Herpetospermum pedunculosum (Ser.) Baill is annual scandent herbs. They are used in the treatment of piles, inflammation of the stomach and the intestines. It can survive the extreme environment of the Tibetan Plateau (TP). However, the underlying mechanisms of this adaptation to H. pedunculosum from TP remain unclear. Here, we combined physiological and metabolomics methods to analyze H. pedunculosum response to altitude gradient differences.H. pedunculosum. In addition, a metabolic network was constructed based on metabolomic datasets using a weighted correlation network analysis (WGCNA) approach. The network analysis uncovered 4 distinguished metabolic modules highly associated with I, II, III and IV respectively. Furthermore, the analysis successfully classified 50 samples into seven groups: carbohydrates, amino acids, organic acids, lipid components, polyamine, secondary metabolism and others.At high altitude, increases in the activities of Ascorbate peroxidase (APX), Glutathione reductase (GR), Dehydroascorbate reductase (DHAR), Monodehydroascorbate reductase (MDHAR), Superoxide dismutase (SOD) have been observed in leaves. Total Glutathion content, total Ascorbate content and the ASA (ascorbic acid)/docosahexaenoic acid (DHA) ration were highly elevated from low altitude to high altitude. In addition, high altitude induces decrease of the Anthocyanidin content (ANTH) and increase of abscisic acid content (ABA). The GC-MS analyses identified of 50 metabolites from leaves of H. pedunculosum response to high altitude. The results of this study laid the foundation of the molecular mechanism on H. pedunculosum from high altitude.In the present study, the content of parts of amino acid components increased in samples collected at higher altitudes, and most of metabolites, including carbohydrates and organic acids were assigned to the carbon metabolic pathway comprising reductive pentose phosphate pathway, glycolysis and TCA cycle, indicating the direct relationship between adaptability and the carbon metabolic pathway and amino acids in The online version of this article (10.1186/s12864-019-5778-y) contains supplementary material, which is available to authorized users. Herpetospermum pedunculosum (Ser.) Baill is key cucumber species distributing in the Tibetan plateau of China. As annual herbs, it can survive in the Tibetan Plateau (TP) extreme environment, including violent winds, low temperatures, drought and low oxygen concentration [H. pedunculosum to TP has been unclear. H. pedunculosum is also distributed in southwest China, Nepal, and northeast India [H. pedunculosum as a natural medicine for liver disease [Herpetospermum pedunculosum against carbon tetrachloride-induced liver injury in rats [ntration . Therefontration . Howeverst India , 4. In Tst India . As a drst India . The lat disease . Researc in rats . HoweverRhododendron (Ericaceae) size and the wings of seeds become smaller at higher altitudes than at lower altitudes [P. saundersiana has multiple strategies to adapt to the high-altitude environment of the Northwestern TP [H. pedunculosum from TP remain unclear.Increasing evidence supported that altitude gradients are a useful environmental factors to investigate ecological and evolutionary responses of plants to geographic conditions , 9. Diffltitudes . P. saunstern TP . Resproustern TP . TherefoH. pedunculosum in response to a different altitude. However, comparative metabolomics methods have not been extensively used to study H. pedunculosum plant ecological adaptation. Therefore, the underlying physiological and molecular mechanisms of this adaptation of H. pedunculosum to the extremely Tibetan environment remain unclear.Metabolomics is a powerful tool to study the dynamic of small molecule metabolites. With advances in mass spectrometry, metabolomics have been used in ecological investigations and measurement the impact of factors such as climate change, disease and infection in plants . In geneH. pedunculosum response to altitude gradient differences. Based on these results, we hypothesized that the H. pedunculosum plants use multiple strategies to acclimate to environmental stress from TP. The results of this study lay the foundation for H. pedunculosum research on the molecular mechanism of high altitude.Here, we combined physiological and metabolomics methods to analyze The phenotypes of seeds and leaves from different samples at different altitudes were observed and compared Fig. . There i2O2 content was found to show decrease with increased altitude, but higher content of O2− and MDA was found to be produced in high altitude  and chlorophyll fluorescence of H. pedunculosum. we measured the dynamic Pn, Gs, Ci, Tr and the ratio of variable and maximal fluorescence (Fv/Fm)  compared with the samples from low and high altitude (2800 m and 3300 m). In addition, Ci was decreased with increased altitude. Tr and Fv/Fm was not significantly changed between different altitudes. Additionally, the anthocyanidin content (ANTH) decreased and abscisic acid content (ABA) increased with elevated altitude  for samples collected at different altitudes. After Orthogonal projection to latent structures discriminant analysis (OPLS-DA) was conducted to characterize changes in metabolite concentrations according to both identified and unidentified metabolites. OPLS-DA modeling could separate all samples into four distinct groups  exhibited increases in abundance in high altitude   approach was used to identify metabolic modules related to a different altitude by the GC-MS data. Thus, networks were constructed based on all identified metabolites. Accordingly, all the correlated metabolites were grouped into 4 distinct metabolic modules  Fig. . Among tP. saundersiana and A. alpina decreased at high altitude [A. annua plants had a minor promoting effect on more numerous glandular trichomes [H. pedunculosum at high altitude.To understand the mechanical properties in response to high altitude, the stomatal size and stomatal density in leaf epidermis was measured. The pollutant ozone and pathogenic microbes can result in stomatal closure and thereby prevent damaging entry to the leave –17. As saltitude . Howeverrichomes . In contrichomes . Thus, i2O2 and O2·- can result in subsequent oxidative stress, which can lead to reduction in photosynthetic CO2, pigment loss and decreased protein [2O2 and O2·- varied slightly with elevated altitud. These observations confirmed that H. pedunculosum plants at high altitude suffer less oxidative stress, which is potentially linked to plant adaptation and tolerance to biotic and abiotic stressed in Tibet. However, there results were not consistent with the finding for an indicator of membrane lipid peroxidation (MDA). Additionally, previous research found that increased altitude resulted in marked increases in H2O2 and O2·- in plant Lamiophlomis rotata and Populus kangdingensis [H. pedunculosum at high altitude can take advantage of various antioxidative defense systems to defense oxidative stress.Overproduction of ROS such as H protein . In Fig.ingensis , 22. TheIn Fig. H. pedunculosum at high altitude all showed decreased tendency with elevated altitude. As shown in Fig. H. pedunculosum to high altitude. Our conclusion is consist with the study of Habibi et al. [Pn and Gs were lower at low altitude (2800 m), then increased at moderate altitude (3000 m and 3100 m), and last decreased at high altitude (3300 m). Parts of results are in agreement with previous reports alpine Phrynocephalus erythrurus [P. saundersiana [2 concentration (Ci) exhibited low levels with increased altitude, which would affect photosynthetic activity. Indeed, our metabolomic analysis showed the content of ribulose-5-phosphate was decreased. As an important component, maintenance of ribulose-5-phosphate was linked to photoprotection under stress [The energy required for biosynthesis is solely generated from respiration in plants . Thus, ti et al. . Therefoaltitude 00 m, ther stress . This phExtensive studies have also revealed that plants can adapt to different environmental stressed by ABA regulation –33. TherH. pedunculosum. Generally, primary metabolites are directly involved in plant growth and development. Amino acids also play a role in the defense mechanisms and stress responses of plants [H. pedunculosum, which were indicated in Additional file H. pedunculosum. However, further studies will be needed to clarify the functions of these amino acids in high altitude tolerance. By contrast, the levels of some amino acids such as oxoproline, carbamoyl-aspartic acid and 5-hydroxytryptophan were reduced in plant samples collected at high altitudes. The reductions in these amino acids may contribute to the production of new essential stress proteins or repair damaged or misfolded proteins to facilitate H. pedunculosum for acclimation high altitude. Generally, for central role in nitrogen metabolism, biosynthesis, degradation, and transport of amino acid are tightly regulated to meet demand to nitrogen and carbon availability [H. pedunculosum from high altitude, as this different accumulation of these amino acids under high altitude is energetically more efficient when the biosynthesis and degradation of amino acids occur in leaves simultaneously. However, the metabolism, transport, and stress signal integration of these amino acids can influence each other in an unpredictable pattern up to now.In the study, we firstly reported that metabolomic involvement in response to altitude in f plants –39. The lability . Or thesThe dose-dependent elevation in amino acid levels suggest that it is more accumulated than other metabolites, and their accumulation is a more sensitive response to altitude changes. Among all identified metabolites from different altitudes, four secondary metabolites also showed increased level. An extreme metabolite was varied in this study. For instance, tricetin exhibited the largest fold-change (83.85 ×). In plants, the flavones tricetin is relatively rare and was only reported to occur in the myrtaceae pollen . HoweverH. pedunculosum response to high altitude. Physiologically, some of these metabolites can play a role act as osmolytes in cytoplasm under extreme environment in Tibet, resulting in an increase of osmotic potential of the cytoplasm in leaf cells to maintain appropriate cell status, while the accumulation of organic acids may contribute to keeping ionic balance [The metabolic profile analyses demonstrated that the high altitude of resulted in substantial Among them, tricarboxylic acid (TCA) cycle intermediates supporting diverse functions including defense, developmental stages and tissue types in plants , 50. Int balance .H. pedunculosum to Tibetan environment is completely clear. Therefore, we should synthetically consider these factors in the next work.Although much is known about the regulation mechanism of biosynthesis pathways by metabolites themselves, it should be pointed out that only parts of metabolites were measured in this study. Therefore, the regulation mechanisms at the transcriptional, post-transcriptional, and protein levels to more metabolites need to be identified. In addition, it is somewhat regrettable that we can′t present the detailed data including temperature, rainfall and soil because of the special geographical environment. Accordingly, the relationship between environment and adaptability of H. pedunculosum can survive harsh condition in the Tibetan Plateau, however the adaptation mechanism to adverse environment remains unknown. To gain a comprehensive physiological and biochemical and metabolomic understanding of H. pedunculosum, we compared physiological and comparative metabolomic traits of different altitudes H. pedunculosum. Differences in seed and leaf phenotypes at different altitudes. H2O2, O2−, MDA, SOD, APX, GR, DHAR, MDHAR, GSSG, AsA content was found to show increase with increased altitude. In addition, high altitude was found to cause a decrease in the total respiratory rate, at respiration rate, cyt respiration rate and residual respiration rate.To date, H. pedunculosum response to high altitude.Most of the metabolic species identified by metabolomics are carbohydrates, amino acids and organic acids. To obtain a detailed and clear overview on the abundance of differences, we have drawn metabolic pathways Figs. H. pedunculosum are collected in August 2016 Gangdese Mountains  near Bayi District, in central Tibet Autonomous Region, China.The experiments were carried out at the Harbin Northeast Forestry University Genetic laboratory. The leaf and seeds of the The surface texture observation of 30 individual seed at each altitude randomly collected by digital camera . And leaves in seedlings were also investigated by photograph. Three exomorphic parameters including seed length, seed width, seed thickness were detected and thousand of seed weight was also calculated , the randomly selected leaves were from plants at the same and different altitudes, washed double-distilled water and were fixed in formaldehyde. The sample was dehydrated with ethanol  and then sprayed with gold. Then, observations and photographs were obtained using a SEM . The stomatal shape and size including stomatal length, stomatal density and trichomes density was calculated with Image J 1.4.7 software .2O2) was detected spectrophotometrically according to PV Sergiev et al. [g for 10 min the supernatant was aspirated. 0.5 ml PBS and 1 ml potassium iodide were added to the 0.5 ml extracted solution for dark reaction for 5 min. The absorbance of the mixture was measured at 390 mm and H2O2 concentration was obtained according to the standard curve of hydrogen peroxide.Hydrogen peroxide  and 0.5 ml hydroxylamine hydrochloride (10 mM) were added to 1 ml extracted solution and keep 25 °C for 0.5 h. Add developer 1 ml α-naphthylamine to the mixture and dark reaction for 15 min. The absorbance of the mixture was measured at 530 mm and O2• − generation rate was obtained according to the standard curve of sodium nitrite.0.5 g fresh leaves are ground into powder in liquid nitrogen and added into PBS buffer, after centrifugation at 6, 000×g for 10 min the supernatant was aspirated. Add 2 ml TBA (0.5%) to 1 ml extracted solution and then boil in water for 15 min and centrifuged at 12, 000×g for 5 min. Absorbance was measured at 450, 532 and 600 nm. Statistical analysis of measurement results by SPSS software.MDA content was estimated by the method of Wang et al. . 1.5 g fg for 10 min the supernatant was aspirated. The extracted solution of 60 μl was added with 0.2 ml MET, 0.2 ml EDTA, 0.2 ml NBT and 0.3 ml riboflavin reaction for 15 min. The absorbance of the mixture was measured at 560 mm. Ascorbate peroxidase  activity was measured by the method of Y, Nakano and K Asada [2O2. The absorbance of the mixture was measured at 290 mm. Glutathione reductase  was measured following the method of Carlberg [Superoxide dismutase  activity was determined according to the method of Beauchamp and Fridovich . PeroxidCarlberg . The extCarlberg . MonodehCarlberg . StatistThe content of total ascorbic acid (AsA + DHA) was determined following the method of Kampfenkel et al. . The absTotal glutathione  were determined following the method of Edwards et al. . The absGs), maximum efficiency of PSII (Fv/Fm), Net photosynthetic rate (Pn) and intercellular CO2 (Ci)) of leaves at 25 °C, photon f1ux density (PFD) 900 μmol m− 2 s− 1 [At 9:30 am Li-Cor 6400 portable photosynthesis measuring systems  was used to measure the gas exchange parameters . After the total respiration rate, SHAM-resistant respiration (Cyt pathway) rate and KCN-resistant respiration  were measured.The Leaf respiration rate was determined following the method of Tomaz et al. . 1 cm2 l0.5 g frozen samples were ground into powder by liquid nitrogen and added into buffer  ice water bath for 4 h and the supernatants were obtained by centrifugation for 15 min . The supernatant was separated by C18 columns to obtain the hormone. Endogenous MeJA level was determined following the method of Bai et al. . The totN,O-bis (trimethylsilyl) -trifluoroaceta by mid (BSTFA) and 1% TMCS), GS-MS analysis for metabolitic analysis.The metabolites from each sample were extracted by the methanol/chloroform method. 60 mg leaves were ground into powder by liquid nitrogen and added into extract buffer. Then the homogenate was vortexed and sonicated for 30 min, twice and centrifuged . 700 μL supernatant was air-dried, added by 80 μL pyridine amine hydrochloride (15 mg/ml) and vortexed for 1.5 h at 37 °C. Then the mixture was added buffer  with non-polar DB-5 capillary column . The mass spectrometer was set to scan a mass range of 50–600 ron beam .HROMO TOF 4.34 (LECO Corp.) software package. Metabolites  were identified based on available reference standards in our lab and the NIST 11 mass spectra library  and the Fiehn library linked to CHROMO TOF 4.34 software. Identification required a minimum forward match percentage of 70% or higher [All unprocessed data from GC-MS were analyzed by Cr higher .www.genome.jp/kegg) database to identify metabolic pathways influenced by altitude. All metabolic correlation networks were carried out by the standard procedure of WGCNA [The SIMCA-P + 14.0 software package analyzes the data set generated from GC-MS. Orthogonal projections to latent structure discriminant analysis (OPLS-DA) were carried out to visualize the metabolic alterations among experimental groups. Heat map was used to visualize metabolite response. Metabolites  annotated to metabolic pathway using the KEGG , Stomatal length (B), Seed width (C), Stomatal density (D), Seed thickness (E) and Trichomes density (D) of Herpetospermum pedunculosum (Ser.) Baill. (DOC 1830 kb)Additional file 2:Table S1. Effects of altitude gradient on net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci), transpiration (Tr) and the maximum efficiency of PSII (Fv/Fm) in leaves of Herpetospermum pedunculosum (Ser.) Baill. (DOCX 1630 kb)Additional file 3:Figure S2. Orthogonal projections to latent structures discriminant analysis (OPLS-DA) score scatter plot derived from the GC-MS data set for leaves of Herpetospermum pedunculosum (Ser.) Baill. (DOCX 98 kb)Additional file 4:Table S2. The relative contents of the identified metabolites in leaves of Herpetospermum pedunculosum (Ser.) Baill. (DOC 97 kb)Additional file 5:Table S3. on metabolites identified in Herpetospermum caudigerum Wall., showing their molecular masses, KEGG compound codes, suggested metabolic functions, and the corresponding KEGG pathway codes. (XLS 31 kb)Additional file 6:Figure S3. Changes in metabolite concentrations in leaves of Herpetospermum caudigerum Wall. at four altitudes including 2800 m, 3000 m, 3100 m and 3300 m . B/A: The ratio of the B concentration to the A concentration is shown by red colored bars. (DOC 1120 kb)Additional file 7:Figure S4. Classification of metabolites according to different altitudes. (DOCX 100 kb)Additional file 8:Table S4–1. All of the metabolites identified in leaves of Herpetospermum pedunculosum (Ser.) Baill. Table S4–2. The difference of metabolites in Herpetospermum pedunculosum (Ser.) Baill leaves between 3000 m and 2800 m altitude. Table S4–3. The difference of metabolites in Herpetospermum pedunculosum (Ser.) Baill leaves between 3100 m and 2800 m altitude. Table S4–3. The difference of metabolites in Herpetospermum pedunculosum (Ser.) Baill leaves between 3300 m and 2800 m altitude. (XLSX 336 kb)


Apparent treatment-resistant hypertension (aTRH) is a common condition associated with risk of cardiovascular events. However, the risk of cardiovascular mortality associated with aTRH in the US population is unknown. We aimed to assess the risk of cardiovascular disease (CVD) mortality associated with aTRH in the US population.We analyzed data from 6357 adult hypertensive participants of the National Health and Nutrition Examination Survey (1988–1994 and 1999–2010) linked to the National Death Index. Based on presence of uncontrolled hypertension [blood pressure (BP) ≥140/90 mmHg] and the number of antihypertensives prescribed, we classified participants into the following groups: non-aTRH (BP < 140/90 mmHg and ≤ 3 antihypertensives); controlled aTRH (BP < 140/90 mmHg and ≥ 4 antihypertensives); and uncontrolled aTRH (BP ≥140/90 mmHg and ≥ 3 antihypertensives).p < 0.001). In fully adjusted models, aTRH was associated with a 47% higher risk of CVD mortality compared with the non-aTRH group [1.47 (1.1–1.96)]. Similar increase in risk of CVD mortality was noted across aTRH subgroups compared with the non-aTRH group: controlled aTRH [1.66 (1.03–2.68)] and uncontrolled aTRH [1.43 (1.05–1.94)]. Among non-aTRH subgroups, those on 3 antihypertensive medications had a 35% increased risk of CVD mortality than those on < 3 medications [1.35 (0.98–1.86)].Of the 6357 participants, 1522 had aTRH, representing a US prevalence of 7.6 million. Of the participants with aTRH, 432 had controlled aTRH and 1090 had uncontrolled aTRH. During follow-up (median 6 years), there were 550 CVD deaths. The cumulative incidence of CVD mortality was significantly higher in the aTRH group compared with non-aTRH group  or ≥ 4 medications (controlled aTRH), groups not generally considered at high risk. Future risk reduction interventions should consider focusing on these high-risk groups.The online version of this article (10.1186/s12882-019-1315-0) contains supplementary material, which is available to authorized users. Question: What is the risk of cardiovascular disease (CVD) mortality associated with apparent treatment resistant hypertension (aTRH) in the US population?Findings: In this retrospective case-control study that included 6357 adults, aTRH was associated with a 47% higher risk of cardiovascular mortality compared with the non-aTRH group. aTRH has a US prevalence of 7.6 million people.Meaning: Regardless of BP control, people with aTRH remain at a higher risk of cardiovascular outcomes; future risk reduction interventions should consider focusing on these high-risk groups.Hypertension affects 29% of the US adult population, corresponding to approximately 90 million Americans . HyperteApparent treatment-resistant hypertension (aTRH) is defined as hypertension that remains uncontrolled (≥140/≥90 mmHg) despite treatment with ≥3 antihypertensive medications or hypertension that is controlled (< 140/90 mmHg) on ≥4 antihypertensives . In compThe goal of our study was to assess the risk of cardiovascular mortality amongst individuals with aTRH in the US using data from the National Health and Nutrition Examination Survey. Furthermore, we sought to compare the risk of cardiovascular mortality amongst aTRH subgroups, stratified by BP control.The NHANES are cross-sectional, multistage, stratified, clustered probability sample of the noninstitutionalized US civilian population conducted by the National Center for Health Statistics (NCHS), a branch of the Center for Disease Control and Prevention. NHANES III was conducted from 1988 to 1994 and the continuous NHANES were conducted from 1999 to 2010 with data released in 2 year cycles. We excluded participants that were < 18 years of age, had no recorded BP measurements, no follow-up, no antihypertensive use, and no known diagnosis of hypertension  treated with ≤3 medications. We further subdivided this group into those on 3 antihypertensives and those on < 3 antihypertensives.Controlled aTRH: Hypertensive participants with controlled hypertension (BP < 140/< 90 mmHg) treated with ≥4 antihypertensives.Uncontrolled aTRH: Hypertensive participants with uncontrolled hypertension (BP ≥140/90 mmHg) treated with ≥3 antihypertensives.BP was measured after the participant had been resting in a seated position for 5 min with feet on the floor. Three consecutive BP readings were obtained and reported as the mean of all systolic readings over the mean of all diastolic readings . Antihyp2 and modeled as a linear spline with knots at 18 kg/m2 and 25 kg/m2. Serum creatinine was measured using a kinetic rate Jaffe method and used to calculate estimated glomerular filtration rate (eGFR) . In the fully adjusted model (Model 4), there was no significant increased risk of all-cause mortality of the aTRH group in comparison to the non-aTRH group [1.15 (0.97–1.36)]  is suggested as a significant value. Using this threshold, there were no significant differences within subgroups.Pre-specified subgroup analyses are presented in Table In this U.S. nationally-representative study, we found that 7.6 million US adults with diagnosed and treated hypertension had aTRH, which was associated with 47% higher risk of cardiovascular mortality compared to people without aTRH. Those with controlled aTRH and uncontrolled aTRH had 66 and 43% higher risk of cardiovascular death, compared with the non-aTRH group. Among participants without aTRH, those on 3 antihypertensive medications had a trend toward higher risk of cardiovascular mortality than those on < 3 antihypertensive medications. Our findings suggest that patients with aTRH and non-aTRH requiring 3 medications for BP control represent high cardiovascular disease risk groups and should be considered as targets for risk reduction interventions.Hypertension increases the risk of coronary artery disease, myocardial infarction, stroke, accelerated atherosclerosis, congestive heart failure, and ESRD . While mOver the past three decades, significant efforts have been focused on improving awareness, treatment, and control of hypertension. According to Egan et al., the percentage of hypertensive patients with uncontrolled hypertension decreased from 73.2% in 1988 to 52.5% in 2008 [In prior studies, aTRH was found to be associated with higher risk of ESRD, ischemic cardiac events, congestive heart failure, and stroke in comparison to patients with non-resistant hypertension , 17. ImpAlthough the pathophysiology of aTRH is not well-understood, it is known that patients with aTRH have higher levels of brain-type and natriuretic peptides as well as aldosterone . Additio2) as compared to the non-aTRH group (82 ml/min|1.73 m2). Clinical implication of these findings is that in patients with aTRH (whether controlled or uncontrolled) careful attention must be paid to reducing cardiovascular risk and preserving kidney function. From a population health perspective, aTRH identifies a patient population at 2.5-fold higher risk of cardiovascular mortality. This high-risk population can be easily identified using electronic health records, and population health management strategies could target this population for focused interventions such as lifestyle modification, and use of medications with cardioprotective and renoprotective effects.The observed associations between aTRH and cardiovascular mortality in our study provides some interesting insights Table . In the Several limitations of our study deserve mention. First, we only had participant reported prescriptions and did not have information on prescription adherence which limits our ability to differentiate between true treatment-resistant hypertension and uncontrolled hypertension as the result of medication noncompliance . Second,In conclusion, in the US population, aTRH is common with 7.6 million affected adults and is associated with higher risk of cardiovascular disease mortality. In particular, the risk of cardiovascular disease mortality remains high among those with controlled BP on 3 medications (non-aTRH) or ≥ 4 medications (controlled aTRH), groups not generally considered at high risk. Future risk reduction interventions should consider focusing on these high-risk groups.Additional file 1:Table S1. Cumulative Incidence of All-Cause Mortality among NHANES participants with aTRH and controlled hypertension. (DOCX 16 kb)


Many patients with primary immunodeficiency (PID) require immunoglobulin G (IgG) replacement therapy, delivered as intravenous IgG (IVIG) or subcutaneous IgG (SCIG). We aim to identify trends in efficacy and safety that would not be evident in individual studies of small patient numbers. Seven open-label, Phase 3, prospective, multicenter studies of the efficacy and safety of Hizentra® (a SCIG), conducted in Japan, Europe, and the US were summarized. Overall, 125 unique patients received 15,013 weekly infusions during a total observation period of 250.9 patient-years. Mean weekly doses of Hizentra® were 83.22–221.3 mg/kg body weight; infusion rates per patient  were 25.2–49.3 mL/h across studies. The rates of infections and serious bacterial infections were 3.10 and 0.03 events per patient/year, respectively. Annualized rates of days hospitalized due to infection, out of work/school, and prophylactic antibiotic use were 0.95, 5.14, and 36.78 per patient, respectively. For the equivalent monthly dose, weekly Hizentra® SCIG administration resulted in expectedly-increased serum IgG trough levels in patients switching from IVIG, and maintained levels in patients switching from previous SCIG. Adverse events (AEs) totaled 5039 (events/infusion 0.094–0.773), almost all of which were mild/moderate. Three thousand one hundred ninety-seven were considered treatment-related, the most common of which were injection site reactions . Systemic AEs were very uncommon. The results from these seven studies indicate that Hizentra® therapy was both efficacious and well tolerated during long-term treatment. This is particularly important in patients with PID, who may require lifelong IgG replacement therapy.The online version of this article (10.1007/s10875-018-0560-5) contains supplementary material, which is available to authorized users. The majority of patients with primary immunodeficiency (PID), including common variable immunodeficiency (CVID) and X-linked agammaglobulinemia (XLA), require immunoglobulin G (IgG) replacement therapy –3. IntraSCIG and IVIG are equally effective , 6; howeHizentra®  was the first 20% liquid IgG product approved for SCIG administration. The high IgG concentration permits administration of smaller volumes, while the relatively low viscosity facilitates high infusion rates , 12. FivData were reviewed from seven open-label, Phase 3, prospective, multicenter studies as follows: (1) Japan  or 3 months ; the European pivotal study included patients administered IVIG at 3–4 weekly intervals or SCIG at regular weekly intervals, both for at least 6 months. The patient ages ranged from 2 to 75 years , 2 to ≤ 65 years , and 16–65 (UK sites within the European study).Major exclusion criteria included the following: (1) newly diagnosed PID ; (2) serious bacterial infection (SBI) at the time of screening or first infusion; (3) lymphoreticular malignancies including chronic lymphocytic leukemia, non-Hodgkin’s lymphoma, or thymoma with immunodeficiency; (4) a positive PCR result at screening for any of the following viral markers: human immunodeficiency virus, hepatitis C virus, or hepatitis B virus.The duration of each of the seven studies is shown in Fig. The majority of doses were administered at home following patient self-infusion or their caregiver’s infusion technique training. During site visits, infusions were performed under the supervision of study site personnel. In the Japanese and European pivotal studies, Hizentra® dosages were equivalent to those received during their previous treatment regimen . In the US pivotal study, a conversion factor of 1.53 was used to calculate a monthly SCIG dose bioequivalent to the previous IVIG dose. This conversion factor was based on previous pharmacokinetic studies that showed a dose ratio of SCIG (Hizentra®):IVIG of 1.53 (range 1.26–1.87) led to an area under the curve (AUC) . In the In all follow-up and extension studies, the doses used remained the same on a mg/kg basis as in the previous pivotal or follow-up study. Doses in all studies could be adjusted if medically indicated, or if a patient’s weight changed by more than 5% at any time during the study period. In the US extension study, dose adjustment was permitted if an individual’s steady-state IgG trough level resulted in a trough level ratio (ratio of trough on Hizentra® to the last steady-state trough on the pre-study IVIG therapy) that differed by more than 15% from the pre-specified value of 1.29.The objectives and endpoints of each study are described in Table Safety endpoints included the type, number, rate, severity and treatment-relatedness of any AEs per infusion and per patient, and local tolerability of subcutaneous Hizentra® administration. AEs were coded using the Medical Dictionary for Regulatory Activities (MedDRA) version 14.1 in the Japanese and European extension studies, version 12.0 in the European pivotal study, version 11.0 in the US pivotal study, and version 13.1 in the US extension study.The initial US Hizentra® study procedures included injection site reaction (ISR) evaluations by the patient and the physician, using a variety of methods and multiple mandatory time points during and after infusion. Most ISRs in the US pivotal study were transient, mild, expected, and spontaneously resolving, with approximately 50% of ISRs resolving within 24 h post infusion. Subsequently, less stringent patient/caregiver evaluations  were used to assess ISRs.A population pharmacokinetic (PK) analysis was conducted using IgG serum concentration data from the clinical trials described above and in Table Definitions of each data set are summarized in Table χ2). The annualized rate of SBI was compared with the target rate of <1.0 SBI per patient per year, as recommended by the US Food and Drug Administration (FDA) [Annualized rates were calculated by dividing the number of episodes observed (y) by the total exposure days (T) and multiplying by 365. Upper limits of 99% confidence intervals (CIs) were calculated using the following formula, where T and y are defined as above: (365/T) × (0.5 × on (FDA) .Total serum IgG trough levels during Hizentra® therapy were compared with those achieved during the mandatory IVIG treatment periods by calculating the geometric mean ratios (GMR) and respective 90% CIs.The individual rates of AEs per infusion were calculated for each patient by counting all AEs experienced by one patient and dividing by the total number of Hizentra® infusions administered to this patient. Similar calculations were performed for overall rates of AEs per infusion, using the total number of AEs divided by the total number of infusions.The analysis of the number of infections per calendar month utilized a logistic regression model based on a Poisson distribution using SAS® PROC GENMOD . The possibility of a seasonal effect was assessed based on pairwise differences between months. No adjustment for multiplicity was made in the exploratory analysis of differences between months and the level of significance was set to 5% for each comparison. Seasonal effect was further assessed by fitting a LOESS regression to obtain a reasonable fit of the observed values. Based on this result, a GENMOD model was built to test the model parameters.Comparisons between the rates of AEs in patients receiving an IVIG product, Privigen®  and Hizentra® were performed using the safety data from the Japanese follow-up and extension studies, all European and US studies, and two multicenter studies of Privigen (NCT00168025 and NCT00322556). Specific MedDRA preferred terms for reactions typical for IgG replacement therapy,  were identified and searches performed using the MedDRA data collected from each study. Event rates per compound were calculated as total events over the total number of infusion . The ratio of rates and its 95% CIs were calculated as outlined in Graham et al. .All analyses were carried out using SAS® software version 9.4 .n = 20), AEs (n = 12) and “other” reasons, such as loss to follow-up, termination of study site, non-compliance etc. (n = 11).A total of 125 unique patients in the ITT/AT populations received 15,013 weekly infusions; treatment characteristics are presented in Table In Japan, Europe, and the US, patients were enrolled for up to 183, 208, and 168 consecutive weeks, respectively  in the Japanese pivotal study to 221.3 mg/kg  in the US extension study . The LOESS regression revealed a smooth parabolic curve; therefore, a generalized model with a linear and quadratic term for calendar month was used to determine statistical significance. Both terms were significant , indicating a significant drop in the frequency of infections during the middle of the calendar year  days per patient Table . AntibioIn the Japanese, European, and US pivotal studies, switching to weekly Hizentra® SCIG resulted in an increase in serum IgG trough levels. Patients who received IVIG prior to Hizentra®  had median (range) baseline serum IgG trough levels of 5.90 (4.67–10.01), 6.48 (5.26–11.71), and 10.47 (6.54–19.0) g/L, respectively. Median (range) serum IgG trough levels at follow-up studies were 6.64 5.20–10.43), 8.09 (5.2–11.2), and 11.43 (7.24–22.04) g/L in the Japanese, European, and US pivotal studies, respectively with dose adjustment in the US study  than in patients with XLA (7.61 g/L during infusions 12 to 17); however, the increase was comparable (6.9% in CVID compared to 8.7% in XLA). The primary objective of the Japanese pivotal study was also met, as the GMR of serum IgG trough levels was similar to those of the preceding IVIG treatment period . All three pivotal studies, therefore, met their respective primary objectives.There were no clinically relevant differences between median serum IgG trough concentrations at baseline and during SCIG maintenance dosing in the follow-up and extension studies Table .The final structural population PK (pharmacometric) model was a two-compartment model with inter-individual variability on CL and V2 on all patients. The median values of all non-covariate parameters from bootstrap resampling were consistent with the original population PK estimates Table . The effAnalysis of pooled data revealed no differences in IgG metabolism between ethnic groups despite the numerically different results. The effect of the Japanese population was also tested in various sensitivity models, results from which showed that there was no significant race-related covariate effect from the inclusion of Japanese patients Fig. . After aThere were 5039 AEs in total across all studies. Overall, there were no relevant differences in the frequency of AEs between the different age groups receiving Hizentra® in any of the studies, and there was no increase in the rate of AEs with increasing age, as might be expected. The rates of AEs per infusion were also similar in male and female patients. The incidence of patients with AEs at least possibly related to the study medication in the European pivotal study appeared higher in patients with CVID compared with patients with XLA .Events per infusion ranged from 0.094 in the European extension study to 0.773 in the US pivotal study  reported  and Japanese extension study  discontinued the study due to AEs. In the European studies, 6 patients (11.8%) in the pivotal study discontinued due to AEs (at least possibly related to study drug in 3 patients) and 1 patient died (due to AE unrelated to study drug) in the extension study. In the US studies, 2 patients (4.1%) in the pivotal study discontinued due to AEs (one of which was deemed at least possibly related to study drug), and 1 patient (4.8%) in the US extension study discontinued due to an AE, which was not considered related to study drug.ISRs were more common with Hizentra® than with Privigen®  suggests that adherence to effective dosing and delivery was excellent in these cohorts. Furthermore, the low numbers of patients discontinuing treatment suggested that Hizentra® SCIG was also well tolerated.There were some differences between the Japanese, European, and US studies. These include distribution of primary endpoints, study designs , study length, demographics, and SCIG dose . Furthermore, the use of prophylactic antibiotics appeared higher in the Japanese studies, most likely as a result of local prescribing practices.Long-term tolerability of IgG replacement therapy in PID is an important consideration, as patients often require IgG throughout life. The results of these studies indicate that repeated, self-administered SCIG Hizentra® therapy up to 4 years is efficacious and well tolerated.ESM 1(DOCX 348 kb)


The microbiome has been implicated in the initiation and persistence of inflammatory bowel disease. Despite the fact that diet is one of the most potent modulators of microbiome composition and function and that dietary intervention is the first-line therapy for treating pediatric Crohn’s disease, the relationships between diet-induced remission, enteropathy, and microbiome are poorly understood. Here, we leverage a naturally-occurring canine model of chronic inflammatory enteropathy that exhibits robust remission following nutritional therapy, to perform a longitudinal study that integrates clinical monitoring, 16S rRNA gene amplicon sequencing, metagenomic sequencing, metabolomic profiling, and whole genome sequencing to investigate the relationship between therapeutic diet, microbiome, and disease.Escherichia coli and Clostridium perfringens), reduced severity of dysbiosis, and increased levels of the secondary bile acids, lithocholic and deoxycholic acid. Physiologic levels of these bile acids inhibited the growth of E. coli and C. perfringens isolates, in vitro. Metagenomic analysis and whole genome sequencing identified the bile acid producer Clostridium hiranonis as elevated after dietary therapy and a likely source of secondary bile acids during remission. When C. hiranonis was administered to mice, levels of deoxycholic acid were preserved and pathology associated with DSS colitis was ameliorated. Finally, a closely related bile acid producer, Clostridium scindens, was associated with diet-induced remission in human pediatric Crohn’s disease.We show that remission induced by a hydrolyzed protein diet is accompanied by alterations in microbial community structure marked by decreased abundance of pathobionts  contains supplementary material, which is available to authorized users. Human inflammatory bowel disease (IBD), including Crohn’s disease and ulcerative colitis, is a multifactorial and debilitating disease characterized by chronic immune-pathology, disruption of intestinal homeostasis, and altered composition of the gut microbiome (dysbiosis). Several lines of evidence point to resident gut bacteria as important factors in the etiology of IBD. First, disease is often more severe in areas of the intestine with the highest microbial biomass, and antibiotics are frequently used as an adjunct therapy with immunosuppressants or monoclonal antibodies for managing IBD , 2. SecoAlthough a range of environmental factors have been shown to influence the microbiome, diet is regarded as one of the most potent modulators of the composition and function of the gut-resident microbial community in healthy humans and other mammals , 8, and The relationship between diet, microbiome structure, and enteropathy is complicated to dissect from human subject research where diet is challenging to control, necessitating either retrospective studies in conjunction with extensive food intake surveys , controlC. hiranonis, are associated with the diet-induced remission of canine CE and exert antibacterial activity against isolates of E. coli and C. perfringens in vitro, suggesting these metabolites may modulate disease. These findings improve our understanding of how dietary therapy can modulate microbial communities and reduce GI disease.Despite the fact that the gut microbiome has been implicated in IBD pathogenesis and that diet can be used to manage symptoms of IBD, there is limited insight into how this occurs. In this study, we examined dogs with CE and monitored changes in their fecal microbial community structure and metabolites in response to treatment with a therapeutic hydrolyzed protein diet. By comparing changes over time in dogs with food-responsive chronic enteropathy (FRE), versus animals that failed diet therapy and required subsequent combination therapy, we showed that the hydrolyzed protein diet induces rapid remission that is associated with a structural and functional remodeling of the microbial community in the gut. Notably, we observed that secondary bile acids, likely produced by P < 0.05 at day 0) and did not show a significant reduction 2 weeks after starting diet therapy . Animals with active disease were switched from their current diet to a commercially available therapeutic hydrolyzed protein diet Fig. a. Impactapy Fig. c. NDR anapy Fig. ” sectionapy Fig. c. These n = 20), NDR (n = 9), and healthy control animals at baseline (n = 24). Consistent with previous reports [Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Fusobacteria   and which has been implicated in large bowel diarrhea/colitis in dogs [E. coli and C. perfringens. Next, we asked whether the microbiome in DR and NDR animals differed prior to starting treatment (day 0). Although no differences were observed between the two groups in community diversity, evenness, or distance from healthy controls (unweighted or weighted UniFrac), we identified 20 OTUs that were differentially abundant between DR and NDR animals, 12 of which were enriched in animals that ended up responding to diet treatment  Fig. f. OTUs f in dogs . Taken t in dogs , 41, theTo assess whether the diet-induced remission was accompanied by alterations in dysbiosis, we compared the microbial community structure before and after administration of therapeutic diet in DR animals. Although global phylogenetic distance and Shannon diversity were unchanged  between DR animals before and after diet-induced remission (days 0 vs. 14)  Fig. a, b. Dif14) Fig. d, the se14) Fig. e and lit14) Fig. f were re14) Fig. g, h, sugEscherichia-Shigella, Clostridium, and Fusobacterium)  or C. perfringens (n = 3) isolates derived from dogs with active CE in our ENTiCE study. These species were selected since either they or their parent genera were associated with disease in our animal model. Deoxycholic acid blocked the growth of both species at a concentration comparable to what we detected in the fecal samples   operon—an activity unique to a limited number of anaerobes representing a small fraction of the microbiome, including some Clostridial and Eubacterial species [Clostridium species  and two Eubacterium species (Eubacterium biforme and E. dolichum) present in these samples at a relative abundance ≥ 0.01% in at least 10% samples. Of these species, C. hiranonis was among the most abundant . Eighty-two percent (31/39) of the clostridial isolates from animals with active disease were C. perfringens, consistent with the reported involvement of this organism in canine [C. hiranonis isolates were obtained from independent DR animals in remission at day 42. We selected these C. hiranonis isolates and three C. perfringens isolates for whole genome sequencing. Reads were aligned to the reference C. hiranonis genome, revealing an intact bai operon in canine C. hiranonis, but not C. perfringens  in drinking water [C. hiranonis  at day -3, DSS treatment resulted in significant bacterial expansion (dysbiosis) in mice by day 8 . To test this, we analyzed publicly available data from the Pediatric Longitudinal Study of Elemental Diet and Stool Microbiome Composition (“PLEASE”), a study that examined approximately 20 patients before and after treatment with EEN [C. scindens, which like C. hiranonis is also recognized for having high 7-dehydroxylation activity [C. scindens was estimated using the proportion of total reads that map to the reference genome, revealing significant increases in this species and the bai operon from pretreatment to 8 weeks post-EEN  but not those that failed therapy  , a biomarker of disease activity for IBD [R = − 0.3515, P = 0.03287), but not in non-responders . Similarly, a significant negative correlation between bai operon and FCP was observed in diet-responsive , but not non-responsive patients   Fig. a, b. In  for IBD , indicatClostridium species (C. hiranonis and C. scindens) are key features associated with diet-induced remission in humans and dogs. Previous studies reported dysregulated bile acid metabolism in IBD patients [C. hiranonis and E. coli were biomarkers of disease status in dogs [C. hiranonis as a remission-associated taxon that may serve as a source of secondary bile acids. Furthermore, we observed that C. hiranonis preserved deoxycholic acid levels and ameliorated disease in DSS-treated mice, further highlighting a link between bile acid-producing clostridia and remission of gastrointestinal diseases. Our in vitro inhibition experiments with lithocholic and deoxycholic acids on the C. perfringens and E. coli isolates, together with previous findings of in vitro antibacterial activities of lithocholic acid on E. coli [C. hiranonis abundance could potentially contribute to the control of pathobionts in the gut. Indeed, as dogs entered remission following dietary therapy, we observed a remarkable decrease of C. perfringens and E. coli, coincident with increased levels of lithocholic and deoxycholic acid. In addition to being antimicrobial, physiological levels of these bile acids have been shown to modulate intestinal inflammation, in part due to their ability to signal through the farnesoid X receptor and other bile acid receptors to inhibit pro-inflammatory signaling [Using an animal model of food-responsive chronic inflammatory enteropathy, we showed that secondary bile acids and bile acid-producing patients  and dogspatients , and sho in dogs . Our res E. coli , 55, sugignaling . Our datC. difficile infection [C. difficile infections frequently arise after antibiotic treatment, a phenomenon attributed to the effect of antibiotics on secondary bile acid levels [E. coli  and resulted in more mucosal healing  [Rates of remission achieved with diet therapy in this and prior studies of canine CE  mirror r levels) . HoweverE. coli and C. perfringens), host immunity, or both, thereby reducing pathology. Alternatively, diet may induce remission through mechanisms that are independent of the microbiome, in which case alterations to the microbiome and restoration of normal bile acid production would be consequences rather than causes of remission. Finally, diet may impact disease through both microbiome-dependent and microbiome-independent mechanisms. Nevertheless, given that certain bacteria and bile acids have been implicated in IBD pathogenesis and host inflammation [The precise role that diet and dietary components play in remission has yet to be defined. One interpretation of our data is that therapeutic diet modifies the composition and/or function of the microbiome, which subsequently impacts pathobionts . Levels of lithocholic and deoxycholic acid in stool rose rapidly after the diet therapy and inhibited the in vitro growth of disease-associated taxa. Mining public data from diet therapy in human pediatric Crohn’s disease showed similar results, supporting a model by which dietary interventions lead to remission. These results warrant further investigations in other animal models and human studies and constitute an important first step in establishing a framework for evaluating the efficacy of dietary interventions, which could help guide the rational design of more effective therapeutic diets.In summary, these data show that remission induced by a therapeutic hydrolyzed protein diet is linked to improved microbiota structure in canine chronic inflammatory enteropathy, marked by decreased relative abundance of pathobionts and increased abundance of a secondary bile acid producer , complete blood count, serum biochemical profile, serum measurement of canine trypsin-like immunoreactivity, cobalamin and folate, urinalysis, abdominal ultrasound examination, and disease severity scoring using the Canine Chronic Enteropathy Clinical Activity Index (CCECAI) [Client-owned animals presenting with clinical signs of canine chronic enteropathy (CCE) were screened at the Ryan Veterinary Hospital of the University of Pennsylvania for enrollment in a study to determine the Effect of Nutritional Therapy on Microbiome in Canine Enteropathy (“ENTiCE”). Dogs were screened if they had any one of the following clinical signs for ≥ 3 weeks’ duration: vomiting, diarrhea, or weight loss despite adequate caloric intake. Dogs were excluded from screening if they had been treated with a hydrolyzed protein diet, antibiotics, corticosteroids, or probiotics within the previous 2 weeks. At the time of screening, the following were performed on each animal: complete physical examination, routine fecal screening  . If thesThe ENTiCE study included three 14-day treatment tiers  following the manufacturer’s recommendations. A mock community pool containing purified genomic DNA from 12 known bacterial isolates was amplified and sequenced as a quality control. Additional controls included extraction of blank-processed samples , and water only, to determine background microbial signal. A dual-index amplicon sequencing method was employed for PCR amplification of the V4 region of the 16S RNA gene . Pico-grRNA gene  with filRNA gene . ChimeriRNA gene  against RNA gene , 75. QuaRNA gene , the bioRNA gene , and theRNA gene . SingletRNA gene .P value < 0.05) using un-rarefied reads. To estimate the effect from outliers, separate fold changes of relative abundances (1 read was added to avoid taking log of zero) were also calculated by excluding the 10% outliers (5th–95th) in each group. The Spearman correlation was computed between the abundance of each microbial composition log-transformed) and the values of different factors . To avoid taking log of the zero value, we added 1 read to the abundance for each composition before calculating the Spearman correlation. All P values in the above analysis were adjusted by the FDR (Benjamini-Hochberg) method for multiple comparisons except where noted. In order to get a taxonomic assignment at species level for the OTUs from Clostridium sensu stricto 1, the corresponding representative sequences in SILVA database were used to search against NCBI “nr” database. Species were temporarily assigned by the best hits (P < 1e−5), and further confirmations were done by comparing the relative abundances of these species determined by metagenomic shotgun sequencing method and by 16S sequencing method. The OTU “New.ReferenceOTU131” represents C. perfringens, which is the most dominant OTU in some dogs, and OTU “HQ776819.1.1426” corresponds to C. hiranonis.For differential abundance analysis and association analysis, filtering on un-rarefied OTU table of samples with > 10,000 reads was carried out to remove taxa with a max abundance < 0.1% across all samples and present in < 10% of all samples. DESeq2  implemenbai operon in microbiome were identified from the gene family search result of HUMAnN2 according the reference cluster IDs of these genes in UniRef50. Low abundance gene pathways with max abundance < 0.01% across all samples or present in < 25% samples were filtered out. Two-sided Wilcoxon signed-rank test was used for comparisons of UniPathway pathways at different timepoints (FDR < 0.05). Principal component analysis for pathways was performed by the R package factoextra. The identified Clostridium spp. and Eubacterium spp. were further searched for the existence of genes involved in secondary bile acid production (bai operon) using tBlastn against the reference genomes of these species in GenBank with the protein sequence of genes in 7α-dehydroxylation pathway  .Sequencing libraries were prepared using Illumina Nextera XT with 1 ng DNA extracted from canine stool collected at days 0, 14, and 42 from 18 out of the 20 diet-responsive dogs in the study. Sizing and quantification of libraries were carried out using a Tapestation 4200 (Agilent) and Qubit 3 (Thermo Fisher), respectively. Equimolar amounts of each library were pooled and sequenced on an Illumina NextSeq 500 instrument to produce 150 bp paired-end sequences. Sequencing adapters and low-quality reads were trimmed and filtered by Trimmomatic (v0.36) (leading:3 trailing:3 slidingwindow:4:15 minlen:36). High-quality reads were mapped to the canine reference genome (CanFam3.1), using Bowtie2 v2.3.4.1 (--very-sensitive), and aligned reads were removed using SamTools . After hClostridium. Among them, Clostridium scindens has been well known for the secondary BA-producing ability. Paired reads with PCR duplicates removed by samtools [C. scindens reference genome  as well as strain VE202-05 (ASM47184v1) using bwa-mem (v0.7.17-r1188) [Metagenomic data from pediatric Crohn’s disease patients before and after exclusive enteral nutrition (EEN) have been described previously  and weresamtools  were ali7-r1188)  with def7-r1188) .−5) were plated on brain-heart infusion (BHI), yeast casitone fatty acid with carbohydrate (YCFAC) [C. hiranonis, C. perfringens, and E. coli isolates, 1 ng of DNA was used to construct sequencing libraries using Illumina Nextera XT. Libraries were sized and quantified as described above for metagenomic sequencing. Sourmash was used to detect contamination reads from metagenomic sequencing for the isolates [C. hiranonis (ASM15605v1) using Stampy [bai operon was calculated for each isolate to show the existence of genes in 7α-dehydroxylation pathway.Rectal swabs freshly collected from dogs with active disease (day 0) and/or in remission at the end of the study (day 42) were transferred to an anaerobic chamber  within 1 h of collection. The tip of each swab was homogenized in 1 mL of pre-reduced PBS with 1% cysteine (PBSc). Serial dilutions made in PBSc (down to 10 (YCFAC) , gut mic (YCFAC) , and De  (YCFAC)  agars , the value 0.5  was given for analysis. Canine isolates of C. perfringens (n = 3) and E. coli were revived from glycerol stocks in Modified Reinforced Clostridial Broth  or Luria broth , respectively, and grown overnight in the anaerobic chamber at 37 °C. Lithocholic and deoxycholic acids (Sigma) were dissolved in 100% ethanol (30 mg/mL). Growth inhibition by deoxycholic acid was determined by microbroth dilution and assessed by OD630 after overnight growth. Due to low solubility (< 1 mg/L), inhibition by lithocholic acid was assessed by counting colonies on agar plates with LCA  that were incubated anaerobically, at 37 °C for 24 (E. coli) or 48 h (C. perfringens). Growth curves  were run for bacterial strain under each condition. All procedures were conducted in an anaerobic chamber .Bile acids were quantified in stool using a Waters Acquity uPLC System with a QDa single quadrupole mass detector and an autosampler as described previously . Brieflyn = 5 mice per cage) at baseline, and drinking water was replaced with either filter-sterilized water (mock-treatment) or a filter-sterilized solution of 2.5% (w/v) DSS  in water. The mice treated with mock or DSS were orally gavaged C. hiranonis  or anaerobic PBS (control) from days 0 to 4. C. hiranonis was grown overnight in MRCB, anaerobically, at 37 °C. Culture density was assessed via optical density (630 nm) and the required volume of culture was spun at 10,000 g for 15 min. The consumption of DSS water was monitored every day, and no significant difference was observed between the groups. Stool samples were collected at day -1 and day 3 for metabolomic analysis. Mice were euthanized on day 8, and colon tissues were collected. Distal colons were cut, fixed in formalin, embedded in paraffin, and stained with hematoxylin and eosin (H&E) or Periodic acid–Schiff (PAS). Histologic sections were evaluated by a board-certified veterinary pathologist (CB). Colitis was scored according to previously published criteria [Female 7-week-old C57BL/6 J mice (specific pathogen-free) were purchased from Jackson Laboratories and maintained for 2–3 weeks in the animal facility at University of Pennsylvania prior to the to the start of dextran sulfate sodium (DSS) treatment. Animals were randomly assigned to groups  in LB media at day -3. Then, the mice were subjected to the same procedure as described above . At day 8, colonic content was harvested, weighed, and plated on LB agar plates under kanamycin selection (100 μg/mL). E. coli colonies were counted and normalized by sample weight for each mouse.For nts Fig. k, C57BL/Additional file 1:Figure S1. Detailed clinical design of the ‘ENTiCE’ canine chronic enteropathy study. Figure S2. Community structure of microbiomes in the dogs with CE and in the healthy dogs. Figure S3. Differentially abundant OTUs between healthy animals and CE animals. Figure S4. Microbiota community structure changes induced by diet therapy. Figure S5. OTU level  dynamics of microbiota structure for DR animals throughout the study. Figure S6. Concentrations of bile acids detected in fecal samples from diet-responsive dogs. Figure S7. Bile salt hydrolase (BSH) abundance in DR animals. Figure S8. Heatmap of the top 25 most abundant species across the samples in DR animals based on metagenomic data. Figure S9. Relative abundance of C. hiranonis in diet responsive dogs calculated from metagenomic data. Figure S10. Representative H&E staining sections of distal colon tissues at day 8 (40 x objective). Figure S11. Disease score-matched analysis. (PDF 5033 kb)Additional file 2:Table S1. OTUs with differential abundances between samples of healthy dogs and dogs with CE at day 0 . Table S2. OTUs with differential abundances between day 0-samples of diet responsive dogs and non-diet responsive dogs. Table S3. Genera with differential abundances between samples of day 14 and day 0 (day 14 versus day 0) for diet responsive dogs. Table S4. OTUs with differential abundances between samples of day 14 and day 0 for diet responsive dogs (day 14 versus day 0). Table S5. OTUs with differential abundances between samples of day 14 and day 0 (day 14 versus day 0) for non-diet responsive dogs. Table S6. Differentially abundant pathways between day 14 and day 0 after diet treatment from metagenomic analysis for diet-responsive dogs . Table S7. Comparisons of bile acids in samples at different timepoints for diet responsive dogs. Table S8. Spearman correlations between abundance of OTUs and concentration of Bile acids in diet responsive dogs. Table S9. Spearman correlations between bile acids and bile salt hydolases (BSHs) in diet-responsive dogs (P < 0.05). (XLSX 135 kb)


Accumulating evidence suggests that breastfeeding benefits children’s intelligence, possibly due to long-chain polyunsaturated fatty acids (LC-PUFAs) present in breast milk. Under a nutritional adequacy hypothesis, an interaction between breastfeeding and genetic variants associated with endogenous LC-PUFAs synthesis might be expected. However, the literature on this topic is controversial.FADS2 polymorphisms rs174575 and rs1535 coded assuming a recessive effect of the G allele, and intelligence quotient (IQ) in Z scores.We investigated this gene × environment interaction through a collaborative effort. The primary analysis involved >12 000 individuals and used ever breastfeeding, There was no strong evidence of interaction, with pooled covariate-adjusted interaction coefficients  of 0.12 and 0.06  for the rs174575 and rs1535 variants, respectively. Secondary analyses corroborated these results. In studies with ≥5.85 and <5.85 months of breastfeeding duration, pooled estimates for the rs174575 variant were 0.50  and 0.14 , respectively, and 0.27  and −0.01  for the rs1535 variant.FADS2 polymorphisms. However, subgroup analysis suggested that breastfeeding may supply LC-PUFAs requirements for cognitive development if breastfeeding lasts for some (currently unknown) time. Future studies in large individual-level datasets would allow properly powered subgroup analyses and further improve our understanding on the breastfeeding × FADS2 interaction.Our findings did not support an interaction between ever breastfeeding and  Breastfeeding is considered to improve children’s intelligence, possibly due to long-chain polyunsaturated fatty acids (LC-PUFAs).FADS2 on intelligence quotient (IQ) is controversial.The literature on the interaction between breastfeeding and variants in the de novo collaborative meta-analysis did not find support for this interaction when comparing ever vs never breastfed individuals.Our Subgroup analyses, although underpowered, were compatible with a role of breastfeeding duration in this interaction. This finding requires replication. Breastfeeding has well-established short-term benefits on children’s health. There is also accumulating evidence that breastfeeding may benefit cognitive development.,,One of the possible biological mechanisms underlying the effect of breastfeeding on IQ is through long-chain polyunsaturated fatty acids (LC-PUFAs), such as docosahexaenoic acid (DHA). Meta-analyses of randomized controlled trials of supplementation of DHA and other LC-PUFAs in infants reported improved cognitive development,,The role of LC-PUFAs in the association between breastfeeding and IQ can be investigated through a gene × environment (G × E) interaction analysis. Here, we framed this G × E interaction as a nutritional adequacy hypothesis. A brief and general definition of nutritional adequacy is that, once an individual’s nutritional requirement is met, further intake of the given nutrient yields no additional benefit.FADS2 gene.,,FADS2 gene were associated with lower levels of PUFAs in plasma and erythrocyte phospholipids.et al. were the first to evaluate the interaction between genetic variation in FADS2 and breastfeeding, with IQ in children as the outcome.2 metric ranging from 0.33 to 0.68 in European populations in 1000 Genomes Phase 3. For both SNPs, having ever being breastfed was positively associated with IQ in all genetic groups, except in G-allele homozygotes where there was no association.This G × E interaction has been investigated using single nucleotide polymorphisms (SNPs) in the et al. presented results consistent with our nutritional adequacy hypothesis, with breastfed individuals presenting similar mean values of IQ across FADS2 genotypes.et al.However, in a replication study, Steer FADS2 genotypes ?; (ii) is the association of breastfeeding with IQ different between subgroups of FADS2 genotypes (i.e. is there a G × E interaction)?; and (iii) do study-level characteristics explain between-study heterogeneity in this G × E interaction?The contradictory results observed in the literature may be due to lack of statistical power and/or contextual differences that lead to heterogeneity between studies, as discussed in detail elsewhere.The protocol of this study has been published elsewhere.r2 or INFO quality below 0.3); (ii) twin studies; and (iii) lack of appropriate ethical approval.Eligibility criteria were: (i) availability of at least a binary breastfeeding variable , intelligence measured using standard tests and at least rs174575 or rs1535 SNPs (either genotyped or imputed); and (ii) European ancestry studies, or multi-ethnic studies capable of defining a subsample of European ancestry individuals. Exclusion criteria were: (i) only poorly imputed genetic data available  Project,Småbørn Kost Og Trivsel-I (SKOT-I),,,,A total of 10 eligible studies were identified, all of which were included in the meta-analysis: the 1982 Pelotas Birth Cohort Study,IJE online.In an attempt to improve statistical power, a subsample of 32 842 individuals from the UK BiobankIJE online). IQ measures were converted to Z scores (mean = 0 and variance = 1) within each participating study. The primary analysis involved breastfeeding (coded as never = 0 and ever = 1), FADS2 polymorphism assuming a recessive genetic effect of the G allele  and an interaction term between them. Different genetic effects, different categorizations of breastfeeding, and exclusive breastfeeding  were evaluated in pre-planned secondary analyses. Unless explicitly stated, all analyses refer to any quality of breastfeeding (i.e. combining exclusive and non-exclusive breastfeeding).The main outcome variable was IQ. IQ tests varied between studies  unadjusted (i.e. no covariates); (ii) adjusted 1: controlling for sex and age (linear and quadratic terms) when IQ was measured, ancestry-informative principal components (when available) and genotyping centre (for studies involving multiple laboratories); (iii) adjusted 2: same covariates in adjusted 1 model, as well as maternal education (linear and quadratic terms) and maternal cognition (linear and quadratic terms); if only one of the maternal variables was available, adjusted model 2 controlled only for that variable. Continuous covariates, as well as sex , were mean-centred before analysis, and squaring was performed before mean-centring. Covariate adjustment was performed by including not only a ‘main effect’ term, but also FADS2 interaction in this analysis is indicative that those maternal variables may confound the main breastfeeding × FADS2 interaction analysis having participant’s IQ as the outcome variable. This is a form of negative control analysis.As a sensitivity analysis, the role of gene-environment correlation was evaluated by repeating models (i) and (ii), but having maternal cognition (in Z scores) or maternal schooling (in years) as outcome variables rather than the participant’s IQ. Maternal cognition or schooling are important predictors of an individual’s IQ, and cannot be consequences of the participant’s genotype. Therefore, any evidence of breastfeeding × Analyses were performed using linear regression with heteroskedasticity-robust standard errors. Results from all studies were pooled using fixed and random effects meta-analysis. Stratified meta-analysis and random effects meta-regression were used to evaluate the potential moderating role of the following variables (one meta-regression model per moderator): IQ test; adjustment for ancestry-informative principal components; age at IQ measurement; timing of breastfeeding measurement; continental region; mean year of birth; prevalence of having ever being breastfed; mean breastfeeding duration; and sample size. Adjusted R² values, which can be interpreted as the amount of between-study heterogeneity explained by the moderator, were obtained from the meta-regression models.IJE online, for details). This complements the sample size calculations presented in the protocol by allowing inclusion of estimated between-study heterogeneity in the calculations.The power of our primary analysis, focusing on the rs174575 polymorphism, was quantified via simulations  measured IQ using the Wechsler Intelligence Scale.As shown in IJE online, provides a description of the two FADS2 SNPs in each study. The SNPs rs174575 and rs1535 were directly genotyped in three and five studies, respectively. The minimum value of imputation quality was 0.984. The frequency of the G allele ranged from 20.5% to 30.8% for the rs174575 variant, and from 28.5% to 39.1% for the rs1535 variant. There was no strong statistical evidence against Hardy-Weinberg equilibrium, with the smallest P-values being 0.058 (Generation R) and 0.074 (SKOTI-II) for rs174575, and 0.085 (1982 Pelotas Birth Cohort), 0.044 (Raine) and 0.089 (SKOTI-II) for rs1535. Although these results may be suggestive of some population substructure  or batch effects , it is unlikely that such phenomena substantially influenced the results because ancestry-informative principal components, computed using genome-wide genotyping data, were adjusted for in these four studies.IJE online. Among eligible studies (i.e. excluding the UK Biobank), the mean age, maternal education and breastfeeding duration ranged from 2.5 to 30.2 years, 11 to 19 years and 2.3 to 8.2 months, respectively. Each sex represented approximately half of the participants in all studies. All IQ measures produced a variable with mean close to 100 and similar standard deviations . The exception was the one used in SKOT-I and SKOT-II (i.e. third edition of the Ages and Stages Questionnaire), which produced a variable with mean close to 50. The median of the prevalence of ever breastfeeding was 91% (ranging from 50% to 95%), and the median of the median of any breastfeeding duration was 4.3 months (ranging from 0 to 7.9). Among all individuals included in primary analysis for at least one of the SNPs (n = 13 292), 11 055 were ever breastfed.Additional study characteristics are displayed in FADS2 genotype, ever breastfeeding was associated with increases of 0.37  and 0.30  Z scores in IQ in fixed and random effects meta-analyses, respectively. Assuming that IQ has a standard deviation (SD) of 12.2 points (the median of the standard deviation of IQ measures among participating studies), these coefficients correspond to 4.5 and 3.7 points in IQ, respectively. In the fully adjusted model (adjusted 2), the respective coefficients were 0.26  and 0.17 , or 3.2 and 2.1 points in IQ.In analyses without stratification according to FADS2 interaction term of 0.18  and 0.12 , respectively. These coefficients can be interpreted as the mean difference in IQ Z scores comparing ever with never breastfed individuals among GG carriers, minus the mean difference in IQ Z scores comparing ever with never breastfed individuals among carriers of other genotypes—e.g. a positive interaction coefficient indicates that the benefit of breastfeeding on IQ is stronger among GG individuals. Similar results were obtained when using fixed effects meta-analysis.Results for the rs1535 variant presented a similar trend, but were even less suggestive of interaction. When using random effects meta-analysis, the estimates of the interaction term were −0.04  in the unadjusted and 0.06  in the fully-adjusted model (i.e. adjusted model 2). Using fixed effects meta-analysis yielded similar results.IJE online, there was no strong indication of interaction when analysing other categorizations of breastfeeding duration and FADS2 SNPs coded assuming a recessive effect. This was also the case when FADS2 variants were coded assuming additive (IJE online), dominant (IJE online) and overdominant (IJE online) effects. The same was observed for exclusive breastfeeding (IJE online).As shown in IJE online, displays the results obtained when including the UK Biobank, which was analysed as two independent samples according to the genotyping platform (Biobank_Axiom and Biobank_BiLEVE). Its inclusion resulted in a combined sample size of more than 45 000 individuals. When FADS2 variants were coded assuming recessive effects, the pooled estimates from the unadjusted model were −0.02  and 0.08  for fixed and random effects meta-analysis, respectively. The corresponding estimates from the adjusted (1) model were −0.04  and 0.00 , respectively. There was also no strong statistical evidence supporting an interaction when other genetic effects were assumed.Regarding the rs1535 variant, the following subgroup-specific estimates were consistent with those of the rs174575 SNP: adjustment for principal components, with pooled estimates of 0.09  and −0.03  among studies that did and did not perform this adjustment, respectively; age at IQ measurement, with pooled estimates of 0.04  and 0.07  among studies that measured IQ when individuals were ≥10 and <10 years old, respectively; and sample size, with pooled estimates of 0.11  and 0.01 (95% CI: −0.43 and 0.45) among larger and smaller studies, respectively. However, in all those cases the adjusted R² values were 0%. Prevalence of ever breastfeeding presented adjusted R² values of 0% and 8.3% when dichotomized and when analysed continuously, respectively. The pooled estimates for the rs1535 variant were 0.15  and 0.01  among studies with prevalences of ever breastfeeding of ≥90% and <90%, respectively. The most consistent moderator between SNPs was breastfeeding duration, with pooled estimates for the rs1535 SNP of 0.27  and −0.01  among studies with ≥5.85 and <5.85 months of average duration, respectively; adjusted R² values were 22.2% and 4.9% when breastfeeding duration was dichotomized and analysed continuously, respectively.There was no strong evidence in support of gene-environment correlation involving maternal education or maternal cognition . RegardiIJE online, displays the results of the power calculations. Assuming no between-study heterogeneity, the power of the primary analysis was ≥80% to detect a G × E coefficient between 0.219 and 0.263 IQ Z scores . This finding is against the notion that there is a FADS2 genetic subgroup where breastfeeding is not associated with IQ . Other limitations were: small sample size for some analyses, such as those involving exclusive breastfeeding; heterogeneity in important study characteristics, such as age (including pooling children and adult studies), type of IQ test and timing of breastfeeding measurement; and small number of studies in the meta-regression analyses. Another potential limitation was lack of adjustment for maternal genotypes, which may confound the association between participant’s genotype and IQ because maternal genotypes may influence fatty acid composition in breast milk.,Our study also had limitations. Some of them relate to the small numbers of individuals in some categories, which we addressed by adapting the protocol, such as by re-defining the ‘never breastfed’ group and by excluding some categories of breastfeeding from the analysis . We believe there are three main reasons for this. One of them is substantial between-study heterogeneity, which was mitigated both by design  and analysis , but could not be fully eliminated (as described above). There is a trade-off between heterogeneity and sample size, and in the case of this study, including further restrictions in the eligibility criteria would be prohibitive. The second is overestimation of the G × E coefficient in the sample size calculations, where we used the point estimate reported by Steer et al.,A final limitation of our study is lack of power to detect G × E coefficients of relevant magnitude . Since such analysis would involve many subgroupings, the best alternative would likely be to perform them in a large dataset of individual-level data, which may be achieved by a consortium-based effort such as this collaborative meta-analysis. This and other future investigations will be important to further refine our understanding on the role of LC-PUFAs on the association between breastfeeding and intelligence. This will also have practical implications, such as identifying whether current breastfeeding recommendations provide for the upper limit of cognitive benefits related to LC-PUFAs intake (if such limit exists), and the potential benefits (if any) of supplementing a lactating infant with LC-PUFAs.Although our primary findings were not supportive of an interaction between breastfeeding and IJE online) for study-specific funders and grant numbers. This work was coordinated by researchers working within the Medical Research Council (MRC) Integrative Epidemiology Unit, which is funded by the MRC and the University of Bristol .This work was supported by several funding agencies: see the Supplementary MaterialClick here for additional data file.Supplementary TablesClick here for additional data file.


The relationship between visceral adiposity and acute pancreatitis (AP) has not been completely elucidated. This study evaluated the significance of visceral adipose tissue (VAT) and the ratio of VAT to skeletal muscle tissue (VAT/SMT) in the prognosis of AP patients.Based on a 1:2 propensity score matching, 306 hospitalized patients were enrolled in the study analysis from 2010 to 2017. VAT, subcutaneous adipose tissue (SAT), and SMT were measured using unenhanced computed tomography (CT). Cox proportional hazards models were applied for the analysis.p < 0.001). Intensive care transfer, AP severity, systemic complications, and prognostic scores  significantly correlated with VAT and the VAT/SMT ratio in AP patients. The multivariate adjusted hazard ratios (HRs) for VAT and the VAT/SMT ratio in the relationship of body parameters and AP mortality were 1.042 , 1.019–1.066) and 7.820 , respectively. Compared with other prognostic scores, VAT had the highest area under the curve of receiver operating characteristics (ROC) .VAT and the VAT/SMT ratio were significantly higher in the severe AP (SAP) and moderately severe AP (MSAP) groups compared to the mild AP (MAP) group (both High VAT and VAT/SMT ratio are independent negative prognostic indicators of AP.NCT03482921. Date of registration: 03/23/2018.Clinical study registration number:  Acute pancreatitis (AP) is cause by inflammation of the pancreas. In the majority of cases, AP is mild and self-limited. However, about 20% of AP patients experience severe complications with a high risk of mortality , 2. It i2 by the World Health Organization) is a risk factor for AP severity and increases the incidence of systemic complications and mortality [It is well-established that obesity (defined as body mass index (BMI) > 30 kg/mortality –9. BMI mortality –13. Someortality , 14–16. ortality . There aWe analyzed 1662 consecutive hospitalized patients with AP between August 1st, 2010 and August 31st, 2017 at Ningbo First Hospital, China. This study was approved by the Human Ethics Committee. The clinical study registration number is NCT03482921. Severe AP (SAP) and moderately severe AP (MSAP) were defined in 111 patients according to the Revised Atlanta Classification of AP  AP severity was defined using the Revised Atlanta Classification criteria. The clinical classification defined 3 grades of severity: MAP, MSAP, and SAP.Systemic complication was defined as a score ≥ 2 in one of three assessed organ systems  according to the modified Marshall scoring system .Local complications of AP were divided into: acute peri-pancreatic fluid collection (APFC), acute necrotic collection (ANC), pancreatic pseudocyst, and walled-off necrosis (WON). Diagnosis of local complication on unenhanced computed tomography (CT) was evaluated by two experienced radiologists who were blinded to detailed information about the cases. When necessary, follow-up contrast-enhanced CT (CECT) and magnetic resonance imaging (MRI) are performed based on the clinical status of patients, including persistence or recurrence of abdominal pain, increasing organ dysfunction, and development of clinical signs of sepsis. Once patients are diagnosed with APFC, ANC, WON, or pseudocyst in the follow-up imaging examinations, they are considered to present with local complications.Creatinine, calcium, and albumin levels obtained within the first 24 h were collected for each patient. Additional variables included intensive care unit (ICU) requirement, length of hospital stay, and in-hospital mortality.The APACHE-II, BISAP, and SIRS scores were calculated within the first 24 h of admission, and Ranson’s score was recorded within 48 h of admission.A dual 16-slice CT scanner  was used for entire / upper abdominal CT at 250 mA; tube voltage, 120 kV; data collection thickness 5 mm; reconstruction thickness 1 mm; and reconstruction interval 1 mm. CT scans were performed within 24 h after patient admission. Entire abdomen CT was performed to 165 patients. Upper abdomen CT was performed to 141 patients. Two consecutive, axial images on unenhanced CT at the L3/4 intervertebral space were retrospectively reviewed by two radiologists who were blinded to patient information. We calculated fat and muscle cross-sectional areas, which have been validated as the best proxies of SAT and VAT independent of age . Skeletap value < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS software .Continuous variables are expressed as mean ± standard deviation (SD) and median (interquartile range). Categorical outcomes are presented as frequencies and proportions. Comparisons of different variables were performed using one-way analysis of variance (ANOVA) and Likelihood Ratio. Cox regression analysis adjusted for confounders were used to identify the relationships. Discrimination of the predicting factors, including VAT and VAT/SMT ratio, were accomplished via receiver operating characteristic (ROC) curves. A two-tailed 2. The mean SAT value was 131.1 ± 23.1 cm2. The mean VAT value was 136.1 ± 27.3 cm2. The mean SMT value was 138.2 ± 25.8 cm2.In this study, every MSAP and SAP patient was propensity score matched with another MAP patient at a ratio of 1:2. Finally, 306 hospitalized patients were enrolled for further analysis. Among the 306 patients, causative factors of AP included gallstone-related , idiopathic , hypertriglyceridemia , alcohol abuse , and others . The mean age of the patients was 50.6 ± 18.7 years. The mean estimated BMI was 24.4 ± 4.5 kg/mp < 0.001). The mean SMT values decreased as AP severity increased (p < 0.001).Age, sex, and BMI distribution were not statistically different among groups after propensity matching. There was a significant association between AP severity and individual fat parameters, except for SAT Table . VAT andBased on the VAT value at the L3/4 intervertebral space, patients were separated into five groups. ICU transfer, AP severity, and systemic complications  were significantly related to VAT values in the AP patients  had significant relationship with the VAT/SMT ratio (Table p < 0.001).Further analyses were conducted for creatinine, calcium, and albumin levels using an adjusted model Table . The mulThe AUCs were measured to evaluate individual fat parameters of VAT and the VAT/SMT ratio for predicting the incidence of SAP and MSAP, ICU stay, and the incidence of WON Fig. .Fig. 3ROVAT had the highest area under the curve of ROC  for predicting the incidence of SAP and MSAP, among independent predictors. The AUC of the VAT/SMT ratio was also significantly higher compared to the predictive scoring systems . The optimal cut-off value of VAT for the prognosis of AP was 145.0. The sensitivity and specificity of this cut-off value were 90.2 and 93.1%, respectively. The optimal cut-off point of the VAT/SMT ratio for the prognosis of AP was 0.706. The sensitivity and specificity of this cut-off value were 80.4 and 90.2%, respectively. The cut-off values for VAT and the VAT/SMT ratio can be used to predict the incidence of SAP and MSAP.We also demonstrated that VAT and the VAT/SMT ratio had high sensitivity and specificity for predicting the incidence of ICU stay and WON compared to other predictive scoring systems. The AUC of VAT for predicting the incidence of ICU stay was 0.900 . The second highest AUC of ROC was the VAT/SMT ratio . The AUC of the VAT/SMT ratio was the highest predictor of the incidence WON .This propensity score-matched, case-control study assessed the significance of VAT and the VAT/SMT ratio as AP prognostic factors. VAT and the VAT/SMT ratio increased as AP severity increased, including the incidence of SAP and MSAP, the incidence of ICU transfer, and the incidence of WON. This study also calculated the optimal cut-off values, which can be used to predict AP severity.AP involves sudden inflammation in the pancreas. Early prediction of SAP will allow more aggressive management at early stages of the disease. There are many SAP predictive scoring systems. However, none can predict the severity at early stages of AP. If initial CT scans in AP patients are routinely performed at an early stage, the values of VAT and the VAT/SMT ratio could be quickly calculated and used as predictors.2. However, Asians tend to have a significantly lower BMI, at approximately 2–3 kg/m2 lower compared to age and sex matched whites with the same amount of body fat [Obesity is a well-established risk factor of severity and mortality in AP patients , 26. In body fat . Currentbody fat –33. In abody fat , 14–16. Previous studies reported gender-based variations in fat distribution, especially in obese patients , 35. RisThere are four studies that have presented similar results to the finding described here. A study of 62 cases  reportedThere are several limitations in our study. First, at present, it is difficult to perform a CT scan on a patient within 24 h after admission in some countries and regions. Second, due to its retrospective design, the statistical power of analysis is relatively weak. Third, individuals from the Chinese population were included in our study. We did not recruit AP patients in western countries, which may increase potential bias.This is the largest study exploring the relationship between different body parameters and severity of AP after taking several covariates into account. Furthermore, we calculated the optimal cut-off value of VAT and the VAT/SMT ratio. Therefore, VAT and the VAT/SMT ratio should be considered as independent predictors in AP patients. To extend our results to clinical-decision making, further randomized, multi-center, prospective studies that include different countries and regions are needed to better assess the association between visceral adiposity with severity of AP.The results of this study suggest that VAT and the VAT/SMT ratio are strong predictors of severity, mortality, and systemic complications in AP patients.