstates fucosylation and recovery of homeostasis. A mutant of B. fragilis (Δgmd-fclΔfkp) unable to utilize fucose does not restore fut2 expression, mucosal fucosylation, or recovery. The concordance of fucosylation, colonization, and mucosal resilience implies a strong and active mutualism that underlies healthy homeostasis of the gut and recovery from a variety of mucosal insults. Defining the mechanisms of this protection is an objective of this proposal.
Human milk oligosaccharides (HMOS) are prebiotic. Prebiotics are dietary carbohydrates indigestible by mammalian gut that promote growth of beneficent (mutualist) microbes whose small organic acid fermentation products acidify the gut, inhibit pathogens, and confer health benefits to the host. We found that HMOS in the urine and feces of breastfed infants match the pattern in the milk of their mothers, consistent with HMOS not being fully digested or absorbed in infants. Oligosaccharides isolated from human milk promote growth of representative mutualists Bifidobacterium longum and Lactobacillus acidophilus in vitro comparable to the most popular plant-derived prebiotics. Prebiotics are utilized by mutualists B. longum and L. acidophilus, but not by non-mutualists Campylobacter jejuni or Escherichia coli. After 48 hours of fermentation in vitro, numerous abundant organic acids, measured by our novel LC/MS technique, accumulate in the medium of the mutualists, which becomes acidic, but not so by the non-mutualists. C. jejuni and E. coli are not inhibited by the presence of oligosaccharides in their media, but are by the acidic media from mutualist Bifidobacteria incubated with HMOS, 2’-FL, 3-FL, GOS or HMOS. Thus, acidic fermentation products of these prebiotics by resident probiotic mutualists help mediate health benefits to the host. We developed instrumental analytic methods to measure individual organic acid fermentation products relibly at the low concentrations produced by microbes and optimized the linearity, range, precision, and accuracy for each measureable fatty acid. Isolated mutualists ferment individual oligosaccharides to produce specific patterns of small organic acids. Putative mechanisms whereby specific fatty acids modulate distinct facets of microbial and intestinal homeostasis can be tested. 
HMOS directly inhibits inflammation. HMOS indirectly inhibit inflammation by blocking pathogen attachment and by stimulation of beneficial microbiota. We found that HMOS also directly inhibit inflammatory processes: a) Inflammation secondary to campylobacter infection was inhibited by 2’-FL in HT 29, a human intestinal epithelial cell line. In a murine model that exhibits typical acute transient enteric infection by Campylobacter, a robust immune response, and spontaneous clearing of infection, 2-FL attenuated secondary infection of mesenteric lymph nodes, liver, and spleen, and induction of inflammatory signaling molecules of the acute phase mucosal immune response. b) Mice fed dextran sulfate sodium (DSS) exhibit severe irritation of the colon that generally resolves without permanent damage to the rodent, but we found that pretreating mice with a mixture of antibiotics, which reduces and disrupts their microbiota, results in a more severe colitis that is lethal to approximately half of the animals. Feeding 2’-FL results in significantly improved recovery from the insult, and preliminary data indicate a combined prebiotic and anti-inflammatory effect. c) ETEC, UPEC, and AIEC infection of human intestinal epithelial cells in vitro induce CD14-dependent induction of IL-8, and this pro-inflammatory response is inhibited by 2’-FL but not by other HMOS. This type I pilli invasion of T84 and H4 intestinal epithelial cells induces inflammation through CD14 induction, which activates portions of the ‘Macrophage Migration Inhibitory Factors’ inflammatory pathway via SOCS2/STAT3/NF-?B, which is specifically inhibited by 2’-FL. d) Intact immature (fetal) human intestinal mucosa exhibit the hyper-reactive immune signaling response typical of neonates. HMOS from colostrum (cHMOS) modulated release of cytokines; the genes most strongly modulated were classified by into 4 networks controlling immune cell communication, mucosal immune system differentiation, and homeostasis. cHMOS attenuate PAMP-stimulated acute phase inflammatory cytokine secretion, while elevating cytokines involved in tissue repair and homeostasis. Of the cHMOS, 3’-galactosyllactose (3’-GL) specifically quenches polyinosine-polycytidylic acid (TLR3) -induced IL-8 levels, and is a major component of commercial GOS. We propose to study the mechanism of these phenomena using the distinct prebiotics 2’-FL (and 3-FL in non-secretor milk), commercial GOS, and GMOS, to define specific mechanisms whereby endogenous mucosal glyconjugates interact with prebiotics to shape microbiota and mucosa toward homeostasis and resilience.
Synthesis of 2’-FL and 3-FL. In nature, copious quantities of HMOS are only found in the form of the mixtures contained in human milk. This limited the amounts of pure HMOS available for testing. Thus, a major hurtle for translating the above findings into clinical practice had been the synthesis of enough pure oligosaccharide for studies on efficacy and mechanism in preclinical in vitro studies, and ultimately enough GMP 2’-FL for human trials. Chemical syntheses of HMOS are limited by cost and toxic byproduct impurities. In vitro enzymatic syntheses of HMOS are limited by the expense of nucleotide-sugar precursors. We designed a strategy whereby the nucleotide precursor, GDP-fucose, is synthesized within a microbe, whereupon a fucosyltransferase transfect donates fucose to lactose. This was refined and brought to fruition by a company that we founded, Glycosyn LLC. A custom designed metabolically-engineered E. coli allows scalable fermentation and purification of 2’-FL at high specific yields. The enhanced lactose and GDP-fucose pools were utilized for 2’-FL production through the action of an heterologous a1,2 fucosyltransferase (a1,2FT) inserted into the E. coli ampC locus. Analogous manipulations of an a1,3 fucosyltransferase allow the synthesis of  3-FL. The pure 2’-FL and 3-FL provided by Glycosyn allow ongoing experiments on their individual functions. 
Current research. The focus continues to be on glycobiology of human milk and of the infant gut, and the relationship of these glycans to the microbiota. Developmental changes in gut and milk glycans influence the early colonization of the infant gut, and differences in colonization of the gut influence the ontogeny of glycans on the surface of the intestinal mucosa. This interkingdom reciprocal interaction is typical of a mutualistic symbiotic relationship. We study how colonization strongly relates to the maturation of the mucosal immune system, and how dysbiosis in the premature infant is associated with inflammatory and infective diseases. Our expertise in instrumental analysis is being applied toward defining fermentation products from mutualist bacteria that contribute toward favorable outcomes, and from less favorable colonization patterns where the fermentation products may contribute toward loss of homeostasis. Other projects include isolating glycans from fungi that have anti-inflammatory activity in the gut. Thus, our current projects include studies on the biological role of human milk glycans include their ability to inhibit pathogens, promote healthy microbiota, and suppress inflammation. This research program has been fully supported by the NIH without interruption from 1985 through 2015.

1976 -1984 Assistant to Associate Professor (tenured), Teaching and research Nutrition, Biochemistry, Advanced Normal and Therapeutic Nutrition, Graduate Courses in research and seminars., University of Kentucky, Lexington, KY

1984 -2004 Associate to Senior Scientist (Biochemist), NIH funded Research, Eunice Kennedy Shriver Center for Mental Retardation, Waltham, MA

1988 -2000 Research Fellow to Instructor, Neurology, Faculty of Medicine, Harvard University, Boston, MA

1988 -2000 Assistant to Associate Biochemist, Neurology, Massachusetts General Hospital, Boston, MA

1998 -2004 Director, Program in Glycobiology, NIH funded Research, Eunice Kennedy Shriver Center for Mental Retardation, Waltham,MA

1998 -2010 Investigator, Clinical Nutrition Research Center, Harvard Medical School, Boston, MA

2000 -2004 Professor, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA

2001 -2004 Professor, Graduate School of Biomedical Sciences, University of Massachusetts Medical School, Worcester, MA

2004 -2010 Glycobiologist, Pediatric Gastroenterology and Nutrition , Massachusetts General Hospital, Boston, MA

2004 -2010 Director, Program in Glycobiology, Developmental Immunology Laboratory, Massachusetts General Hospital, Boston, MA

2006 -2010 Associate Professor (tenured), Department of Pediatrics, Harvard Medical School, Boston, MA

2010 -2016 Professor, Department of Biology, Boston College, Chestnut Hill, MA

2010 -2016 Director, Program in Glycobiology, Boston College, Chestnut Hill, MA

2016 - Director, DSN Medical Consulting, NK Labs, Cambridge, MA

2020 - Adjunct Professor, Department of Environmental and Public Health, University of Cincinnati College of Medicine, Cincinnati, OH

Grant: #R01HD109915 Investigators:DeFranco, Emily; Greis, Kenneth; Meller, Jaroslaw; Morrow, Ardythe; Newburg, David; Rivers, Laurie 08-23-2022 -05-31-2027 National Institute of Child Health and Human Development Defining the systems biology of human milk and lactation and the impact of maternal health and environmental exposures Role:Collaborator 789533.00 Awarded Level:Federal

Investigators:Newburg, David 04-01-2023 -04-30-2024 Friesland Campina Nederland BV Validation of an in silico model of infant gut microbiome development Role:PI 73374.40 Hold Level:Industry

Grant: #R01AI173245 Investigators:Huaman Joo, Moises; Huang, Shouxiong; Newburg, David; Niu, Liang 06-01-2023 -05-31-2028 National Institute of Allergy and Infectious Diseases M. tuberculosis metabolites to activate human mucosal-associated invariant T cells Role:Collaborator 559263.00 Awarded Level:Federal

Research - Kettering Laboratories
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Phone: (617)835-3236
newburdd@ucmail.uc.edu

Home - 15 Harrington Street
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Phone: (617)835-3236
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