New Developments in Irritable Bowel Syndrome

New Developments in Irritable Bowel Syndrome Mark Pimentel, MD, FRCP(C) Director, GI Motility Program Cedars-Sinai Medical Center

IBS Forks in the Road Not all decisions in how to handle IBS made things better. What seems like the easiest road to diagnose, characterize and treat IBS may have led us into problems which now have to be fixed.

Stress and IBS: Cause and Effect? Level 1 Evidence: Apply severe psychological stress to people and watch them develop IBS over time Level 2 Evidence: Compare psychological scores in IBS and compare to a disease comparable to IBS. Level 3 Evidence: Compare psychological scores between IBS and healthy people GOLD SILVER JUNK BONDS

Stress and IBS Military study: Shooting gun in combat Shooting another human Active combat Injured in combat Only Gastroenteritis was associated with IBS Porter CK, et al. Dig Dis Sci. 2013.

Stress Due to Chronic Disease Not IBS? Shah E, et al. Ann Gastroenterol. 2014.

IBS Timeline 1950 s 1960 s 1970 s 1980 s 1990 s 2000 s -Spastic Colitis -Spastic Colon -Irritable Bowel -Irritable bowel syndrome -Functional Bowel Disease IBS type -IBS IBS-D, IBS-C, IBS-A -IBS IBS-D, IBS-C, IBS-M, IBS-U

IBS Microbial Hypothesis Acute Gastroenteritis CdtB Toxin Autoimmunity to vinculin Gut ICC/neuronal Changes Bacterial Overgrowth IBS

D-IBS M-IBS C-IBS Non-Constipation-IBS

Breath Testing is Abnormal in IBS Forest plot of all age-sex matched studies Type of % % Author Breath Test OR (95% CI) CI) Weight Weight Grover sucrose 2.29 (0.89, 5.87) 5.87) 18.65 18.65 Lupascu Pimentel glucose lactulose 10.89 (3.52, 33.71) 33.71) 16.82 16.82 20.67 (5.29, 80.69) 80.69) 14.68 14.68 Parodi glucose 4.30 (1.24, 14.98) 14.98) 15.71 15.71 Scarpellini Collin lactulose lactulose 24.27 (7.35, 80.15) 80.15) 16.20 16.20 18.04 (6.55, 49.71) 49.71) 17.94 17.94 Overall (I-squared = 67.9%, p = 0.008) 9.64 (4.26, 21.82) 21.82) 100.00 100.00 NOTE: Weights are from random effects analysis.1.2.5 1 2 5 10 20 Shah. et al. Dig Dis Sci. 2010.

Small Bowel Culture in IBS P<0.05 P<0.001 43% 24% 4% 12% Posserud. et al. Gut. 2007;56:802-8. N=165 IBS, 26 controls

SIBO and IBS 60% 27.3% P=0.004 Pyleris. et al. DDS. 2012. N=77 non-d-ibs, N=35 D-IBS

Bacteria quantity Log10 Single Organism PCR in IBS DUODENAL ASPIRATES P<0.05 P<0.05 N=77 non-d-ibs, N=35 D-IBS Pyleris. et al. Scand J Gastroenterol. 2015. N=77 non-d-ibs, N=35 D-IBS

Agilent Plots of Normal and IBS IBS Healthy Control Pyleris. et al. Scand J Gastroenterol. 2015.

Sequencing Results- Normal Small Bowel Normal Subjects Pyleris. et al. Scand J Gastroenterol. 2015.

Sequencing Results- IBS Small Bowel IBS Subjects Pyleris. et al. Scand J Gastroenterol. 2015.

Primary Outcome (4 Weeks After Tx) Primary Primary Primary Key Secondary Primary Key Secondary Key Secondary Other Key Secondary Other Secondary Other Secondary Other Secondary FDA Proposed FDA Proposed FDA Proposed FDA Proposed Pimentel. et al. NEJM. 2011. Efficacy Odds Outcome Study Ratio (95% CI) p-value TARGET 1 1.53 (1.10,2.12) 0.0125 SGA-IBS Efficacy TARGET 2 Weekly Odds 1.45 (1.05,2.01) 0.0263 Outcome Efficacy Combined Study Odds Ratio 1.49 (1.18,1.88) (95% CI) p-value 0.0008 Efficacy Outcome TARGET Study 1 Odds Ratio 1.53 (1.10,2.12) (95% CI) p-value 0.0125 Outcome SGA-IBS Study Ratio (95% CI) p-value TARGET 2 1 1.62 1.45 1.53 (1.16,2.27) (1.05,2.01) (1.10,2.12) 0.0045 0.0263 0.0125 SGA-IBS IBS Weekly SGA-IBS Bloating Combined TARGET 1 2 1.53 1.49 1.45 (1.10,2.12) (1.18,1.88) (1.05,2.01) 0.0125 0.0008 0.0263 Weekly TARGET 1.45 (1.05,2.01) 0.0263 Weekly Combined 1.49 1.49 (1.08,2.06) (1.18,1.88) 0.0167 0.0008 Combined 1.49 1.56 (1.18,1.88) (1.23,1.96) 0.0008 0.0002 TARGET 1 1.62 (1.16,2.27) 0.0045 IBS Bloating TARGET 2 1 1.49 1.62 (1.08,2.06) (1.16,2.27) 0.0167 0.0045 Weekly IBS Bloating 1.62 (1.16,2.27) 0.0045 IBS Bloating Combined TARGET 1 2 1.76 1.56 1.49 (1.26,2.47) (1.23,1.96) (1.08,2.06) 0.0009 0.0002 0.0167 Weekly 1.49 (1.08,2.06) 0.0167 Weekly SGA-IBS Daily TARGET Combined 2 1.59 1.56 (1.13,2.24) (1.23,1.96) 0.0072 0.0002 Combined 1.56 (1.23,1.96) 0.0002 TARGET 1 1.61 1.76 (1.28,2.04) (1.26,2.47) <0.0001 0.0009 SGA-IBS Daily TARGET 2 1 1.41 1.59 1.76 (1.01,1.97) (1.13,2.24) (1.26,2.47) 0.0486 0.0072 0.0009 SGA-IBS Bloating Daily 1 1.76 (1.26,2.47) 0.0009 Combined TARGET 2 1.76 1.61 1.59 (1.26,2.44) (1.28,2.04) (1.13,2.24) <0.0001 0.0008 0.0072 SGA-IBS Daily Daily TARGET 2 1.59 (1.13,2.24) 0.0072 Combined TARGET Combined 1 1.61 1.52 1.41 1.61 (1.28,2.04) (1.21,1.92) (1.01,1.97) (1.28,2.04) <0.0001 <0.0001 0.0004 0.0486 IBS Bloating TARGET 2 1 1.41 1.45 1.76 1.41 (1.01,1.97) (1.05,2.02) (1.26,2.44) (1.01,1.97) 0.0486 0.0255 0.0008 0.0486 Daily IBS Bloating IBS Bloating Ab Pain Combined TARGET 2 1.52 1.76 (1.21,1.92) (1.26,2.44) 0.0004 0.0008 Daily TARGET 1.76 1.46 (1.26,2.44) (1.05,2.03) 0.0008 0.0232 Daily TARGET Combined 1 1.45 1.52 (1.05,2.02) (1.21,1.92) 0.0255 0.0004 IBS Ab Pain Combined TARGET 2 1 1.52 1.42 1.46 1.45 (1.21,1.92) (1.13,1.78) (1.05,2.03) (1.05,2.02) 0.0004 0.0028 0.0232 0.0255 Daily IBS Ab Pain 1 1.45 (1.05,2.02) 0.0255 IBS Ab Pain Combined TARGET 2 1.42 1.46 (1.13,1.78) (1.05,2.03) 0.0028 0.0232 Daily TARGET 2 1.46 (1.05,2.03) 0.0232 Daily TARGET Combined 1 1.40 1.42 (1.02,1.92) (1.13,1.78) 0.0401 0.0028 Ab Pain & Stool Combined 1.42 (1.13,1.78) 0.0028 TARGET 2 1 1.55 1.40 (1.12,2.13) (1.02,1.92) 0.0077 0.0401 Daily Ab Pain (FDA) & Stool Combined TARGET 2 1 1.47 1.55 1.40 (1.17,1.84) (1.12,2.13) (1.02,1.92) 0.0009 0.0077 0.0401 Daily Ab Pain (FDA) & Stool 1.40 (1.02,1.92) 0.0401 Ab Pain & Stool Combined TARGET 1 2 1.48 1.47 1.55 (1.08,2.03) (1.17,1.84) (1.12,2.13) 0.0157 0.0009 0.0077 Ab Daily Pain (FDA) Daily 1.55 (1.12,2.13) 0.0077 Daily (FDA) TARGET Combined 2 1 1.46 1.48 1.47 (1.06,2.00) (1.08,2.03) (1.17,1.84) 0.0194 0.0157 0.0009 (FDA) Ab Pain Daily Combined 1.47 (1.17,1.84) 0.0009 Combined TARGET 2 1 1.46 1.48 (1.17,1.83) (1.06,2.00) (1.08,2.03) 0.0009 0.0194 0.0157 (FDA) Ab Pain Daily 1 1.48 (1.08,2.03) 0.0157 Ab Pain Daily Combined TARGET 2 1.46 (1.17,1.83) (1.06,2.00) 0.0009 0.0194 (FDA) TARGET 2 1.80 (1.06,2.00) (1.25,2.59) 0.0194 0.0015 (FDA) Stool Consist. TARGET Combined 1 1.80 1.46 (1.25,2.59) (1.17,1.83) 0.0015 0.0009 Stool Consist. Combined 1.46 1.57 (1.17,1.83) (1.12,2.21) 0.0009 0.0096 Daily (FDA) TARGET 2 1 1.57 1.80 (1.12,2.21) (1.25,2.59) 0.0096 0.0015 Daily Stool (FDA) Consist. Combined 1 1.80 1.67 (1.25,2.59) (1.31,2.14) <0.0001 0.0015 Stool Consist. Combined TARGET 2 1.67 1.57 (1.31,2.14) (1.12,2.21) <0.0001 0.0096 Daily (FDA) TARGET 2 1.57 (1.12,2.21) 0.0096 Daily (FDA) Combined 1.67 (1.31,2.14) <0.0001 Combined 1.67 (1.31,2.14) <0.0001 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 0.0 0.5 Odds 1.0 1.0 Ratio and 95% 1.5 1.5 CI 2.0 2.5 2.0 2.5 Favors PlaceboOdds Ratio and 95% Favors CI Rifaximin Favors PlaceboOdds Ratio and 95% Favors CI Rifaximin Favors Placebo Favors Rifaximin

Durability of Response (3 Months) Primary Efficacy Outcome SGA-IBS Weekly Study TARGET 1 TARGET 2 Combined Odds (95% CI) Ratio p-value 1.35 (1.00, 1.82) 0.0477 1.52 (1.13, 2.03) 0.0053 1.44 (1.17, 1.77) 0.0007 Key Secondary IBS Bloating Weekly TARGET 1 TARGET 2 Combined 1.28 1.56 1.42 (0.95, 1.73) (1.16, 2.09) (1.15, 1.75) 0.1042 0.0031 0.0011 Other Secondary SGA-IBS Daily IBS Bloating Daily IBS Ab Pain Daily TARGET 1 TARGET 2 Combined TARGET 1 TARGET 2 Combined TARGET 1 TARGET 2 Combined 1.60 1.47 1.48 1.50 1.67 1.53 1.35 1.35 1.31 (1.18, 2.18) (1.09, 1.99) (1.20, 1.83) (1.10, 2.04) (1.24, 2.25) (1.24, 1.89) (1.00, 1.83) (1.01, 1.81) (1.06, 1.61) 0.0025 0.0127 0.0003 0.0103 0.0008 <0.0001 0.0495 0.0435 0.0118 FDA Proposed TARGET 1 Ab Pain & Stool TARGET 2 Daily (FDA) Combined TARGET 1 Ab Pain Daily TARGET 2 (FDA) Combined TARGET 1 Stool Consist. TARGET 2 Daily (FDA) Combined 1.36 1.44 1.40 1.31 1.37 1.33 1.70 1.48 1.58 (1.01, 1.83) (1.08, 1.92) (1.14, 1.72) (0.98, 1.75) (1.03, 1.83) (1.09, 1.64) (1.24, 2.33) (1.09, 2.00) (1.27, 1.97) 0.0396 0.0141 0.0014 0.0725 0.0298 0.0058 0.0009 0.0114 <0.0001 0.0 0.5 1.0 1.5 2.0 2.5 Odds Ratio and 95% CI Favors Placebo Favors Rifaximin Pimentel. et al. NEJM. 2011.

Patients With Adequate Relief, % Alosetron 60 50 * Treatment * * * * * * * * Follow-up 40 30 20 Alosetron (n=279 ) Placebo (n=290 ) 10 0 *P<.05; Diarrhea-predominant intention to treat (ITT) population. Chey WD, et al. Am J Gastroenterol. 2004;99:2195-2203. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Months

Randomize 1:1 TARGET 3 TRIAL Open-label Treatment Phase Double-blind Treatment Phases Screening 7-13 d PBO Study Day 1 Open-Label Rifaximin 550 TID x 2 weeks 2w RFX 4w f/u Maintenance Phase Variable up to 18w Non-Responders Withdrawn and proceed to EOS Phase First Repeat Treatment 2w RFX Primary Evaluation Period 2w PBO 4w f/u 4w f/u Maintenance Phase 6w 6w Second Repeat Treatment 2w RFX 2w PBO 4w f/u 4w f/u Follow up * * * * * Stool sample collection * Only patients with recurrent symptoms randomized 4 W EOS

Weekly Average Comparability of Symptom Severity TARGET 3 6 Open Label TARGET 3: All DBR Subjects (n=636) Worst abdominal pain over the last 24hr Double-blind 5 4 3 2 BL Wk 1 Wk 2 Wk 3 Wk 4 Wk 5 Wk 6 BL Wk 1 Time Subjects Do Not Return to Baseline Symptom Severity Following Initial Therapy with Rifaximin Wk 2 Wk 3 Wk 4 Wk 5 Wk 6 Wk 7 Wk 8 Wk 9 Wk 10 Wk 11 Wk 12 Wk 13 Wk 14 Wk 15 Wk 16 Wk 17 Wk 18

D-IBS M-IBS C-IBS Constipation-IBS

Methane- Important in C-IBS Meta-analysis of studies % % Author Year OR (95% (95% CI) CI) Weight Weight Peled 1987 0.83 (0.20, (0.20, 3.56) 3.56) 8.43 8.43 Fiedorek 1990 4.32 (1.60, (1.60, 11.68) 11.68) 12.03 12.03 Pimentel 2003 5.58 (2.22, (2.22, 14.03) 14.03) 12.68 12.68 Pimentel 2003 44.23 (2.48, (2.48, 788.51) 788.51) 3.18 3.18 Majewski 2007 1.81 (0.70, (0.70, 4.67) 4.67) 12.46 12.46 Bratten 2008 2.22 (1.14, (1.14, 4.33) 4.33) 15.14 15.14 Parodi 2009 1.89 (0.79, (0.79, 4.51) 4.51) 13.17 13.17 Hwang 2009 47.67 (8.73, (8.73, 260.41) 260.41) 6.99 6.99 Attaluri 2009 3.70 (2.06, (2.06, 6.66) 6.66) 15.92 15.92 Overall (I-squared = 64.6%, p = 0.004) 3.51 (2.00, (2.00, 6.16) 6.16) 100.00 100.00 NOTE: Weights are from random effects analysis Kunkel. et al. Dig Dis Sci. 2011..1.2.5 1 2 5 10 20

Methane Slows Intestinal Transit n=5, p<0.0001 69% mean slowing of transit with CH 4 Pimentel. et al. Am J Physiol. 2006.

Methanobrevibacter Smithii- the Cause of Constipation Kim. et al. Dig Dis Sci. 2013. Hydrogen Producing IBS Stool Methane Producing IBS Stool

Stool M. smithii level by qpcr M. Smithii Is the Source of Methane in C-IBS 10 6 Breath Methane Positive (>3ppm) Constipated 10 4 Breath Methane Negative (<3ppm) 10 2 0 Normal

Methanobrevibacter Smithii or Methane as a Test Dictates Treatment CH4 Eradication P=0.001 Clinical Response P=0.01 Low. et al. Gastroenterol and Hepatol. 2010.

Methane Positive C-IBS Double Blind Placebo Controlled Trial Constipation VAS score P=0.01 Pimentel. et al. Dig Dis Sci. 2014.

VAS score After Treatment Bloating Level by Group P<0.01 Pimentel. et al. Dig Dis Sci. 2014.

VAS score Final Visit Constipation Severity Based on Methane >3ppm Analysis of the Neomycin + Rifaximin Group P=0.04 Pimentel. et al. ACG. 2013.

SIBO Treatment Strategy IBS SYMPTOMS LACTULOSE BREATH TEST Hydrogen Positive Methane Positive Rifaximin Rifaximin+Neomycin Symptom Re-evaluation/Retest No Response Pimentel. Am J Gastroenterol. 2010. Prokinetic Investigate

IBS Microbial Hypothesis Acute Gastroenteritis CdtB Toxin Autoimmunity to vinculin Gut ICC/neuronal Changes Bacterial Overgrowth IBS

Risk of PI-IBS Increases 7-Fold After Infectious Gastroenteritis* Study (year/bacteria) Ji (2005/Shigella) Mearin (2005/Salmonella) Wang (2004/Unspecified) Okhuysen (2004/Unspecified) Cumberland (2003/Unspecified) llnyckyj (2003/Unspecified) Parry (2003/Bacterial NOS) Rodriguez (1999/Bacterial NOS) Pooled estimate Protective Effect Increased Risk 0.1 0.5 1 10 50 OR (95% Cl) 2.8 (1.0-7.5) 8.7 (3.3-22.6) 10.7 (2.5-45.6) 10.1 (0.6-181.4) 6.6 (2.0-22.3) 2.7 (0.2-30.2) 9.9 (3.2-30.0) 11.3 (6.3-20.1) 7.3 (4.8-11.1) 9.8% IBS in cases vs 1.2% IBS in controls OR *Systematic review of 8 studies involving 588,061 subjects; follow-up ranged from 3 to 12 months. Halvorsen HA, et al. Am J Gastroenterol. 2006;101:1894-1899.

OR for IBS After Acute Gastroenteritis* Characteristics of Acute Illness Identify Patients at Risk for PI-IBS 10 8 P=.000 6 P=.013 4 2 P=.029 P=.006 P=.001 P=.000 0-2 Age Female Diarrhea >7 d Bloody Stools Abdominal Cramps *Identified from multiple logistic regression analysis from 2069 participating in the Walkerton Health Study. Marshall JK, et al. Gastroenterology. 2006;131:445-450. Weight Loss >10 lbs

Post-Infectious IBS = IBS Shah, et al. J Neurogastroenterol Motil, 2012.

Mechanism: Rat Model (SIBO = IBS) n=33 n=33 C. jejuni Stool = Campy- No Acute Gastroenteritis Stool = Campy+ Acute Gastroenteritis 3 Months After Recovery Stool Consistency Evaluation Stool = Campy- Recovery Pimentel. et al. Dig Dis Sci, 2008. Bacterial Quantitation by RT-PCR of Duodenum, Jejunum, Ileum

Mechanism: Rat Model (SIBO = IBS) 17% 21% 27% 6.7% Pimentel. et al. Dig Dis Sci, 2008. 3 months after Campylobacter jejuni 81-176 infection

Rectal Lymphocytes % in Rats Mechanism: Rat Model (SIBO = IBS) P<0.001 Persistent altered stool consistency P<0.01 Increased rectal lymphocytes P=0.84 P<0.01 Pimentel. et al. Dig Dis Sci, 2008.

8 C+/SIBO+ 8 C+/SIBO- 8 C- CD117 immunostain of duodenal, jejunal, ileal cross-sections (Dako Cytomation, Inc; Carpinteria, CA) Randomized and coded 2 independent, blinded readers reported as DMP ICC per villus

ICC Level and SIBO * P<0.05 vs. C- # P<0.001 vs. C- * # * Jee. et al. World J Gastroenterol. 2010.

Ileal ICC in Controls Jee. et al. World J Gastroenterol. 2010. 20x

Ileal ICC in C+/SIBO- Jee. et al. World J Gastroenterol. 2010. 20x

Ileal ICC in C+/SIBO+ Jee. et al. World J Gastroenterol. 2010. 20x

Cytolethal Distending Toxin (CDT) C. jejuni E. Coli Salmonella Shigella Cytolethal Distending Toxin (CDT) A B All cause IBS Cell Entry G2 Phase Unknown Arrest

Cytolethal Distending Toxin (CDT) Wild type Campy CdtB (-) Campy Campy + Rifaximin Daily stool for Campy (length of colonization) 3 months later stool evaluation Bacterial quantitative PCR of small bowel

CDT and Rifaximin in IBS: Rat Model Campy CdtB- P-value Rifaximin P-value Stool % wet weight 60.1±6.8 60.8±3.6 0.47 61.1±3.8 0.33 Consistency 1.51±0.37 1.23±0.27 <0.00001 1.15±0.30 <0.0000001 Standard Deviation Proportion with normal bowel form all 3 days Note: No significant differences were seen between CDT- and Rifaximin treated arms of the study. Neurogastroenterol Motil 2013. 8.4±6.4 4.2±2.4 <0.0001 4.1±2.3 <0.0001 17.8% 50.0% <0.01 OR=4.63 59.3% <0.00001 OR=6.7

Molecular Mimicry Ganglia DMP ICC Pre-immune Serum Anti-CDT Antibodies

IBS Mechanism/Nerve Damage GANGLIA OF HUMAN ILEAL SECTIONS wab S-100 S-100 + wab Anti-CdtB is also Anti-glial in humans Morales. et al. Dig Dis Sci. 2015.

Vinculin- The Link to IBS

Vinculin Focal adhesion plaques Actin filaments

Molecular Mimicry/Autoimmunity Cytolethal Distending Toxin B Human Vinculin

Molecular Mimicry/Autoimmunity Cytolethal Distending Toxin B Human Vinculin

Vinculin Expression and SIBO Rats with single or double infection, SIBO or no SIBO

Immunization Trial Recombinant CdtB Campylobacter Sprague-Dawley Rats

Vinculin Expression and Small Bowel Bacterial Levels % of rats with Elevated Small Bowel Bacteria Triantafyllou. DDW, 2014.

Blood Test For IBS D-IBS subjects (N=2375) Subjects with IBD (N=142) which included Crohn s disease (N=73) and ulcerative colitis (N=69) Subjects with celiac disease (N=121) Healthy subjects (N=43)

Blood Test For IBS 1 AUC=0.81 (95% CI, 0.77-0.84) marker Anti-CdtB Anti-vinculin AUC=0.62 (95% CI, 0.58-0.67) Anti-vinculin Ab Anti-CdtB Ab 0 0 1 1-specificity

IBS Pathophysiologic Sequence Food Poisoning Bacterial Toxin? Autoimmunity Gut Nerve Damage Bacterial Overgrowth IBS E. Coli C. jejuni Shigella Salmonella Cytolethal Distending Toxin (CDT B) Anti-vinculin -Reduced ICC -Reduced MMC -Breath testing -Culture -qpcr -deep sequencing Antibiotics

IBS/SIBO WORKSHEET 3.0 STRESS Molecular Mimicry PROKINETIC CRF Acute Gastroenteritis Immune response Anti-CDT Ab Other Ab Anti-vinculin antibody Reduced DMP-ICC SMALL BOWEL DYSMOTILITY COLON Immunity to Infection METHANE SLOW TRANSIT C-IBS SIBO/ IBS ANTIBIOTIC HYDROGEN D- AND M-IBS

Conclusions Small intestinal bacterial changes are seen in at least 60% of IBS. M. smithii (methane production) slows transit and is associated with constipation Breath testing strategizes the treatment for IBS Acute gastroenteritis causes IBS C. jejuni gastroenteritis causes SIBO in rats through CdtB neurotoxicity CdtB can cause autoimmunity to vinculin via molecular mimicry Serum anti-cdtb and anti-vinculin can distinguish IBS from IBD IBS is an organic disease

Acknowledgements Motility Research Team/Endocrine Team Gene Kim Stacy Weitsman Walter Morales Shari Chua Gillian Barlow, PhD Chris Chang, MD, PhD Sequencing Team Vincent Funari, PhD Jie Tang, PhD Jordan Brown Ruchi Mathur, MD, FRCP(C) Mark Goodarzi, MD, PhD Michelle Jones, PhD Zachary Marsh Emily Marsh Venkata Pokkunuri Ali Rezaie, MD, MPH Pat Guerry, PhD Mark Riddle, MD Michael Prouty, MD Brooks Cash, MD Evangelos Giamarellos-Bourboulis, MD Emmanouill Pyleris, MD Katerina Pistikis, MD Kostas Triantafyllou, MD