1

The irritable infantThe irritable infant

J. Marc Rhoads, M.D.J. Marc Rhoads, M.D.

I have the following financial relationships to disclose:

BioGaia, Inc.*$15,000 unrestricted gift for basic science research

* Products or services produced by this company are relevant to my presentation.

Agenda

1. What makes babies irritable?

2. What is colic? Etiology: Theories

3. Current treatment

4. Potential role of probiotics

Causes of cryingCauses of crying

Cow milk protein Cow milk protein allergyallergy

Esophageal refluxEsophageal reflux

DysphagiaDysphagia DysphagiaDysphagia

DyscheziaDyschezia

Normal infant Normal infant behaviorbehavior

2. What is colic?Wessel, “The Rule of Threes” Wessel, “The Rule of Threes”

Pediatrics 1954Pediatrics 1954

•• > 3 hours/day> 3 hours/day

•• >3 days/week>3 days/week>3 days/week>3 days/week

•• >3 weeks>3 weeks

•• Gone during 3rd month of ageGone during 3rd month of age

9/27/2011

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Colic

Affects 8-33% of all infants Matheson I. Tidsskr Nor Laegeforen 115: 2386-2389, 1995

Associated with parental exhaustion and postpartum depression

(OR 1.79, 95% CI 1.29, 2.50) H ll EA M t Child H lth J 2006 M 10(2) 149 57 Howell EA. Matern Child Health J. 2006 Mar;10(2):149-57

Six of 23 mothers had thoughts of infanticide during colic episodes

Levitzky S and Cooper R. Clin Pediatr (Phila) 39: 395-400, 2000

Infant homicides = homicide rates among teens• Rate has doubled since 1970, from 4.3 to 9.1 per 100,000

children under age one http://www.childtrendsdatabank.org/whatsNew.cfm

Crybabies: Crybabies: Solving the Colic Solving the Colic

ConundrumConundrum

Jerome Jerome GroopmanGroopman, M.D., M.D.

The New Yorker, Sept 17, 2007The New Yorker, Sept 17, 2007

ColicColic

“The sound of a crying baby…is just about “The sound of a crying baby…is just about

the most disturbing, demanding, shattering the most disturbing, demanding, shattering g g gg g g

noise we can hear.” noise we can hear.”

KitzingerKitzinger

Colic: Theories ProliferatedColic: Theories Proliferated

•• Immaturity of nerves, muscles in GI tractImmaturity of nerves, muscles in GI tract

•• Allergy to dietary proteinAllergy to dietary protein

P ti b l i tP ti b l i t•• Poor parenting by overly anxious parentsPoor parenting by overly anxious parents

•• Wastebasket term that's used when we can't Wastebasket term that's used when we can't explain what's going on inside a babyexplain what's going on inside a baby

•• Bryan Bryan VartabedianVartabedian, M.D. , M.D. Colic SolvedColic Solved

Colic and infant breath H2 test

Colic infants produce more gas

Less gas at 3 mos.

Moore DJ et al. J Pediatr 1988;113:979-84

Increased macromolecular permeability in babies with colic

Lothe L et al. Acta Paediatr Scand 1990; 79: 417-21

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3. Current Treatment

Behavioral interventions.

Controlled trials of counseling and a car-ride simulation device: these interventions are nosimulation device: these interventions are no better than reassurance alone

• Parkin PC, Schwartz CJ and Manuel BA. Pediatrics 92: 197-201, 1993.

World’s Happiest Baby: H. Karp, MD• 5 S’s

QuizQuiz

•• Which medical treatment(s) have been Which medical treatment(s) have been shown to be beneficial in a randomized, shown to be beneficial in a randomized, controlled trial for babies with colic?controlled trial for babies with colic?

Si hiSi hi•• SimethiconeSimethicone

•• Acid blockersAcid blockers

•• Casein/whey Casein/whey hydrolysateshydrolysates

•• LactobacilliLactobacilli

•• Herbal teasHerbal teas

QuizQuiz

•• Which treatment(s) have been shown to be beneficial Which treatment(s) have been shown to be beneficial in a randomized, controlled trial for babies with colic?in a randomized, controlled trial for babies with colic?

•• SimethiconeSimethicone

A id bl kA id bl k•• Acid blockersAcid blockers

•• Hypoallergenic formulaeHypoallergenic formulae

•• LucassenLucassen PL and PL and AssendelftAssendelft WJ. Systematic WJ. Systematic review … infant colic. review … infant colic. PediatricsPediatrics 108: 1047108: 1047--1048, 20011048, 2001

••Lactobacillus Lactobacillus reuterireuteri

••Herbal teasHerbal teas

Is Is L. reuteriL. reuteri an effective treatment an effective treatment for infant colic?for infant colic?

•• 90 breast90 breast--fed infants with colic fed infants with colic Age: 11Age: 11--80 days80 days

•• L. reuteri L. reuteri (10(1088 cfu/day) in sunflower oilcfu/day) in sunflower oil versus simethicone placeboversus simethicone placebo

•• L. reuteri L. reuteri decreased crying time decreased crying time 156 to 51 min./day156 to 51 min./day

Savino et al, Pediatrics 2007

Effectiveness of L reuteri versus placebo Study #2

Savino F et al. Pediatrics 2010;126:e526-e533

Evidence that microbiota may contribute to colic

Babies with colic have elevated fasting breath H2 values

Culture techniques identified increased Enterobacteriaceae and reduced Lactobacilli in infants with colic

Therapeutic administration of L. reuteri improved colic symptoms

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ALTERED FECAL MICROFLORA AND ALTERED FECAL MICROFLORA AND INTESTINAL INFLAMMATION IN BABIES INTESTINAL INFLAMMATION IN BABIES

WITH COLICWITH COLIC

Nicole Y Fatheree, BBA1, Michael J Ferris, PhD2, Johana Norori, MS2, Yuying Liu, PhD1, Joseph F Lucke, PhD1, Jon E Tyson, MD1 and Jon Marc Rhoads, MD1

1Pediatrics, University of Texas Health Sciences Ctr., Houston, Texas, United States and 2Pediatric Research Institute, Children's Hospital, New Orleans, LA, United States.

HypothesesHypothesesIn Babies with ColicIn Babies with Colic

•• Molecular analysis will show specific bacteria Molecular analysis will show specific bacteria

associated with colic and elevated breath Hassociated with colic and elevated breath H22

•• Fecal Fecal calprotectincalprotectin will show intestinal will show intestinal

inflammation inflammation

•• Barr Diary Crying AnalysisBarr Diary Crying Analysis

•• Hydrogen Breath TestHydrogen Breath Test

Colic StudyColic StudyMethods

y gy g

•• Denaturing Gradient Gel Denaturing Gradient Gel

Electrophoresis (DGGE) AnalysisElectrophoresis (DGGE) Analysis

•• Fecal Fecal CalprotectinCalprotectin

Barr Diary Analysis

Barr Diary Crying Results

400

500

600

me (m

in) 400

500

600

NormalColic

1 2

0100

200

30

0

Day

Cry

ing T

im

1 2

0100

200

30

0

Box Plot Analysis. Box Plot Analysis. Increased min. of crying in colicky infants over a 48-hour period.

p=0.001

Denaturing Gradient Gel Electrophoresis (DGGE) Analysis

•• DNA extracted from stool specimens of 36 DNA extracted from stool specimens of 36 patientspatients

•• 16S RNA gene segments PCR amplified16S RNA gene segments PCR amplified

•• AmplificatesAmplificates were separated into bands by were separated into bands by DGGE according to sequencesDGGE according to sequences

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Kodak 1D Image Analysis

Fecal bands on DGGEFecal bands on DGGE

Reduced microbial diversity in colic

p=0.01

C O L I C C O N T R O L

Species Identified % similarity

P1 P2 P3 P4 P5 P6 P7 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C17

Akkermansia muciniphila 100-99 X X X XBacteroides vulgatus 99-97 X X* X X*Bacteroides thetaiotaomicron 98 X XBifidobacterium adolescentis 99 XBifidobacterium bifidum 99 XBifidobacterium breve 100-99 X XBifidobacterium longum 96-99 X X X X X XBifidobacterium scardovii 100 XClostridium butyricum 97 XClostridium nexile 99 XClostridium ramosum 100 XClostidium clostridiiformis 96 XClostridium paraputrificum 100 XClostridium orbiscindens 96 XCl t idi f i X

Denaturing Gradient Gel Electrophoresis Analysis

Clostridium perfringens 100 XCoprobacillus catenaformis 99 X

Escherichia/Shigella/Enterobacter 100-99 X X X X X* X X* X X X X X X XEnterobacter sp. 100 XEnterobacter/Pantoea 99-98 X X X X* X X X X

Enterococcus faecalis 97 X X

Enterococcus sp. 98 X X* X X

Lactobacilli gasserii 100-98 X X

Lachnospiraceae sp. 95 X

Klebsiella sp. 99 X X*

Klebsiella pneumoniae 100-98 X X X X X X X

Proteus mirabilis 100 XStreptococcus salivarius 100-99 X X X X X XStreptococcus thermophilus 100 XTM7 phylum bacterium 99 X

Veillonella ratti 99 X X*

C O L I C C O N T R O L

Species Identified % similarity

P1 P2 P3 P4 P5 P6 P7 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C17

Akkermansia muciniphila 100-99 X X X XBacteroides vulgatus 99-97 X X* X X*Bacteroides thetaiotaomicron 98 X XBifidobacterium adolescentis 99 XBifidobacterium bifidum 99 XBifidobacterium breve 100-99 X XBifidobacterium longum 96-99 X X X X X XBifidobacterium scardovii 100 XClostridium butyricum 97 XClostridium nexile 99 XClostridium ramosum 100 XClostidium clostridiiformis 96 XClostridium paraputrificum 100 XClostridium orbiscindens 96 XCl t idi f i 100 X

Denaturing Gradient Gel Electrophoresis Analysis

Clostridium perfringens 100 XCoprobacillus catenaformis 99 X

Escherichia/Shigella/Enterobacter 100-99 X X X X X* X X* X X X X X X XEnterobacter sp. 100 X

Enterobacter/Pantoea 99-98 X X X X* X X X X

Enterococcus faecalis 97 X X

Enterococcus sp. 98 X X* X X

Lactobacilli gasserii 100-98 X X

Lachnospiraceae sp. 95 X

Klebsiella sp. 99 X X*

Klebsiella pneumoniae 100-98 X X X X X X X

Proteus mirabilis 100 XStreptococcus salivarius 100-99 X X X X X XStreptococcus thermophilus 100 XTM7 phylum bacterium 99 X

Veillonella ratti 99 X X*

Fecal Calprotectin

•• Fecal calprotectin, a neutrophil product, is a Fecal calprotectin, a neutrophil product, is a

marker for gastrointestinal inflammation marker for gastrointestinal inflammation

•• EnzymeEnzyme--Linked Immunosorbent Assay Linked Immunosorbent Assay

(ELISA) used in analysis of the stool samples (ELISA) used in analysis of the stool samples

Fecal Calprotectin

750

1000

1250

tin

(

g/g

)

Control Colic0

250

500

Cal

pro

tect

Suggests inflammation of the intestines in control vs. colic.Suggests inflammation of the intestines in control vs. colic.

p = 0.042

Formula Breath H2 test

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Colic StudyColic StudySummary

•• Barr Diary shows increased crying times Barr Diary shows increased crying times

of colic vs. control infantsof colic vs. control infants

•• DGGE analysis suggest lower diversity DGGE analysis suggest lower diversity

of phylotypes in colicky infantsof phylotypes in colicky infants

•• Klebsiella Klebsiella spp. as possible pathogenspp. as possible pathogen

Colic StudyColic StudySummary

•• Breath HBreath H22 test nondiscriminatory, with many in test nondiscriminatory, with many in

both groups with elevated levelsboth groups with elevated levels

•• Fecal calprotectin levels were comparable to Fecal calprotectin levels were comparable to

those of inflammatory bowel disease in babies those of inflammatory bowel disease in babies

with colicwith colic

Factors influencing Bacterial Factors influencing Bacterial DiversityDiversity

•• Developed Developed v. v. developingdeveloping countriescountries•• Country Country v. v. citycity•• Birth at home Birth at home v. v. inin--hospitalhospital•• Premature Premature v. v. termterm•• Antibiotics Antibiotics v. v. nonenone•• Diarrheal illnessDiarrheal illness

Reduced bacterial diversity

Crohn’s disease

Celiac disease

Obesity

Subsequent development of eczema

Klebsiella

Pneumonia in the elderly alcoholic

Neonatal sepsis

Infant diarrhea

Ecchymotic colitis

Klebsiella

Rashid T and Ebringer A. Clin Rheumatol 2007;26:858-864

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Molecular identification of coliform bacteria from colicky breastfed

infants (Turin, Italy)

Savino F et al. Acta Paediatrica 2009; 98:1582-8

Phase II: Infant Colic StudyPhase II: Infant Colic Study

L reuteriflora

Lactobacilli

Klebsiella

? Other

Happy baby

↑

↓

L. reuteri Colonic

L. reuteri

PlaceboLactobacilli

Klebsiella

? Other

Infant with colic:•Flatulence•Abdominal distension•Insatiable appetite•Borborygmi•Poor sleep

↑

→

Placebo

• Lactobacillus reuteri▫ Biogaia as drops in sunflower oil▫ Orally qd for 8 weeks▫ Dose▫ Dose 0.2 ml, 1 x 108 c.f.u.

Placebo: sunflower oil

Potential mechanisms of probiotics?Potential mechanisms of probiotics?

• Competition with endogenous microbiota

• Bacterial products:• Reuterin

• Immune system “modulation”

• Barrier function• Mucin

• Tight junctions

• Improved gastric motility

Indrio F, J. Pediatr 2008;152:801-6

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Blood studiesToll-like receptors 2 and 4

Regulatory T-cells (Tregs)

In vitro stimulation of cytokine production in isolated peripheral blood mononuclear cells PBMC stimulation in vitro: via phorbol myristate PBMC stimulation in vitro: via phorbol myristate

acetate + ionomycin treatment.

MSD Multi-Plex Cytokine Assay: 9 cytokines • (GM-CSF, IFN-, IL-1β, IL-10, IL-12p70, IL-2, IL-6, Il-8, TNF)

Our HypothesisOur Hypothesis

LR produces increased TLR (LR produces increased TLR (--2 or 2 or --9) 9)

signaling and reduces inflammationsignaling and reduces inflammation

Mild Mild immunostimulatoryimmunostimulatory response to response to LRLR

Increase in circulating Increase in circulating TregsTregs

Fill in diary for 2 months

Supplementation of Probiotic (Lactobacillus

reuteri)

Supplementation of placebo

4- Month Observation Period

N=30

N=10

N=40

Phase 1 safety trial: 2008Phase 1 safety trial: 2008--1111

Time: Visit 1Screening

Visit 2

Day 0

Visit 3

Day 14

Visit 4

Day 28

Visit 5

Day 42

Visit 6

Day 56

Visit 7

Day 86

Visit 8

Day 176

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Nicole Fatheree

Limin ZhuYuying Liu

Hossein Rahbar

Mike Ferris Johana Noiri

Marcela Zozaya-HinchliffeNisha

MangalatJohn Chapman

Ten-year f/u of colicky infants

Savino F et al. Acta Paediatr Suppl 2005;94:129-32

Summary

1. Causes of irritability

2. Colic defined Theories

3 Current treatment3. Current treatment

4. Probiotics

Patience is the art of concealing your impatience.Guy Kawasaki

GraciasGracias