Early probiotic supplementation and the risk of celiac disease in children at genetic risk

nutrients Article Early Probiotic Supplementation and the Risk of Celiac Disease in Children at Genetic Risk Ulla Uusitalo 1,*,†, Carin Andren Aronsson 2,†, Xiang Liu 1, Kalle Kurppa 3,4, Jimin Yang 1, Edwin Liu 5, Jennifer Skidmore 6, Christiane Winkler 7,8,9, Marian J. Rewers 10, William A. Hagopian 6, Jin-Xiong She 11, Jorma Toppari 12,13 , Anette-G. Ziegler 7,8,9, Beena Akolkar 14, Jill M. Norris 15, Suvi M. Virtanen 3,16,17, Jeffrey P. Krischer 1, Daniel Agardh 2and on behalf of the TEDDY Study Group ‡ 1Health Informatics Institute, Department of Pediatrics, Morsani College of Medicine, University of South Florida, Tampa, FL 33612, USA 2The Diabetes and Celiac Disease Unit, Department of Clinical Sciences, Lund University, 214 28 Malmö, Sweden 3Tampere Center for Child Health Research, University of Tampere, 33014 Tampere, Finland 4The University Consortium of Seinäjoki, 60320 Seinäjoki, Finland 5Digestive Health Institute, Children’s Hospital Colorado, University of Colorado Denver, Aurora, CO 80045, USA 6Pacific Northwest Research Institute, Seattle, WA 98122, USA 7Institute of Diabetes Research, Helmholtz Zentrum München, 85764 Munich-Neuherberg, Germany 8Forschergruppe Diabetes, Klinikum rechts der Isar, Technische Universität München, 80804 Munich, Germany 9Forschergruppe Diabetes e.V., Helmholtz Zentrum München, 85764 Munich-Neuherberg, Germany 10 Barbara Davis Center for Childhood Diabetes, University of Colorado School of Medicine, Aurora, CO 80045, USA 11 Medical College of Georgia, Augusta University, Augusta, GA 30912, USA 12 Research Centre for Integrative Physiology and Pharmacology, Institute of Biomedicine, University of Turku, 20014 Turku, Finland 13 Department of Pediatrics, Turku University Hospital, 20521 Turku, Finland 14 NIDDK, National Institute of Health, Bethesda, MD 20892, USA 15 Department of Epidemiology, Colorado School of Public Health, University of Colorado, Aurora, CO 80045, USA 16 Unit of Nutrition, National Institute for Health and Welfare, 00271 Helsinki, Finland 17 Tampere University Hospital, and the Science Center of Pirkanmaa Hospital District, 33520 Tampere, Finland *Correspondence: [email protected]; Tel.: +1-813-396-9516 †The two authors contributed equally. ‡Membership of the TEDDY Study Group is provided in the Acknowledgments. Received: 22 June 2019; Accepted: 29 July 2019; Published: 2 August 2019   Abstract: Probiotics are linked to positive regulatory effects on the immune system. The aim of the study was to examine the association between the exposure of probiotics via dietary supplements or via infant formula by the age of 1 year and the development of celiac disease autoimmunity (CDA) and celiac disease among a cohort of 6520 genetically susceptible children. Use of probiotics during the first year of life was reported by 1460 children. Time-to-event analysis was used to examine the associations. Overall exposure of probiotics during the first year of life was not associated with either CDA (n=1212) (HR 1.15; 95%CI 0.99, 1.35; p=0.07) or celiac disease (n=455) (HR 1.11; 95%CI 0.86, 1.43; p=0.43) when adjusting for known risk factors. Intake of probiotic dietary supplements, however, was associated with a slightly increased risk of CDA (HR 1.18; 95%CI 1.00, 1.40; p=0.043) compared to children who did not get probiotics. It was concluded that the overall exposure of probiotics during the first year of life was not associated with CDA or celiac disease in children at genetic risk. Nutrients 2019,11, 1790; doi:10.3390/nu11081790 www.mdpi.com/journal/nutrients Nutrients 2019,11, 1790 2 of 14 Keywords: probiotics; dietary supplements; infant formula; celiac disease autoimmunity; celiac disease 1. Introduction Probiotics have been defined as live organisms which confer a health benefit to the host when administered in adequate amounts [ 1 ]. There is a long history of safe use of selected microbes in foods, but the vulnerability of certain immunocompromised populations should be taken into consideration before probiotic supplementation [ 2 ]. Administration of probiotics to healthy infants is considered to be safe [ 3 , 4 ] and is assumed to have a positive effect on the regulation of the immune system. Probiotic supplementation has long been used for its preventive effects, e.g., on atopic eczema [ 5 , 6 ] and to improve and maintain gastrointestinal health [ 7 ]. Whether probiotics are beneficial against colic pain and decrease the related crying time in young infants remains a controversial topic [ 8 – 11 ]. Nevertheless, supplementation of infant diets with probiotics has become more common worldwide during the recent years. For instance, a considerable increase in probiotic supplement use has been observed in Sweden since 2004 [ 12 , 13 ], while infants in Finland have been commonly given probiotics since the late 1990s [ 14 ]. Despite the increase in probiotic use, their mechanisms of action have not entirely been established. Furthermore, it has been suggested that dead bacteria and their components can also exhibit probiotic properties, in addition to probiotics and probiotic metabolites [15]. Celiac disease is a chronic autoimmune disease of the small bowel, characterized by villous atrophy and inflammation of the intestinal mucosa [ 16 ]. Early infant feeding, with emphasis on gluten intake, influences risk [ 17 ]. While gluten is the necessary antigen for celiac disease to develop, there is some evidence that various environmental exposures such as repeated gastrointestinal infections may also trigger the disease [ 18 – 20 ], either on their own or in interaction with gluten exposure [ 21 ]. Certain changes in gut microbiota composition, as well as the development and maturity level of the gut microbiota, have been linked to celiac disease [ 21 , 22 ]. Some previous studies have also shown that probiotics are beneficial for children who suffer from this autoimmune disorder [23,24]. We previously demonstrated that early exposure of probiotics may decrease the risk of type 1 diabetes (T1D) related autoimmunity among at-risk children in the ongoing prospective, The Environmental Determinants of the Diabetes in the Young (TEDDY) birth cohort study [ 12 ] which is a multi-national longitudinal observational study with the goal of identifying environmental factors associated with T1D and celiac disease. To date there is no prospective study showing that probiotics may prevent celiac disease. The aim of this study was to examine whether the timing of initial probiotic exposure during the first year of life is associated with the risk of celiac disease autoimmunity (CDA) and celiac disease in the TEDDY study. 2. Materials and Methods 2.1. Study Population TheTEDDYstudyinvolves6clinicalresearchcenterslocatedinColorado,Georgia,andWashington in the U.S. and in Finland, Germany, and Sweden in Europe. All sites follow the same study protocol including scheduled visits every 3rd month until the age of 4 years and every 6th month thereafter until 15 years of age [ 25 ]. Between September 2004 and February 2010, the TEDDY study screened 424,788 newborns infants of whom 21,589 infants fulfilled the inclusion criteria based on the Human Leukocyte Antigen (HLA) genotyping (Supplemental Table S1). Among those eligible children, 8676 children were enrolled in the prospective cohort study. For this study we only included children with selected HLA genotypes: DR3/3, DR3/4, DR4/4, and DR4/8 who had been screened for tissue transglutaminase autoantibodies (tTGA) (n=6520) (Figure S1). As of July 2017, 6520 children had been followed to a median (interquartile range (IQR)) age of 8.7 (7.4–10.2) years and included for this study. Nutrients 2019,11, 1790 3 of 14 Forall studyparticipants, separatewritten informedconsentforgenetic screening andparticipation in the follow-up study were obtained from a parent or primary caregiver before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by local Ethics Committees and Institutional Review Boards and monitored by an External Evaluation Committee formed by the National Institutes of Health. 2.2. Screening for Celiac Disease Related Autoimmunity (CDA) and Celiac Disease Annual screening for celiac disease starts with tTGA at 2 years of age, as previously described [ 26 ]. ChildrenwhoaretTGA positive arere-testedafter3–6monthsanddefined as having CDA if persistently tTGA positive in two consecutive samples. In addition, children who tested tTGA positive had their serum samples retrospectively analyzed. Samples from as early as 3 months of age were available in order to determine the closest time-point of seroconversion. Caregivers to children with CDA were referred to their health care provider for further evaluation of celiac disease. For the purpose of this study, celiac disease was defined as biopsy proven (i.e., an intestinal biopsy showing a Marsh score ≥2 ) or having a persistently tTGA level of ≥100 units if a biopsy was not performed [27]. 2.3. Information on Characteristics, Diet and Health of the Study Population Information about basic demographic characteristics of the mother and her newborn baby was received from the infant screening form. A questionnaire was mailed to the home of the mother prior to the first clinic visit (3 to 4.5 months postpartum). This questionnaire contained questions regarding illnesses during pregnancy, mother’s use of medications and dietary supplements, smoking status, and maternal body mass index (BMI) before pregnancy. After enrollment, families received a questionnaire on the mode of delivery and the child’s early diet, including use of probiotics between 0 to 3 months of age. Information about the mother’s education and the birth order of the child was received from the primary caretaker questionnaire at the 9-month clinic visit. Parents were advised to consistently maintain a diary after the first clinic visit in order to collect information on child illnesses and diet. The child’s age at the start and end of probiotic supplementation and/or infant formula, as well as of each type of formula, were recorded. Probiotic exposure was defined as the timing of first probiotic introduction of either via dietary supplement or infant formula. Species of probiotics in supplements and infant formulas were examined based on the composition of the selfreported brand name products. The majority of the probiotic bacteria in dietary supplements and infant formulas taken by the TEDDY children consisted of Lactobacillus reuteri and Lactobacillus rhamnosus, although 17% of the families were not able to identify the brand name of the probiotics they used. The latter was more likely to occur during the first 3 months of age, when the information on probiotic intake was recalled retrospectively. Study nurses reviewed the questionnaires and diaries during the family’s clinic visit or over the phone every 3 months to minimize missing or inaccurate information. 2.4. Statistical Methods Time-to-event analysis with Cox proportional hazards (PH) modeling was performed to examine the overall probiotic exposure and timing in the first year of life in relation to the risk of CDA and celiac disease. The magnitudes of the associations were described by hazard ratios (HR) with 95% confidence intervals (CI). The Cox PH models were adjusted for known risk factors for celiac disease including sex, HLA genotype, family history of celiac disease (first-degree relative (FDR) with celiac disease vs. not), and country (as strata). In addition, Cox PH models were adjusted for potential confounders associated with both probiotics use and CDA or celiac disease, including birth year, mode of delivery, mother’s education, duration of exclusive breastfeeding, and child’s diarrhea status in the first 3 months. Probiotic exposure in the first year of life was incorporated into the Cox PH model in two ways: (a) probiotic exposure was categorized as binary (yes vs. no): probiotics users vs. non-users, and (b) source of probiotic exposure was categorized into three groups: dietary supplements, infant formula, Nutrients 2019,11, 1790 4 of 14 or none. Among probiotics users, the age at first probiotic exposure was examined as a continuous variable in the Cox PH model. Analyses were carried out using the Statistical Analysis System software (version 9.4; SAS Institute, Cary, NC, USA). 3. Results 3.1. Study Population During follow-up, 1212 (18.6%) children were identified as having CDA at a median (IQR) age of 3.3 (2.2–5.0) years (range 0.9–11.5 years), while 455 (7.0%) children were diagnosed with celiac disease at a median (IQR) age of 4.3 (3.2–6.2) years (range 1.2–12.5 years). The characteristics of the children by the status of CDA or celiac disease are presented in Table 1. Table 1. Characteristics of the study participants by status of celiac disease autoimmunity (CDA) and of celiac disease. Developed CDA (n=1212) Did not Develop CDA (n=5308) Developed Celiac Disease (n=455) Did not Develop Celiac Disease (n=6065) Country -US 437 (36.1) 2195 (41.4) 135 (29.7) 2497 (41.2) -Finland 257 (21.2) 1227 (23.1) 85 (18.7) 1399 (23.1) -Germany 58 (4.8) 289 (5.4) 17 (3.7) 330 (5.4) -Sweden 460 (37.9) 1597 (30.1) 218 (47.9) 1839 (30.3) Family member with celiac disease 123 (10.1) 136 (2.6) 76 (16.7) 183 (3.0) Sex, male 517 (42.7) 2815 (53.0) 167 (36.7) 3165 (52.2) HLA DR-DQ genotype -DR4-DQ8/DR4-DQ8 230 (19.0) 2235 (42.1) 73 (16.0) 2392 (39.5) -DR3-DQ2/DR4-DQ8 477 (39.4) 2176 (41.0) 153 (33.7) 2500 (41.2) -DR3-DQ2/DR3-DQ2 505 (41.6) 897 (16.9) 229 (50.3) 1173 (19.3) Birth year 2004–5 217 (17.9) 856 (16.1) 105 (23.0) 968 (16.0) 2006 243 (20.0) 887 (16.7) 76 (16.7) 1054 (17.4) 2007 244 (20.1) 1141 (21.5) 90 (19.8) 1295 (21.3) 2008 220 (18.2) 1104 (20.8) 80 (17.6) 1244 (20.5) 2009–10 288 (23.8) 1320 (24.9) 104 (22.9) 1504 (24.8) Mode of delivery— Cesarean section Yes 262 (21.6) 1394 (26.3) 85 (18.7) 1571 (25.9) No 950 (78.4) 3910 (73.7) 370 (81.3) 4490 (74.1) Mother’s education —more than high school 1016 (85.2) 4203 (81.5) 368 (82.5) 4851 (82.1) Duration of exclusive breastfeeding at least 3 months 363 (30.0) 1275 (24.0) 143 (31.4) 1495 (24.7) Exposure to probiotics by the age of 12 months 281 (23.2) 1179 (22.2) 99 (21.8) 1361 (22.4) Source of first exposure to probiotics by the age of 12 months -Dietary supplement 238 (19.6) 949 (17.9) 83 (18.3) 1104 (18.2) -Infant formula 42 (3.5) 228 (4.3) 16 (3.5) 254 (4.2) -Both 1 (0.1) 2 (0.0) 0 (0.0) 3 (0.0) Age (weeks) at first probiotic exposure among probiotics users median (Q1–Q3) 5 (2, 26) 6 (2, 19) 5 (3, 26) 6 (2, 20) mean (SD) 13.4 (14.7) 12.1 (13.2) 14.5 (15.3) 12.2 (13.3) Nutrients 2019,11, 1790 5 of 14 Table 1. Cont. Developed CDA (n=1212) Did not Develop CDA (n=5308) Developed Celiac Disease (n=455) Did not Develop Celiac Disease (n=6065) Age (weeks) at first probiotic exposure among probiotics users with first exposure from dietary supplements median (Q1–Q3) 4 (2, 20) 5 (3, 17) 4 (2, 26) 5 (2, 17) mean (SD) 12.5 (14.5) 11.8 (13.3) 13.6 (15.4) 11.8 (13.4) Age (weeks) at first probiotic exposure among probiotics users with first exposure from infant formula median (Q1–Q3) 20 (4, 28) 9 (2, 24) 22 (4, 26) 10 (2, 24) mean (SD) 19.0 (14.9) 13.4 (12.6) 19.0 (14.4) 14.0 (13.0) Data are presented as number (percentage) unless otherwise indicated. 3.2. Probiotic Use A total of 1460 children were exposed to probiotics via dietary supplements or infant formula during the first year of life. The characteristics of the children by the source of probiotic exposure in the first year of life are presented in Table 2. The participants’ characteristics that were positively associated with probiotics use during the first year of life were country (p<0.001), later birth year (p<0.001), mode of delivery (other than Cesarean section) (p=0.012), being the first born child (p<0.001), older maternal age (p=0.001), higher maternal education (p<0.001), not smoking during pregnancy ( p=0.005 ), maternal probiotic use during pregnancy (p<0.001), shorter duration of exclusive breastfeeding ( p<0.001 ), antibiotic use (p<0.001), diarrhea during the first 3 months (p<0.001), gastrointestinal infections (p<0.001), and lower incidence of common cold during the first 3 months (p=0.006) (Table 2). There was a considerable increase in probiotics use by birth year in Sweden (Figure S2) when compared to other countries where there was not as much difference in the probiotics use across the study years. Table 2. Characteristics of probiotics users by source of first exposure and non-users during the first year of life. Source of First Probiotic Exposure among Probiotics Users during the First Year of Life a Non-Users of Probiotics (n=5060) pbpc Dietary Supplements (n=1187) Infant Formula (n=270) Country <0.001 <0.001 US 119 (10.0) 49 (18.1) 2464 (48.7) Finland 776 (65.4) 29 (10.7) 678 (13.4) Germany 10 (0.8) 154 (57.0) 182 (3.6) Sweden 282 (23.8) 38 (14.1) 1736 (34.3) Family member with celiac disease 0.736 0.600 Yes 62 (5.2) 8 (3.0) 189 (3.7) No 1125 (94.8) 262 (97.0) 4871 (96.3) Sex 0.814 0.472 Male 614 (51.7) 133 (49.3) 2584 (51.1) Female 573 (48.3) 137 (50.7) 2476 (48.9) Nutrients 2019,11, 1790 6 of 14 Table 2. Cont. Source of First Probiotic Exposure among Probiotics Users during the First Year of Life a Non-Users of Probiotics (n=5060) pbpc Dietary Supplements (n=1187) Infant Formula (n=270) HLA DR-DQ genotype 0.533 0.061 -DR4-DQ8/DR4-DQ8 519 (43.7) 83 (30.7) 1862 (36.8) -DR3-DQ2/DR4-DQ8 446 (37.6) 137 (50.7) 2069 (40.9) -DR3-DQ2/DR3-DQ2 222 (18.7) 50 (18.5) 1129 (22.3) Birth year <0.001 <0.001 2004–5 97 (8.2) 45 (16.7) 930 (18.4) 2006 168 (14.2) 38 (14.1) 924 (18.3) 2007 260 (21.9) 47 (17.4) 1077 (21.3) 2008 272 (22.9) 64 (23.7) 988 (19.5) 2009–10 390 (32.9) 76 (28.1) 1141 (22.5) Mode of delivery: Cesarean section 0.012 0.281 Yes 238 (20.1) 99 (36.7) 1319 (26.1) No 949 (79.9) 171 (63.3) 3737 (73.9) Birth order, first child <0.001 0.314 Yes 575 (50.0) 131 (50.0) 2127 (43.4) No 575 (50.0) 131 (50.0) 2778 (56.6) Mother’s age at delivery (years) 0.001 0.177 ≤24 107 (9.0) 18 (6.7) 604 (11.9) 25–29 402 (33.9) 73 (27.0) 1437 (28.4) 30–34 424 (35.7) 113 (41.9) 1798 (35.5) >34 254 (21.4) 66 (24.4) 1221 (24.1) Mother’s education <0.001 0.133 High school or less 123 (10.6) 36 (13.8) 974 (19.8) More than high school 1038 (89.4) 225 (86.2) 3954 (80.2) Maternal pre-pregnancy body mass index 0.462 0.278 ≤25 776 (66.6) 180 (67.2) 3057 (61.6) >25 390 (33.4) 88 (32.8) 1905 (38.4) Smoking during pregnancy 0.005 0.334 Yes 119 (10.2) 34 (12.6) 552 (11.0) No 1052 (89.8) 236 (87.4) 4460 (89.0) Maternal antibiotic use during pregnancy 0.363 0.605 Yes 289 (24.7) 55 (20.4) 1122 (22.4) No 882 (75.3) 215 (79.6) 3896 (77.6) Maternal probiotics use during pregnancy <0.001 0.317 Yes 91 (7.7) 13 (4.8) 148 (2.9) No 1096 (92.3) 257 (95.2) 4912 (97.1) Duration of exclusive breastfeeding <0.001 <0.001 <3 months or none 887 (74.8) 225 (83.3) 3766 (74.4) ≥3 months 299 (25.2) 45 (16.7) 1293 (25.6) Age at gluten introduction 0.313 0.027 <17 weeks 69 (5.9) 11 (4.2) 312 (6.3) 17–26 weeks 425 (36.1) 76 (28.7) 1818 (36.5) >26 weeks 684 (58.1) 178 (67.2) 2851 (57.2) Nutrients 2019,11, 1790 7 of 14 Table 2. Cont. Source of First Probiotic Exposure among Probiotics Users during the First Year of Life a Non-Users of Probiotics (n=5060) pbpc Dietary Supplements (n=1187) Infant Formula (n=270) Child antibiotic use during the first 12 months <0.001 0.849 Yes 728 (61.3) 125 (46.3) 2259 (44.6) No 459 (38.7) 145 (53.7) 2801 (55.4) Diarrhea during the first 3 months <0.001 0.268 Yes 115 (9.7) 44 (16.3) 419 (8.3) No 1072 (90.3) 226 (83.7) 4641 (91.7) Gastrointestinal infections during the first 12 months <0.001 0.019 Yes 415 (35.0) 92 (34.1) 1496 (29.6) No 772 (65.0) 178 (65.9) 3564 (70.4) Common cold during the first 3 months 0.006 0.055 Yes 677 (57.0) 142 (52.6) 2955 (58.4) No 510 (43.0) 128 (47.4) 2103 (41.6) Age at first exposure to probiotics (weeks) 0.005 Mean (SD) 12 (14) 14 (13) Median (IQR) 5 (2–17) 10 (2–24) Duration of probiotic exposure during the first year of life (weeks) 0.073 Mean (SD) 30 (18) 25 (18) Median (IQR) 35 (11–48) 23 (8–44) a : Three children were exposed to both probiotic dietary supplements and infant formula at the same time and were not included here. b :pvalue from the Cochran–Mantel–Haenszel test for the association of characteristics between probiotics users and non-users during first year of life; analyses adjusted for country. c :pvalue from the Cochran–Mantel–Haenszel test (on categorical variables) or the analysis of covariance (on continuous variables) for the association of characteristics between the sources of first probiotic exposure; analyses adjusted for country. Data are presented as number (percentage) unless otherwise indicated. 3.3. Risk of CDA and Celiac Disease There was no difference in the risk of CDA (HR 1.15; 95%CI 0.99, 1.35; p=0.07) or celiac disease (HR 1.11; 95%CI 0.86, 1.43; p=0.43) between probiotics users and non-users during the first year of life when the models were adjusted for potential confounders: country, sex, HLA-genotype, FDR with celiac disease, birth year, mode of delivery, mother’s education, duration of exclusive breastfeeding, and child’s diarrhea during the first 3 months (Table 3). However, probiotic exposure from dietary supplements alone when compared to no exposure was associated with a slightly increased risk of CDA when adjusted for the potential confounders (HR 1.18; 95%CI 1.01, 1.40; p=0.043) (Table 3). Nutrients 2019,11, 1790 8 of 14 Table 3. Overall probiotic exposure, timing of first probiotic exposure by source, and the risk of celiac disease auto-immunity (CDA) and celiac disease. CDA Celiac Disease HR (95% CI) a p HR (95% CI) b p HR (95% CI) a p HR (95% CI) b p Exposed to probiotics during the first year of life: Yes vs. no 1.11 (0.96, 1.29) 0.177 1.15 (0.99, 1.35) 0.073 1.04 (0.82, 1.33) 0.731 1.11 (0.86, 1.43) 0.432 First exposure to probiotics by source: Supplements vs. none 1.15 (0.98, 1.35) 0.085 1.18 (1.01, 1.40) 0.043 1.03 (0.79, 1.35) 0.804 1.09 (0.83, 1.44) 0.534 Formula vs. none 0.91 (0.64, 1.30) 0.616 0.98 (0.69, 1.41) 0.930 1.12 (0.63, 1.98) 0.695 1.20 (0.68, 2.13) 0.537 Age at first exposure to probiotics among users (/week) c,d 1.01 (1.00, 1.02) 0.047 1.01 (1.00, 1.02) 0.210 1.02 (1.01, 1.04) 0.009 1.02 (1.00, 1.03) 0.055 Age at first exposure to probiotics among users whose first exposure to probiotics were from dietary supplements (/week) c 1.00 (0.99, 1.01) 0.478 1.00 (0.99, 1.01) 0.618 1.01 (1.00, 1.03) 0.102 1.01 (0.99, 1.03) 0.218 Age at first exposure to probiotics among users whose first exposure to probiotics were from infant formula (/week) c 1.03 (1.01,1.05) 0.014 1.02 (0.99, 1.05) 0.285 1.04 (1.00, 1.09) 0.044 1.04 (0.99, 1.09) 0.135 a Hazard ratios adjusted for sex, HLA genotype, first-degree relative (FDR) with celiac disease and country. b Hazard ratios adjusted for sex, HLA genotype, FDR with celiac disease, country, birth year, mode of delivery, mother’s education, duration of exclusive breastfeeding, and child’s diarrhea during first 3 months. (Birth year, mode of delivery, mother’s education, duration of exclusive breastfeeding, and child’s diarrhea during first 3 months were statistically significantly (p-value <0.05) associated with probiotic exposure during the first year of life, and with celiac disease autoimmunity (CDA) and/or celiac disease.) c Hazard ratios describe the change in the risk for every one week delay in the probiotic exposure. dHazard ratios adjusted additionally for the source of probiotics. No association was found between the age of the child at the time of initial probiotic exposure and the risk of the outcomes when adjusting for all the potential confounders (Table 3). However, a time-to-event analysis with smoothing splines [ 28 , 29 ] showed a slightly increased subsequent risk of celiac disease when a probiotic dietary supplement was introduced during the first weeks of life (Figure 1). Nutrients 2019,11, 1790 9 of 14 Nutrients 2019, 11, x FOR PEER 10 of 15 Figure 1. The estimated effects of age at probiotic exposure (by source of probiotics) on the log hazards of celiac disease autoimmunity (CDA, n = 281) (a,c; nonlinearity: p = 0.054 and p = 0.16, respectively) and celiac disease (n = 99) (b,d; nonlinearity: p = 0.16 and p = 0.20, respectively) from time-to-event analysis with smoothing splines on 1460 subjects who were exposed to probiotics during the first year of life. 4. Discussion The present study showed no protective association between overall probiotics use during first year of life and the risks of CDA or celiac disease in children at increased risk of T1D and celiac disease. In fact, probiotic exposure from dietary supplements during the first weeks of life was associated with a small increase in the risk of celiac disease. This finding is in contrast to a recent randomized double-blinded placebo-controlled study in Sweden in which synergistic effects of probiotics on the peripheral autoimmune response were observed in genetically predisposed children with CDA who were receiving two strains of Lactobacillus, as compared to placebo [30]. However, to our knowledge no other study has investigated the association between probiotic use and celiac disease in children in a prospective multicenter study like TEDDY. Probiotic use in the first year of life was positively linked to various infections and antibiotic use in children. This finding could be interpreted as infectious episodes potentially acting as a confounder when studying the association between probiotic exposure and the outcomes. However, we did not find any association between infections or antibiotic use and the risk of CDA or celiac disease in this study. Probiotics use was associated with shorter exclusive breastfeeding, which could have been related to earlier introduction of gluten-containing cereals. However, there is no current evidence that early introduction of gluten could be linked to the risk of celiac disease risk [31,32]. Moreover, Figure 1. The estimated effects of age at probiotic exposure (by source of probiotics) on the log hazards of celiac disease autoimmunity (CDA, n=281) ( a , c ; nonlinearity: p=0.054 and p=0.16, respectively) and celiac disease (n=99) ( b , d ; nonlinearity: p=0.16 and p=0.20, respectively) from time-to-event analysis with smoothing splines on 1460 subjects who were exposed to probiotics during the first year of life. 4. Discussion The present study showed no protective association between overall probiotics use during first year of life and the risks of CDA or celiac disease in children at increased risk of T1D and celiac disease. In fact, probiotic exposure from dietary supplements during the first weeks of life was associated with a small increase in the risk of celiac disease. This finding is in contrast to a recent randomized double-blinded placebo-controlled study in Sweden in which synergistic effects of probiotics on the peripheral autoimmune response were observed in genetically predisposed children with CDA who were receiving two strains of Lactobacillus, as compared to placebo [ 30 ]. However, to our knowledge no other study has investigated the association between probiotic use and celiac disease in children in a prospective multicenter study like TEDDY. Probiotic use in the first year of life was positively linked to various infections and antibiotic use in children. This finding could be interpreted as infectious episodes potentially acting as a confounder when studying the association between probiotic exposure and the outcomes. However, we did not find any association between infections or antibiotic use and the risk of CDA or celiac disease in this study. Probiotics use was associated with shorter exclusive breastfeeding, which could have been related to earlier introduction of gluten-containing cereals. However, there is no current evidence that early introduction of gluten could be linked to the risk of celiac disease risk [ 31 , 32 ]. Moreover, the association between breastfeeding duration and the risk of celiac disease still remains inconclusive [ 33 ].