Intestinal microbial and metabolite profile in infants with small bowel stomas after bowel resection.

BACKGROUND: Infants with small bowel stomas (SBstoma) frequently struggle with absorption and rely on parenteral nutrition (PN). Intestinal absorption is difficult to predict based solely on intestinal anatomy. The purpose of this study was to characterize the microbiota and metabolic by-products within stoma effluent and correlate with clinical features and intestinal absorption. METHODS: Prospective cohort study collecting stoma samples from neonates with SBstoma (N = 23) or colostomy control (N = 6) at initial enteral feed (first sample) and before stoma closure (last sample). Gut bacteriome (16S rRNA sequencing), short-chain fatty acids (SCFAs) and bile acids (BAs) were characterized along with volume and energy content of a 48 h collection via bomb calorimetry (last sample). Hierarchical clustering and linear regression were used to compare the bacteriome and BAs/SCFAs, to bowel length, PN, and growth. RESULTS: Infants with ≤50% small bowel lost more fluid on average than those with >50% and controls (22, 18, 16 mL/kg/d, p = 0.013), but had similar energy losses (7, 10, 9 kcal/kg/d, p = 0.147). Infants growing poorly had enrichment of Proteobacteria compared to infants growing well (90% vs. 15%, p = 0.004). An increase in the ratio of secondary BAs within the small bowel over time, correlated with poor prognostic factors (≤50% small bowel, >50% of calories from PN, and poor growth). CONCLUSION: Infants with SBstoma and poor growth have a unique bacteriome community and those with poor enteral tolerance have metabolic differences compared to infants with improved absorption.

The importance of the timing of microbial signals for perinatal immune system development.

Background: Development and maturation of the immune system begin in utero and continue throughout the neonatal period. Both the maternal and neonatal gut microbiome influence immune development, but the relative importance of the prenatal and postnatal periods is unclear. Methods: In the present study, we characterized immune cell populations in mice in which the timing of microbiome colonization was strictly controlled using gnotobiotic methodology. Results: Compared to conventional (CONV) mice, germ-free (GF) mice conventionalized at birth (EC mice) showed few differences in immune cell populations in adulthood, explaining only 2.36% of the variation in immune phenotypes. In contrast, delaying conventionalization to the fourth week of life (DC mice) affected seven splenic immune cell populations in adulthood, including dendritic cells and regulatory T cells (Tregs), explaining 29.01% of the variation in immune phenotypes. Early life treatment of DC mice with Limosilactobacillus reuteri restored splenic dendritic cells and Tregs to levels observed in EC mice, and there were strain-specific effects on splenic CD4+ T cells, CD8+ T cells, and CD11c+ F4/80+ mononuclear phagocytes. Conclusion: This work demonstrates that the early postnatal period, compared to the prenatal period, is relatively more important for microbial signals to influence immune development in mice. Our findings further show that targeted microbial treatments in early life can redress adverse effects on immune development caused by the delayed acquisition of the neonatal gut microbiome.

Characterization of the peroxide sensitivity of COX-deficient yeast strains reveals unexpected relationships between COX assembly proteins.

A number of distinct cuproproteins of the mitochondrial inner membrane are required for the assembly of cytochrome oxidase (COX), thought to function in a "bucket brigade" fashion to provide copper to the Cu(A) and Cu(B) sites. In yeast, the loss of two these proteins, Sco1p and Cox11p, leads to respiratory deficiency and a specific inability to survive exposure to hydrogen peroxide (H(2)O(2)). Using a quantitative assay, we have identified subtle differences in the peroxide-sensitive phenotypes between sco1 and cox11 mutant strains. Interestingly, the peroxide sensitivity of the sco1 null strain can be suppressed by overexpressing either SCO2 or COX11, although overexpression of neither SCO1 nor SCO2 can rescue the cox11 null strain. We also find that overexpression of either CTT1, encoding the cytosolic catalase T, or CTA1, encoding the mitochondrial matrix catalase, suppresses the peroxide sensitivity in both the sco1 and the cox11 null mutants. Direct measurement of peroxide metabolism shows that sco1 and cox11 null strains fail to degrade a significant amount of exogenously provided H(2)O(2). Taken together, our data demonstrate that although Cox11p and Sco1p play distinct roles in COX assembly, they seem to play overlapping or related roles in peroxide metabolism that require further investigation.

Delivery of rapamycin-loaded nanoparticle down regulates ICAM-1 expression and maintains an immunosuppressive profile in human CD34+ progenitor-derived dendritic cells.

Immune responses of dendritic cells (DCs) can be modulated by delivery of adjuvants to alter their maturation profile. The purpose of this study was to generate DCs from CD34(+) cells of human cord blood and characterize the effects of poly(D,L-lactic-co-glycolic acid) (PLGA)-nanoparticle encapsulated rapamycin in generating an immunosuppressive DC. Expression of ICAM-1 (intercellular adhesion molecule), a key molecule in DC-T cell interaction was increased in mature DCs in response to lipopolysaccharide (LPS). When rapamycin was encapsulated in the nanoparticle to maintain DCs in the immature state, ICAM-1 expression was down regulated. When delivered in the free form, rapamycin did not alter the expression of ICAM-1. Cytokine arrays exhibited an immunosuppressive profile of various cytokines in response to the nanoparticulate delivery of rapamycin. In addition, RT-PCR data demonstrated the presence of toll like receptor (TLR) 9 transcripts, although our DCs are myeloid in nature. In summary, our study demonstrates that DCs may be rendered immunosuppressive upon delivery of rapamycin-containing nanoparticles.

The MHC of the duck (Anas platyrhynchos) contains five differentially expressed class I genes.

MHC class I proteins mediate a variety of functions in antiviral defense. In humans and mice, three MHC class I loci each contribute one or two alleles and each can present a wide variety of peptide Ags. In contrast, many lower vertebrates appear to use a single MHC class I locus. Previously we showed that a single locus was predominantly expressed in the mallard duck (Anas platyrhynchos) and that locus was adjacent to the polymorphic transporter for the Ag-processing (TAP2) gene. Characterization of a genomic clone from the same duck now allows us to compare genes to account for their differential expression. The clone carried five MHC class I genes and the TAP genes in the following gene order: TAP1, TAP2, UAA, UBA, UCA, UDA, and UEA. We designated the predominantly expressed gene UAA. Transcripts corresponding to the UDA locus were expressed at a low level. No transcripts were found for three loci, UBA, UCA, and UEA. UBA had a deletion within the promoter sequences. UCA carried a stop codon in-frame. UEA did not have a polyadenylation signal sequence. All sequences differed primarily in peptide-binding pockets and otherwise had the hallmarks of classical MHC class I alleles. Despite the presence of additional genes in the genome, the duck expresses predominantly one MHC class I gene. The limitation to one expressed MHC class I gene may have functional consequences for the ability of ducks to eliminate viral pathogens, such as influenza.

The dominant MHC class I gene is adjacent to the polymorphic TAP2 gene in the duck, Anas platyrhynchos.

We are investigating the expression and linkage of major histocompatibility complex (MHC) class I genes in the duck ( Anas platyrhynchos) with a view toward understanding the susceptibility of ducks to two medically important viruses: influenza A and hepatitis B. In mammals, there are multiple MHC class I loci, and alleles at a locus are polymorphic and co-dominantly expressed. In contrast, in lower vertebrates the expression of one locus predominates. Southern-blot analysis and amplification of genomic sequences suggested that ducks have at least four loci encoding MHC class I. To identify expressed MHC genes, we constructed an unamplified cDNA library from the spleen of a single duck and screened for MHC class I. We sequenced 44 positive clones and identified four MHC class I sequences, each sharing approximately 85% nucleotide identity. Allele-specific oligonucleotide hybridization to a Northern blot indicated that only two of these sequences were abundantly expressed. In chickens, the dominantly expressed MHC class I gene lies adjacent to the transporter of antigen processing ( TAP2) gene. To investigate whether this organization is also found in ducks, we cloned the gene encoding TAP2 from the cDNA library. PCR amplification from genomic DNA allowed us to determine that the dominantly expressed MHC class I gene was adjacent to TAP2. Furthermore, we amplified two alleles of the TAP2 gene from this duck that have significant and clustered amino acid differences that may influence the peptides transported. This organization has implications for the ability of ducks to eliminate viral pathogens.