Probiotic compositions and methods of use for enhancing childhood growth and social functioning - Patents.com

Abstract

Disclosed herein are methods and compositions useful for treating a subject suffering from Prader-Willi syndrome (PWS). The methods include administering to a subject in need thereof a composition comprising a probiotic, such as Lactobacillus reuteri (L. reuteri) and Bifidobacterium animalis subsp. lactis (B. Lactis).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 63 / 127,936, filed December 18, 2020, the contents of which are incorporated by reference in their entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT N / A

[0003] Sequence Listing A Sequence Listing is attached to this application and has been submitted as an ASCII text file of Sequence Listing entitled "125141_03682_ST25.txt", which is 926 bytes in size and was created on December 6, 2021. The Sequence Listing has been submitted electronically with this application via EFS-Web and is hereby incorporated by reference in its entirety.

[0004] The field of the invention relates to methods and compositions useful for treating subjects suffering from Prader-Willi syndrome (PWS), comprising administering to a subject in need thereof a composition comprising a probiotic, such as Lactobacillus reuteri (L. reuteri) and Bifidobacterium animalis subsp. lactis (B. Lactis). [Background technology]

[0005] Prader-Willi syndrome (PWS) is a rare genetic syndrome affecting approximately one in 15,000 people (Cassidy SB, Irizarry KA). PWS is recognized as the most common genetic cause of life-threatening childhood obesity (Butler MG, Irizarry KA). Morbid obesity and neuropsychiatric complications are the leading causes of death or long-term disability. In addition to some reported efficacy of growth hormone (Bakker NE, Kuppens RJ, Zhu JL), treatments are primarily behavioral.

[0006] The gut microbiota is involved in the pathogenesis of obesity and associated comorbidities in PWS subjects (Olsson LM). Independent of the PWS population, gut microbiota diversity and composition have been reported to affect nutrient metabolism and energy expenditure (Aoun A). Although gut microbiota diversity and composition were found to differ between obese and lean individuals (Lv Y), gut dysbiosis was found to be very similar in PWS-associated and diet-associated obesity (Zhang C).

[0007] Gut microbiota dysbiosis has been shown to activate inflammatory processes and contribute to the development of insulin resistance (Corado Gomes A). Dysbiotic gut microbiota transplanted from PWS patients into rats affected GLP-1 expression and decreased insulin receptor signaling 2 weeks before an increase in body fat composition, indicating that gut microbiota dysbiosis may play a role in the development of obesity (Deng). Recent studies have shown that probiotics may improve gut microbiota and metabolic disorders in diet-induced obese mice (Ke X), and also in a randomized controlled trial of weight management in overweight adults (Hibberd). Microbiota dysbiosis is not only associated with obesity, but is also closely linked to neuropsychiatric disorders, including schizophrenia (Akhondzadeh S), psychotic disorders (Vindegaard N), and autism spectrum disorder (ASD) (Navarro F). Previous studies conducted in our laboratory have even shown that the gut microbiota has the potential to serve as a biomarker to aid in the diagnosis and subtyping of ASD (Kong XJ et al.). Probiotic treatments are already widely used to help people with neuropsychiatric disorders (Liu J, Dickerson F).

[0008] Lactobacillus reuteri (L. reuteri) is a well-studied probiotic bacterium that can colonize many mammals. Direct supplementation or prebiotic modulation of L. reuteri may be an attractive preventive and / or therapeutic measure against inflammatory and metabolic diseases (Navarro F). L. reuteri V3401 has been reported to reduce inflammatory biomarkers, modify gastrointestinal microbiota and motility, and improve metabolic syndrome in adults (Tenorio-Jimenez,West CL). L. reuteri has also been shown to improve incretin and insulin secretion in glucose-tolerant humans (Simons MC). Of note, L. reuteri 263 showed anti-obesity effects by promoting white adipose tissue remodeling in high-energy diet rats (Chen LH). Despite these findings detailing the positive effects of L. reuteri on gut microbiota and metabolism, the direct effect of L. reuteri on obesity in humans is still debated. In fact, one study even found a correlation between endogenous abundance of L. reuteri and adiposity in Mexican children (Huerta-Avila). In addition to these metabolic benefits, L. reuteri has also been shown to exert beneficial effects on the brain and behavior. L. reuteri (DSM-17938) was associated with a significant reduction in the mean crying time in infant colic (Karkhaneh M). L. reuteri NK33, in combination with B. adolescentis NK98, alleviated and prevented the development of immobilization stress-induced anxiety / depression and colitis in mice (Jang HM). A research group at MIT reported that L. reuteri upregulates the neuropeptide hormone oxytocin (OXT), a factor essential for social connection and reproduction, within the vagus nerve-mediated pathway in mice while preventing age-related weight gain (Poutahidis T[1], Varian B).The same group reported that these benefits extend to human subjects, finding that, similar to mice, OXT-producing cells are increased in the caudal paraventricular nucleus (PVN) of the hypothalamus after consumption of L. reuteri lysate (Poutahidis T[2]). Further studies found that L. reuteri acts in a vagus-dependent manner to rescue deficits in social interaction-induced synaptic plasticity in the ventral tegmental area through oxytocin signaling modulation in multiple models of ASD (Sgritta M). L. reuteri treatment was found to improve antisocial behavior in male Shank3 mice and reduce repetitive behavior in both male and female Shank3 KO mice (Sgritta M).

[0009] Bifidobacterium animalis subsp. lactis (B. lactis) is a rod-shaped anaerobic bacterium found in the digestive tract of most mammals, including humans

[16] . Anti-obesity effects have been associated with the administration of several strains of B. lactis, such as A6, CECT8145, Bf141, B420 and BB-12 (Alyousif et al., 2018; Barz et al., 2019; Carreras et al., 2018; Dimidi et al., 2019; Huo et al., 2020; Ibarra et al., 2018; Pedret et al., 2019b; Uusitupa et al., 2020a). Increased abundance of B. lactis in the gut has been associated with systemic health and anti-inflammatory benefits. Many strains of B. lactis are considered to be health-promoting probiotics and are commonly formulated into fermented dairy products. Topical application of B. lactis HN019 was shown to delay the onset of symptoms associated with experimental periodontitis in rats (Oliveira et al., 2017). One study reported that administration of B. lactis BB-12 reduced the risk of respiratory tract infections in early childhood (Taipale et al., 2016). Another study found that the combination of B. lactis with Lactobacillus acidophilus reduced inflammatory signaling in intestinal epithelial cells (S.-C. Li et al., 2019).

[0010] Although the positive effects of probiotics are well documented in the general population, it is unclear whether similar effects are observed in subjects with different genetic backgrounds, such as those suffering from PWS. Summary of the Invention

[0011] Disclosed herein are methods and compositions useful for treating a subject suffering from or suspected of having PWS. The methods include administering an effective amount of a composition comprising one or more probiotics. In some embodiments, the probiotic comprises one or more of Lactobacillus, sp., Saccharomyces, sp., Bifidobacterium, sp., Bacillus, sp., and Eubacterium hallii. In some embodiments, the probiotic comprises Lactobacillus sp. (e.g., Lactobacillus reuteri (L. reuteri), and Bifidobacterium animalis subsp. lactis (B. lactis), and Bifidobacterium animalis subsp.

[0012] In some embodiments, the subject suffers from one or more of the following symptoms or conditions: obesity, small stature, social deficit, fine motor disorder, developmental delay, and abnormal behavioral characteristics, and after treatment, the subject's symptoms or conditions are reduced compared to before treatment. In some embodiments, the developmental delay includes one or more of communication, gross motor control, fine motor control, problem solving, and personal and social interaction by L. reuteri. In some embodiments, the abnormal behavioral characteristics include one or more of restricted repetitive behavior patterns (RRB), abnormal social interaction (SI), abnormal social communication (SC), abnormal emotional response (ER), abnormal cognitive style (CS), and maladaptive speech (MS) by L. reuteri. In some embodiments, the subject suffers from obesity, short stature, and the body mass index (BMI) of the subject after treatment is lower than the BMI of the subject before treatment with L. reuteri, and the height of the subject after treatment is higher than the height of the subject before treatment with B. lactis. In some embodiments, the subject suffers from psychopathology of varying severity as measured by a post-treatment Clinical Global Impression-Improvement (CGI-I), which is lower than the subject's baseline CGI severity before treatment with B. lactis. In some embodiments, the subject suffers from developmental delay, and the subject's Ages and Stages Questionnaires, 3rd Edition (ASQ-3) score is statistically improved in one or more of communication, gross motor function, fine motor function, problem solving, and personal and social interaction after treatment compared to the subject's ASQ-3 score before treatment with L. reuteri. In some embodiments, the subject suffers from abnormal behavioral traits, and the subject's GARS-3, 3rd Edition score (GARS-3) is statistically improved in one or more of RRB, SI, SC, ER, CS, and MS after treatment compared to the subject's GARS-3 score before treatment with L. reuteri.In some embodiments, the subjects suffer from psychopathology of various severity, and the subject's Clinical Global Impression-Improvement (CGI-I) is statistically improved for one or more scores of improvement (CGI-I) and severity (CGI-S) after treatment compared to the subject's CGI-I and CGI-S scores before treatment for B. lactis.

[0013] In some embodiments, the treatment comprises administering an effective dose of a probiotic once a day, twice a day, three times a day, or four times a day. In some embodiments, the treatment comprises administering an effective dose of a probiotic for at least about 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or at least about 12 weeks. In some embodiments, the effective dose is about 1×10 3 , about 2×10 3 , about 3×10 3 , about 4×10 3 , about 5×10 3 , about 6×10 3 , about 7×10 3 , about 8×10 3 , about 9×10 3 , or approximately 10 × 10 3 In some embodiments, the subject is administered one or more additional therapeutic agents.

[0014] In some embodiments, the probiotic comprises either L. reuteri or B. lactis, the probiotic is administered at a dose of about 3×103 CFU twice daily for 12 weeks, and after treatment, the subject shows a statistically relevant improvement in one or more of BMI, fine motor function, and problem-solving ability as measured by ASQ-3 test. In some embodiments, the composition of the subject's microbiota is different after treatment compared to before treatment. In some embodiments, the difference comprises a decrease in one or more of Escherichia-Shigella, Porphyromonas, and Ruminococcus torques by L. reuteri. In some embodiments, the difference comprises an increase in one or more of Bifidobacterium, Lactobacillus, Faecalibacteria, Roseburia, and Alistipes by L. reuteri. In some embodiments, the difference comprises a significant positive association of Rothia with RRB after treatment with B. lactis.

[0015] In some embodiments, compositions are provided comprising an effective dose of one or more probiotics and a growth hormone. In some embodiments, the probiotic comprises Lactobacillus sp. In some embodiments, the probiotic comprises Lactobacillus reuteri and the growth hormone comprises human growth hormone.

[0016] These and other embodiments, aspects, advantages, and features of the present invention are described in part in the following description and will become apparent to those skilled in the art upon examination of the following description of the invention and the referenced drawings, or by the practice of the invention. The accompanying drawings depict one or more embodiments, which do not necessarily represent the full scope of the invention. [Brief description of the drawings]

[0017] [Figure 1]Overview of study conduct and participant enrollment and dropout flow chart for the L. reuteri study. [Diagram 2] Age distribution of study participants for the study on L. reuteri. Participant groups are indicated by the color of the frequency bars. Ages of subjects receiving the placebo control ranged from 1 to 15 years, and ages of subjects receiving the active probiotic ranged from 0.5 to 23 years. [Diagram 3] Tabular overview of estimated marginal means of BMI at each study time point in the L. reuteri trials. [Figure 4] Tabular summary of pairwise comparisons of change in BMI at 6 and 12 weeks compared to baseline for the L. reuteri trials. [Diagram 5] Tabular overview of psychometric measures, including ASQ-3 and GARS-3 measures, at 6- and 12-week study time points in the L. reuteri study. [Figure 6A] Summary of genus-level relative abundance and microbial diversity measures for the L. reuteri study. (A) Plot of gut microbiota relative abundance at genus level, baseline, 6 weeks, and 12 weeks. (B) Mean α-diversity measured via Shannon, Simpson, ACE, and Chao1 indices. (C) β-diversity by plot of principal coordinate analysis (PCoA) scores of gut microbial data based on Bray-Curtis dissimilarity matrix. [Figure 6B] Summary of genus-level relative abundance and microbial diversity measures for the L. reuteri study. (A) Plot of gut microbiota relative abundance at genus level, baseline, 6 weeks, and 12 weeks. (B) Mean α-diversity measured via Shannon, Simpson, ACE, and Chao1 indices. (C) β-diversity by plot of principal coordinate analysis (PCoA) scores of gut microbial data based on Bray-Curtis dissimilarity matrix. [Figure 6C]Summary of genus-level relative abundance and microbial diversity measures for the L. reuteri study. (A) Plot of gut microbiota relative abundance at genus level, baseline, 6 weeks, and 12 weeks. (B) Mean α-diversity measured via Shannon, Simpson, ACE, and Chao1 indices. (C) β-diversity by plot of principal coordinate analysis (PCoA) scores of gut microbial data based on Bray-Curtis dissimilarity matrix. [Figure 7A-C] Fold change in relative abundance at the genus level over the course of the intervention for the probiotic group (green) and placebo (blue) for the trial on L. reuteri. Each bar represents the log2-transformed relative change in gut microbial abundance at 6 and 12 weeks compared to baseline. [Fig. 7D-F] Fold change in relative abundance at the genus level over the course of the intervention for the probiotic group (green) and placebo (blue) for the trial on L. reuteri. Each bar represents the log2-transformed relative change in gut microbial abundance at 6 and 12 weeks compared to baseline. [Figure 7G-I] Fold change in relative abundance at the genus level over the course of the intervention for the probiotic group (green) and placebo (blue) for the trial on L. reuteri. Each bar represents the log2-transformed relative change in gut microbial abundance at 6 and 12 weeks compared to baseline. [Figure 8] Table of predicted KEGG enzyme abundances based on PICSRUSt-2 predictive functional profiling of subjects receiving either active probiotics or placebo control. The mean abundance of KEGG enzymes is differentially enriched at different concentrations in placebo and level 3 probiotics. [Figure 9A]ROC curves of classification between treatment and placebo groups based on selected clinical indicators and functional metagenomic features using logistic regression for the study on L. reuteri. (A) Classification using clinical indicators including ASQ-3 total and fine motor scores and GARS-3 SC and SI scores. (B) Classification using selected functional features of gut metagenome. [Figure 9B] ROC curves of classification between treatment and placebo groups based on selected clinical indicators and functional metagenomic features using logistic regression for the study on L. reuteri. (A) Classification using clinical indicators including ASQ-3 total and fine motor scores and GARS-3 SC and SI scores. (B) Classification using selected functional features of gut metagenome. [Figure 10] A table outlining the clinical logistic regression model indices used in the ROC analysis for the study on L. reuteri is presented. [Figure 11] Provides a table outlining the metagenomic profiling logistic regression model indices used in the ROC analysis for the study on L. reuteri. [Figure 12] Tables showing univariate associations between genus- and family-level bacterial abundance and clinical measures at weeks 6 and 12 combined based on an overall linear model using the MaAsLin2 package are provided. Significant correlations shown are based on active probiotic groups. Taxonomic rankings are shown in brackets with "f" indicating family-level microbiota and "g" indicating genus-level microbiota. [Figure 13] Overview of study conduct and participant enrollment and dropout flow chart for the B. lactis study. [Figure 14] Clinical Global Impression (CGI)-Severity at baseline between the two arms for the B. lactis trial. Comparison of CGI-S at baseline between the probiotic group (blue) and the placebo group (yellow). No differences in the overall level of severity were seen between the groups (p>0.05). [Figure 15]Table showing comorbid conditions of study participants. [Figure 16A-C] Comparison of z-score changes in height (A–C) and weight (D–F) from baseline, weeks 0 to 6, and weeks 6 to 12 between the probiotic group (blue) and placebo (yellow) using Wilcoxon rank-sum tests for the study on B. lactis. [Fig. 16D-F] Comparison of z-score changes in height (A–C) and weight (D–F) from baseline, weeks 0 to 6, and weeks 6 to 12 between the probiotic group (blue) and placebo (yellow) using Wilcoxon rank-sum tests for the study on B. lactis. [Figure 17A-B] Comparison of ABC total score (A), SRS-2 total score (B), ASQ-3 total score (C) and RRB score (D) over the course of the intervention between the probiotic group (blue) and the placebo group (brown) for the study on B. lactis. No group significance was found (P>0.05). [Fig. 17C-D] Comparison of ABC total score (A), SRS-2 total score (B), ASQ-3 total score (C) and RRB score (D) over the course of the intervention between the probiotic group (blue) and the placebo group (brown) for the study on B. lactis. No group significance was found (P>0.05). [Figure 18] Probiotic and placebo CGI-I at week 12 for the study on B. lactis. The percentage of participants given each level of improvement is displayed as a bar graph, with the probiotic group (blue) having significantly better improvement overall than the placebo group (yellow, p<0.05). [Figure 19A] Overview of the relative abundance of gut microbiota at the phylum and genus level in both probiotic and placebo subjects at baseline, 6 weeks, and 12 weeks of the study for B. lactis. [Figure 19B] Overview of the relative abundance of gut microbiota at the phylum and genus level in both probiotic and placebo subjects at baseline, 6 weeks, and 12 weeks of the study for B. lactis. [Figure 19C] Overview of the relative abundance of gut microbiota at the phylum and genus level in both probiotic and placebo subjects at baseline, 6 weeks, and 12 weeks of the study for B. lactis. [Fig. 20A-D] Changes in α and β diversity indices from probiotic intervention for the study on B. lactis. (A) Recognized species index; (B) Phylogenetic diversity of faith; (C) Shannon index; (D) Simpson index. *P<0.05; **P<0.01 by t-test. (E) β diversity by Non-metric Multidimensional Scaling (NMDS) score plot of gut microbial data based on Bray-Curtis dissimilarity matrix. Placebo (red dots) and probiotic (blue dots). [Figure 20E] Changes in α and β diversity indices from probiotic intervention for the study on B. lactis. (A) Recognized species index; (B) Phylogenetic diversity of faith; (C) Shannon index; (D) Simpson index. *P<0.05; **P<0.01 by t-test. (E) β diversity by Non-metric Multidimensional Scaling (NMDS) score plot of gut microbial data based on Bray-Curtis dissimilarity matrix. Placebo (red dots) and probiotic (blue dots). [Figure 21A-C] Fold change in relative abundance at the genus / species level over the course of the intervention for the probiotic group (blue) and placebo (orange) for the study on B. lactis. Each bar represents the relative change in log2 shift in gut microbial abundance at 6 and 12 weeks compared to baseline. Significant differences are marked with * to indicate P<0.05. [Fig. 21D-F] Fold change in relative abundance at the genus / species level over the course of the intervention for the probiotic group (blue) and placebo (orange) for the study on B. lactis. Each bar represents the relative change in log2 shift in gut microbial abundance at 6 and 12 weeks compared to baseline. Significant differences are marked with * to indicate P<0.05. [Fig. 21G-I]Fold change in relative abundance at the genus / species level over the course of the intervention for the probiotic group (blue) and placebo (orange) for the study on B. lactis. Each bar represents the relative change in log2 shift in gut microbial abundance at 6 and 12 weeks compared to baseline. Significant differences are marked with * to indicate P<0.05. [Figure 22A-C] Fold relative change in abundance at family level for the study on B. lactis. Each bar represents the relative change in log2 shift in gut microbial abundance compared to baseline at weeks 6 and 12. [Fig. 22D-F] Fold relative change in abundance at family level for the study on B. lactis. Each bar represents the relative change in log2 shift in gut microbial abundance compared to baseline at weeks 6 and 12. [Fig. 22G-I] Fold relative change in abundance at family level for the study on B. lactis. Each bar represents the relative change in log2 shift in gut microbial abundance compared to baseline at weeks 6 and 12. [Figure 23] Prediction of KEGG enzyme abundance based on functional gene analysis of PICSRUSt2 in probiotic and placebo groups for a study on B. lactis. Mean abundance of KEGG enzymes differentially enriched in placebo and probiotic according to level 3. [Figure 24] Comparison of KEGG pathway predictions in the placebo and probiotic groups for a study on B. lactis. Mean abundance of KEGG pathways enriched in d for placebo and probiotic according to level 1. [Diagram 25] Comparison of KEGG orthologous (KO) predictions between placebo and probiotic groups for a study on B. lactis. Mean abundance of KEGG pathways differentially enriched in placebo and probiotic according to level 2. [Figure 26]Correlation between the abundance of bacterial genus and clinical indicators in the study on B. lactis using the Spearman method was performed for the probiotic (blue) and placebo (yellow) groups at 6 weeks. The probiotic group showed a positive correlation between RRB score and Rothia (R=0.97, P<0.005). No significant correlation was observed in the placebo group. [Figure 27] Epworth Sleepiness Scale (ESS) at baseline between the two groups for the study on B. lactis. Comparison of ESS scores at baseline between the probiotic group (blue) and the placebo group (yellow). No difference in sleepiness levels was found between the groups (p>0.05). [Figure 28] Clustering of gut microbiota communities in fecal samples from PWS subjects receiving placebo or probiotics at baseline, 6 weeks, or 12 weeks in a study on B. lactis, using nonmetric multidimensional scaling (NMDS) on Bray-Curtis dissimilarity matrices. [Figure 29A-B] Family-level relative abundance plots of gut microbiota composition in subjects taking probiotics or placebo at baseline, 6 weeks, and 12 weeks in a study on B. lactis. (A-D) Family-level analysis; (E-J) Gene-level analysis in a study on B. lactis. [Fig. 29C-D] Family-level relative abundance plots of gut microbiota composition in subjects taking probiotics or placebo at baseline, 6 weeks, and 12 weeks in a study on B. lactis. (A-D) Family-level analysis; (E-J) Gene-level analysis in a study on B. lactis. [Fig. 29E-F] Family-level relative abundance plots of gut microbiota composition in subjects taking probiotics or placebo at baseline, 6 weeks, and 12 weeks in a study on B. lactis. (A-D) Family-level analysis; (E-J) Gene-level analysis in a study on B. lactis. [Fig. 29G-H]Family-level relative abundance plots of gut microbiota composition in subjects taking probiotics or placebo at baseline, 6 weeks, and 12 weeks in a study on B. lactis. (A-D) Family-level analysis; (E-J) Gene-level analysis in a study on B. lactis. [Figure 29I-J] Family-level relative abundance plots of gut microbiota composition in subjects taking probiotics or placebo at baseline, 6 weeks, and 12 weeks in a study on B. lactis. (A-D) Family-level analysis; (E-J) Gene-level analysis in a study on B. lactis. [Fig. 30A-C] Key gut microbes associated with obesity at genus level for the study on B. lactis. (A-C) Relative abundance of genera with respect to obesity. (D) The overall composition of gut microbiota in normal and overweight groups showed a significant partition (F statistic = 1.7239; R2 = 0.067015; P = 0.011). PERMANOVA results are labeled. [Figure 30D] Key gut microbes associated with obesity at genus level for the study on B. lactis. (A-C) Relative abundance of genera with respect to obesity. (D) The overall composition of gut microbiota in normal and overweight groups showed a significant partition (F statistic = 1.7239; R2 = 0.067015; P = 0.011). PERMANOVA results are labeled. [Figure 31A] Phylogenetic co-occurrence network analysis showing the predominant bacterial groups associated with the intervention from three time points between placebo and probiotic groups based on the SparCC correlation algorithm for a trial on B. lactis. Each node represents a bacterial genus. Each color represents the relative abundance at different time points (green: baseline, red: 6 weeks, purple: 12 weeks). The size of the node indicates the relative abundance of each genus and the density of the lines represents the SparCC coefficient. Each edge represents the correlation between a pair of taxa. SparCC permutations=100, p-value threshold=0.05, correlation threshold=0.5. [Figure 31B]Phylogenetic co-occurrence network analysis showing the predominant bacterial groups associated with the intervention from three time points between placebo and probiotic groups based on the SparCC correlation algorithm for a trial on B. lactis. Each node represents a bacterial genus. Each color represents the relative abundance at different time points (green: baseline, red: 6 weeks, purple: 12 weeks). The size of the node indicates the relative abundance of each genus and the density of the lines represents the SparCC coefficient. Each edge represents the correlation between a pair of taxa. SparCC permutations=100, p-value threshold=0.05, correlation threshold=0.5. [Figure 32A] Network of correlations between placebo and probiotics taken at baseline, 6 weeks, and 12 weeks (A–C, respectively) for a study on B. lactis . [Figure 32B] Network of correlations between placebo and probiotics taken at baseline, 6 weeks, and 12 weeks (A–C, respectively) for a study on B. lactis . [Figure 32C] Network of correlations between placebo and probiotics taken at baseline, 6 weeks, and 12 weeks (A–C, respectively) for a study on B. lactis . DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Herein, we describe two independent randomized, double-blind, placebo-controlled studies to test whether probiotic intake has beneficial effects on obesity, social behavior, anthropoid growth and neurogenesis in PWS and to determine whether these effects are associated with changes in the gut microbiota. To conduct these studies, we enrolled a total of 71 PWS patients to evaluate the efficacy of a L. reuteri strain (LR-99) and a total of 65 PWS patients to evaluate the efficacy of a B. lactis strain (BB-11) on body mass index (BMI), psychological measures, and gut microbiota composition and function compared to a placebo control. In addition to potentially aiding novel interventions for PWS patients, the microbiota composition data collected from this study may shed light on mechanisms underlying PWS pathology and the gut-brain axis.

[0019] The presently disclosed subject matter is described herein using several definitions, which are set forth below and throughout the application.

[0020] definition

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.

[0022] As used in this specification and the claims, the indefinite articles "a" and "an" should be understood to mean "at least one," unless clearly indicated otherwise.

[0023] The term "and / or" as used herein and in the claims should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are sometimes conjunctively present and other times disjunctively present. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related to the specifically identified elements or not. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with open-ended language such as "comprising", may refer, for example, in one embodiment to only A (optionally including elements other than B), in another embodiment to only B (optionally including elements other than A), and in yet another embodiment to both A and B (optionally including other elements).

[0024] As used herein and in the claims, "or" should be understood to have the same meaning as "and / or" defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, i.e., including at least one of a number or list of elements, but also including two or more, and optionally including additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") when preceded by a term of exclusivity, such as "either," "one of," "only one of," or "exactly one of." When used in the claims, "consisting essentially of" shall have its ordinary meaning as used in the field of patent law.

[0025] Whenever an embodiment is described with the word "comprising," other similar embodiments that are described with the terms "consisting of" and / or "consisting essentially of" are included.

[0026] As used herein, the terms "approximately" or "about" in reference to numbers are generally interpreted as including numbers within a range of 5% in either direction (greater or less) of the number, unless otherwise stated or clear from the context (except where such number exceeds 100% of its possible values).

[0027] Numerical ranges include the numbers that define the range, and any individual value provided herein can serve as an endpoint of a range that includes other individual values ​​provided herein. For example, a set of values ​​such as 1, 2, 3, 8, 9, and 10 is also a disclosure of numerical ranges such as 1-10, 1-8, 3-9, etc. Similarly, a disclosed range is a disclosure of each individual value encompassed within that range. For example, a stated range of 5-10 is also a disclosure of 5, 6, 7, 8, 9, and 10.

[0028] While the invention has been described in terms of one or more preferred embodiments, it should be understood that many equivalents, alternatives, variations, and modifications, aside from those expressly stated, are possible and are within the scope of the invention.

[0029] As used herein, the term "treat" includes arresting, substantially inhibiting, slowing or reversing the progression of a condition, substantially improving the clinical or aesthetic symptoms of a condition, or substantially preventing the appearance of clinical or aesthetic symptoms of a condition. For purposes of this disclosure, "treat" or "treatment" describes the management and care of a patient for the purpose of combating a disease, condition, or disorder. These terms encompass both preventative, i.e., prophylactic and palliative treatment. "Treat" includes administering a composition of the present disclosure to prevent the onset of symptoms or complications, to alleviate symptoms or complications, or to eliminate a disease, condition, or disorder. As used herein, the term "treat" and words derived therefrom do not necessarily imply 100% or complete treatment or prevention. Rather, there are various degrees of treatment or prevention that one of skill in the art will recognize as having potential benefit or therapeutic effect. In this regard, the methods of the present disclosure can result in any amount of treatment or prevention at any level of a disease in a mammal. Furthermore, the treatment or prevention provided by the methods of the present invention can include one or more treatments or preventions of the disease state or symptom being treated or prevented, or disease condition, such as PWS. Also, for purposes of this disclosure, "prevention" can encompass delaying the onset of a disease, or a symptom or condition thereof, and "treating" or "treatment" includes the management and care of a subject for the purpose of combating a disease, condition, or disorder. Treatment includes administration of a probiotic as described herein to prevent the onset of symptoms or complications and / or to alleviate symptoms or complications of a disease, condition, or disorder.

[0030] The term "treatment" may be characterized by one or more of the following: (a) improvement of body mass index (BMI), for example, by weight loss and / or height increase; (b) improvement of developmental delay characteristics; and / or (c) improvement of abnormal behavioral characteristics. By way of example, but not by way of limitation, treatment may be characterized by improvement of one or more characteristics, such as weight, height, BMI, communication, gross motor control / function, fine motor control / function, problem solving, personal and social interaction, restricted repetitive behavior patterns (RRB), abnormal social interaction (SI), abnormal social communication (SC), abnormal emotional response (ER), abnormal cognitive style (CS), and maladaptive voice (MS). Additionally, individuals with PWS are known to have small stature, poor growth, poor muscle tone, food cravings, developmental delays, cognitive impairment, mood and behavioral problems, obesity, sleep apnea, and comorbidities. Relief or favorable changes in such common symptoms may also be considered an improvement of PWS symptoms.

[0031] As used herein, the terms "effective amount" and "therapeutically effective amount" refer to an amount of one or more effective therapeutic agents sufficient to produce a desired therapeutic response without undue adverse side effects such as toxicity, irritation or allergic reaction. The specific "effective amount" will obviously vary depending on factors such as the specific condition being treated, the physical condition of the subject, the duration of treatment, the nature of the concomitant therapy (if any), and the specific formulation and structure of the therapeutic agent or its derivatives used. The exact dosage is selected by the individual physician in view of the patient being treated. Dosage and administration are adjusted to provide sufficient levels of the effective agent or to maintain the desired effect.

[0032] "Subject" or "individual" or "animal" or "patient" refers to any subject, particularly a mammalian subject, for whom diagnosis, prognosis or treatment is desired. Although the present invention generally applies to humans, it can be used for veterinary purposes. For example, it can be desired to treat or test treatments on commercially important livestock animals such as cows, horses, pigs, rabbits, goats and sheep, or related laboratory animals such as rats, mice and rabbits. It can also be desired to treat companion animals such as cats and dogs.

[0033] In some embodiments, the optimal effective amount can be easily determined by those skilled in the art using routine experimentation. In some embodiments, a therapeutically effective amount is achieved by administering multiple therapeutically effective doses, for example, over a day, several days, a week, a few weeks, a few months or a few years.

[0034] Any suitable method can be performed to determine, detect, or monitor a subject's response to a treatment according to the methods provided herein. As used herein, "determining a subject's response to a treatment" refers to evaluating the outcome of a treatment in a subject that responds to administration of a composition provided herein or treatment according to a method provided herein.

[0035] As used herein, body mass index (BMI) refers to a number used as an estimate of an individual's body fat. BMI can be calculated by dividing a person's weight in kilograms by the square of the person's height in meters, or by dividing a person's weight in pounds by the square of the person's height in inches, multiplied by a factor of 703. Furthermore, a high BMI is associated with an increased risk of chronic diseases, such as heart disease, high blood pressure, and type 2 diabetes, in adults. Moreover, BMI also provides a reasonable estimate of body fat in most people.

[0036] The Clinical Global Impression (CGI) is a scale used to measure symptom severity and treatment response. It is a three-item observer-rated scale used by clinicians and researchers to track symptom change, for example, before versus after treatment is started. The three items assessed are 1) severity of illness (CGI-S), 2) overall improvement (CGI-I), and 3) efficacy index (CGI-E), a measure of treatment effects and side effects specific to the drug administered. The CGI was developed for use in NIMH-supported clinical trials to provide a brief, independent assessment of the clinician's view of the patient's overall functioning before and after starting medication in the study. The CGI includes two accompanying one-item scales that assess: (a) severity of psychopathology from 1 to 7 (CGI-S) and (b) change from start of treatment on a similar 7-point scale (CGI-I).

[0037] As used herein, the Ages & Stages Questionnaires®, Third Edition (ASQ®-3) is a questionnaire commonly used to track the developmental progress of children aged 1 month to 5.5 years. The ASQ-3 has five scoring domains: communication, gross motor, fine motor, problem solving, and personal socialization. Each domain contains six age-matched questions. It is one of the most widely available developmental, communication, and behavioral screening tools for young children (Perera et al. 2017, Squires et al. 2009). Although it is one of the most common scales, other similar scales exist that are less common or intended for different forms of research.

[0038] As used herein, the Gilliam Autism Rating Scale, Third Edition (GARS-3) is a questionnaire that helps identify autism in an individual and evaluate its severity. It consists of 56 items that describe characteristic behaviors of individuals with autism. The items are grouped into six subscales: Restricted and Repetitive Behaviors (RB), Social Interaction (SI), Social Communication (SC), Emotional Reactivity (ER), Cognitive Style (CS), and Maladaptive Speech (MS). The GARS-3 is a norm-referenced screening instrument that has been used to identify individuals with autism spectrum disorder, 3rd edition, since 1995 (Gilliam, 1995; Gilliam, 2014). It has been proven to have high rates of validity and reliability, and is therefore particularly utilized in the field of psychology (Benjamin CK 2016, Duffy et al. 2017).

[0039] Prader-Willi syndrome

[0040] As used herein, the term "Prader-Willi syndrome" (PWS) refers to a rare genetic imprinting disorder with an estimated prevalence of 1 / 10,000-1 / 30,000 [1]. Three mechanisms cause this genetic disorder: deletion (DEL) of the 15q11.2-q13 region from the male chromosome (~74% of cases), maternal uniparental disomy (UPD) from the mother (~25%), and defective imprinting (~1%) (Cassidy, 1997). PWS is characterized by severe hypotonia and feeding difficulties in early infancy, followed by hyperphagia and morbid obesity beginning in early childhood (Cassidy, 2012). PWS patients also typically experience generalized neurodevelopmental delays and numerous neuropsychiatric comorbidities (Salehi, 2018).

[0041] There is currently no cure for Prader-Willi syndrome. Of the available symptomatic treatments, growth hormone replacement has proven to be the most effective, especially when administered early in development (WHO Multicentre Growth Reference Study Group, 2006). Growth hormone has been shown to improve height as well as cognitive and motor function. Other treatment options include primarily the treatment of comorbid psychiatric conditions through cognitive behavioral therapy and counseling.

[0042] PWS, like many other neurodevelopmental disorders, exists across a spectrum of signs and symptoms including hypotonia and lack of eye coordination in infancy, hypotonia and abnormal neurological function, hypogonadism, developmental and cognitive delays (e.g., delays in milestones related to communication, gross motor control, fine motor control, problem solving, and personal and social interaction), overeating and obesity, short stature, and abnormal behavioral characteristics (e.g., restricted and repetitive behaviors (RRB), abnormal social interaction (SI), abnormal social communication (SC), abnormal emotional responses (ER), abnormal cognitive style (CS), and maladaptive speech (MS)), as well as psychiatric disorders.

[0043] As used herein, "Autism Spectrum Disorder" (ASD) refers to a developmental condition characterized by deficits in social communication and restricted / repetitive behaviors. ASD, like PWS, exists on a spectrum of diverse sets of signs and symptoms, particularly of variable severity. While most cases of autism are idiopathic, approximately 90% of cases are estimated to be genetically caused. According to the latest estimates from the Centers for Disease Control, ASD has a prevalence of 1 in 54 live births (Maenner et al., 2020). Approximately 25-40% of PWS children have comorbidities with ASD (Bennett et al., 2015). Both PWS and ASD are forms of developmental delay, which is a group of conditions that impair children's learning and functioning during the early developmental stages.

[0044] As used herein, the term "probiotic" refers to organisms, generally bacteria, that are considered to be beneficial rather than harmful to an animal host. With regard to digestive health, the concept of ingesting beneficial bacteria has only become popular in recent years, although the benefits of ingesting certain bacterial strains were first proposed in 1907 by Elie Metchnikoff, who suggested that since lactic acid bacteria can prevent spoilage of stored foods, they may also benefit the digestive tract. Lactobacillus delbruickii subspecies bulgaricus, isolated from fermented dairy products, was of particular interest. Metchnikoff proposed that it was the optimal strain to ingest due to its ability to produce large amounts of lactic acid with little succinic or acetic acid, its ability to rapidly coagulate milk, and its lack of production of alcohol and acetone. With the advent of antibiotics, interest in probiotics waned. However, the emergence of antibiotic-resistant bacteria has led to renewed interest in probiotic bacteria, which are now defined as "live microorganisms that, when administered in adequate amounts, confer a health benefit to the host." It is now a common concept that the accumulation of probiotic organisms in the intestine is beneficial to the overall health of the host organism, and there are reports showing that administration of probiotics is useful for treating intestinal diseases. Surprisingly and unexpectedly, therapeutic probiotic compositions, as disclosed herein, are also useful for treating Prader-Willi syndrome.

[0045] Exemplary probiotic bacteria include, but are not limited to, Lactobacillus sp., Saccharomyces sp., Bifidobacterium sp., Streptococcus sp., Escherichia coli., Bacillus sp., and Eubacterium hallii. Specific examples of such probiotics include L. reuteri V3401, which has been reported in adults to reduce inflammatory biomarkers, modify gastrointestinal microbiota, and subsequently improve metabolic syndrome (Tenorio-Jimenez et al. 2019), L. Reuteri 263, which demonstrated anti-obesity effects associated with energy metabolism remodeling of white adipose tissue in high-energy diet rats (Chen et al. 2018), and L. Reuteri, which has been reported to regulate intestinal motility in mice (West et al. 2020) (DSM-17938). Furthermore, L. Reuteri NK33, together with B. adolescentis NK98, demonstrated immobilization stress-induced anxiety / depression and colitis in mice.

[0046] Bifidobacterium animalis subsp. lactis (B. lactis): Administration of several strains of B. lactis, e.g., A6, CECT8145, Bf141, B420, and BB-12, mainly in animals (Chen et al., 2018; Hibberd et al., 2019; Huerta-Avila et al., 2019; Simon et al., 2015; Tenorio-Jimenez et al., 2019; West et al., 2020). The anti-inflammatory effects of several strains of B. lactis, such as HN019 and BB-12, have also been reported in recent years (Akhondzadeh, 2019; Vindegaard et al., 2020).

[0047] The probiotic composition disclosed herein may contain one type of probiotic organism or a combination of different probiotic organisms.The present probiotic biology study involves the administration of a single strain of probiotic, but there is a growing trend of multi-strain probiotic studies to evaluate the potential additive or synergistic effects between probiotic strains.Specifically, previous studies have shown that the combination of B.lactis and Lactobacillus acidophilus reduces inflammatory signaling in intestinal epithelial cells.

[0048] Disclosed herein is a therapeutic probiotic composition comprising one or more probiotic microorganisms. In some embodiments, the therapeutic probiotic composition is formulated for oral administration, for example as a food or dietary supplement. By way of example, but not by way of limitation, the probiotic composition may be formulated as a milk-based product and may be placed in milk, yogurt, cheese, or ice cream. The food may be formulated as a non-dairy product, such as a fruit-based product or a soy-based product. Such food may be in solid or liquid / drinkable form. Additionally, the food may contain all conventional additives, including but not limited to proteins, vitamins, minerals, trace elements, and other nutritional ingredients.

[0049] In some embodiments, the therapeutic probiotic composition is formulated as a liquid, powder, capsule, tablet or sachet for oral administration. In some embodiments, the capsule or tablet may include an enteric coating and the therapeutic probiotic composition may include one or more pharma- ceutically acceptable carriers. In some embodiments, the carrier may be a capsule for oral administration. In such embodiments, the outer housing of the capsule may optionally be made of gelatin or cellulose. Cellulose has the advantage of maintaining the formulation in the intestinal fluids and precluding premature degradation in the upper gastrointestinal tract, so the product can reach its desired destination. Alternatively, the ingredients may be combined into a tablet. In tablet form, cellulose may also be present to act as a binder to hold the tablet together. The probiotic composition may further include one or more excipients to facilitate the manufacturing process by preventing the ingredients from sticking to machinery. Additionally, such excipients may make the capsule or tablet form easier to swallow and digest through the intestinal tract. The excipients may be vegetable stearates, magnesium stearate, stearic acid, ascorbyl palmitate, retinyl palmitate, or hydroxypropyl methylcellulose. Additional colors, flavors, and excipients known in the art can also be added. The formulated probiotic composition may be administered in the formulated state (e.g., as a capsule or tablet) or may be combined with food or beverages for administration.

[0050] Therapeutic probiotic compositions may include lyophilized microorganisms, live cultures, or combinations thereof, and the microorganisms may be provided in a therapeutically effective dose. In some embodiments, a therapeutically effective dose is about 1×10 per dose. 5 ~1×10 15 of microorganisms, (colony forming units (CFU) per dose), approximately 1 × 10 per dose 6 ~1×10 14 of microorganisms, approximately 1 x 10 per dose 7 ~1×10 13 of microorganisms, approximately 1 x 10 per dose 8 ~1×1012 of microorganisms, approximately 1 × 10 per dose 9 ~1×10 11 of microorganisms, approximately 1 x 10 per dose 10 ~9×10 10 of microorganisms or approximately 3 x 10 per dose 10 For both studies evaluating the effects of L. reuteri and B. lactis in individuals with PWS, each subject randomized to the active probiotic group received 3 × 10 10 They were instructed to take colony forming units (CFU) twice daily.

[0051] An effective dose of the therapeutic probiotic composition can be administered to a subject in need thereof once a day, twice a day, three times a day, four times a day or more. In some embodiments, the therapeutic probiotic composition is administered an effective dose of probiotics for at least about 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or at least about 12 weeks. In some embodiments, the therapeutic probiotic composition is administered continuously or periodically for several years or for the life of the subject, as symptoms require. By way of example, but not limitation, in some embodiments, an effective dose of the therapeutic probiotic composition is administered daily, for example, twice a day, for 12 weeks.

[0052] In some embodiments, a therapeutic composition comprising a probiotic, such as L. reuteri, is administered in combination with one or more additional active agents. By way of example, the additional active agents include growth hormones (e.g., human growth hormone), oxytocin, serotonin, and dopamine. The additional active agents may be administered simultaneously with the probiotic composition (e.g., as part of the same formulation), or may be administered separately, at the same time or at a different time, as the probiotic composition. Thus, in some embodiments, a composition comprising a probiotic and one or more additional active agents is administered to a subject in need thereof (e.g., a subject diagnosed with or suspected of having PWS).

[0053] In some embodiments, the method of the present invention changes the composition of the subject's microbiota, making it different after treatment compared to before treatment. In the examples, the inventors demonstrate that the composition of the gut microbiota undergoes substantial changes after administration of L. reuteri LR-99 probiotics (see, for example, Figures 6 and 7) and correlate the changes in the abundance of the microbiota with clinical indicators (see Figure 13). Specifically, they determined that the abundance of certain bacteria (i.e., Escherichia-Shigella, Porphyromonas, and Ruminococcus torques) decreased after treatment, whereas the abundance of other bacteria (i.e., Bifidobacterium, Lactobacillus, Faecalibacteria, Roseburia, and Alistipes) increased after treatment with L. reuteri. Given that Bifidobacterium is widely considered beneficial for gut health and weight loss (Pedret et al., 2019a; Uusitupa et al., 2020b), we also observed significant changes to the gut microbiota profile and specific gut microbiota of individuals with PWS after B. lactis supplementation (see Figures 18-20). Interestingly, we found that the improvement of Clinical Global Impression-Improvement (CGI-I) was significantly improved after 12 weeks of B. lactis supplementation. These findings demonstrated that both L. reuteri and B. lactis as potent probiotics induce significant favorable gut microbiota composition changes that result in reduced fat deposition via insulin and calcium signaling regulation and improved mental health via the gut-brain axis.

[0054] Lactobacillus is known to have a protective effect against weight gain in humans and has been found to inhibit the activity of pro-inflammatory interleukins associated with obesity and poor obesity-related outcomes (Cox et al., 2015; Rosing et al., 2017a). Surprisingly, we observed improvements in overall development as measured by ASQ-3 total score (P<0.05), fine motor function (P<0.05), and possible problem-solving ability (P=0.051) after supplementation with L. reuteri. Such results have not been found in any of the literature reports of such effects observed in the above mentioned areas. Furthermore, L. reuteri intervention significantly improved social communication (P<0.01) and social interaction (P<0.05) compared to controls aged 3 years and older. L. reuteri rescues social interaction-induced synaptic plasticity in the ventral tegmental area of ​​ASD mice, but not in oxytocin receptor-deficient mice (Sgritta et al., 2019). Oxytocin nasal spray has been used to treat PWS subjects with beneficial effects (Junli Zhu & Xuejun Kong, 2017), and the use of L. reuteri has not yet been reported to improve social function in human studies, and it may be more cost-effective, convenient, and potentially induce longer-lasting endogenous oxytocin release than using oxytocin directly. This finding warrants further investigation of the internal mechanisms mediated by oxytocin or other neurotransmitters / hormones involved in the pathogenesis of PWS and its comorbidities.

[0055] These findings therefore strongly support the use of probiotics as a beneficial early intervention to improve overall developmental level and therefore alter the prognosis of individuals with PWS. Furthermore, such probiotic strains may be applicable to children with developmental delays of other causes, and further research in these areas is urgently indicated. EXAMPLES

[0056] The examples presented herein are not intended to be limiting, but are provided to demonstrate aspects of the present technology.

[0057] Example 1: Supplementation with L. reuteri

[0058] Study design

[0059] We designed and conducted a randomized, double-blind, placebo-controlled clinical trial (flow chart, Figure 1). In this study, we randomly assigned enrolled PWS participants to either a probiotic group or a placebo group in a 1:1 ratio. We expected that a 12-week treatment period would be sufficient for probiotic supplementation to induce detectable changes. A total of 52 participants (26 in each group) were required to achieve 80% statistical power for the primary outcome assuming a large effect size of 0.8 (Cohen's d). We enrolled and randomized 71 subjects (probiotic group=37, placebo group=34), of which 56 (probiotic group=28, placebo group=28) completed the 12-week study and were included in the final intention-to-treat data analysis.

[0060] ethical considerations

[0061] Ethical approval was issued by the Internal Review Board (IRB) of the Second Affiliated Hospital of Kunming Medical University (Review-YJ-2016-06). The probiotic clinical trial was registered in the Chinese Clinical Trial Registry (ChiCTR), number ChiCTR1900022646. Signed informed consent was obtained from the subjects' parents or legal guardians in accordance with IRB requirements. The study was conducted in accordance with the Declaration of Helsinki.

[0062] participants

[0063] Study participants were recruited through the PWS Care & Support Center in Zhejiang, China. Participants were included if they met the following criteria: were genetically confirmed to have PWS; had not received any form of probiotics for at least 4 weeks; had received stable medication for at least 4 weeks; had no plans for changes in medication or psychosocial interventions during the trial; were willing to provide a stool sample in a timely manner; and were willing to cooperate with interview and testing procedures. Potential participants were excluded if they had other known genetic disorders or were pregnant or breastfeeding prior to the study.

[0064] Randomization and blinding

[0065] Randomization and allocation concealment were performed by a statistician who was not part of the study team. A randomization sampling number was generated electronically for each anonymized subject. Identical-looking coded probiotics and placebos were prepared by the Beijing Huayuan Academy of Biotechnology to ensure allocation concealment. Both participants and the study staff / researchers who collected and analyzed outcome data were blinded to treatment condition. Blinding was also maintained by making the probiotic packaging look identical to the placebo sachets.

[0066] intervention

[0067] Probiotics LR-99 (Beijing Huayuan Academy of Biotechnology) in the form of sachets was used in the study. Each sachet of probiotic supplement contained 3 × 10 10The placebo was maltodextrin in a sachet with a similar color, flavor, and taste to the probiotic sachet. Subjects received either the probiotic or the placebo in one sachet twice daily for a period of 12 weeks. Of note, probiotics are supplements with minimal side effects. The placebo maltodextrin also has minimal adverse effects.

[0068] Primary outcomes:

[0069] 1. Weight and height measurements were obtained by parents using a standard scale and collected by study staff. BMI calculated by weight and height was converted to z-score using age-growth standards provided by the WHO (2006).

[0070] 2. Psychological measurements

[0071] (a) Ages and Stages Questionnaires, 3rd Edition (ASQ-3). ASQ-3 is one of the most widely available developmental screening tools for young children. ASQ-3 has five domains: communication, gross motor, fine motor, problem solving, and personal socialization (Squires J 2009). Total scores were calculated. We interviewed all subjects under the age of 5.

[0072] (b) Gilliam Autism Rating Scale, Third Edition (GARS-3) (Gilliam JE). It consists of 56 items that describe the characteristic behavior of autistic individuals. The items are grouped into six subscales: Restricted Repetitive Behaviors (RRB), Social Interaction (SI), Social Communication (SC), Emotional Reactivity (ER), Cognitive Style (CS), and Maladaptive Speech (MS). Total scores and subscales were calculated. We interviewed all subjects over the age of 3.

[0073] Secondary outcomes:

[0074] 1. Fecal microbiota

[0075] (a) Sample handling and collection

[0076] Stool samples were collected in DNA / RNA shielded fecal collection tubes (Zymo, Cat. No. R1101) containing 1 mL of preservation solution, transported to the laboratory by ice bags, and then frozen at -80°C. DNA was extracted using the TIANmap fecal DNA kit (TIANGEN, Cat. No. DP328) according to the manufacturer's instructions, and DNA samples were carefully quantified with a Nanodrop Spectrophotometer. The A260 / A280 ratio was also measured to confirm high purity DNA yield. DNA samples were frozen at -20°C until use.

[0077] (b) 16S rRNA gene amplicon sequencing

[0078] The 16S rRNA V3-V4 library was constructed by two rounds of PCR using the following primers, 341F: 5'TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGAGGCAGCAGCCTACGGGNBGCASCAG3' (SEQ ID NO: 1), and 805R: 5'GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGACTACNVGGGTATCTAATCC3' (SEQ ID NO: 2), with the following reaction sequence: 95°C for 2 min, followed by 25 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 5 min. PCR products were purified with 1x KAPA AMPure beads (KAPA, catalog no. KK8002). The products were then subjected to a second PCR reaction step (95°C for 2 min, followed by eight cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, with a final extension at 72°C for 5 min). PCR products were purified with 1× KAPA AMPure beads and analyzed using a Bioanalyzer DNA kit, followed by quantification by real-time PCR. DNA libraries were pooled and sequenced on an Illumina MiSeq (Illumina; CA) using a 2× 250 bp paired-end protocol with overlapping reads.

[0079] statistical analysis

[0080] All raw data were recorded and processed in Microsoft Excel 2007 and R. Data presentation followed the CONSORT recommendations for reporting the results of randomized clinical trials (RCTs). Statistical procedures were performed using α = 0.05 as the significance level.

[0081] Selected clinical or predictive functional profiling indices were used to construct receiver operating characteristic (ROC) curves via the plotROC package for multiple logistic regression models.

[0082] Wilcoxon rank-sum tests were applied to determine between-group differences in z-scores for weight, height, total scores and subscores of ASQ-3, GARS-3, ABC, and SRS at baseline, subject-specific change from 0 to 6 weeks, and subject-specific change from 6 to 12 weeks.

[0083] Linear mixed-effects models (LME) were used to analyze primary outcomes and assess differences within each primary outcome over the course of the study (0–6 weeks, 6–12 weeks, and 0–12 weeks) for each group. For all LME analyses, time, age, and sex were included as fixed effects and random intercepts to account for within-subject correlations with repeated measures over time. In case of significant main effects, Bonferroni-corrected pairwise comparisons were performed.

[0084] Secondary outcomes were analyzed using the same methods as the primary outcomes. In addition, linear regression was performed to examine correlations between clinical indicators and microbiota composition.

[0085] Microbiota data processing and analysis

[0086] Sequencing reads were filtered using QIIME2 (v2019.10) based on quality scores (Bolyen E 2019). They were denoised using Deblur with default parameters to obtain abundance tables of samples by amplicon sequence variants (ASVs) (Amir A 2017).

[0087] Alpha diversity was calculated using QIIME2. Bray-Curtis distance was used to characterize microbiota beta diversity. ASV taxonomy was assigned using a sklearn-based taxonomy classifier trained on sequences from Greengenes v13.8 with a 99% similarity level. Significant differences in the relative abundance of microbial phyla, genera, and alpha diversity between the placebo and probiotic groups were identified by Kruskal-Wallis test. False positive rate (FDR) based on Benjamini-Hochberg (BH) adjustment was applied for multiple comparisons (Jiang J 2017).

[0088] PICSRUSt-2 was used to infer microbial functional content based on ASV abundance tables, followed by the generation of Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologs (KO), enzyme classification numbers, and pathway abundance tables (Douglas GM, Czech L). Differential analysis was performed on the fold ratios between the probiotic and placebo groups using permutation-based nonparametric tests, and the best differential features were rendered and plotted with Calour (Xu ZZ). All 16S rRNA raw data are pending submission to the NCBI Sequence Read Archive (SRA).

[0089] result

[0090] 1. Demographic characteristics of PWS participants

[0091] A total of 71 subjects aged 64.4 ± 51.0 months (ranging from 6 to 264 months) with a genetically confirmed diagnosis of Prader-Willi syndrome were included in the study. Of these, 37 subjects aged 65.0 ± 53.8 months were randomized to receive the active probiotic L. reuteri, whereas 34 subjects aged 64.0 ± 49.0 months were randomized to receive placebo. An overview of the age distribution of the subjects is shown in Figure 2. Furthermore, due to difficulties in data collection, a total of 56 subjects (n = 28 in each group) had available baseline clinical indicators. Group-wise comparisons of baseline age, sex, genotype and other characteristics did not show any significant differences (P > 0.05). Detailed demographic characteristics of the enrolled participants are summarized in Table 1. [Table 1]

[0092] The study recruitment procedure and dropouts at each study time point are shown as a flow chart in Figure 1. No serious or severe adverse events were observed. No significant differences were found between the two groups in experiencing any of the observed adverse events (P>0.05). Of the 71 subjects initially enrolled, 15 dropped out over the course of the study. Eight subjects dropped out due to antibiotic use, which led to termination. Seven of the declines were due to self-discontinuation. None of the dropouts were due to adverse effects (Figure 1).

[0093] 2. Effects of probiotics on BMI and psychological measures

[0094] To assess longitudinal changes in primary outcomes by group, Bonferroni-corrected pairwise comparisons were applied to linear mixed-effects models that account for repeated measurements over time, using age, sex, and study time points as fixed effects and subject as a random intercept. Estimated marginal means of BMI at each study visit are shown by group as a table in Figure 3. Based on such analysis, the inventors determined that subjects receiving active probiotics showed a significant reduction in BMI. Specifically, such significant differences were uniquely observed in the active probiotic group for BMI between baseline and week 6 and between baseline and week 12 (Figure 4, P<0.05).

[0095] Group comparisons of psychological assessment scores were performed at weeks 6 and 12 by Wilcoxon rank sum test. A summary of psychological assessment scores including GARS-3 and ASQ-3, and related statistics for both groups at weeks 6 and 12 are shown in Figure 5. These results suggest that L. reuteri LR-99 significantly reduced BMI in subjects receiving active probiotic compared to subjects receiving placebo at both 6 (P<0.05) and 12 (P<0.01) weeks of treatment, compared to baseline measurements.

[0096] 3. Changes in microbiota composition and function by probiotic intervention

[0097] After sequencing, we obtained a total of 3,198,401 raw reads and an average of 49,206,169 reads per sample (range 29,501-71,027 reads per sample).The overall phylum- and genus-level variation in gut microbiota composition over the course of the intervention is shown in Figure 6A for both the probiotic and placebo groups.

[0098] Overall, α-diversity, determined using the Shannon, Simpson, ACE and Chao1 indices, did not show any significant differences between groups (Figure 6B). However, β-diversity showed significant separation between probiotic treatments by permutational multivariate ANOVA (PERMANOVA, F-statistic = 1.9018; R 2 = 0.022667; P < 0.05, Figure 6C).

[0099] To characterize the changes in abundance of potentially clinically significant bacteria over the course of the intervention, we calculated the Log 2-fold change of the detected and identified gut microbiota. The fold change of gut microbiota abundance aggregated at the genus level is shown in Figure 7.

[0100] In an attempt to elucidate the changes in the functional profile of the gut microbiota between those receiving active probiotics and those receiving placebo, we applied predictive functional profiling and performed group-by-group comparisons of the mean abundance differences for each identified functional pathway. Several functional pathways were determined to be differentially expressed between subjects receiving active probiotics (Figure 8, Q value < 0.1).

[0101] Subsequently, receiver operating characteristic (ROC) curve analysis was used to identify significant clinical parameters (BMI, social communication, social interaction, total ASQ, fine motor) that can be used as biomarkers of response to treatment to characterize subjects receiving either probiotics or placebo (Figure 9A). The fitted logistic regression model is summarized in the table presented as Figure 10. Next, we identified several important metagenomic functional pathways that can be used to characterize subjects receiving either active probiotics or placebo (Figure 9B). The fitted logistic regression model is summarized in the table presented as Figure 11. Classification using clinical indices including ASQ-3 total and fine motor scores and GARS-3 SC and SI scores resulted in an AUC of 0.9 (95% CI = 0.7 to 1). Similarly, classification using selected functional features of gut metagenome resulted in an AUC of 0.801 (95% CI = 0.713 to 0.899).

[0102] Since some strains of Lactobacillus are probiotics known to have protective effects against weight gain in humans and have been found to inhibit proinflammatory interleukin activity associated with obesity and poor obesity-related outcomes (Cox et al., 2015; Rosing et al., 2017a), the observed reduction in BMI and improved social functioning in subjects administered the active probiotic are consistent with expectations. However, in the absence of supporting literature, the improved fine motor function, overall development, and predicted changes in metagenomic functional profiles are unexpected and surprising results.

[0103] 4. Correlation between gut microbiota abundance and clinical indicators

[0104] The association between family and genus level microbiota abundance and clinical indicators was evaluated via MaAsLin2 as a univariate linear correlation. The significant correlations at family and genus level with clinical indicators for the combined measurements at 6 and 12 weeks are reported in the table presented as Figure 12.

[0105] Consideration In our 12-week randomized, double-blind, placebo-controlled study of 71 PWS subjects, L. reuteri LR-99 significantly reduced BMI in those receiving the active probiotic compared with those receiving placebo at both 6 (P<0.05) and 12 (P<0.01) weeks of treatment, compared with measurements at baseline.

[0106] In the past, intervention with L. reuteri failed to induce improvement in BMI in humans (Maes M et al., Agusti A et al.). Such novel findings in this study provide new avenues for early intervention in PWS to prevent obesity and associated complications. This is important for early intervention because among all subjects in this study cohort, more than half of the subjects are under 5 years old. In past studies, other strains of L. Reuteri, such as L. Reuteri 263, demonstrated anti-obesity effects associated with energy metabolism remodeling of white adipose tissue in high-energy diet rats (Chen LH). L. Reuteri SD5865 has been shown to improve incretin and insulin secretion in glucose-tolerant humans (Simons MC). Another strain, L. reuteri V3401, was reported to improve metabolic syndrome in adults as it reduces inflammatory biomarkers and modifies the gastrointestinal microbiota (Tenorio-Jimenez). Individuals with PWS are found to have absolute or functional growth hormone (GH) deficiency, and GH replacement is currently the most effective treatment for PWS (Zhu JL, Bakker NE). GH has been found to increase height as well as reduce body fat and improve cognitive, motor and mental function (Bakker NE, Kuppens RJ). Increased efficacy and prognostic benefits have been observed with early initiation of GH treatment (Bakker NE). One study found that probiotic L. reuteri can increase growth hormone levels in mice (Varian BJ), demonstrating a potential mechanism by which probiotics may reduce BMI and treat PWS patients, promoting endogenous growth hormone release. Our findings warrant further investigation of the biological mechanisms of probiotics, a promising intervention for PWS that is better tolerated and easier to use than GH replacement (Onubi OJ).

[0107] Interestingly, we found that L. reuteri intervention significantly improved social communication (P<0.01) and social interaction (P<0.05) compared to controls over 3 years of age. Furthermore, we found significant increases in total ASQ-3 scores (P<0.05) and fine motor subscales (P<0.05) in the L. reuteri intervention group compared to the placebo control group when compared at the final study visit (week 12).

[0108] These novel and important findings pave the way for the use of probiotics to improve BMI, social function, fine motor function, and overall developmental indicators in PWS-affected children. As mentioned above, researchers have previously reported that L. reuteri upregulates the neuropeptide hormone oxytocin (OXT), an essential factor for social bonding and reproduction (Poutahidis T). OXT-producing cells were found to increase in the caudal paraventricular nucleus (PVN) of the hypothalamus after feeding on a sterile lysed preparation of L. reuteri (Varian B). Further studies showed that L. reuteri rescues social interaction-induced synaptic plasticity in the ventral tegmental area of ​​ASD mice, but not oxytocin receptor-deficient mice (Sqritta M). Oxytocin nasal spray has been used to treat PWS subjects with beneficial effects (Zhu J), and using L. reuteri, which can induce endogenous oxytocin release, is more cost-effective, convenient, and potentially longer-lasting than using oxytocin directly. This finding warrants further research into the internal mechanisms mediated by oxytocin or other neurotransmitters / hormones involved in the pathogenesis of PWS and its comorbidities. Regarding the observed improvements in overall development (by BMI, P<0.05), fine motor function (P<0.05), and possible problem-solving ability (P=0.051) with probiotics, we found no literature reports on these. These findings strongly support the use of probiotics as a beneficial early intervention to improve overall developmental level and thus change the prognosis of individuals with PWS. Further research in these areas is indicated.

[0109] The changes in the composition of the microbiota observed with the intervention by the inventors have previously been associated with weight loss and attenuation of inflammation. Of note, the inventors found a significant separation of gut microbiota β diversity between the probiotic and placebo groups after treatment. Baseline β diversity is directly correlated with long-term weight loss when adherent to a controlled diet (Grembi JA). Thus, probiotic supplementation may have a preventive effect or promote diet-induced weight loss. Such changes in gut microbial diversity after supplementation with L. reuteri are consistent with expectations, since previous studies on Lactobacillus probiotic supplementation in healthy individuals have shown it to modulate the overall gut microbiota composition (Ferrario et al., 2014).

[0110] Following administration of L. reuteri, the inventors also noted a trend towards a decrease in the abundance of several bacteria, including Escherichia-Shigella, Porphyromonas and Ruminococcus torques. Escherichia-Shigella is a well-recognized pathogenic bacterium that is abundant in individuals with obesity and type 2 diabetes (Anhe, FF, Thingolm LB), and also in autism, and is associated with constipation (Eshraghi RS). A role for periodontal pathogens, especially Porphyromonas gingivalis (P. gingivalis), in the development or exacerbation of systemic disease has been proposed (Mulhall H). Ruminococcus torques is one of the prominent species in IBD that is abundant in intestinal dysbiosis (Lloyd-Price, J). Bacteroides was found to be abundant in subjects with type 1 and type II diabetes (Alkanani AK, Remely M), although some controversial results were reported for the anti-inflammatory effects of Bacteroides (Hiippala K). These findings were only demonstrated in the probiotic treatment group, not in the placebo group, of PWS patients. This indicated that the probiotics we used could significantly alter the gut microbiota composition and further alter gut and brain function through their anti-inflammatory effects and gut-brain axis signaling.

[0111] Conversely, Bifidobacterium, Lactobacillus, Faecalibacteria, Roseburia and Alistipes were increased in the gut after LR-99 treatment. Lactobacillus, the genus to which the interventional probiotic belongs, has also been found to have a protective effect against weight gain in humans and inhibit the activity of pro-inflammatory interleukins that are associated with obesity and poor obesity-related outcomes (Rosing JA, Cox AJ). Bifidobacterium is widely considered to be beneficial for gut health and weight loss (Pedret A, Uusitupa HM). The abundance of Alistipes was inversely correlated with adiposity, lipid and glucose homeostasis parameters (Garcia-Ribera S, 2020). Roseburia was more abundant in the microbiota of ketonuric pregnant women, which correlated with increased maternal lipid metabolism and lower blood glucose levels (Robinson H), and Roseburia and Faecalibacteria are butyrate-producing bacteria that are anti-inflammatory, and Faecalibacteria were found to reduce intestinal permeability and lower inflammation (MorklS et al.). These findings were only demonstrated in the probiotic treatment group, not the placebo group, of PWS patients. This indicated that the probiotics we used significantly altered the gut microbiota composition and that their active metabolites could further alter gut and brain function by affecting fat metabolism and gut-brain axis signaling.

[0112] Furthermore, using predictive functional genetic analysis, we identified significant upregulation of calcium signaling pathway, flavonoid biosynthesis, carotenoid biosynthesis, steroid biosynthesis, N-glycan biosynthesis, photosynthesis, valine, leucine, isoleucine biosynthesis, and fission (yeast), with both P and Q values ​​of <0.05. Calcium signaling pathways are important in regulating obesity (Song Z). Flavonoids are a signature class of secondary metabolites formed from the assembly of relatively simple scaffolds. They are extensively modified by chemical reactions including glycosylation, methylation, and acylation (Tohge T, Jiang T). Carotenoids, antioxidants, have previously been found to have beneficial effects on obesity and obesity-related pathologies (Mounien L). Steroid biosynthesis favors anti-inflammation and stress response (Chatuphonprasert W). N-glycan biosynthesis promotes immune regulation and anti-inflammation (Reily C). Dietary supplementation with Leu or Ile reduced body weight by regulating lipid metabolism-related genes, and insulin sensitivity and fatty liver impaired by HFD were alleviated after Leu or Ile supplementation (Ma Q). Valine, leucine, and isoleucine refer to branched-chain amino acids (BCAAs). BCAA supplementation has been used in patients with BCKDK deficiency (Garcia-Cazorla A) to successfully reduce ASD symptoms and improve cognitive function. These findings were only demonstrated in the probiotic treatment group, not the placebo group, of PWS patients. This indicated that the probiotics we used could significantly change the gut microbiota composition and further alter gut and brain function through their anti-inflammatory effects, reducing gut permeability, decreasing cytokines, and affecting gut-brain axis signaling.

[0113] The insulin signaling pathway and starch and sucrose metabolism were also found to be upregulated with P<0.05 but Q>0.1. The insulin transduction pathway is a biochemical pathway through which insulin increases glucose uptake into fat and muscle cells and decreases glucose synthesis in the liver, thus participating in the maintenance of glucose homeostasis (Bevan P). The starch and sucrose metabolic pathways have been found to be downregulated in ASD (Rose DR 2018). The human gut microbiota is a key component of digestion as it promotes the breakdown of complex carbohydrates and proteins (Oliphant,K 2019). Thus, it is likely that such functional changes in the fecal metagenome could be beneficial to the host, but the underlying mechanistic role of such changes in the gut microbiota remains unclear.

[0114] Predictive functional gene analysis also showed a significant downregulation of arachidonic acid metabolism, with P and Q both <0.05. Arachidonic acid metabolism is involved in inflammatory processes (Violette Said Hanna, FA Kuehl Jr). Lipopolysaccharide (LPS) and phosphotransferase system (PTS) were also found to be downregulated with P<0.05 but Q>0.05. Lipopolysaccharide (LPS), an endotoxin from gram-negative pathogens such as Escherichia Shigella, has been reported to be involved in the causes of obesity (Hersoug LG), autism and the gut-brain axis (Srikantha P). The phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) is the major carbohydrate transport system in bacteria. The PTS catalyzes the phosphorylation of incoming sugar substrates, coupled with translocation across the cell membrane, making the PTS the link between sugar uptake and metabolism (Postma PW, Meadow ND). Taken together, microbiota compositional data and predictive functional gene analyses indicate that the segregation of diversity caused by treatment with the LR-99 probiotic favors protection against obesity and obesity-related pathologies.

[0115] Furthermore, using receiver operating characteristic (ROC) curve analysis, we found that clinical indices including ASQ-3 total, fine motor score, and GARS-3 SC and SI scores yielded an AUC of 0.9 (95% CI = 0.7 to 1). Similarly, classification using selected functional features of gut metagenome yielded an AUC of 0.801 (95% CI = 0.713 to 0.899). These further confirmed that our novel findings on improved clinical indices and gut microbiota by L. reuteri have high sensitivity and specificity for predicting treatment response, which was not seen in the placebo group.

[0116] RRB is one of the core symptoms of autism spectrum disorder (ASD) and is reported in as many as 25-40% of PWS cases (Salehi P, Bennett JA). Alitipes was found to be negatively correlated with RRB, and Subdoligranulum was found to be positively correlated with BMI.

[0117] Faecalibacterium was negatively correlated with BMI. A decrease in the relative abundance of Alistipes (Strati F, Srikantha P) was found in ASD. Subdoligranulum was found to be increased in obese mice (Elmassry MM). Obese individuals had lower abundance of Faecalibacterium (Crovesy, L). Bifidobacterium was negatively correlated with BMI as expected.

[0118] In conclusion, this randomized, double-blind, placebo-controlled study for children with PWS showed that treatment with the probiotic LR-99 for 12 weeks significantly reduced BMI at week 6, with more pronounced effects when examined after 12 weeks of treatment, significantly improved social communication and social interaction, and developed fine motor skills, especially at week 12. These novel findings have important implications for the early treatment of PWS. Probiotic treatment also altered the composition and function of the microbiota in an anti-obesity and anti-inflammatory manner.

[0119] There are several limitations of the study that are worth considering. First, although we employed an appropriate recruitment and retention strategy, enrollment and retention of PWS participants in this study was difficult, the sample size was relatively small, and further subgroup analysis was limited. Second, the wide age range used in this study resulted in high subject population heterogeneity and potentially variable treatment efficacy. Third, the assessment of the fecal microbiota was not controlled for dietary habits that may affect microbial abundance at the individual level. Thus, future studies with larger sample sizes, improved control for environmental factors, and subgroup stratification are justified. Due to the limitations of the study listed above, further studies are justified to investigate the mechanisms and efficacy of LR-99 probiotic treatment in PWS.

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[0121] Example 2: Supplementation with B. lactis

[0122] Study design

[0123] We designed and conducted a randomized, double-blind, placebo-controlled clinical trial (flow chart, FIG. 13). In this study, we randomly assigned eligible PWS participants to either the probiotic group or the placebo group in a 1:1 ratio. We assume that a 12-week treatment period is sufficient for probiotic supplementation to induce detectable changes. A total of 52 participants (26 in each group) were required to achieve 80% statistical power for the primary outcome assuming a large effect size of 0.8 (Cohen's d).

[0124] ethical considerations

[0125] Ethical approval was issued by the Internal Review Board (IRB) of the Second Affiliated Hospital of Kunming Medical University (Review-YJ-2016-06). The probiotic clinical trial was registered in the Chinese Clinical Trial Registry (ChiCTR), number ChiCTR1900022646. Signed informed consent was obtained from the subjects' parents or legal guardians in accordance with IRB requirements. The study was conducted in accordance with the Declaration of Helsinki.

[0126] participants

[0127] We enrolled 65 subjects (69.1% male, 30.9% female) aged 52.5±38.2 months with a genetically confirmed diagnosis of Prader-Willi syndrome. Study participants were recruited through the PWS Care&Support Center in Zhejiang, China. Participants were included if they met the following criteria: were genetically confirmed to have PWS; had not been administered any form of probiotics for at least 4 weeks; had received stable medication for at least 4 weeks; had no plans for changes in medication or psychosocial interventions during the study; were willing to provide a stool sample in a timely manner; and were willing to collaborate on the interview and study procedures. Potential participants were excluded if they had other known genetic disorders or were pregnant or breastfeeding prior to the study.

[0128] Randomization and blinding

[0129] Randomization and allocation concealment were performed by a statistician who was not part of the study team. Randomization sampling numbers were generated electronically for each anonymized subject. Identical-looking coded probiotics and placebos were prepared by the Beijing Huayuan Academy of Biotechnology to ensure allocation concealment. Both participants and the study staff / researchers who collected and analyzed outcome data were blinded to treatment condition. Blinding was also maintained by making the probiotic packaging look identical to the placebo sachets.

[0130] intervention

[0131] Probiotics BL-11 (Beijing Huayuan Academy of Biotechnology) was used in the study in the form of sachets containing probiotics BL-11 in powder form. Each sachet of probiotic supplement contained 3 × 10 10 The sachets contained colony forming units (CFU). The placebo was maltodextrin in a sachet with a similar color, flavor, and taste to the probiotic sachet. Subjects received one sachet of either the probiotic or the placebo twice daily for a period of 12 weeks and were instructed to take the contents of the sachet orally with water.

[0132] Primary outcomes:

[0133] 1. Weight and height measurements were obtained by parents using a standard scale and collected by study staff. Weight, height, and BMI were converted to z-scores using age-growth standards provided by the WHO (WHO Multicentre Growth Reference Study Group, 2006).

[0134] 2. Psychological measurements

[0135] 1) Ages and Stages Questionnaires, 3rd Edition (ASQ-3) (Parent-Completed, nd). ASQ-3 is one of the most widely available developmental screening tools for young children. ASQ-3 has five domains: communication, gross motor, fine motor, problem solving, and interpersonal socialization. A total score was calculated. We interviewed all subjects under the age of 5 years.

[0136] 2) Aberrant Behavior Checklist (ABC) (Bolyen et al., 2019). The ABC is a 58-item behavioral rating scale used to measure behavior problems across five subscales: irritability, lethargy / social withdrawal, stereotypy, hyperactivity / noncompliance, and inappropriate speech. A total score was calculated. We interviewed all subjects over the age of 5.

[0137] 3) Social Responsiveness Scale (SRS) (Constantino & Gruber, 2005) The SRS consists of 65 items used to quantitatively evaluate the severity of social behavior. A total score was calculated. We interviewed all subjects over the age of 5.

[0138] 4) Restricted Repetitive Behaviors (RRB) were based on a 4-point scale (0-3) adopted from the Gilliam Autism Rating Scale, Third Edition (GARS-3) (Gilliam, 2014). A total score was calculated. We interviewed all subjects over the age of 3.

[0139] Secondary outcomes:

[0140] 1. Fecal microbiota

[0141] 1) Sample handling and collection

[0142] Stool samples were collected at three study time points: pre-intervention (0 weeks), 6 weeks, and 12 weeks. Sample collection was performed using DNA / RNA shielded stool collection tubes (Zymo, Cat. No. R1101) containing 1 mL of preservation solution, transported to the laboratory by ice bags, and then frozen at -80°C. DNA was extracted using the TIANmap fecal DNA kit (TIANGEN, Cat. No. DP328) according to the manufacturer's instructions, and DNA samples were carefully quantified with a Nanodrop Spectrophotometer. The A260 / A280 ratio was also measured to confirm high purity DNA yield. DNA samples were frozen at -20°C until use.

[0143] 2) 16S rRNA gene amplicon sequencing

[0144] The 16S rRNA V3-V4 library was constructed by two rounds of PCR using the following primers, 341F: 5'TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGAGGCAGCAGCCTACGGGNBGCASCAG3' (SEQ ID NO: 1), and 805R: 5'GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGACTACNVGGGTATCTAATCC3' (SEQ ID NO: 2), with the following reaction sequence: 95°C for 2 min, followed by 25 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 5 min. PCR products were purified with 1x KAPA AMPure beads (KAPA, catalog no. KK8002). The products were then subjected to a second PCR reaction step (95°C for 2 min, followed by eight cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, with a final extension at 72°C for 5 min). PCR products were purified with 1× KAPA AMPure beads and analyzed using a Bioanalyzer DNA kit, followed by quantification by real-time PCR. DNA libraries were pooled and sequenced on an Illumina MiSeq (Illumina; CA) using a 2× 250 bp paired-end protocol with overlapping reads.

[0145] 2. The Clinical Global Impression (CGI) was developed for use in clinical trials to provide a brief, independent assessment of the clinician's view of the patient's overall functioning before and after starting medication in the trial. The CGI includes two accompanying one-item scales that assess: (a) severity of psychopathology on a scale of 1–7 (CGI-S) and (b) change from the start of treatment on a similar 7-point scale (CGI-I).

[0146] 3. GI symptoms were assessed based on the total number of existing GI symptoms at baseline, including constipation, diarrhea, abdominal pain, excessive flatulence, bloody stools, nausea, difficulty swallowing, anorexia, dyspepsia, and acid reflux.

[0147] statistical analysis

[0148] All raw data were recorded and processed in Microsoft Excel 2007 and R. Data presentation followed the CONSORT recommendations for reporting the results of randomized clinical trials (RCTs). Statistical procedures were performed using α = 0.05 as the significance level.

[0149] We applied Wilcoxon rank-sum tests to determine between-group differences in z-scores for weight, height, total scores and subscores of ASQ-3, ABC and SRS at baseline, subject-specific change from week 0 to week 6, and subject-specific change from week 6 to week 12. Linear mixed models were also used to account for repeated measures.

[0150] Because we had several primary outcomes, we used the false discovery rate (FDR) to adjust for multiple comparisons. Secondary outcomes were analyzed using methods similar to those for the primary outcomes. In addition, linear regression was performed to examine correlations between clinical indicators and microbiota composition.

[0151] Microbiota data processing and analysis

[0152] Sequencing reads were filtered using QIIME2 (v2019.10) based on quality scores (Bolyen et al., 2019). They were denoised using Deblur with default parameters to obtain abundance tables of samples by amplicon sequence variants (ASVs) (Amir et al., 2017).

[0153] Alpha diversity was calculated using QIIME2. Bray-Curtis distance was used to characterize microbiota beta diversity. ASV taxonomy was assigned using a sklearn-based taxonomy classifier trained on sequences from Greengenes v13.8 with a 99% similarity level. Significant differences in the relative abundance of microbial phyla, genera, and alpha diversity between the placebo and probiotic groups were identified by Kruskal-Wallis test. False positive rate (FDR) based on Benjamini-Hochberg (BH) adjustment was applied for multiple comparisons (Jiang et al., 2017).

[0154] PICSRUSt2 was used to infer microbial functional content based on ASV abundance tables, followed by the generation of Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologs (KO), enzyme classification numbers, and pathway abundance tables (Czech et al., 2020; Douglas et al., 2020). Differential analysis was performed on the fold ratios between the probiotic and placebo groups using permutation-based nonparametric tests, and the best differential features were rendered and plotted with Calour (Xu et al., 2019). All raw data from 16s rRNA Illumina amplicon sequencing have been deposited in The National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA, PRJNA643297).

[0155] result

[0156] 1. Demographic characteristics of PWS participants A total of 65 subjects with a genetically confirmed diagnosis of Prader-Willi syndrome were enrolled. Of these, 31 subjects aged 49.4±34.4 months were randomized to receive the active probiotic BB-11, whereas 34 subjects aged 55.5±41.9 months were randomized to receive placebo. Group-wise comparison of baseline age and gender distribution did not show any significant differences (P>0.05). Detailed demographic characteristics and comorbid GI symptoms of enrolled participants are summarized in Table 1. Overall severity as indicated by CGI-S scores at baseline compared between groups is shown in Figure 14. No group differences were noted (P>0.05). 47.5% of subjects have one or more GI symptoms in the study population, as shown in Table 2 below. [Table 2]

[0157] No serious or severe adverse events were observed. All observed adverse events and the main causes of dropouts are tabulated in Figure 15. No significant differences were found between the two groups (P>0.05).

[0158] 2. Effects of probiotics on weight, height, psychological measurements and CGI-I

[0159] Anthropometric measurements were collected and analyzed throughout the course of treatment. Height gain from 6 to 12 weeks was significantly greater in the probiotic group than in the placebo group (mean difference = 2.58 cm, P < 0.05, Figure 16A-C). No significant change in weight was observed over time in either group (Figure 16D-F).

[0160] The results obtained from the psychological measures including ASQ-3, ABC, SRS, and RRB are shown in Figure 17. No significant differences were found in the linear mixed effects model (P>0.05) for ASQ-3, ABC, SRS, and RRB scores.

[0161] Global improvement in symptoms over the course of treatment was measured using the CGI-I scale, and we found significantly greater symptom improvement in the probiotic group compared to the placebo group (Figure 18, P<0.05).

[0162] 3. Changes in microbiota composition and function by probiotic intervention

[0163] After sequencing, we obtained a total of 3,088,722 raw reads and an average of 49,818 reads per sample (range = 29,329-119,440 reads). The overall phylum- and genus-level variation in gut microbiota composition over the course of the intervention is shown in Figure 19 for both the probiotic and placebo groups.

[0164] Alpha diversity was slightly but significantly increased in the probiotic group compared to the placebo group after 6 weeks (Figures 20A-D). Beta diversity, analyzed by permutation multivariate ANOVA (PERMANOVA), showed significant separation among probiotic treatments (F statistic = 2.2526; R 2 = 0.035613; P < 0.05, NMDS stress = 0.19048, Figure 20E).

[0165] To characterize the changes in abundance of potentially clinically significant bacteria over the course of the intervention, we present the fold changes of several selected bacterial genera and families in Figure 21. The relative abundances of Lachnospiraceae ND3007, Ruminococcaceae UCG-003, Streptococcus mutans, Comamonadaceae, Alistipes, and Rothia showed a trend toward a decrease from baseline levels in the probiotic group at both 6 and 12 weeks (Figures 21A-F). Among such bacterial taxa, only Comamonadaceae showed a significant decrease among probiotic group subjects at 6 weeks compared to baseline levels (Figure 21D, P<0.05). In contrast, Bifidobacterium, Lactobacillus, and Prevotella 9 increased from baseline at 12 weeks in the probiotic group (Figures 21G-I). At the family level, similar trends were observed in both groups (Figure 22).

[0166] Functional gene prediction analysis showed that several genes had differential abundance in the probiotic group after the 12-week treatment period. Notably, genes encoding ubiquinone biosynthesis protein (ubiB, k03688), phytoene desaturase (EC:1.3.99.29), phytoene desaturase (lycopene formation) (EC:1.3.99.31) and all-trans-zeta-carotene desaturase (EC:1.3.99.26) were all upregulated, whereas genes encoding dimethylargininase (k01482) and acid phosphatase (phoN, k09474, EC:3.1.3.2) were downregulated (Figure 23). These findings do not meet the false discovery criteria for significance with multiple comparisons. Analysis results from predicted KEGG pathways shown in Figure 24 and predicted KOs shown in Figure 25 further compare gene expression between the probiotic and placebo groups.

[0167] 4. Correlation between gut microbiota abundance and clinical indicators

[0168] Clinical indicators were correlated with the abundance of bacterial genera. One correlation was found to be significant in the probiotic group, while no significant correlation was found in the placebo group. Specifically, a positive correlation was found between the RRB score and Rothia in the probiotic group at week 6 (Figure 26, R = 0.97, P < 0.005).

[0169] Consideration

[0170] In our 12-week randomized, double-blind, placebo-controlled study of 65 PWS patients, BL-11 increased the height of PWS subjects without changing their weight. During the treatment period, the probiotic group was found to have a significantly higher height gain than the placebo group (p<0.05). Other past probiotic interventions have failed to induce improvements in height (Onubi et al., 2015). This study presents novel evidence for the use of BL-11 as an early intervention in PWS patients. An intervention resulting in height gain in PWS may be most beneficial for patients in the early developmental stages and substantially improve long-term prognosis. Individuals with PWS are found to have absolute or functional growth hormone (GH) deficiency, and GH replacement is currently the most effective treatment for PWS (Bakker, Lindberg, Heissler, Wollmann, Camacho-Hubner, Hokken-Koelega, et al., 2017; Junli Zhu & Xuejun Kong, 2017). GH has been found to increase height as well as reduce body fat and improve cognitive, motor and mental function. Better efficacy and prognostic benefits have been observed with early initiation of GH treatment (Bakker, Lindberg, Heissler, Wollmann, Camacho-Hubner, & Hokken-Koelega, 2017). One study found that the probiotic L. reuteri could increase growth hormone levels in mice (Varian et al., 2018), highlighting a potential mechanism by which probiotics may increase height and treat PWS patients: promotion of endogenous growth hormone release. Our findings justify further investigation of the biological mechanisms of probiotics, a promising intervention for PWS that is better tolerated and easier to administer than GH supplementation (Onubi et al., 2015).

[0171] We did not observe significant weight loss during the intervention period, likely because most of our participants were under 5 years old, an age range in which obesity has not yet become a major problem. Interestingly, the changes in microbiota composition observed by us with B. lactis intervention have previously been associated with weight or fat loss (Amat-Bou et al., 2020; Barz et al., 2019; Carreras et al., 2018; Huo et al., 2020; Mekkes et al., 2014; Pedret et al., 2019a; Uusitupa et al., 2020b), improving fasting insulin sensitivity (Amat-Bou et al., 2020) and attenuating inflammation (Ibarra et al., 2018; Meng et al., 2017). Notably, we found a significant separation of gut microbiota β diversity between the probiotic and placebo groups after treatment. Baseline beta diversity is directly correlated with long-term weight loss when adherent to a controlled diet (Grembi et al., 2020). Thus, probiotic supplementation may have a preventive effect or promote diet-induced weight loss.

[0172] After administration of BL-11, we also noted a decrease in the abundance of several bacterial genera and species that are involved in the pathology of obesity and associated inflammation. Ruminococcaceae UCG-003, associated with VLDL and metabolic syndrome, is also involved in inflammatory bowel disease (Hall et al., 2017; Vojinovic et al., 2019). Lachnospiraceae ND3007 is associated with elevated cholesterol, signs of insulin resistance, and infantile obesity (Liang et al., 2020; Tun et al., 2018; J. Wang et al., 2020). Elevated Streptococcus is associated with inflammatory GI disorders, maternal inflammation, bacteremia, and antibiotic use during pregnancy (Iakovlev et al., 2020; N. Li et al., 2019). Rothia was found to have a higher abundance in gestational diabetes cohorts than in healthy pregnancy cohorts (Crusell et al., 2018). The family Comamonadaceae is generally considered to be pathogenic in humans (Willems, 2013).

[0173] Conversely, Bifidobacterium, Lactobacillus and Prevotella were each found to be significantly increased in the intestine after BL-11 treatment. Bifidobacterium, the genus to which interventional probiotics belong, is widely considered to be beneficial for gut health and weight loss (Alyousif et al., 2018; Barz et al., 2019; Carreras et al., 2018; Dimidi et al., 2019; Huo et al., 2020; Ibarra et al., 2018; S.-C. Li et al., 2019; Oliveira et al., 2017; Pedret et al., 2019a; Taipale et al., 2016; Uusitupa et al., 2020b). In addition to having a protective effect against weight gain in humans, Lactobacillus has been found to inhibit the activity of pro-inflammatory interleukins, which are associated with obesity and poor obesity-related outcomes (Ayyanna et al., 2018; Cox et al., 2015; Rosing et al., 2017b). The effect of Prevotella on the gut microbiota remains uncertain as evidence linking this genus to health benefits and disease has both been reported. Wang et al. (2019) reported that Prevotella-9 was found to be significantly decreased in both mice fed a high-fat diet, and Zeng et al. (2018) reported the same in women with PCOS who were insulin resistant (X.Wang et al., 2019; Zeng et al., 2019). Furthermore, Park et al. (2013) reported increased abundance of Prevotella in obese mice, and Kovatcheva-Datchary et al. (2015) found that dietary fiber-induced improvements in postprandial blood glucose and insulin were positively associated with Prevotella abundance (Kovatcheva-Datchary et al., 2015; Parks et al., 2013).On the other hand, one study found that the family Provetellaceae had a greater relative abundance in three obese patients compared to three normal weight patients (Zhang et al., 2009). Another study investigating the stool bacterial composition of HIV-positive patients found that Prevotella was positively correlated with BMI, although most participants in this study had a BMI in the normal range (Pinto-Cardoso et al., 2017). The conflicting findings regarding Prevotella in gut health and obesity may indicate the importance of balancing the abundance of this genus in the microbiota.

[0174] Furthermore, by using predictive functional gene analysis, we found an enhancement of antioxidant production-related pathways that exert anti-inflammatory and anti-obesity effects. The gene encoding the ubiquinone biosynthesis protein (ubiB, k03688), responsible for the biosynthesis of ubiquinone (CoQ10), was found to be increased in abundance after probiotic treatment. CoQ10 supplementation may be useful for the treatment of various chronic cardiovascular, inflammatory and obesity-related diseases (Zozina et al., 2018). We also found increased abundance of genes encoding phytoene desaturase (EC:1.3.99.29), phytoene desaturase (lycopene formation), (EC:1.3.99.31), and all-trans-zeta-carotene desaturase (EC:1.3.99.26), all of which contribute to carotenoid biosynthesis and have previously been found to have beneficial effects against obesity and obesity-related pathologies (Mounien et al., 2019; Paes-Silva et al., 2019; Wiese et al., 2019). We also found downregulation of two enzymes, dimethylargininase (k01482) and acid phosphatase (phoN, k09474, EC:3.1.3.2), which are associated with the development of obesity and elevated cholesterol and triglyceride levels in human patients (Arlouskaya et al., 2019; Bottini et al., 2002; Lang et al., 2011).

[0175] Taken together, microbiota compositional data and predictive functional gene analyses indicate that the segregation of diversity caused by treatment with BL-11 probiotic favors protection against obesity and obesity-related pathologies.

[0176] We found no significant changes in psychological measures (ASQ-3, ABC, SRS, and RRB), but the CGI-I showed a significant overall improvement in the probiotic group after the treatment period compared with the placebo group (P<0.05).

[0177] Interestingly, we found that the RRB score was positively correlated with Rothia at the genus level (P<0.005). RRB is one of the core symptoms of ASD, which has been reported in as many as 25-40% of PWS cases (Bennett et al., 2015; Salehi et al., 2018). In addition to being associated with diabetes (Crusell et al., 2018), Rothia has been reported to be more common in children with ASD than typically developing children (12.2-fold change; FDR, P<0.05) (Forsyth et al., 2020). Although the mechanism by which BL-11 improves the clinical impression of PWS patients is unclear, the correlation found between Rothia and RRB indicates that BL-11 may regulate signaling in the gut-brain axis. Further investigation of Rothia and other microbiota markers may reveal powerful and actionable targets for neuropsychiatric therapy.

[0178] Our randomized trial showed that treatment with a probiotic B. Lactis strain (BL-11) for 12 weeks significantly increased height, a novel finding with important implications for the early treatment of PWS. Probiotic treatment also improved overall clinical symptoms as indicated by the CGI-I and altered the composition and function of the microbiota in an anti-obesity favorable manner. There are several limitations of the study that are worth considering. First, although we employed an appropriate recruitment and retention strategy, enrollment and retention of PWS participants in this study was difficult, the sample size was relatively small, and further subgroup analyses were limited. Second, although there were no statistical differences in clinical indicators between the probiotic and placebo groups at baseline, the wide age range used in this study resulted in a high heterogeneity of the subject population and potentially variable treatment efficacy. Third, the assessment of the fecal microbiota was not controlled for dietary habits that may affect microbial abundance at the individual level. Thus, future studies with larger sample sizes, improved control for environmental factors, and subgroup stratification are justified. Due to the limitations of the study listed above, further studies are justified to investigate the mechanisms and efficacy of BL-11 probiotic treatment in PWS.

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[0180] In the foregoing description, it will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein may be suitably implemented in the absence of any element or elements, or any limitation not specifically disclosed herein. The terms and expressions used are used as terms of description rather than limitation, and in using such terms and expressions, it is not intended to exclude any equivalents of the features shown and described or any portion thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, although the invention has been illustrated by specific embodiments and optional features, it should be understood that modifications and / or variations of the concepts disclosed herein may be made by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention.

[0181] In this specification, a number of patent and non-patent literature are cited. The cited references are incorporated herein by reference in their entirety. If there is a discrepancy in the definition of a term in this specification compared to the definition of the term in the cited reference, the term shall be interpreted based on the definition in this specification.

Claims

1. Use of an effective amount of probiotics for the treatment of a person diagnosed with or at risk of Prader-Willi syndrome (PWS), wherein the probiotics comprises Lactobacillus reuteri LR-99 (L. reuteri LR-99) and / or Bifidobacterium animalis subsp. lacticis BL-11 (B. lacticis BL-11).

2. The use according to claim 1, further comprising one or more of the following: additional Lactobacillus sp., Saccharomyces sp., additional Bifidobacterium sp., Bacillus sp., and Eubacterium hallii.

3. The use according to claim 1 or 2, wherein the probiotics comprises L. reuteri LR-99 and B. lactis BL-11.

4. The use according to claim 1, wherein the subject suffers from one or more of the following: obesity, short stature, social deficit, fine motor impairment, developmental delay, and abnormal behavioral characteristics, and after treatment, the subject's symptoms or condition are reduced compared to before treatment.

5. The use according to claim 4, wherein the developmental delay includes one or more of the following: communication, gross motor control, fine motor control, problem solving, and interpersonal social interaction.

6. The use according to claim 4, wherein the abnormal behavioral characteristics include one or more of restrictive repetitive behaviors (RRB), abnormal social interactions (SI), abnormal social communication (SC), abnormal emotional responses (ER), abnormal cognitive styles (CS), and maladaptive speech (MS).

7. The use according to claim 4, wherein the subject suffers from obesity and / or short stature, and the subject's body mass index (BMI) after treatment is lower than the subject's BMI before treatment with L. reuteri LR-99, and / or the subject's height after treatment is higher than the subject's height before treatment with B. lactis BL-11.

8. The use according to claim 4, wherein the subject shows improvement in psychopathological severity as measured by the Clinical Global Impression Improvement (CGI-I) and / or Clinical Global Impression Severity (CGI-S) after treatment with B. lactis BL-11.

9. The use according to claim 5, wherein the subject suffers from developmental delay, and after treatment with L. reuteri LR-99, the subject's Ages and Stages Questionnaires, 3rd Edition (ASQ-3) score is statistically improved compared to the pre-treatment ASQ-3 score in one or more of the following: communication, gross motor function, fine motor function, problem solving, and post-treatment interpersonal social interaction.

10. The use according to claim 6, wherein the subject suffers from abnormal behavioral characteristics, and after treatment with L. reuteri LR-99, the subject's Gilliam Autism Rating Scale, Third Edition (GARS-3) score is statistically improved for one or more of the RRB, SI, SC, ER, CS, and MS compared to the pre-treatment GARS-3 score.

11. The use according to claim 1, comprising administering an effective amount of probiotics once, twice, three times, or four times a day.

12. The use according to claim 1, comprising administering an effective amount of probiotics for at least about 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or at least about 12 weeks.

13. The use according to claim 1, wherein the effective amount comprises approximately 1 × 10⁵ to approximately 1 × 10¹⁵, approximately 1 × 10⁶ to approximately 1 × 10¹⁴, approximately 1 × 10⁷ to approximately 1 × 10¹³, approximately 1 × 10³ to approximately 1 × 10¹², approximately 1 × 10⁹ to approximately 1 × 10¹, approximately 1 × 10¹⁰ to approximately 9 × 10¹⁰, or approximately 3 × 10¹⁰.

14. The use according to claim 1, further administered together with one or more additional therapeutic agents.

15. The use according to claim 1, wherein the probiotic is administered twice daily for 12 weeks at a dose of about 3 × 10³ CFU, and after treatment, shows a statistically relevant improvement in one or more of BMI, fine motor function, and problem-solving ability as measured by the ASQ-3 test.

16. The use according to claim 1, wherein the composition of the target microbiome is different after treatment compared to before treatment.

17. The use according to claim 16, wherein the difference between before and after treatment includes a decrease in one or more of Escherichia-Shigella, Porphyromonas, and Ruminococcus torques after treatment with L. reuteri LR-99.

18. The use according to claim 16 or 17, wherein the difference between before and after treatment includes an increase in one or more of Bifidobacterium, Lactobacillus, Faecalibacteria, Roseburia, and Alistipes after treatment with L. reuteri LR-99.

19. The use according to claim 16, wherein the difference between pre-treatment and post-treatment includes a significant positive association of Rothia with RRB.

20. A composition comprising an effective amount of probiotics and growth hormone, wherein the probiotics comprises one or more of Lactobacillus reuteri LR-99 (L. reuteri LR-99) and Bifidobacterium animalis subsp. lactis BL-11 (B. lactis BL-11).

21. The composition according to claim 20, wherein the growth hormone is human growth hormone.

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