CD8+ T cell-derived Fgl2 regulates immunity in a cell-autonomous manner via ligation of FcγRIIB.

The regulatory circuits dictating CD8+ T cell responsiveness versus exhaustion during anti-tumor immunity are incompletely understood. Here we report that tumor-infiltrating antigen-specific PD-1+ TCF-1- CD8+ T cells express the immunosuppressive cytokine Fgl2. Conditional deletion of Fgl2 specifically in mouse antigen-specific CD8+ T cells prolongs CD8+ T cell persistence, suppresses phenotypic and transcriptomic signatures of T cell exhaustion, and improves control of the tumor. In a mouse model of chronic viral infection, PD-1+ CD8+ T cell-derived Fgl2 also negatively regulates virus-specific T cell responses. In humans, CD8+ T cell-derived Fgl2 is associated with poorer survival in patients with melanoma. Mechanistically, the dampened responsiveness of WT Fgl2-expressing CD8+ T cells, when compared to Fgl2-deficient CD8+ T cells, is underpinned by the cell-intrinsic interaction of Fgl2 with CD8+ T cell-expressed FcγRIIB and concomitant caspase 3/7-mediated apoptosis. Our results thus illuminate a cell-autonomous regulatory axis by which PD-1+ CD8+ T cells both express the receptor and secrete its ligand in order to mediate suppression of anti-tumor and anti-viral immunity.

Human intestinal stromal cells promote homeostasis in normal mucosa but inflammation in Crohn's disease in a retinoic acid-deficient manner.

Intestinal stromal cells (SCs), which synthesize the extracellular matrix that gives the mucosa its structure, are newly appreciated to play a role in mucosal inflammation. Here, we show that human intestinal vimentin+CD90+smooth muscle actin- SCs synthesize retinoic acid (RA) at levels equivalent to intestinal epithelial cells, a function in the human intestine previously attributed exclusively to epithelial cells. Crohn's disease SCs (Crohn's SCs), however, synthesized markedly less RA than SCs from healthy intestine (normal SCs). We also show that microbe-stimulated Crohn's SCs, which are more inflammatory than stimulated normal SCs, induced less RA-regulated differentiation of mucosal dendritic cells (DCs) (circulating pre-DCs and monocyte-derived DCs), leading to the generation of more potent inflammatory interferon-γhi/interleukin-17hi T cells than normal SCs. Explaining these results, Crohn's SCs expressed more DHRS3, a retinaldehyde reductase that inhibits retinol conversion to retinal and, thus, synthesized less RA than normal SCs. These findings uncover a microbe-SC-DC crosstalk in which luminal microbes induce Crohn's disease SCs to initiate and perpetuate inflammation through impaired synthesis of RA.

The Entangled World of Memory T Cells and Implications in Transplantation.

Memory T cells that are specific for alloantigen can arise from a variety of stimuli, ranging from direct allogeneic sensitization from prior transplantation, blood transfusion, or pregnancy to the elicitation of pathogen-specific T cells that are cross-reactive with alloantigen. Regardless of the mechanism by which they arise, alloreactive memory T cells possess key metabolic, phenotypic, and functional properties that render them distinct from naive T cells. These properties affect the immune response to transplantation in 2 important ways: first, they can alter the speed, location, and effector mechanisms with which alloreactive T cells mediate allograft rejection, and second, they can alter T-cell susceptibility to immunosuppression. In this review, we discuss recent developments in understanding these properties of memory T cells and their implications for transplantation.

Apolipoprotein E genotype affects innate susceptibility to Listeria monocytogenes infection in aged male mice.

Apolipoprotein E (ApoE) is a lipid transport protein that is hypothesized to suppress proinflammatory cytokine production, particularly after stimulation with Toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS). Studies using transgenic ApoE human replacement mice (APOE) expressing one of three different allelic variants suggest that there is a hierarchy in terms of responsiveness to proinflammatory stimuli such as APOE4/E4 > APOE3/E3 > APOE2/E2. In this study, we test the hypothesis that APOE genotype can also predict susceptibility to infection with the facultative intracellular gram-positive bacterium Listeria monocytogenes. We found that bone-marrow-derived macrophages isolated from aged APOE4/E4 mice expressed elevated levels of nitric oxide synthase 2 and were highly resistant to in vitro infection with L. monocytogenes compared to APOE3/E3 and APOE2/E2 mice. However, we did not find statistically significant differences in cytokine or chemokine output from either macrophages or whole splenocytes isolated from APOE2/E2, APOE3/E3, or APOE4/E4 mice following L. monocytogenes infection. In vivo, overall susceptibility to foodborne listeriosis also did not differ by APOE genotype in either young (2 mo old) or aged (15 mo old) C57BL/6 mice. However, we observed a sex-dependent susceptibility to infection in aged APOE2/E2 male mice and a sex-dependent resistance to infection in aged APOE4/E4 male mice that was not present in female mice. Thus, these results suggest that APOE genotype does not play an important role in innate resistance to infection with L. monocytogenes but may be linked to sex-dependent changes that occur during immune senescence.

ST6GAL1 sialyltransferase promotes acinar to ductal metaplasia and pancreatic cancer progression.

The role of aberrant glycosylation in pancreatic ductal adenocarcinoma (PDAC) remains an under-investigated area of research. In this study, we determined that ST6 ß-galactoside α2,6 sialyltransferase 1 (ST6GAL1), which adds α2,6-linked sialic acids to N-glycosylated proteins, was upregulated in patients with early-stage PDAC and was further increased in advanced disease. A tumor-promoting function for ST6GAL1 was elucidated using tumor xenograft experiments with human PDAC cells. Additionally, we developed a genetically engineered mouse (GEM) model with transgenic expression of ST6GAL1 in the pancreas and found that mice with dual expression of ST6GAL1 and oncogenic KRASG12D had greatly accelerated PDAC progression compared with mice expressing KRASG12D alone. As ST6GAL1 imparts progenitor-like characteristics, we interrogated ST6GAL1's role in acinar to ductal metaplasia (ADM), a process that fosters neoplasia by reprogramming acinar cells into ductal, progenitor-like cells. We verified ST6GAL1 promotes ADM using multiple models including the 266-6 cell line, GEM-derived organoids and tissues, and an in vivo model of inflammation-induced ADM. EGFR is a key driver of ADM and is known to be activated by ST6GAL1-mediated sialylation. Importantly, EGFR activation was dramatically increased in acinar cells and organoids from mice with transgenic ST6GAL1 expression. These collective results highlight a glycosylation-dependent mechanism involved in early stages of pancreatic neoplasia.

Early Life Stress in Mice Leads to Impaired Colonic Corticosterone Production and Prolonged Inflammation Following Induction of Colitis.

BACKGROUND: Early life stress (ELS) is an environmental trigger believed to promote increased risk of IBD. Our goal was to identify mechanisms whereby ELS in mice affects susceptibility to and/or severity of gut inflammation. METHODS: We utilized 2 published animal models of ELS. In the first model, newborn mice were separated from the dam daily for 4 to 8 hours starting on postnatal day 2 and then weaned early on postnatal day 17. Control mice were left undisturbed with the dams until weaning on postnatal day 21. In the second model, dams were fed dexamethasone or vehicle ad libitum in drinking water on postpartum days 1 to 14. Plasma and colonic corticosterone were measured in juvenile and adult mice. Colitis was induced in 4-week-old mice via intraperitoneal injection of interleukin (IL)-10 receptor blocking antibody every 5 days for 15 days. Five or 15 days later, colitis scores and transcripts for Tnf, glucocorticoid receptors, and steroidogenic enzymes were measured. RESULTS: Mice exposed to ELS displayed reduced plasma and colonic corticosterone. Control animals showed improvements in indices of inflammation following cessation of interleukin-10 receptor blockade, whereas ELS-exposed animals maintained high levels of Tnf and histological signs of colitis. In colitic animals, prior exposure to ELS was associated with significantly lower expression of genes associated with corticosterone synthesis and responsiveness. Finally, TNF stimulation of colonic crypt cells from ELS mice led to increased inhibition of corticosterone synthesis. CONCLUSIONS: Our study identifies impaired local glucocorticoid production and responsiveness as a potential mechanism whereby ELS predisposes to chronic colitis in susceptible hosts.

Using 2 distinct animal models, this study shows that in mice, early life stress leads to reduced colonic corticosterone and that induction of colitis after stress removal results in reduced transcription of glucocorticoid synthesis genes, increased Tnf, and enhanced chronicity of intestinal inflammation.

Leveraging technology to make parent training more accessible: Randomized trial of in-person versus online executive function training for parents of autistic children.

LAY ABSTRACT: This study compared the first online parent training program for executive function intervention for autism to in-person parent training on the same content. Participants were parents of autistic children, who were between 8 and 12 years of age and did not have intellectual disability. Parents were randomized to the in-person (n = 51) or online (n = 46) training conditions. Both trainings were developed with stakeholder (parents and autistic people) guidance. In this trial, most parents reported that they liked both trainings and that they were able to implement what they learned with their children. Parents in both groups spent equivalent amounts of time (about 8 hours) with the training materials, but while 94% of parents in the in-person training attended both parent trainings, only 59% of parents in the online group completed all 10 online modules. Parents reported that it was difficult to stay motivated to complete the online trainings over the 10-week trial. Parent and child outcomes did not differ significantly between the groups. Overall, parents reported that the trainings resulted in a reduction in their own parenting strain and improvements in their child's flexibility, emotional control, and global executive function, but not planning and organization. These findings indicated brief in-person and online training can help parents learn to support and improve their autistic children's executive function abilities, reducing their own experience of parenting strain. The finding that the online training was equivalent to the in-person trainings is important because it is accessible to parents who encounter barriers to in-person care.

Sialyltransferase ST6GAL-1 mediates resistance to chemoradiation in rectal cancer.

Locally advanced rectal cancer is typically treated with chemoradiotherapy followed by surgery. Most patients do not display a complete response to chemoradiotherapy, but resistance mechanisms are poorly understood. ST6GAL-1 is a sialyltransferase that adds the negatively charged sugar, sialic acid (Sia), to cell surface proteins in the Golgi, altering their function. We therefore hypothesized that ST6GAL-1 could mediate resistance to chemoradiation in rectal cancer by inhibiting apoptosis. Patient-derived xenograft and organoid models of rectal cancer and rectal cancer cell lines were assessed for ST6GAL-1 protein with and without chemoradiation treatment. ST6GAL-1 mRNA was assessed in untreated human rectal adenocarcinoma by PCR assays. Samples were further assessed by Western blotting, Caspase-Glo apoptosis assays, and colony formation assays. The presence of functional ST6GAL-1 was assessed via flow cytometry using the Sambucus nigra lectin, which specifically binds cell surface α2,6-linked Sia, and via lectin precipitation. In patient-derived xenograft models of rectal cancer, we found that ST6GAL-1 protein was increased after chemoradiation in a subset of samples. Rectal cancer cell lines demonstrated increased ST6GAL-1 protein and cell surface Sia after chemoradiation. ST6GAL-1 was also increased in rectal cancer organoids after treatment. ST6GAL-1 knockdown in rectal cancer cell lines resulted in increased apoptosis and decreased survival after treatment. We concluded that ST6GAL-1 promotes resistance to chemoradiotherapy by inhibiting apoptosis in rectal cancer cell lines. More research will be needed to further elucidate the importance and mechanism of ST6GAL-1-mediated resistance.

Human Microbiota Flagellins Drive Adaptive Immune Responses in Crohn's Disease.

BACKGROUND AND AIMS: Crohn's disease and ulcerative colitis are characterized by dysregulated adaptive immune responses to the microbiota in genetically susceptible individuals, but the specificity of these responses remains largely undefined. Therefore, we developed a microbiota antigen microarray to characterize microbial antibody reactivity, particularly to human-derived microbiota flagellins, in inflammatory bowel disease. METHODS: Sera from healthy volunteers (n = 87) at the University of Alabama at Birmingham and from patients recruited from the Kirklin Clinic of University of Alabama at Birmingham Hospital, including patients with Crohn's disease (n = 152) and ulcerative colitis (n = 170), were individually probed against microbiota bacterial flagellins of both mouse and human origin and analyzed for IgG and IgA antibody responses. Circulating flagellin-reactive T effector (CD4+CD154+) and T regulatory (CD4+CD137+) cells were isolated and evaluated in selected patients. Resulting adaptive immune responses were compared with corresponding clinical data to determine relevancy to disease behavior. RESULTS: We show that patients with IBD express selective patterns of antibody reactivity to microbiota flagellins. Patients with Crohn's disease, but not patients with ulcerative colitis, display augmented serum IgG to human ileal-localized Lachnospiraceae flagellins, with a subset of patients having high responses to more than 10 flagellins. Elevated responses to CBir1, a mouse Lachnospiraceae flagellin used clinically to diagnose CD, correlated with multi-Lachnospiraceae flagellin reactivity. In this subset of patients with CD, multi-flagellin reactivity was associated with elevated flagellin-specific CD154+CD45RA- T memory cells, a reduced ratio of flagellin-reactive CD4+ T regulatory to T effector cells, and a high frequency of disease complications. CONCLUSIONS: Patients with Crohn's disease display strong adaptive immune response to human-derived Lachnospiraceae flagellins, which may be targeted for prognosis and future personalized therapies.

CD4+ T cell activation and concomitant mTOR metabolic inhibition can ablate microbiota-specific memory cells and prevent colitis.

Microbiota-reactive CD4+ T memory (TM) cells are generated during intestinal infections and inflammation, and can revert to pathogenic CD4+ T effector (TE) cells, resulting in chronicity of inflammatory bowel disease (IBD). Unlike TE cells, TM cells have a low rate of metabolism unless they are activated by reencountering cognate antigen. Here, we show that the combination of cell activation and metabolic checkpoint inhibition (CAMCI), by targeting key metabolic regulators mTORC and AMPK, resulted in cell death and anergy, but enhanced the induction of the regulatory subset. Parenteral application of this treatment with a synthetic peptide containing multiple flagellin T cell epitopes (MEP1) and metabolic inhibition successfully prevented the development of CD4+ T cell-driven colitis. Microbiota-specific CD4+ T cells, especially the pathogenic TE subsets, were decreased 10-fold in the intestinal lamina propria. Furthermore, using the CAMCI strategy, we were able to prevent antigen-specific TM cell formation upon initial antigen encounter, and ablate existing TM cells upon reactivation in mice, leading to an altered transcriptome in the remaining CD4+ T cells after ablation. Microbiota flagellin-specific CD4+ T cells from patients with Crohn's disease were ablated in a similar manner after CAMCI in vitro, with half of the antigen-specific T cells undergoing cell death. These results indicate that parenteral activation of microbiota-specific CD4+ T cells with concomitant metabolic inhibition is an effective way to ablate pathogenic CD4+ TM cells and to induce T regulatory (Treg) cells that provide antigen-specific and bystander suppression, supporting a potential immunotherapy to prevent or ameliorate IBD.

Neurotropic Lineage III Strains of Listeria monocytogenes Disseminate to the Brain without Reaching High Titer in the Blood.

Listeria monocytogenes is thought to colonize the brain using one of three mechanisms: direct invasion of the blood-brain barrier, transportation across the barrier by infected monocytes, and axonal migration to the brain stem. The first two pathways seem to occur following unrestricted bacterial growth in the blood and thus have been linked to immunocompromise. In contrast, cell-to-cell spread within nerves is thought to be mediated by a particular subset of neurotropic L. monocytogenes strains. In this study, we used a mouse model of foodborne transmission to evaluate the neurotropism of several L. monocytogenes isolates. Two strains preferentially colonized the brain stems of BALB/cByJ mice 5 days postinfection and were not detectable in blood at that time point. In contrast, infection with other strains resulted in robust systemic infection of the viscera but no dissemination to the brain. Both neurotropic strains (L2010-2198, a human rhombencephalitis isolate, and UKVDL9, a sheep brain isolate) typed as phylogenetic lineage III, the least characterized group of L. monocytogenes Neither of these strains encodes InlF, an internalin-like protein that was recently shown to promote invasion of the blood-brain barrier. Acute neurologic deficits were observed in mice infected with the neurotropic strains, and milder symptoms persisted for up to 16 days in some animals. These results demonstrate that neurotropic L. monocytogenes strains are not restricted to any one particular lineage and suggest that the foodborne mouse model of listeriosis can be used to investigate the pathogenic mechanisms that allow L. monocytogenes to invade the brain stem.IMPORTANCE Progress in understanding the two naturally occurring central nervous system (CNS) manifestations of listeriosis (meningitis/meningoencephalitis and rhombencephalitis) has been limited by the lack of small animal models that can readily distinguish between these distinct infections. We report here that certain neurotropic strains of Listeria monocytogenes can spread to the brains of young otherwise healthy mice and cause neurological deficits without causing a fatal bacteremia. The novel strains described here fall within phylogenetic lineage III, a small collection of L. monocytogenes isolates that have not been well characterized to date. The animal model reported here mimics many features of human rhombencephalitis and will be useful for studying the mechanisms that allow L. monocytogenes to disseminate to the brain stem following natural foodborne transmission.

Modulation of glycosyltransferase ST6Gal-I in gastric cancer-derived organoids disrupts homeostatic epithelial cell turnover.

Programmed cell death promotes homeostatic cell turnover in the epithelium but is dysregulated in cancer. The glycosyltransferase ST6Gal-I is known to block homeostatic apoptosis through α2,6-linked sialylation of the death receptor TNFR1 in many cell types. However, its role has not been investigated in gastric epithelial cells or gastric tumorigenesis. We determined that human gastric antral epithelium rarely expressed ST6Gal-I, but the number of ST6Gal-I-expressing epithelial cells increased significantly with advancing premalignancy leading to cancer. The mRNA expression levels of ST6GAL-I and SOX9 in human gastric epithelial cells correlated positively with one another through the premalignancy cascade, indicating that increased epithelial cell expression of ST6Gal-I is associated with premalignant progression. To determine the functional impact of increased ST6Gal-I, we generated human gastric antral organoids from epithelial stem cells and differentiated epithelial monolayers from gastric organoids. Gastric epithelial stem cells strongly expressed ST6Gal-I, suggesting a novel biomarker of stemness. In contrast, organoid-derived epithelial monolayers expressed markedly reduced ST6Gal-I and underwent TNF-induced, caspase-mediated apoptosis, consistent with homeostasis. Conversely, epithelial monolayers generated from gastric cancer stem cells retained high levels of ST6Gal-I and resisted TNF-induced apoptosis, supporting prolonged survival. Protection from TNF-induced apoptosis depended on ST6Gal-I overexpression, because forced ST6Gal-I overexpression in normal gastric stem cell-differentiated monolayers inhibited TNF-induced apoptosis, and cleavage of α2,6-linked sialic acids from gastric cancer organoid-derived monolayers restored susceptibility to TNF-induced apoptosis. These findings implicate up-regulated ST6Gal-I expression in blocking homeostatic epithelial cell apoptosis in gastric cancer pathogenesis, suggesting a mechanism for prolonged epithelioid tumor cell survival.

Evaluator Training and Reliability for SMA Global Nusinersen Trials1.

BACKGROUND: Training methodology was established to optimize reliability of outcome measures in the nusinersen clinical trials. The Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP INTEND), Hammersmith Functional Motor Scale Expanded (HFMSE), and Revised Upper Limb (RULM) were primary or secondary outcomes. METHODS: Video review, quarterly conference calls, and item scoring checks supported evaluator competence. Baseline and screening along with video review established intra and inter-rater reliability. RESULTS: Inter and intra-rater reliability were both excellent. Intraclass correlation coefficients (ICC) ranged between 0.906-0.994 across initial training meetings and 0.824-0.996 across annual retraining meetings. This was similar for CHOP INTEND (ICC = 0.824-0.951), HFMSE (ICC = 0.981-0.996), and RULM (ICC = 0.966-0.990). Intra-rater reliability for the CHOP INTEND, HFMSE, and RULM were ICC = 0.895 (95% CI: 0.852-0.926; n = 116), ICC = 0.959 (95% CI: 0.942-0.971; n = 125), and ICC = 0.948 (95% CI: 0.927-0.963; n = 126) respectively. CONCLUSIONS: Rigorous evaluator training ensures reliability of assessment of subjects with spinal muscular atrophy (SMA) in multicenter international trials.

Chemistry of the 8-Nitroguanine DNA Lesion: Reactivity, Labelling and Repair.

The 8-nitroguanine lesion in DNA is increasingly associated with inflammation-related carcinogenesis, whereas the same modification on guanosine 3',5'-cyclic monophosphate generates a second messenger in NO-mediated signal transduction. Very little is known about the chemistry of 8-nitroguanine nucleotides, despite the fact that their biological effects are closely linked to their chemical properties. To this end, a selection of chemical reactions have been performed on 8-nitroguanine nucleosides and oligodeoxynucleotides. Reactions with alkylating reagents reveal how the 8-nitro substituent affects the reactivity of the purine ring, by significantly decreasing the reactivity of the N2 position, whilst the relative reactivity at N1 appears to be enhanced. Interestingly, the displacement of the nitro group with thiols results in an efficient and specific method of labelling this lesion and is demonstrated in oligodeoxynucleotides. Additionally, the repair of this lesion is also shown to be a chemically feasible reaction through a reductive denitration with a hydride source.

Genome Sequences of Subcluster K5 Mycobacteriophages AlleyCat, Edugator, and Guillsminger.

Bacteriophages AlleyCat, Edugator, and Guillsminger were isolated on Mycobacterium smegmatis mc2155 from enriched soil samples. All are members of mycobacteriophage subcluster K5, with genomes of 62,112 to 63,344 bp. Each genome contains 92 to 99 predicted protein-coding genes and one tRNA. Guillsminger is the first mycobacteriophage to carry an IS1380 family transposon.

CBirTox is a selective antigen-specific agonist of the Treg-IgA-microbiota homeostatic pathway.

Cultivating an environment of mutualism between host cells and the microbiota is vital, and dysregulation of this relationship is associated with multiple immune disorders including metabolic and skin diseases, asthma, allergy, and Inflammatory Bowel Disease (IBD). One prominent mechanism for maintaining homeostasis is the protective regulatory T cell (Treg)- Immunoglobulin A (IgA) pathway toward microbiota antigens, in which Tregs maintain homeostasis and provide critical survival factors to IgA+ B cells. In order to amplify the Treg-IgA pathway, we have generated a fusion protein, CBirTox, comprised of a portion of the carboxy terminus of CBir1, a microbiota flagellin, genetically coupled to Cholera Toxin B subunit (CTB) via the A2 linker of CT. Both dendritic cells (DCs) and B cells pulsed with CBirTox selectively induced functional CD4+Foxp3+ Tregs in vitro, and CBirTox augmented CD4+Foxp3+ cell numbers in vivo. The induced Foxp3 expression was independent of retinoic acid (RA) signaling but was inhibited by neutralization of TGF-ß. CBirTox treatment of B cells downregulated mammalian target of rapamycin (mTOR) signaling. Furthermore, CBirTox-pulsed DCs induced substantial production of IgA from naïve B cells. Collectively these data demonstrate that CBirTox represents a novel approach to bolstering the Treg-IgA pathway at the host-microbiota interface.

SERS and SERRS Detection of the DNA Lesion 8-Nitroguanine: A Self-Labeling Modification.

Rapid and sensitive methods to detect DNA lesions are essential in order to understand their role in carcinogenesis and for potential diagnosis of cancers. The 8-nitroguanine DNA lesion, which is closely associated with inflammation-induced cancers, has been characterized for the first time by surface-enhanced Raman spectroscopy (SERS). This lesion has been studied as the free base, as well as part of a dinucleotide and oligodeoxynucleotides (ODNs) at 5 different excitation wavelengths in the range 785-488 nm. All nitrated samples produced distinctly different spectra from their control guanine counterparts, with nitro bands being assigned by DFT calculations. Additional resonance enhancement was observed at the shorter excitation wavelengths, these SERRS measurements allowed the detection of one nitrated guanine in over 1,300 bases. In addition, SER(R)S can be used to detect whether the unstable lesion is covalently attached to the ODN or has been released by hydrolytic depurination.

Tautomerism in 8-Nitroguanosine Studied by NMR and Theoretical Calculations.

The guanine base in DNA, due to its low oxidation potential, is particularly sensitive to chemical modifications. A large number of guanine lesions have been characterized and studied in some detail due to their relationship with tissue inflammations. Nevertheless, one example of these lesions is the formation of 8-nitro-guanosine, but the NMR data of this compound was only partially interpreted. A comprehensive study of the two possible tautomeric forms, through a detailed characterization of this compound, has implications for its base pairing properties. The target compound was obtained through a synthetic sequence of five steps, where all intermediates were fully characterized using spectral data. The analysis of the two tautomers was then evaluated through NMR spectroscopy and theoretical calculations of the chemical shifts and NH coupling constants, which were also compared with the data from guanosine.

Host-microbiota interactions in the intestine.

The comprehensive collection of bacterial species, termed microbiota, within human and other mammalian hosts has profound effects on both innate and adaptive immunity. Multiple host innate mechanisms contribute to intestinal homeostasis, including epithelial production of protective mucin layers maintaining spatial segregation in the intestine as well as epithelial cell secretion of a broad range of antimicrobial peptides. Additionally, epithelial cells employ autophagy to contain and eliminate invading bacteria; interestingly, genetic variants in specific autophagy genes are linked to susceptibility to Crohn's disease. Innate lymphoid cells, which rapidly respond to cytokine and microbial signals, have emerged as important regulators of the intestinal immune response to the microbiota. With regard to adaptive immunity, specific microbial species stimulate induction of regulatory T cells while others induce effector T cells within the gut. Such stimulation is subject to dysregulation during inflammation and disease, contributing to 'dysbiosis' or an abnormal microbiota composition that has been associated with a variety of immune-mediated inflammatory disorders, including celiac disease. The microbiota communicates with the immune system and vice versa; thus, an abnormal microbiota composition likely translates into an altered host immune response, though the exact mechanisms of such are not yet clear. Immunoglobulin A plays a critical role in limiting bacterial access to the host and in maintaining mutualism with the microbiota. Perturbation of the mucosal barrier via infection or other means can induce effector T cells reactive to the intestinal microbiota, and these cells can persist as memory cells for extended periods of time and potentially serve as pathogenic effector cells upon re-encounter with antigen. Health is associated with a diverse microbiota that functions to maintain the balance between T effector and T regulatory cells in the intestine. Whether dysbiosis can be reversed in immune-mediated disease, thus restoring health, is a question of intense interest for this active area of research.