research goal is to use noninvasive approaches to define how early nutrition shapes host-microbe interactions and influences intestinal development in breast-fed versus formula-fed infants. She hopes that the knowledge gained can be used to identify selective additives, such as bioactive proteins, prebiotics (including HMO), and probiotics that can be added to infant formula to provide some of the health benefits afforded by breast-feeding.

Differential Expression of Microbial Genes in Breast-Fed Versus Formula-Fed Infants

In what Donovan described as a “proof-of-concept” study, she and colleagues used a method developed by Robert Chapkin (Davidson et al., 1995) for isolating exfoliated epithelial cells from stool to identify genes differentially expressed in breast-fed versus formula-fed infants (Chapkin et al., 2010). Specifically, they analyzed stool samples collected at 3 months of age from vaginally delivered term infants who were medically certified as healthy and who were either exclusively breast-fed (N = 12) or formula-fed (N = 10) (Chapkin et al., 2010). The researchers gained institutional review board (IRB) approval to train the mothers themselves to collect the samples at home. The initial messenger RNA (mRNA) expression microarray analysis yielded 4,250 genes that were expressed in all infants. Of those, about 1,200 were significantly differentially expressed between breast-fed and formula-fed infants. Due to the small sample size and thus greater potential for false discovery, the scientists compared these 1,200 genes to a list of 546 that they had predicted could be differentially expressed based on their known roles in intestinal biology. This yielded 146 differentially expressed genes, to which researchers applied a linear discriminant analysis and coefficient of determination analyses developed by Edward Dougherty and colleagues (Dougherty et al., 2009; Kim et al., 2000) to identify the genes that best classified breast-fed versus formula-fed infants and those that were master regulators, respectively.

The strongest classifier was EPAS1, which encodes a protein involved in cellular response to hypoxia. Given that necrotizing enterocolitis (NEC) is associated with tissue hypoxia and that human milk has been shown to protect preterm infants from NEC, Donovan speculated that upregulation of EPAS1 in breast-fed infants might be helping those babies’ guts to tolerate hypoxic episodes.

Other genes that qualified as good classifiers are summarized in Table 5-1. The linear discriminant analysis methodology used allowed investigators to identify not just single genes that could be considered good classifiers of breast-fed versus formula-fed infants, but also two- and three-gene combinations (Chapkin et al., 2010).

Donovan speculated that these gene expression differences might ex-



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