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Robert H. Schiestl

Intestinal Microbiota: A Key Player in Longevity, Genomic Instability, and Lymphoma in Atm deficient mice. Robert H. Schiestl. Professor of Pathology, Environmental Health and Radiation Oncology UCLA. Bacteria in our body.

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Robert H. Schiestl

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  1. Intestinal Microbiota: A Key Player in Longevity, Genomic Instability, and Lymphoma in Atm deficient mice Robert H. Schiestl Professor of Pathology, Environmental Health and Radiation Oncology UCLA

  2. Bacteria in our body There are 10 fold more bacteria than human cells in our body, most of them contained in our intestines

  3. Intestinal microbiota and inflammation In 1995 Dr. Barry Marshall and Dr Robin Warren received the Nobel Price in Medicine for their discovery of H. pylori as a cause for stomach ulcers Mammals w/o intestinal microbiota are immunodeficient

  4. ATAXIA TELANGIECTASIA (AT) Clinical manifestation: • Autosomal recessive disease (1 in 40.000-100.000 people affected) • Early-onset progressive cerebellar ataxia • High incidence of tumors (30% develop lymphoma or leukemia) • Growth retardation • Immunodeficiency • Biological markers: • Chromosomal instability • Hypersensitivity to radiation • Imbalance in antioxidant levels and antioxidative enzymes

  5. Different median survival rates of Atm-/- mice

  6. homologous recombination (in embryonic life) HR leads to a deletion of exons 6-18 wild type p Exons 1-5 6-18 19-23 How DNA deletions are scored in vivo Mouse strain: pun mouse (C57BL/6J-pun/pun) • dilute gray fur color • pink eyes • pun mutation 70 kb pun Exons1-5 6-18 6-18 19-23

  7. 70 kb deletion at the pun locus results in pigmented spots on the fur • Fur spot assay reverted premelanocytes expand clonally to form a fur spot DNA deletion spot

  8. pun reversion results in pigmented spots on the fur and retinal pigment epithelium (RPE) DNA deletion spots on the fur of pun mice The eye and the Retinal Pigment Epithelium (RPE) RPE neural retina RPE choroid DNA deletion spots on the RPE optic nerve

  9. 1 eye spot = 1 deletion event in the RPE 1-cell spot 4-cells spot 34-cells spot an eye spot is a group of pigmented cells next to each other or separated by no more than one unpigmented cell a single cell deletion event can be detected among 50.000 RPE cells

  10. Eye spot frequency in Atm deficient mice Distribution of spots,% Number of spots/RPE Atm-/- mice have high number of eye spots as compared to wild type 8.1 ± 3.1 (n=28) vs 5.9 ± .9 (n=36) spots/eye, respectively; P=0.001

  11. Difference in the frequency of genetic instability Harvard - UCLA

  12. Semi-conventionalized Atm-/- mice in a non-sterile facility have increased DNA deletions compared to Atm-/- mice in a sterile environment p<0.01 * p<0.05 *

  13. Ribosomoal intergenic spacer analysis (RISA) shows that mice in different facilities have different spectra of 18S rRNA Semi-conventialized mice Mice in a sterile environment “Conventional” mice Markers

  14. RISA results show that antibiotic treatment followed by re-inoculation with fecal samples from donor mice reconstitute the intestinal flora Semi-conventialized mice Markers After 4 weeks of antibiotic treatment Conventionalized mice Donor mice

  15. Older Atm-/- mice

  16. Older wildtype mice

  17. Micronucleus Assay * Indicates p<0.05 compared to wt control and as indicated

  18. Semi-conventionalized mice in a non-sterile facility have a decreased median lifespan compared to mice housed in a sterile facility Kaplan-Meier survival curve of Atm-/- mice housed in normal and sterile facilities. The survival curves of mice living in normal and sterile facilities are significantly different (p<0.05). n=34 and 31 for the sterile SPF facility and non-sterile SPF facility, respectively.

  19. Lymphoma latency is shorter in semi-conventional mice in a non-sterile environment Lymphoma latency is shorter in a non-sterile environment. The latency of lymphoma development in a non-sterile environment is significantly shorter than in a sterile environment (p<0.01). n=15 and 13 for the sterile SPF facility and non-sterile SPF facility, respectively

  20. Lactobacillus johnsonii 456 treatment reduces DNA damage in ATM -/- mice

  21. Changes in Lymphocytes in peripheral blood populations caused by Lactobacillus inoculation

  22. Changes in Lymphocyte populations in spleen caused by Lactobacillus inoculation

  23. When treated with LBJ, mice showed a marked decreased in T cell infiltration in the liver

  24. LBJ treatment decreases inflammatory cytokine levels in both blood and liver : IL-1beta, IL-12, and IFN-g • LBJ treatment increases levels of IL-4, IL-10, and TGF-beta, which enable inflammatory control, especially in the liver. • Inflammatory diseases and oxidative stress: Cancer, heart disease, neurological disease, arthritis and ageing etc.

  25. „Restricted“ mice have very distinctive microbiota PCoA of Bray-Curtis difference between gut communities (all data) Each data point is a bacterial community from the gut of one mouse ATM-/- ATM +/- ATM +/+ Sterile Conventional DLAM-Conventional Semi-conventional Restricted

  26. Group 1: Indicator phylotypes • 32 indicator phylotypes for DLAM mice (both ATM-/- and wt). • Few genotype-specific phylotypes, consistent with the ANOVA result that the genotype is less important. • Diversity of indicators, including some putative opportunstic pathogens, e.g. in the Helicobacteraceae

  27. Comparing bacterial communities with PCA of unifrac score, a phylogenetic similarity metric Restricted Sterile Semi-conventional Conventional DLAM Conventional Weighted unifrac (considers relative abundance of taxa) Unweighted unifrac (presence/absence of taxa)

  28. Identification of bacteria that causeor suppress genetic instability and lymphoma in mice

  29. Candidate protective bacteria that are statistically (P < 0.000) more abundant Candidate causative bacteria that are statistically (P < 0.000) more abundant in RM than CM in CM than RM 1. Lactobacillus johnsonii: 2, Clostridium polysaccharolyticum; 3, Clostridium populeti; 4, Eubacterium hadrum; 5, Clostridium oroticum; 6, Barnesiella intestinihominis; 7, Clostridium fimetarium; 8, Acetanaerobacterium elongatum; 9, Porphyromonadaceae bacterium C941; 10, Butyrivibrio crossotus; 11, Butyricimonas synergistica; 12, Clostridium chauvoei; 13, Lachnospiraceae bacterium DJF_VP30; 14, Porphyromonas sp. C1075; 15, Prevotella sp. oral clone CY006; 16, Rumen bacterium NK4A66; 17, Filifactor alocis; 18, Cyanobacterium sp. MS-B-20; 19, Clostridium tyrobutyricum; 20, Alistipes onderdonkii; 21, Barnesiella viscericola. 1, Dysgonomonas gadei; 2, Prevotellaceae bacterium P4P_62; 3, Belliella sp. MIM10; 4, Parabacteroides merdae; 5, Clostridium sp. AN-AS17; 6, Capnocytophaga ochracea; 7, Pedobacter koreensis; 8, Eubacterium sp. BU014; 9, Riemerella anatipestifer; 10, Helicobacter typhlonicus; 11, Petrimonas sulfuriphila; 12, Caminicella sporogenes; 13, Nubsella zeaxanthinifaciens; 14, Porphyromonas sp. MI10-1288x; 15, Sphingobacterium sp. NBRC 15338; 16, Proteiniphilum acetatigenes; 17, Parabacteroides goldsteinii; 18, Bacteroidetes bacterium P073B; 19, Porphyromonas catoniae; 20, Bacteroides nordii.

  30. Who did the work? Ramune Reliene Irene Maier Lynn Yamamoto Angeline Tilly Jared Liu David Berry Alexander Loi Mike Davoren Yelena Rivina

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