HIV progression is characterized by immune activation and microbial translocation. One factor that may be contributing to HIV progression could be a dysbiotic microbiome. We therefore hypothesized that the GI mucosal microbiome is altered in HIV patients and this alteration correlates with immune activation in HIV. 121 specimens were collected from 21 HIV positive and 22 control human subjects during colonoscopy. The composition of the lower gastrointestinal tract mucosal and luminal bacterial microbiome was characterized using 16S rDNA pyrosequencing and was correlated to clinical parameters as well as immune activation and circulating bacterial products in HIV patients on ART. The composition of the HIV microbiome was significantly different than that of controls; it was less diverse in the right colon and terminal ileum, and was characterized by loss of bacterial taxa that are typically considered commensals. In HIV samples, there was a gain of some pathogenic bacterial taxa. This is the first report characterizing the terminal ileal and colonic mucosal microbiome in HIV patients with next generation sequencing. Limitations include use of HIV-infected subjects on HAART therapy.

OBJECTIVES:Bowel preparations (BPs) taken before colonoscopy may introduce a confounding effect on the results of gastrointestinal microbiota studies. This study aimed to determine the effect of bowel preparation on the mucosa-associated and luminal colonic microbiota in healthy subjects (HC) and inflammatory bowel disease (IBD) patients.METHODS:Biopsy samples (n=36) and fecal samples (n=30) were collected from 10 HC and 8 IBD subjects pre- and post-BP. 16S rRNA gene was pyrosequenced using 454 Titanium protocols. We compared the differences between the pre- and post-BP samples (i.e., comparisons-across-bowel-prep); we examined the effect of BP on the expected separation of the mucosal vs. the luminal compartments (i.e., comparisons-across-compartments). Last, we compared the baseline differences between the HC vs. IBD groups (a secondary analysis), and examined whether the differences between the HC vs. IBD changed after BP.RESULTS:In comparisons-across-bowel-prep, the Shannon's index (SI) decreased only in the biopsy samples of IBD subjects post-BP (P=0.025) and phylogenetic diversity-whole tree (PD-WT) metric decreased in biopsy samples of HC subjects post-BP (P=0.021). In secondary comparisons, the subtle differences between the fecal samples of the HC vs. IBD groups, in terms of evenness and the SI, were not apparent post-BP. In terms of β-diversity, in comparisons-across-bowel-prep, the proportion of shared operational taxonomic units (OTUs) in pre- and post-BP samples was low (~30%) and unweighted Unifrac distances between pre- and post-BP specimens ranged from 0.52 to 0.66. HC biopsies were affected more than IBD biopsies with BP (P=0.004). In comparisons-across-compartments, the proportion of shared OTUs between biopsy and fecal samples increased and Unifrac distances decreased post-BP in IBD subjects, reducing the differences between the mucosal and luminal compartments of the gut microbiota. Interindividual differences in Unifrac distances were preserved even with BP effects, although the effects were greater on weighted Unifrac distances. Bacteroidetes and its subtypes increased post-BP in both the luminal and mucosal compartments.CONCLUSIONS:Bowel preparations affect the composition and diversity of the fecal and luminal microbiota in the short term, introducing potential bias into experiments examining the gut microbiota. The magnitude of the effect of BP is not greater than that of interindividual variation. Both the luminal and mucosal compartments of the gut microbiota get affected, and samples from controls and IBD subjects may get affected differently. Studies of the colonic microbiota should take into account the direction and the magnitude of the change introduced by BP during the design stage of the experiments, and consider sample sizes so that potential bias is minimized.

Objectives To examine the changes in bacteria in hospitalized preterm infants over the first month of life. Methods Rectal swabs were collected daily from 12 preterm infants. DNA was extracted from swabs from day of birth and weekly thereafter. Bacterial taxa were identified with next generation sequencing using universal bacterial primers targeted at the 16S rDNA on a 454 Roche titanium platform. Sequences were clustered into operational taxonomic units (OTUs), and taxonomy was assigned against the Greengenes databank using Qiime1.4. Quantitative PCR was used to determine the abundance of Bifidobacterium spp. Functional assessment of the microbiome was performed with PICRUSt. Results Average birth weight and gestational age were 1055g and 28 weeks, respectively. There were 6-35 different bacterial families identified in the day of birth samples, unrelated to mode of delivery. Richness decreased over hospitalization (week 1: 16.9±7.7 vs. weeks 3-5: 10.7±3.4, p<0.001). The Shannon diversity index demonstrated lowest diversity at birth, an increase at week 2, followed by a rapid decline at weeks 3-5, suggesting development of a more uniform microbiota composition after 2 weeks of NICU stay. Enterobacteriaceae, Staphylococcaceae, and Enterococcaceae constituted the majority of the bacterial families. Bifidobacterium spp. were infrequently detected at very low levels. PICRUSt analysis revealed enhancement of peroxisome, PPAR and adipocytokine signaling; plant-pathogen interaction; and aminobenzoate degradation pathways in week 1 samples. Conclusions Our results suggest that while preterm infants have individualized microbiota that are detectable at birth, the differences decrease during the neonatal intensive care unit hospitalization with increasing prominence of pathogenic microbiota.

Retinoic acid-inducible gene-I (RIG-I) is a cytoplasmic immune receptor sensing viral RNA. It triggers the release of type I interferons (IFN) and proinflammatory cytokines inducing an adaptive cellular immune response. We investigated the therapeutic potential of systemic RIG-I activation by short 5′-triphosphate-modified RNA (ppp-RNA) for the treatment of acute myeloid leukemia (AML) in the syngeneic murine C1498 AML tumor model. ppp-RNA treatment significantly reduced tumor burden, delayed disease onset and led to complete remission including immunological memory formation in a substantial proportion of animals. Therapy-induced tumor rejection was dependent on CD4 + and CD8 + T cells, but not on NK or B cells, and relied on intact IFN and mitochondrial antiviral signaling protein (MAVS) signaling in the host. Interestingly, ppp-RNA treatment induced programmed death ligand 1 (PD-L1) expression on AML cells and established therapeutic sensitivity to anti-PD-1 checkpoint blockade in vivo. In immune-reconstituted humanized mice, ppp-RNA treatment reduced the number of patient-derived xenografted (PDX) AML cells in blood and bone marrow while concomitantly enhancing CD3 + T cell counts in the respective tissues. Due to its ability to establish a state of full remission and immunological memory, our findings show that ppp-RNA treatment is a promising strategy for the immunotherapy of AML.

Analysis of autoinflammatory and immunodeficiency disorders elucidates human immunity and fosters the development of targeted therapies. Oligoadenylate synthetase 1 is a type I interferon–induced, intracellular double-stranded RNA (dsRNA) sensor that generates 2′-5′-oligoadenylate to activate ribonuclease L (RNase L) as a means of antiviral defense. We identified four de novo heterozygous OAS1 gain-of-function variants in six patients with a polymorphic autoinflammatory immunodeficiency characterized by recurrent fever, dermatitis, inflammatory bowel disease, pulmonary alveolar proteinosis, and hypogammaglobulinemia. To establish causality, we applied genetic, molecular dynamics simulation, biochemical, and cellular functional analyses in heterologous, autologous, and inducible pluripotent stem cell–derived macrophages and/or monocytes and B cells. We found that upon interferon-induced expression, OAS1 variant proteins displayed dsRNA-independent activity, which resulted in RNase L–mediated RNA cleavage, transcriptomic alteration, translational arrest, and dysfunction and apoptosis of monocytes, macrophages, and B cells. RNase L inhibition with curcumin modulated and allogeneic hematopoietic cell transplantation cured the disorder. Together, these data suggest that human OAS1 is a regulator of interferon-induced hyperinflammatory monocyte, macrophage, and B cell pathophysiology.