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					Bacterial Infection Promotes
  Colon Tumorigenesis in
       Apcmin/+ Mice
  Joseph V. Newman, Takeo Kosaka,
  Barbara J. Sheppard, James G. Fox,
         and David B. Schauer
     Background on Diet and
           Microflora
• Louis Pasteur (1822-1895)

• Adult Humans have more prokaryotic than
  eukaryotic cells
• Symbiotic relationship in GI tract for more
  efficient nutritional benefit
• Epidimiological studies have linked high
  incidence rates for colon cancer to a western
  diet (high in animal products)
             Digestive Anatomy
  Small vs. Large intestine:

• Different developmental
layers
• Epithelium structure
• Commensal digestive
flora
• The occurrence of
intestinal tumors in mice vs.
humans
    Common Digestive Flora
• Stomach               • Colon
                          –   Enterobacteria
  – (Heliobacter sp.)     –   Enterococcus faecalis
• Small Intestine         –   Bacteroides *
                          –   Bifidobacterium *
  – Enterococci
                          –   Clostridium
  – Lactobacilli          –   Lactobacillus *
  – (E. coli,             –   Streptococcus
    Psesudomonads)        –   Staphylococcus
                          –   Ruminococcus
                          –   Peptostreptococcus
                          –   Peptococcus
              Large Intestine
• 3 distinct regions   •   1011 to 1012 cells/g wet feces
                       •   >500 species
   – Cecum             •   Lactobacilli
   – Colon             •   Bacteria have a few key
   – Rectum                roles:
                            – Nutrient breakdown
                            – Preventing pathogenic
                              colonization
                            – Maintaining overall
                              physiological conditions
 Dietary Fiber breakdown
Sloughed cells and dietary fiber




  Fermentation


                           Glycolysis                         ATP

                                                      propionate
                                          Pyruvate                    Liver
                   SO2
                                                          ATP
                                                                              methanogenesis
  H2S                        H2                                    CO2 + H2                    CH4
          sulfidogenesis



                              acetate                     butyrate
                Skin                    Acetyl- CoA                    Gut Epithelium

                             ATP                            ATP
  Diet and its effect of gut flora
• Fermentation of SCFA  H2 + CH4
• Efficient mechanism for H2 disposal has evolved along 2 major
  pathways:
    – Methanogenic achea
    – SRB
    – Acetogenic*
• Low in colon cancers: high levels of methanogens
• western diet: higher levels of colon cancer
• Difference: Meat in the diet leads to an increase in SRB
• The composition of diet not only impacts the substrates for gut
  flora, but also sets up a predictable competitive relationship
Large Intestine
        • Colon mucosa has flat
          epithelium with crypt
          complexes
           – Differentiated cells
           – Proliferative stem and
             precursor cells
        • SCFA degredation:
          proliferation  differentiation
         Intestinal Epithelium
• Mucosal epithelium are bound by tight
  junctions, the most luminal cell-cell junctions
• 2 major functions:
   – Permeability barrier
   – Protein Separation
• Tight junctions
   – Occludin
   – Claudin
   Bacteria, inflammation, …
• Analyze KO mice to       • Hosting a bacterial
  germ-free conditions       population is not without
  – TCR/p53 Dbl. KO         consequence
                              – Maintain gene to protect
  – IL-10 deficient mice        against bacterial stress:
  – Apc Min mice  50%          peroxidative stress,
    tumor                       bacterial antigen,
                                inflammation
                              – Intact mucosal barrier
         Possible models of
           tumorigenesis
• Inflammation/cancer depends on aggregate
  interactions
  – Quorum sensing
  – Alterations in flora due to diet
• Weak genetic defects and polymorphisms in
  hosts might allow normal flora to induce
  tumors over extended period of time
           Diseases of the Colon/Large
                    Intestine
 Crohn’s Disease       Inflammatory Bowel Disease (IBD)    Ulcerative Colitis     Diverticulitus
Chronic Inflammation        Inflammation, Rigidity and    Chronic Inflammation   Colon Develops a
                                Thickening of Colon                                   pocket




                                               Colon Caner
          Relevance of studying
           Bacterial infection

Helicobater pylori         Increased gastric cancer
H. hepaticus               Hepatocellular carcinoma,
                           Liver
Lawsonia intracellularis   Intestinal epithelium
                           proliferation (cancer
                           biomarker)
Group D Streptococcus      Inflammation, dysplasia,
                           rectal carcinoma
                Introduction
• C. rodentium - naturally gram (-) occurring
  bacterial pathogen of lab mice
• Infection:
  – Epithelial cell hyperproliferation (IBD, Chron’s,
    colitis) & thickening/rigidity of colon
  – Diarrhea and weight loss (suckling mice)
  – Colonic hyperplasia and limited inflammation
    (adult)
Transmissible Murine Colonic
    Hyperplasia (TMCH)
• Colonic crypts are 2   • No direct evidence
  to 3 times longer        linking C. rodentium
  compared to normal       to tumorigenesis
  mice                   • Increased colonic
• Epithelium contain       adenoma counts in
  twice the number of      presence of
  dividing cells           carcinogens
               AE Lesions
• Attaching and Effacing lesions during colon
  infection
  – Dissolution of brush border,
  – cupping of adherent bacteria,
  – cytoskeleton rearrangements of epithelium
• Enteropathogenic and Enterohemorrhagic E.
  coli (EPEC & EHEC) infections
• Similar gene locus is required for AE
  formation
    C. rodentium animal model of infection
 AE Lesions (Chicken and the
            egg)
• Is inflammation causing the altered
  epithelium, which allows for bacterial
  association?
• Does bacterial attachment cause these
  lesions, which then induce
  inflammation?
• AE pathogens have been shown to attach to
  surface epithelial cells via type III secretion
  pathway, possibly causing the release of
  some inflammatory mediators
   – 7 day post infection
   – 21 days post infection
            ApcMin/+ Mouse
• Nonsense mutation of adenomatous
  polyposis coli gene
• Apc:
  – Regulates cellular division frequency
  – Regulates cellular attachment/movement
• Mice are pre-disposed to multiple intestinal
  neoplasms (Min)
                          Methods
• Inoculated 4 week old
  mice
                                               100 L Culture
   –   ApcMin/+   w/ 100L o/n
     culture                                   or
   – Apc+/+ w/ 100L sterile                   100 L sterile media
     media
• Confirmed infection 7
  days post infection w/            10 days
                                    or
  CFU counts                        5 months
• Sacrificed mice 10 days
  and 5 months post
  inoculation
            the messy steps
Pathology                    Immunohistochemistry
• The colon was removed      • Representative samples
  and examined for             were frozen and stained
  hyperplasia                  for:
                                smooth muscle
• Adenomas were                    actin
  counted and measured
                                – F4/80 (macrophage
• Grossly altered tissue           marker)
  was excised and               – COX-2
  mounted for histological
  analysis
Results




    • A: Mucosal epithelium,10
      days post infection
    • B: Intact basement
      membrane and hyperplasia
    • C: Dysplastic tissue with
      adenoma
            Colonic Adenoma
              from infected
                Min mouse




• D. COX-2
• E. COX-2 + F4/80
• F. COX-2 + actin
High magnification of
     Adenoma




       G. COX-2
       H. COX-2 + F4/80
       I. COX-2 +Actin
                   Results
ApcMin/+ mice              Wt mice
• Visible thickening       • Visible thickening
  and rigidity of colon      and rigidity of colon
  (10 days post              (10 days post
  infection)                 infection)
• Mean crypt column        • Mean crypt column
  height 2x that of          height 2x that of
  uninfected Min mice        uninfected Min mice
  (significant)              (significant)
             No significant difference
               Discussion
• Infection promotes adenoma formation in Min
  mice
• Could promotion be due to hyperproliferative
  state induced by infection?
• COX-2 levels were not detected in colon
  tissue from infection (10 day post-infection)
   Is COX-2 involved in earliest stages of
  tumor promotion in Min mice?
           Future Direction
• Is microbiota required for colon
  tumorigenesis?
• Do A/E pathogens produce alterations of
  epithelial cell cytokinetics?
• What “chemical signals” are secreted by
  bacteria associated with aberrant crypts?
• Do bacterial signals influence gene activities
  of colon mucosal cells?
• Heat-stable
  enterotoxin
• GC-C pathway
  specific to intestinal
  epithelium
• Carcinoma cell
  proliferation was
  inhibited

				
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