The Vicious Cycle of Diarrhea and Malnutrition:           Effect on Growth and Development        Cognition, Obesity, Biom...
DisclosuresCofounder of AlGlutamine, LLCMember Probiotics Scientific Advisory Board
The Vicious Diarrhea-MN Cycle          Diarrhea        Inc. incidence +          Inc. duration            & severity      ...
Malnutrition increases incidence         and duration of diarrhea                         >-3 WAZ <-3 WAZ %inc. p valueEpi...
ECD /Enteric Infections Stunt Growth („60s-‟80s)           •Guatemala•“Matagram” and dysentery/bpnuem Scrimshaw Taylor Gor...
“Matagram” showing that repeated diarrheal andother illnesses pull children off their growth curves       Mata L. The chil...
Starting ok                  WHZ                   but losing                         WAZ          at 3-24 months!        ...
Diarrhea ablates catch-up growth                                         0.5                Weight gain (kg/month)        ...
Early childhood diarrhea (0-2 years old) impairs height-for-               age at 2-7 years old (8.2cm)                   ...
Multi-country analysis confirms that   diarrhea increases stunting (HAZ-2 <-2)                 Every 5 episodes stunts 13%...
The Vicious Cycle of Diarrhea and Malnutrition:           Effect on Growth and Development        Cognition, Obesity, Biom...
Infectious Diseases Impair IQ   The Economist, July 1, 2010.                                 When controlling for :       ...
Vicious Cycles of the Diseases of Poverty                      Enteric & Other                    Anti-      Water,     Sa...
Albendazole Improves Physical and CognitiveDevelopment (hookworm, Ascaris &Trichuris)  •Kenyan schoolchildren had better F...
Global Burden of Infections/MN:             Decreasing Mortality, but Increasing Morbidity                     Of 7.6-10.6...
Diarrhea increases stunting &            Stunting impairs Cognitive            Development & SchoolingMod-sev HAZ-2yo     ...
Nutrition @ 0-2yo increases IQ and earning Atole veg/prot suppl (2/4 Guatemalan villages ‟69-‟77)   (163kcal/11.5g protein...
Magnitude of lasting disability effectsfrom early childhood diarrhea (ECD):          1. Growth shortfalls (esp. HAZ-2; 8.2...
Low HAZ-2 correlates with:    •Adult stunting    •Less attained schooling (1y=12-14% lifetime earnings)    •Reduced econom...
The cognitive deficit most impaired with diarrhea         is Semantic Fluency (as in Alzheimer‟s Disease)        (and ApoE...
Neurocognitive markers of cortical function (>5yo):•TONI/Raven Prog. Matrices•WRAML Verbal learning•WISC Coding•NEPSY Flue...
Most (>75%) of Human Brain Growth & Development         Occurs in the First 2 Years of Life                    Human brain...
Importance of a Healthy Gut in Early Childhood             To full growth and healthy development:          Key in first 2...
Might ECD/Stunting also risk Obesity?!     [Bad water colliding with bad diets]    Of 117/532 Adults in Maceio Slum (NE Br...
Low HAZ-2 correlates with:    •Adult stunting    •Less attained schooling (1y=12-14% lifetime earnings)    •Reduced econom...
Long-term epigenetic effects                 & evolution of imprinting(Dense cytosine methylation of CG dinucleotides sile...
Early MN     Later Obesity & Heart Disease• 300k 19yo men more obese if mothers pregnant  in Dutch Hunger Winter of 1944-5...
Chronic consequences of enteric infections:                                      Physical and cognitive development       ...
JID 148: 986, 1983.CID 28: 966, 1999.
Markers and Impact of Environmental Enteropathy        Assessing:               Urine                   Stool             ...
Prolonged or Persistent Diarrhea    (ie any     diarrhea >7d) signals heavy diarrhea burdens and weight and height growth ...
Emerging causes of Persistent Diarrhea/MN             in Fortaleza, Brazil                                     Cases      ...
Emerging causes of Persistent Diarrhea/MN             in Fortaleza, Brazil                                                ...
Evidence for lasting disability effectsfrom early childhood diarrhea/enteric infections* Growth shortfalls (esp. HAZ-2; 8....
Cryptosporidium, EAEC, Giardia          or Intestinal InflammationStunt Growth and/or Psychomotor Development •Fecal neopt...
ECD or Giardiasis Stunt Growth and Cognition•Stunting or Giardia Peru <2yo                         WISC-R IQ @ 9yo        ...
Enteroaggregative E. coli (EAEC) causesPersistent Diarrhea, Intestinal Inflammation, Growth Shortfalls•EAEC is associated ...
MalED Case-Control Site, Fortaleza, Ceará, Brazil•Serves 313km2 ; 13kmfrom UPC•21k children           IPREDE Nutrition Cli...
Brazil case control: EAEC with MN       Microorganism             Control           Cases *           p values            ...
Cryptosporidiosis impairs growth:•Even “Asyx” (63 vs 37%)               162-342g less @3m•Stunting (persisting @ 1y) if:  ...
Murine model of weanling malnutrition     & Cryptosporidium infection                             C57BL/6J mice at 7 days...
Cryptosporidium infection causes Malnutrition                                        ie. MN is an ID AND                  ...
Synergistic effects of malnutrition andCryptosporidium infection on ileal architecture                  (H&E; 10X; at 14do...
Malnutrition is associated with a 1 to 2.3 log (ie 10 to                                             200-fold) heavier inf...
Cryptosporidium causes and worsens MN    Unexcysted oocysts in weaned, 2% protein MN mice     N, Uninfected     P<0.03    ...
“Peace Corps Gut”                        Normal                        PCV w Diarrhea and Malabsorption (d-xylose & B12)40...
Candidates to help “repave”     the small bowel absorptive            tennis court                          (every 3 days!...
Vicious Cycles of the Diseases of Poverty                      Enteric & Other                    Anti-      Water,     Sa...
Diarrhea (Ac, Pro, PD) predisposes to   growth decrements (WAZ or HAZ)                                                 *  ...
Glutamine and Ala-Gln ORRT       improve the repair of intestinal epithelium                  in IEC6 cells and in        ...
Diarrhea impairs Cognitive          Development & Growth               independentlyECD0-2, HAZ2 or WAZ2 predicts TONI & C...
Prolonged and persistent diarrhea account for only        100%                     17% of episodes, yet half of all diarrh...
Proposed model for Ala-Gln coupling to EGFR signaltransduction during enteric infections and malnutrition.                ...
AlaGln improves Wt, morphometry, apoptosis &   proliferation in MN weaned C57Bl/6 mice          Ueno… Moore.          Am J...
AlaGln improves barrier function (lactuloseexcretion) and weight gains in malnourished             children in Brazil     ...
Gln or Ala-Gln enhance absorption (even with Crypto infections when Glucose fails)         3                              ...
Another vicious cycle: Barrier disruption & Inflammation TJ (TNF, MLCK)(lo cap; nonselect)Claudin, Occludin,     ZO1, acti...
Urine Metabonome in MN vs Control Childrenshowing increased NAG, DMA, DMG; less Mannitiol      (by NMRS and PLS-DA coeffic...
Enteroaggregative E. coli (EAEC)    Infection Impairs Growth     & MN worsens infection   DMEM / HS (Nourished Control) n=...
Induces   Glutamine                     HSP70, which:•Attenuates iNOS, IL-6, IL-8    (in rat CP bypass)•Protects against n...
Cryptosporidium and Giardia genotypes in        Brazil and South Africa •C. hominis predominates in NE Brazil with:    gre...
Conclusions (re Crypto and MN)•Crypto infections, +diarrhea, ProD or PD impair growth inyoung children (or mice);•Likely v...
Disruption of Intestinal Barrier Function in Children with Acute and Persistent Diarrhea in Fortaleza, Brazil             ...
EndoCAb correlates with disrupted intestinal  barrier function and with stunted growth                Campbell DI, Elia M,...
Enteroaggregative E. coli (EAEC) causesPersistent Diarrhea, Intestinal Inflammation, Growth Shortfalls and          is the...
HAZ correlates with Grades and Rank (among 242 college schoolgirls in S. India)                                r2=.037    ...
Infant MN       CVD  Barker Hypothesis                      Barker D. et al. Lancet 1986, „89, ‟90.                      H...
“Thrifty epigenotype”Candidate genes for environmental „canalization‟Stoger R. BioEssays 30: 156, 2008.
Infants with ProD are high risk                   for future episodes of PD                         (+) Prolonged Diarrhea...
Cryptosporidium: A Leading cause of     Prolonged and Persistent Diarrhea &              Growth Shortfalls                ...
Long-term Consequences of     Cryptosporidium Infection•Malnutrition             Sallon ‟88; Sarabia-Arce ‟90; Checkley ‟9...
ApoE4 Upregulates an    Arginine-selective Cat-1 Transporter•Transgenic mice expressing the human APOE4allele have express...
Arginine improves wt gain & reduces ilealCryptosporidium infection by over 10-fold by both iNOS          & arginase-depend...
MN-Infected ApoE4/4ki Mice have better growth and              Villus/Crypt Architecture, and shed less Cryptosporidia    ...
Recurrent diarrhea impairs height           and weight gains                               Ht gain (cm)          Wt gain (...
The Vicious Cycle of Diarrhea and Malnutrition: Effect on Growth and Development Cognition, Obesity, Biomarkers, Pathogens
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The Vicious Cycle of Diarrhea and Malnutrition: Effect on Growth and Development Cognition, Obesity, Biomarkers, Pathogens

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The Vicious Cycle of Diarrhea and Malnutrition: Effect on Growth and Development Cognition, Obesity, Biomarkers, Pathogens

  1. 1. The Vicious Cycle of Diarrhea and Malnutrition: Effect on Growth and Development Cognition, Obesity, Biomarkers, Pathogens Richard L Guerrant Aldo AM Lima, Reinaldo Oria, Jim Roche, Rebecca Scharf, Mark DeBoer, Jim Nataro, Orleancio Azevedo, Noronha Diniz, Pranay Sinha, et al Infectious Diseases and International Health Centers for Global Health University of Virginia-Federal University of Ceara-ICIDR-NIAID/FIC/FNIH Charlottesville, VA-Fortaleza, Brazil The Global Crisis of Childhood Diarrhea PROGRAMME FOR GLOBAL PAEDIATRIC RESEARCH SYMPOSIUM Boston - April 30, 2012            
  2. 2. DisclosuresCofounder of AlGlutamine, LLCMember Probiotics Scientific Advisory Board
  3. 3. The Vicious Diarrhea-MN Cycle Diarrhea Inc. incidence + Inc. duration & severity Malnutrition Dec. Wt gain (short) Dec. Ht gain (long)
  4. 4. Malnutrition increases incidence and duration of diarrhea >-3 WAZ <-3 WAZ %inc. p valueEpisodes/2m 1.9 2.6 37% 0.001Duration (d) 6.7 11.6 73% 0.004Total d of D. 12.1 24.2 100% <0.001•By Wilcoxon rank sum test•Also valid for moderate MN (<90% HAZ) Schorling JB et al, Int J Epi 19: 728-735, 1990.
  5. 5. ECD /Enteric Infections Stunt Growth („60s-‟80s) •Guatemala•“Matagram” and dysentery/bpnuem Scrimshaw Taylor Gordon; Mata etal. Am J Clin Nutr24:249, 1971.•ECD (not fever/pneumonia) stunts Ht 6.5%, irresp of feeding Martorell R et al. Am J Dis Child129: 1296, 1975. •Gambia •ECD (not ARI) impairs Ht & Wt gains Rowland MG et al. Br J Nutr 37: 441, 1977. •ECD (& ARI) <1yo/nonBF impairs Wt gains Rowland et al. Am J Clin Nutr 47:134, 1988. •Mexico •ECD (not ARI) impairs Wt gains to 10% @3yo Condon-Paoloni et l. AJPH 67: 651, 1977. •Brazil •ECD impairs Wt (32% @2wks), Ht (41% @ 3.5m) Guerrant et al. JID 148: 986, 1983. •ECD blunts catchup growth & PD (not fever/pneumonia) stunts Wt & Ht Leslie J, de Souza MA. Ch 15 in Edge of Development 1996; Schorling, Guerrant Lancet 335: 599, 1990; Moore et al Gastro 139: 156, 2011. •Bangladesh •ECD (not ARI) stunts Wt @ 2m & 2/10cm Ht @ 5yo, esp. Shigella, ETEC but irresp of etiol. Black RE et al, Pediatr 73: 799, 1984.
  6. 6. “Matagram” showing that repeated diarrheal andother illnesses pull children off their growth curves Mata L. The children of Santa Maria Cauque. MIT Press, Cambridge MA. 1978.
  7. 7. Starting ok WHZ but losing WAZ at 3-24 months! HAZVictora…Shrimpton,Pediatrics 125: e473, 2010. Latin America AfricaPediatrics 107: e75, 2001. S.Asia
  8. 8. Diarrhea ablates catch-up growth 0.5 Weight gain (kg/month) 0.4 0.3 Weight for age <-3 Weight for age >-3 0.2 0.1 0 <15 15-30 31-45 >45 % of Days with DiarrheaSchorling JB, Guerrant RL. Diarrhea and catch-up growth. Lancet 335: 599-600, 1990;Petri et al. JCI 118: 1278, 2008; Guerrant et al. Nutr Rev 66: 487, 2008; Checkley et al. IJE 37: 816, 2008.
  9. 9. Early childhood diarrhea (0-2 years old) impairs height-for- age at 2-7 years old (8.2cm) Moore SR et al, Int J Epi 30: 1457, 2001.
  10. 10. Multi-country analysis confirms that diarrhea increases stunting (HAZ-2 <-2) Every 5 episodes stunts 13% (over incidence range of 3.6-13.4 episodes/child-yr!)** >5 episodes of diarrhea before 2yo accounts for 25% of all stunting. Checkley et al, Int‟l J Epi 37: 816-30, 2008.
  11. 11. The Vicious Cycle of Diarrhea and Malnutrition: Effect on Growth and Development Cognition, Obesity, Biomarkers, Pathogens •Vicious cycles of the diseases of poverty; •ECD lasting stunting and cognitive impairment; •Potential effects of early infections and stunting on obesity/DM/MetS/CVD; •Biomarkers/Pathogens-esis/Novel Interventions
  12. 12. Infectious Diseases Impair IQ The Economist, July 1, 2010. When controlling for : •GDP per capita; • Education (WB lit.AVED) •Temperature; Nutritional deficiencies correlation w IQ was lost when ID removed ie ID, not MN or poverty may cause the “Flynn Effect” of IQ:Development Eppig C, CL Fincher, R Thornhill. Proc Biol Sci 277: 3801, 2010.
  13. 13. Vicious Cycles of the Diseases of Poverty Enteric & Other Anti- Water, Sanitation Infections +Diarrhea Microbials; Vaccines EnvironmentWorsened InfectionIntensity & Damage Intestinal Damage Repeated & & Inflammation Microbiome Persisting Impaired Innate & Infections Nutrient Acquired Mucosal Malabsorption Defenses &/or Loss Hs genome Probiotic Malnutrition Repair Nutrients Microflora Impaired Vaccine Responses Cognitive Impairment Obesity DM, HT, CVD, Poverty Met. Synd. $$Health Care
  14. 14. Albendazole Improves Physical and CognitiveDevelopment (hookworm, Ascaris &Trichuris) •Kenyan schoolchildren had better Fitness, Growth (W+L) and Appetite 2-4m after single Albendazole. Stephenson L, et al. AJTMH 41: 78, 1989; TRSTMH 84: 277, 1990; J Nutr 123: 1036, 1993. •Jamaican schoolchildren had better short & long- term memory (WISC digit span), Fluency and scanning 2 m after Albendazole x3d & arithmetic 10y w HAZ-2. Nokes C, Grantham McGregor S, Bundy D, et al. Proc R Soc Lond B 247: 77, 1992 ; Chang SM et al J Child Psychol Psychiat 43:775, 2002.
  15. 15. Global Burden of Infections/MN: Decreasing Mortality, but Increasing Morbidity Of 7.6-10.6m Child Deaths/yr: Diarrhea specific mortality 25Mortality rate per thousand 20 1955-1979 15 YLL 10 5 1982-1990 1990-2000(Yrs of Life Lost) 0 <1y 1-4y 0-4y Malnutrition Age group 53% Diarrhea 15-18% (>3100/d)Morbidity 6 Episodes per Person Year 5 YLD 178m stunted (HAZ<-2) 4 3 1955-1979 1980-1990(Yrs Lost to Disability) MN is THE leading risk 2 1990-2000 1 20% of ALL children; factor for health loss! 0 32% DC children! 11-16% of all DALYs ; 0-5m 6-11m 1y 2y 3y 4y Age group 35% of all <5yo DALYs!*DALYs (Disability Adjusted Life Yrs) Black , Bryce et al. Lancet 375: 1969, 2010; *Black et al Lancet; 371:243, ‟08; (YLL + YLD) Murray, Lopez Lancet 349: 1436, „97; 367: 1747, „06. 365:1147, 2005. Kosek et al. Bull WHO 81: 197, 2003; . Bhutta et al Lancet 371: 417, „08. JHPN 27:319, 2009. Guerrant et al Tr Parasitol 18: 191, 2002.
  16. 16. Diarrhea increases stunting & Stunting impairs Cognitive Development & SchoolingMod-sev HAZ-2yo 25-61% IQ z lower @ 8-11yo (Philippines Nonverbal Intel. - Guthrie test; p<0.001) •Both severity and timing dependent; only 1/3 catch up •Partially reversible •Compounded with indirect effects on schooling. 30% lower IQ @ 11yo Stunting impairs cognition, but schooling still helpsStunting delays schooling Mendez M & Adair L. J Nutr 129: 1555-62, 1999.
  17. 17. Nutrition @ 0-2yo increases IQ and earning Atole veg/prot suppl (2/4 Guatemalan villages ‟69-‟77) (163kcal/11.5g protein vs 59kc/0prot “Fresco” -INCAP Martorell, et al)•10% better Raven IQ (AFTER adjusting for schooling) Stein AD et al. Arch Ped Adolesc Med 162: 612-8, 2008.•46% higher wages @ 25-42yo m •8-20% higher reading, Raven, schooling f (But not after 3yo!) Hoddinott J et al. Lancet 371: 411-6, 2008.
  18. 18. Magnitude of lasting disability effectsfrom early childhood diarrhea (ECD): 1. Growth shortfalls (esp. HAZ-2; 8.2cm by 7yo) HAZ-2 is the best predictor of human capital! (schooling, economic productivity, offspring wt!) Victora C. et al, 5 cohorts (Bz, Guat, India, Philipp, S Africa) Lancet 371: 340, 2008. 2. Fitness impairment (=17% decr. work prod.) 3. Cognitive impairment (c. 10 IQ points) 4. School performance (c. 1 yr) (increased age at starting school and age-for-grade) These effects >DOUBLE the global diarrhea DALYs.Moore et al. Int J Epi 30: 1457, 2001. Guerrant DI et al. Am J Trop Med Hyg 61: 707, 1999.Niehaus et al Am J Trop Med Hyg 66: 590, 2002. Guerrant et al Tr Parasitol 18: 191, 2002. Lorntz et al PIDJ 2006.
  19. 19. Low HAZ-2 correlates with: •Adult stunting •Less attained schooling (1y=12-14% lifetime earnings) •Reduced economic productivity (Monthly income) •Lower offspring birthwt (in females) •Hi glucose, BP, lipids (p BMI controlled for, implicating subsequent wt gains; ?Barker hypothesis; “Thrifty epigenotype” & genetic „canalization‟)HAZ-2 is the best predictor of human capital! (schooling, economic productivity, offspring wt!) Victora C. et al, 5 cohorts (Bz, Guat, India, Philipp, S Africa) Lancet 371: 340, 2008.
  20. 20. The cognitive deficit most impaired with diarrhea is Semantic Fluency (as in Alzheimer‟s Disease) (and ApoE4 protects children‟s cognition with heavy diarrhea; “thrift allele”) 125 R2 = 0.135, p=0.012 100 Test of Non-verbalIntelligence-III quotient 75 IQ at 6-9yo 50 0 5 10 15 20 25 30 Guerrant DI et al. Am J Trop Med Hyg 61: 707, 1999. Niehaus et al. Diarrhea rates at 0-2yo AJTMH 66: 590, 2002. Patrick et al. Child Neuropsych 11: 233, 2005. Oria R, Patrick P, Lima A, et al Pediatr Res. 57: 310, 2005.
  21. 21. Neurocognitive markers of cortical function (>5yo):•TONI/Raven Prog. Matrices•WRAML Verbal learning•WISC Coding•NEPSY Fluency (Sem/Ph) (vs Developmental Psychology; nature + nurture: Bailey, HOME; McArthur Language)
  22. 22. Most (>75%) of Human Brain Growth & Development Occurs in the First 2 Years of Life Human brain Human brain wt/age growth Human postnatal visual cortex dendritic tree birth 15 m 2 yo + only 1 opportunity for neuronal connections or „forever lost‟
  23. 23. Importance of a Healthy Gut in Early Childhood To full growth and healthy development: Key in first 2 years of life, because that‟s when: 1.Synaptic “tracks” are laid; 2.Most vulnerable Mendez M & Adair L. J Nutr 129: 1555-62, 1999.       Stein AD et al. Arch Ped Adolesc Med 162: 612-8, 2008.   Hoddinott J et al. Lancet 371: 411-6, 2008.
  24. 24. Might ECD/Stunting also risk Obesity?! [Bad water colliding with bad diets] Of 117/532 Adults in Maceio Slum (NE Brazil) who were stunted:• 30% (vs 23.6% of nonstunted) were overweight or obese vs • 16% (vs 23% of nonstunted) were underweight ?via increased cortisol:insulin or decreased IGF-1 levels ??Transgenerational epigenetic DNA methylation/silencing Sawaya AL et al. Nutr Rev 62: S127-133, 2004; Varela-Silva et al. Coll Anthropol 1: 39-46, 2007.
  25. 25. Low HAZ-2 correlates with: •Adult stunting •Less attained schooling (1y=12-14% lifetime earnings) •Reduced economic productivity (Monthly income) •Lower offspring birthwt (in females) •Hi glucose, BP, lipids (p BMI controlled for, implicating subsequent wt gains; ?Barker hypothesis; “Thrifty epigenotype” & genetic „canalization‟)HAZ-2 is the best predictor of human capital! (schooling, economic productivity, offspring wt!) Victora C. et al, 5 cohorts (Bz, Guat, India, Philipp, S Africa) Lancet 371: 340, 2008.
  26. 26. Long-term epigenetic effects & evolution of imprinting(Dense cytosine methylation of CG dinucleotides silences promoters) • Heavy LEP promoter methylation occurs during postzygotic development in mice & humans. Stoger R. Epigenetics 1: 155, 2006. Stoger R. BioEssays 30: 156, 2008. • LEP (and IGF2, IL10, ABCA1, GNASAS, MEG3) methylation greater in WBC of periconceptually DHW exposed vs sibs. Tobi, Stein et al. Hum Mol Gen 18: 4046, 2009.
  27. 27. Early MN Later Obesity & Heart Disease• 300k 19yo men more obese if mothers pregnant in Dutch Hunger Winter of 1944-5. Ravelli GP et al. NEJM 295: 349, 1976. Whitelaw NC & Whitelaw E. Hum Mol Genet. 15: R131, 2006.• 7845 women in DHW Famine (@10-17yo) have 27% more HD. vanAbeelen A et al. BBC News & Europ Heart J. Aug 25, 2011.• Early Childhood Stunting (or lo BWt) has 29-65% greater Abd obesity (waist:hip/eaHAZ-3) Schroeder, Martorell, Flores. AJEpi 149: 177, 1999.
  28. 28. Chronic consequences of enteric infections: Physical and cognitive development Margolis „10 Niehaus „02 Pinkerton „11 Eppig „10 Mendez, Adair „99 Moore ‟01 Cognitive ECD Checkley „08 Early Stein, Martorell „08 Sinha P „11 Impairment Enteropathy Stunting (Early Childhood HAZ-2 Van Abeelen ‟11 Diarrhea) Victora ‟08 ; Martins 06; Sesso 04; Ferreira „09 BMI-2 Preg/LBWt Grillol 05; Barker NEJM „05 Adult HD/HT Schroeder „99 Ferreira „01, „09 Obesity DM/GiTT Bhargava „04 Ravelli (DHWinter) „76 Abd (waist:hip) Met. Synd Barker „86, „88, „90 , ‟92 Fall ‟08 HiLDL; LoHDL Victora „08 Poor Later childhood obesity Poor Later growth childhood 2h G>140@30yo CHD<64yo Z growth obesity 1.5k Delhi 8k NorwayBMI Bhargava „04 Barker „05
  29. 29. JID 148: 986, 1983.CID 28: 966, 1999.
  30. 30. Markers and Impact of Environmental Enteropathy Assessing: Urine Stool Blood Barrier/Abs Damage: L/M ** Alpha1-AT EndoCAb** Cr ?Zonulin* Zonulin ?Intestinal FABP* ?Intestinal FABP* Intestinal FABP Inflammation: MeBlue Microscopy (Hemoccult) Lactoferrin (nonBF)** MPO Nitric Oxide (ie IL-8 et al nitrates+nitrites) * ?IL-8 mRNA* ?Calprotectin* ??Calprotectin mRNA* **Documented to be associated ?Neopterin* with stunting. ?SAA3* Repair: ?Citrulline* ?Citrulline *Research (?Reg-1*) questions. Small Bowel Overgrowth Lactulose Breath Test Tissue Biopsy +quantitative culture Gut Integrity METABONOME Meeting-Seattle, Field History: Hx of ProD >7d** December, 2010
  31. 31. Prolonged or Persistent Diarrhea (ie any diarrhea >7d) signals heavy diarrhea burdens and weight and height growth shortfalls; ie. provides a “field biomarker” for impending malnutrition! Moore, Lima et al. Gastroenterol. 139: 1156, 2010.
  32. 32. Emerging causes of Persistent Diarrhea/MN in Fortaleza, Brazil Cases Controls (n=127) (n=331) Enteroaggregative E. coli AA probe + 32%* 14% AA probe - 36* 17 Cryptosporidium 25** 5 Giardia lamblia 21** 8 Ricci et al. Nature S1: 29, 2006 *P<0.05; **P<002 Fang et al J Ped Gastro Nutr 1995 Koopmans et al Ped ID J 16:504, 1997 Lima et al. J Infect Dis 181: 1643, 2000 Steiner T et al. J Infect Dis 177: 88, 1998.
  33. 33. Emerging causes of Persistent Diarrhea/MN in Fortaleza, Brazil Cases Controls (n=127) (n=331)Enteroaggregative E. coli(-0.76 HAZ; p<0.05; Steiner JID 177: 88,‟98 EAEC w MN in MalED CC Lima „12) AA probe + 32%* 14% AA probe - 36* 17Cryptosporidium 25** 5(Checkley „97; ‟98; Agnew JID 177: 754,„98)Giardia lamblia 21** 8 (Newman TMIH 6: 624, ‟01 Sx w MN; PD>AD) Ricci et al. Nature S1: 29, 2006 Fang et al J Ped Gastro Nutr 1995 Koopmans et al Ped ID J 16:504, 1997 Lima et al. J Infect Dis 181: 1643, 2000 *P<0.05; **P<002 Steiner T et al. J Infect Dis 177: 88, 1998.
  34. 34. Evidence for lasting disability effectsfrom early childhood diarrhea/enteric infections* Growth shortfalls (esp. HAZ-2; 8.2cm by 7yo) •Crypto Infections increase diarrhea morbidity and nutritional shortfalls to 18m [Agnew 98; Lima 00; Newman 99] •Crypto infections +diarrhea = dec. wt gain @1m [Checkley 97] •Crypto infections <6m/stunted = .95-1.05 cm deficits @1y [Checkley 98] •EAggEC infections + inflammation = growth shortfalls [Steiner 98] •Diarrrhea<2yo = 3.6cm stunted @7yo (8.2cm w helminths) [Moore 01; + Checkley et al, 08] Fitness impairment (=17% decr. work prod.) •Albendazole =7% inc. HST @4m [Stephenson 93] •Diarrhea or Crypto <2yo = 4-8% dec. HST @4-7yo [Guerrant 99] •4.3% inc. HST = 16.6% inc. work prod. [Ndamba 93] Cognitive impairment (c. 10 IQ points) •Diarrhea <2yo dec. WISC coding/digit @5-9yo [Guerrant 99] •Diarrhea <2yo dec. TONI @6-10yo [Niehaus 02] •Giardia or stunting = 4-10 pts dec. WISC-R @9yo [Berkman 02] School performance (c. 1 yr))) •Diarrhea <2yo = inc. AASS; AFG [Lorntz 06; Guerrant 02] * Petri et al JCI 118: 1277-1290, 2008; Guerrant et al Nutr Rev 66: 487-505, 2008.
  35. 35. Cryptosporidium, EAEC, Giardia or Intestinal InflammationStunt Growth and/or Psychomotor Development •Fecal neopterin (inflammation) in 72 children followed 15m in rural Gambia had impaired long-term ht and wt gains (r=-0.29 and -0.36; p<0.001) [not Giardia or L/M] Campbell DI et al. J Ped Gastro Nutr 39: 153, 2004.
  36. 36. ECD or Giardiasis Stunt Growth and Cognition•Stunting or Giardia Peru <2yo WISC-R IQ @ 9yo Berkman DS et al. Lancet 359: 564, 2002.•Of 160 Children <5yo in Turkey, those w Giardia were stunted (OR=7.7) and had psychomotor impairment (OR=2.7) Simsek Z et al. J Trop Pediatr 50: 90, 2004.•Giardia Egypt <4yo had cognitive development if E4- Yahya RS et al. Internet J Parasit Dis 4:1, 2009.•Heavy ECD or Giardia <2yo Sem. Flu @ 6-12yo E4- Newman TMIH 6: 624, „01 PD; Patrick PD et al. Child Neuropsych 11: 1-12, 2005 Oria R et al. Ped Res 57: 310, „05; Bz J Biol Res 43: 249, 2010.•Giardia 6-13yo Iran had WISC Fluency, Span (indep of WAZ) Partovi F et al. Iran JPH 36: 73, 2007.•Asxy Giardia Salvador <4yo have stunting 1y later Prado MS et al. Parasitol 131: 51, 2005.•Sx Giardia S India predict wasting and cognition@ 3yo Ajjampur SSA et al. Trop Paediatr 31: 203, 2011. Guerrant, Oria, Moore, Scharf, Lima “ 201, 2011.
  37. 37. Enteroaggregative E. coli (EAEC) causesPersistent Diarrhea, Intestinal Inflammation, Growth Shortfalls•EAEC is associated with 36% of PD; 30-74% w HIV•EAEC-PD has elevated fecal LF, IL-8, IL-1 [Fli-C; TLR5]•EAEC-no D. also had elevated LF & Growth Shortfalls Steiner T et al JID 177: 88, 1998; Wanke et al JID 178: 185, 1998; Samie A et al. 77: 142, 2007; Steiner T et al JCI 105: 1769, 2000. Nataro et al and Steiner et al CID: 2006. EAEC Turista (26%) Only with IL-8 -251 AA or AT Polymorphism•0/23 w EAEC Diarrhea had TT vs 9-18% of 46 others without Sxor without EAEC (OR = 208; p<0.01)•Also those with -251 AA had greater fecal IL-8 (vs AT or TT;p<0.01) Jiang ZD et al. JID 188: 506, 2003.
  38. 38. MalED Case-Control Site, Fortaleza, Ceará, Brazil•Serves 313km2 ; 13kmfrom UPC•21k children IPREDE Nutrition Clinic, Fortaleza•2000 newchildren/yr•30% of <3yohave HAZ <-2 n=380 children enrolled Aldo Lima, Alvaro Madeiro, Sulivan Mota, et al. 2010-12.
  39. 39. Brazil case control: EAEC with MN Microorganism Control Cases * p values Total N (%) N (%) N (%) EPEC 3/62 (4.8) 1/88 (1.1) ns 4/150 (2.7) aEPEC 1/62 (1.6) 5/88 (5.7) ns 6/150 (4) EAEC 4/62 (6.5) 22/88 (25) p = 0.0147 26/150 (17.3) EHEC 3/62 (4.8) 3/88 (3.4) ns 6/150 (4) EIEC 5/62 (8.1) 8/88 (9.1) ns 13/150 (8.7) ETEC 2/62 (3.2) 4/88 (4.5) ns 6/150 (4) Campylobacter 14/70 (20) 12/104 (11.5) ns 26/174 (14.9) Adenovirus 0/42 (0) 1/72 (1.4) ns 1/114 (0.9) Astrovirus 0/42 (0) 1/72 (1.4) ns 1/114 (0.9) E. histolytica 2/38 (5.3) 2/63 (3.2) ns 4/101 (4) Giardia 7/38 (18.4) 12/63 (19) ns 19/101 (18.8) Cryptosporidium 2/38 (5.3) 1/63 (1.6) ns 3/101 (3)EPEC: enteropathogenic E. coli; EHEC: enterohemorragic E. coli; EAEC: enteroadherence E. coli (aaiC + aatA);EIEC: enteroinvasive E. coli.Case: child <-2 WAZ; and control: >-1 WAZ; * Fisher‟s exact test. Aldo Lima, A Havt et al 2011.
  40. 40. Cryptosporidiosis impairs growth:•Even “Asyx” (63 vs 37%) 162-342g less @3m•Stunting (persisting @ 1y) if: •Infection @ <5m (Agnew <1y 1-2 Z @ 2yo) •Stunted @ time of infection•Also in Africa (Molback) if <1yo, no catchup• And Asia (Ajjampur) Checkley et al. Am J Epi 145: 156, 1997; multiple Crypto common “ “ 148: 497, 1998; Agnew et al. JID 177: 754, 1998; and lower HAZ-2 Molback et al. Am J Clin Nutr. 65: 149, 1997; Ajjampur et al. AJTMH 83: 1110, 2010.
  41. 41. Murine model of weanling malnutrition & Cryptosporidium infection  C57BL/6J mice at 7 days old. Note the difference in the body weight between the nourished and malnourished mice. 4h of separation 8h of separation 12h of separation C57BL/6J Wild types 4 5 6 = Crypto infection 14 Age (days)
  42. 42. Cryptosporidium infection causes Malnutrition ie. MN is an ID AND MN worsens Crypto infection 275 NC (n=11) 250 NI 106 (n=16) % initial weight (g) MC (n=10) 225 41% loss MI 105 (n=14) 200 MI 106 (n=16) 175 further 48% loss 150 125 ** 100 **p<0.01 vs all groups by 6 7 8 9 10 11 12 13 14 ANOVA‟s Bonferroni test Age (days) Coutinho, Oria, Meton, Sevilleja, et al. J Parasitol 94: 1225, 2008.
  43. 43. Synergistic effects of malnutrition andCryptosporidium infection on ileal architecture (H&E; 10X; at 14do; 8d p infection) Nourished Non-infected Malnourished Non-infected Nourished Infected Malnourished Infected Coutinho, Oria, Meton, Sevilleja, et al. J Parasitol 94: 1225, 2008.
  44. 44. Malnutrition is associated with a 1 to 2.3 log (ie 10 to 200-fold) heavier infection by fecal shedding or in colon or ileum in 107 Cryptosporidium-infected mice at peak infection (d8) 2.50 2.00 log Mean (# of parasites / mg of ileal tissue)log Mean (# of parasites / mg of stool) 2.00 1.50 1.50 1.00 1.00 log Mean (# of parasites / mg of colonic tissue) 2.00 0.50 0.50 1.50 0.00 1.00 -0.50 0.00 MN (n=12) N (n=14) MN (n=6) N (n=3) Group Group 0.50 Error bars: +/- 1 SE Error bars: +/- 1 SE Fecal Shedding 0.00 Parasites/gm ileum (MN = 1.55 vs N = 0.54 MN (n=7) N (n=3) (MN = 2.02 ± 0.11 vs N = -0.25 ± 0.13, log/mg; N=26 mice in 3 paired Group Error bars: +/- 1 SE n=9 mice, p < 0.001) experiments; p=0.015) Parasites/gm colon (MN = 1.79 vs N = -0.14, n=10 mice, p= 0.001) Detected by Real-time PCR Coutinho, Oria, Meton, Sevilleja, et al. J Parasitol 94: 1225, 2008.
  45. 45. Cryptosporidium causes and worsens MN Unexcysted oocysts in weaned, 2% protein MN mice N, Uninfected P<0.03 14% P<0.05 MN MN-Infected N, infected MN, Uninfected 23% P<0.03 MN, infected P=0.029 Infection @ d58 Costa L, Noronha, Roche, et al. JID 205: 1464, 2012.
  46. 46. “Peace Corps Gut” Normal PCV w Diarrhea and Malabsorption (d-xylose & B12)40% (of 114) had d-xylose <5g52% (of 23) had low Schilling B12 Lindenbaum et al. AIM 65: 1201, 1966.Bx nl in 2/17 (12%); 8 had mild ;7 moderate “jejunitis”
  47. 47. Candidates to help “repave” the small bowel absorptive tennis court (every 3 days!) Normal HIV+ Patient with Cryptosporidium infection Yamada et al. (1995) Conlon et al. (1984)Zinc, Vitamin A, Glutamine, Alanylglutamine, Arginine, ?ApoE peptides, targeted antimicrobials, adjuvants, vaccines Carneiro, Bushen, Brito, Lima, Guerrant. Curr. Infect Dis Rep. 5: 114, 2003. Bushen et al. CID 38: 1764, 2004; Lima N et al. J Pediatr Gastroent Nutr 44: 365, 2007. Ueno… Moore. Am J Physiol GI Liver Physiol 301: 2011. Castro I et al Nutrition, 2011.
  48. 48. Vicious Cycles of the Diseases of Poverty Enteric & Other Anti- Water, Sanitation Infections +Diarrhea Microbials; Vaccines EnvironmentWorsened InfectionIntensity & Damage Intestinal Damage Repeated & & Inflammation Microbiome Persisting Impaired Innate & Infections Nutrient Acquired Mucosal Malabsorption Defenses &/or Loss Hs genome Probiotic Malnutrition Repair Nutrients Microflora Impaired Vaccine Responses Cognitive Impairment Obesity DM, HT, CVD, Poverty Met. Synd. $$Health Care
  49. 49. Diarrhea (Ac, Pro, PD) predisposes to growth decrements (WAZ or HAZ) * Moore, Lima et al. Gastroenterol. 139: 1156, 2010.
  50. 50. Glutamine and Ala-Gln ORRT improve the repair of intestinal epithelium in IEC6 cells and in children and HIV+ pts with diarrhea. Control with Glutamine with Ala-GlnBrito, Carneiro, Lima, Guerrant. 2004.Carneiro, Bushen, Brito, Lima, Guerrant. Curr. Infect Dis Rep. 5: 114, 2003.Bushen et al. CID 38: 1764, 2004; Lima N et al. J Pediatr Gastroent Nutr 44: 365, 2007.Ueno… Moore. Am J Physiol GI Liver Physiol 301: 2011.
  51. 51. Diarrhea impairs Cognitive Development & Growth independentlyECD0-2, HAZ2 or WAZ2 predicts TONI & Coding @ 6-12yo (Brazil cohort of 131 followed from birth; UV p<0.05) •Excluding household income, maternal education, child sex, birth wt, breastfeeding •ECD remained a predictor of IQ even after adjusting for HAZ and WAZ (multivariate p< 0.006-0.029) Pinkerton, Oria, Lima, Patrick et al. 2012.
  52. 52. Prolonged and persistent diarrhea account for only 100% 17% of episodes, yet half of all diarrhea days AND predict heavy diarrhea burdens and growth shortfalls! 83% Acute (<7d) 75% Prolonged (7-13d) Persistent (≥14d)% of diarrhea morbidity 50% 50% 25% 25% 25% 12% 5% 0% By episodes By days Moore, Lima et al. Gastroenterol. 139: 1156, 2010.
  53. 53. Proposed model for Ala-Gln coupling to EGFR signaltransduction during enteric infections and malnutrition. Moore S, Polk B et al. 2009.
  54. 54. AlaGln improves Wt, morphometry, apoptosis & proliferation in MN weaned C57Bl/6 mice Ueno… Moore. Am J Physiol GI Liver Physiol 301: 2011.
  55. 55. AlaGln improves barrier function (lactuloseexcretion) and weight gains in malnourished children in Brazil * p < 0.05 0.4 A * Peso-para-Estatura 0.3 Cumulativo WHZ Escore z Gain 0.2 0.1 0.0 Glicina Alanil-Glutamina Lima N et al. J Pediatr Gastroent Nutr 44: 365, 2007.
  56. 56. Gln or Ala-Gln enhance absorption (even with Crypto infections when Glucose fails) 3 3 Ringers Control Ringers Control 10 mM Gln 10 mM Gln 10 mM AlaGln 2 10 mM AlaGln 2 JnetNaJnetNa 1 1 0 0 n=9 n=9 n=6 n=18 n=18 n=6 Cryptosporidium Control Infected Alanyl-glutamine compares favorably to glutamine in Control and Crypto-infected calves in Ussing Chambers in driving net sodium absorption Blikslager et al, Am J Physiol 281: G645, 2001.
  57. 57. Another vicious cycle: Barrier disruption & Inflammation TJ (TNF, MLCK)(lo cap; nonselect)Claudin, Occludin, ZO1, actin, myosin AJ (Th2, IL13)(hi cap; ionselect) E-cadherin, Lunn P. Proc Nutr Soc abCatenin, myosin 59: 147, 2000. Turner JR Nature Rev, Immunol 9: 799, 2009. Salazar-Lindo E.... D Brewster… A Fasano....P Tarr. JPGN 39: S662, 2004. Sears C. Clin Micro Rev 22: 349, 2009.
  58. 58. Urine Metabonome in MN vs Control Childrenshowing increased NAG, DMA, DMG; less Mannitiol (by NMRS and PLS-DA coefficient plot) Projection to Latent Structure-Discriminant Analysis Cases vs Control s p=0.0248 n=75 CA2-3 Swann J, Lima AAM, et al. 2011.
  59. 59. Enteroaggregative E. coli (EAEC) Infection Impairs Growth & MN worsens infection DMEM / HS (Nourished Control) n=11 EAEC-JM221 (Nourished infected) n=12 EAEC-JM221 (Malnourished infected) n=7 EAEC 29% less p<0.03 p d8 Further 32% less with MN P<0.02 ** Peak shedding = 102-2.8 Nourished/ 103.1-7.5 Malnourished Roche, Cabel, Sevilleja, Nataro et al, JID 202: 506, 2010.
  60. 60. Induces Glutamine HSP70, which:•Attenuates iNOS, IL-6, IL-8 (in rat CP bypass)•Protects against neurodegeneration (in spinocerebellar degeneration, SCA-1 mice) Hayashi Y et al. Circ. 106: 2601, 2002. Cummings CJ et al. Hum Mol Genet 10: 1511, 2001.
  61. 61. Cryptosporidium and Giardia genotypes in Brazil and South Africa •C. hominis predominates in NE Brazil with: greater shedding, lactoferrin & growth shortfalls Bushen, Lima et al. TRSTMH, 2006. •C. hominis predominates in Limpopo, S. Africa; assoc. w diarrhea (57%); lactoferrin (59%) Samie et al. Expt Parasitol 114: 314, 2006. •74% of Giardia are Assemblage B, with greater shedding (3.6 v 1.4 x 105/ml); lactoferrin (especially with first infection; 74 40 20%; also longer) Kohli, Lima et al., TRSTMH 102: 718-725, 2008.
  62. 62. Conclusions (re Crypto and MN)•Crypto infections, +diarrhea, ProD or PD impair growth inyoung children (or mice);•Likely via disrupted intestinal mucosal function (L/M; V/C);•MN worsens Crypto (in stunted children or MN mice), thuscompleting the vicious cycle (that also impairs cognitivedevelopment, with an “Alzheimer-like” deficit).•ApoE4 protects against these deficits only in children withheavy diarrhea burdens.•ApoE4/4 targeted transgenic Eko mice grow better and haveless barrier disruption & infection.•Arginine, like ApoE4, protects mice and may protect childrenfrom the vicious diarrhea-MN cycle.
  63. 63. Disruption of Intestinal Barrier Function in Children with Acute and Persistent Diarrhea in Fortaleza, Brazil 0.8 ** 0.7 0.6 * 0.5 MeanLactulose: Mannitol 0.4Excretion Ratios 0.3 0.2 0.1 0 Control AD PD n=20 n=18 n=2 [vs controls: *p=0.001; **p<0.001] Guerrant, Lima, Barboza, et al Adv Exp Med Biol 473:103-11, 1999. Barboza et al BrasJ Med Biol Res 32:1499-1504, 1999.
  64. 64. EndoCAb correlates with disrupted intestinal barrier function and with stunted growth Campbell DI, Elia M, Lunn PG. J Nutr 133:1237, 2003. Petri, Ahmed, Haque et al 2010.
  65. 65. Enteroaggregative E. coli (EAEC) causesPersistent Diarrhea, Intestinal Inflammation, Growth Shortfalls and is the Leading Bacterial Enteropathogen in US •EAEC is associated with 36% of PD; 30-74% w HIV •EAEC-PD has elevated fecal LF, IL-8, IL-1 •EAEC-no D. also had elevated LF & Growth Shortfalls •EAEC filtrates induce IL-8 release in Caco-2 cells. •EAEC Fli-C (unique flagellin) cloned/sequenced is responsible for the IL-8 release. •EAEC signif assoc with 4.5% diarrhea in Baltimore and New Haven (vs 2-3% Cj, Salm; 10-12% Rota, Noro) Steiner T et al JID 177: 88, 1998; Wanke et al JID 178: 185, 1998; Samie A et al. 77: 142, 2007; Steiner T et al JCI 105: 1769, 2000. Nataro et al and Steiner et al CID: 2006.
  66. 66. HAZ correlates with Grades and Rank (among 242 college schoolgirls in S. India) r2=.037 r2=.030 p =.003 p =.007 (Independent of also significant # siblings, paternal education.) Pranay Sinha, B.S. Ramakrishna, et al. August, 2011.
  67. 67. Infant MN CVD Barker Hypothesis Barker D. et al. Lancet 1986, „89, ‟90. Hertfordshire & Southampton, UK
  68. 68. “Thrifty epigenotype”Candidate genes for environmental „canalization‟Stoger R. BioEssays 30: 156, 2008.
  69. 69. Infants with ProD are high risk for future episodes of PD (+) Prolonged Diarrhea prior to age 1 year *% of children with p=0.002 Log-rank test Persistent Diarrhea No Prolonged Diarrhea prior to age 1 year Age (months) Moore, Lima et al. Gastroenterol. 139: 1156, 2010.
  70. 70. Cryptosporidium: A Leading cause of Prolonged and Persistent Diarrhea & Growth Shortfalls % w Crypto P (vs no D)* •Persistent Diarrhea (n=71) 23.9%* 0.0002 •Prolonged Diarrhea (n=98) 12.2%* 0.018 •Acute Diarrhea (n=402) 11.4%* 0.0043 •No Diarrhea (n=289) 5.2% -Also Giardia with 20.5% of 88 with PD (p=0.0003); and fecal WBC or LF in 43-77% (p<0.02). Lima AAM et al. JID 181: 1643, 2000. Moore S et al. Gastroenterol 139: 1156, 2010.
  71. 71. Long-term Consequences of Cryptosporidium Infection•Malnutrition Sallon ‟88; Sarabia-Arce ‟90; Checkley ‟99; (Peru) Lima ‟92; (Brazil); Molback ‟93; ‟97; (G-Bissau)•Even Asxy Crypto Stunting Checkley ‟97; (Peru) C. hominis > parvum, c 2x shedding Bushen ‟07 (Brazil)) (3.5x106); LF; longer growth delay•Early Diarrhea or Crypto DI Guerrant et al „99 (Brazil)) Fitness + Cognitive impairment 4-7y later•Reactive Arthritis/Reiter‟s Hay ‟87; Ozgul ‟99; Shepherd ‟89; Cron ‟95; Hunter „04 Chalmers & Davies Expt Parasitol 124: 138, 2010.
  72. 72. ApoE4 Upregulates an Arginine-selective Cat-1 Transporter•Transgenic mice expressing the human APOE4allele have expression of mRNA for theconstituitive Arg selective y+ transporter (Cat1) inmicroglia.•Hence NO (not iNOS) and cell proliferation. Colton, et al. Neurobiol Aging 23: 777, 2002 Czapiga & Colton. J Neuroimmunol. 134: 44, 2003.
  73. 73. Arginine improves wt gain & reduces ilealCryptosporidium infection by over 10-fold by both iNOS & arginase-dependent pathways Oocyst shedding in Ileum 200000 * p < 0.05 175000 * p < 0.05 150000 Oocysts per mg of tissue * p < 0.05 125000 100000 75000 50000 25000 0 PBS Arg 200mM Arg 100mM Arg 200mM + Arg 200mM + L-NAME 20mM BEC 2.2mM L-NAME = NG-nitroarginine methyl ester (blocks iNOS); BEC = S-(2-boronoethyl)-L- cysteine (blocks Arginase) Castro I. et al. Nutrition, 2012.
  74. 74. MN-Infected ApoE4/4ki Mice have better growth and Villus/Crypt Architecture, and shed less Cryptosporidia Post-infection 110% of Weight 105 * # ApoE 4/4 MNI (n=16) WT MNI (n=13) 100 ApoE KO MNI (n=14) 95 0 1 2 3 4 5 6 7 Days * p<0.05 ApoE TR 4/4 MNI vs Wild type MNI # p<0.0001 ApoE TR 4/4 MNI vs ApoE KO MNI Shedding p<0.05 10 6 p<0.05 Wild type MNI (n=12) p<0.05 ApoE KO MNI (n=12) p<0.05 nº parasites/mg of stool 10 5 ApoE 3/3 TR MNI (n=8) p=0.05 ApoE 4/4 TR MNI (n=17) 10 4 10 3 10 2 1 3 5 7 Groups Apoe4 mice had no shedding on day 7 De Azevedo, Oria, et al. 2012.
  75. 75. Recurrent diarrhea impairs height and weight gains Ht gain (cm) Wt gain (kg)No recurrent diarrhea 1.74 cm 0.44 kgRecurrent diarrhea* 1.38 cm** 0.25 kg*** * >30% prevalence over subsequent 2m ** 21% less; p=0.1 *** 43% less; p=0.01 Schorling & Guerrant. Diarrhea and catch-up growth. Lancet 335: 599-600, 1990.

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