Cardiovascular (CV) morbidity is the major cause of death in patients with Systemic Lupus Erythematosus (SLE). Previous studies on mannose-binding lectin (MBL) gene polymorphisms in SLE patients suggest that low levels of complement MBL are associated with cardiovascular disease (CVD). However, as large studies on MBL deficiency based on resulting MBL plasma concentrations are lacking, the aim of our study was to analyze the association of MBL concentrations with CVD in SLE patients. Plasma MBL levels SLE patients included in the Swiss SLE Cohort Study were quantified by ELISA. Five different CV organ manifestations were documented. Of 373 included patients  62 patients had at least one CV manifestation. Patients with MBL deficiency (levels below 500 ng/ml or 1000 ng/ml) had no significantly increased frequency of CVD . After adjustment for traditional CV risk factors, MBL levels and positive antiphospholipid serology (APL+) a significant association of CVD with age, hypertension, disease duration and APL+ was demonstrated. In our study of a large cohort of patients with SLE, we could not confirm previous studies suggesting MBL deficiency to be associated with an increased risk for CVD. The prevalence generally ranges from 20 to 70 per 100.0004. Associated with the presence of immune complexes, ongoing complement system activation leads to inflammation and consumption of complement proteins5. This chronic inflammatory state predisposes SLE patients to premature cardiovascular disease (CVD) and infections.The etiology of SLE is multifactorial and the complexity of different factors  matches the diversity of clinical manifestations in SLE patients6, is acknowledged as the major cause of death in SLE patients, regardless of the time after diagnosis8, resulting in a bimodal mortality curve, first described by Urowitz et al. in the 1970s9. The 5-year survival after diagnosis nowadays exceeds 90%12, but this survival rate has not improved since the 1980s.Premature CVD, mostly related to accelerated atherosclerosis18, a 10-fold higher prevalence compared to the general population19. Moreover, the risk of myocardial infarction in young women with SLE has been found to be 50 times higher compared to women of similar age20 and the risk of stroke was found to be increased by 2–8 fold22.The prevalence of ischemic heart disease in SLE patients is estimated to be between 3.8 and 16%23.As traditional Framingham risk factors , hypertension and overweight) cannot fully explain the high rates of ischemic events, SLE is nowadays considered an independent risk factor for CVD26. Among those, MBL is a liver-derived serum protein that binds certain sugars on the surface of pathogenic micro-organisms and cellular debris and, as it cannot directly opsonize pathogens, triggers complement activation via the lectin pathway27. MBL deficiency (<1000 ng/ml) is mostly caused by a three-point mutation  in exon 1 of the MBL gene  that disrupts the assembly of the oligomers and also by a promotor polymorphism (LX) that is associated with low MBL production. However, despite carrying the same genotype, actual MBL levels can vary significantly between individuals29.Several studies suggest that components of the innate immune system, which includes pattern-recognition molecules of the complement system such as mannose-binding lectin (MBL), may play an additional role in the pathogenesis of atherosclerosis and CVD30, more precisely that functional MBL deficiency is associated with an increased susceptibility to SLE31. Furthermore, Panda et al. described that patients with low MBL-producing genotype have a predisposition to develop SLE32. However, data remains controversial as indicated by Losada Lopez et al. who only described a tendency to a higher allele B incidence in SLE patients33.Previous studies indicate that genetic variability in MBL may be involved in the pathogenesis of SLE26 and coronary artery disease24 in the general population, and therefore, to be mostly disadvantageous in this regard34. Of note, Limnell et al. even described that MBL deficiency might lead to venous bypass graft occlusion in patients with coronary heart disease35. This observation is supported by another report on high MBL levels having a cardioprotective effect, i.e. being associated with a decreased risk for myocardial infarction (MCI) among patients with diabetes36. However, data are controversial as some studies show an association of cardiovascular (CV) manifestations with MBL deficiency in the general population but others found no such link39. This controversy has also recently been summarized by Larsen et al.40. In addition, an association of deficiency of complement MBL with reduced mortality in patients with MCI undergoing percutaneous coronary intervention41 as well as with smaller cerebral infarcts and a favorable outcome42 has been described previously. These studies suggest that MBL deficiency – being rather disadvantageous with regard to atherosclerosis and the occurrence of CVD - has an advantageous effect on the outcome after CV events.Independent of its association with SLE, MBL deficiency has been reported to be associated with an increased risk of atherosclerosis43 and thyroid function44. Thus, as studies on MBL deficiency based on resulting MBL plasma concentrations were lacking, this study was undertaken to determine the potential association between low MBL levels and the risk for CV events in SLE.Inconsistencies among study results might in part be due to the retrospective character, the relatively small study populations or the focus on MBL2 gene polymorphisms but not on actual MBL plasma levels, which would implicate the MBL deficiency and which are also influenced by non-genetic factors such as inflammation45 were included in this study. SSCS is a nationwide, multicenter and ethics committee-approved longitudinal study of SLE patients living in Switzerland, conducted in 11 different institutions in 9 Swiss cities. All participants had to fulfill at least 3 out of 11 American College of Rheumatology (ACR) criteria for the classification as SLE at the time of inclusion and had to give written informed consent. In SSCS, the Systemic Lupus Erythematosus Disease Activity (SLEDAI) score with the Safety of Estrogens in Lupus Erythematosus National Assessment (SELENA) modification and Physician’s Global Assessment (PGA) keeps record of the disease activity, while the Systemic Lupus International Collaborating Clinics (SLICC) damage index is used to document chronic organic damage that occurred after diagnosis.373 SLE patients included in the Swiss Systemic Lupus Erythematosus Cohort Study (SSCS)45, more specifically as positive finding of antiphospholipid antibody based on an abnormal serum level of IgG or IgM anticardiolipin antibodies or IgG or IgM beta 2-glycoprotein antibodies or a positive test result for lupus anticoagulant using a standard method.For the analysis of potential confounders, we included common disease activity scores (SELENA-SLEDAI and PGA) and CV risk factors , hypercholesterolemia, arterial hypertension, overweight, and positive antiphospholipid serology (APL+)). Since data on packyears (PY) in some cases were missing we additionally compared ever-smokers with non-smokers. We assumed hypercholesterolemia if patients had any form of treated hyperlipidemia, either familial or acquired, and/or met the common laboratory analytic criteria (cholesterol > 5.2 mmol/l and/or low-density lipoprotein (LDL) > 3.4 mmol/l). Similarly, patients were classified as being hypertensive if any treatment with antihypertensive medication was documented. Overweight was classified using the body-mass-index (BMI) that was calculated using the information that was available at study visits but did not take weight changes over time into account. APL+ was defined based on the specification of the SSCSThis study was approved by the Ethical Committee of the Canton Basel, Switzerland (Ref. No. EK 262/06), all further involved ethical committees and Swissethics . All subjects gave written informed consent in accordance with the Declaration of Helsinki.Patients solely were selected based on the availability of complete disease activity score , SLICC damage index, and available plasma sample at the time of study visit. Demographic , clinical, laboratory and treatment information were extracted from the cohort database.Patients were defined as having CVD if at least one CVD of the SLICC index was present. The following arterial thrombotic events were included: (a.) cerebrovascular ischemia (CVI), (b.) coronary heart disease (CHD), (c.) myocardial infarction (MCI), (d.) peripheral artery disease (PAD) and (e.) mesenteric insufficiency.46. This assay measures the ability of MBL to bind to a mannan-coated surface. Microtitre plates  were coated with 10 µg/ml of mannan  in fresh carbonate–bicarbonate buffer , pH 9.6, and incubated overnight at 4 °C. Plasma samples were tested as in a sandwich ELISA but with Tris-buffered saline with 0.05% Tween-20 (TBST)  and Calciumchlorid-dihydrate (CaCl2) , pH 7.5, as both the diluent and wash buffer. All incubations were carried out at 22 °C. First, the microtitre plates were incubated with samples at a 1:100 dilution for 90 minutes, followed by a biotinylated monoclonal anti-MBL antibody  for again 90 minutes to detect the bound MBL. After incubation with ExtrAvidin peroxidase conjugate  for 40 minutes the plates were developed with BD TMB substrate solutions , stopped after 10 minutes with 0.5 M sulfuric acid (H2SO4)  and read immediately at 450 nm on a microplate biokinetics reader . MBL levels were calculated against a standard serum pool .Plasma samples from the 373 SLE patients were collected between 2008 and 2018, which had been exclusively stored at −70 °C and had never been thawed before use. MBL levels were quantified using a mannan-binding enzyme-linked immunosorbent assay (ELISA), as described previously47 and 1000 ng/ml48, respectively, as described previously.Taking the total number of CV events into account, we predefined two different cut-off levels for MBL deficiency, at 500 ng/mlAs a control, measurement of MBL level was repeated in 120 of our 373 included patients using samples from different study visits and serum samples instead of plasma samples to investigate the long-term stability and intra-individual variability of MBL levels in our patients and the potential influence of biosampling on our study results.First, we examined the baseline characteristics of patients that developed or did not develop CVD using standard descriptive statistical tests. Data are presented as medians (interquartile range (IQR)) or frequencies with percentages when suitable. Beyond the use of predefined cut-offs for the definition of MBL deficiency, a ROC curve was established to explore the data for a potential cutoff level.Analyzing the long-term stability and intra-individual variability of MBL levels we used Wilcoxon Signed-Ranks Test and Spearman rank correlation for the comparison of the two MBL concentrations that were measured in the same patients at two time points.2  test and Fisher’s exact test, respectively, dependent on the group size, were used when comparing the occurrence of CVD and categorical variables. We used the Mann-Whitney U Test and Kruskal-Wallis Test, respectively, when comparing continuous with categorical variables as well as linear regression when comparing two continuous variables. The frequencies of CVD dependent on MBL cut-off levels and MBL levels itself were compared for disease activity index and common CV risk factors as well as APL+. Subsequently, we repeated the analysis for all CVD subcategory.Pearson’s quiLast, we analyzed the association of possible risk factors with the presence of CVD using logistic regression models. A multiple logistic regression analysis was performed, taking the dichotomized (yes/no) CVD in total as the dependent variable and those as independent variables that reached statistical significance in the univariate analysis and all variables, corresponding to traditional CV risk factors.Two-tailed tests and a 5% significance level were used in all analyses. All reported P values of less than 0.05 were considered to indicate statistical significance. The analysis was performed with the statistical package IBM SPSS Statistics 25 for MAC OS X, graphs were performed using the publicly available statistical program R.In a first exploratory analysis of 177 SLE patients, a strong trend towards a significant difference in plasma MBL concentrations between patients with and without CVD was observed. We calculated an adequately powered study (80%) to require at least 362 SLE patients  in order to be able to detect a difference in MBL plasma levels of at least 450 ng/ml in patients with versus without CVD. Based on this calculation, we selected a SLE patient population of 373 patients to account for potentially missing data.https://www.sgaim.ch/fileadmin/user_upload/Adaptionen/Congress/BilderHighres/Bilder_FK_2019/Abstracts_2019.pdf. This article is not published nor is under publication elsewhere.The abstract of this article is present on a university repository website and can be accessed on This study was approved by the Ethical Committee of the Canton Basel, Switzerland (Ref No EK 262/06), all further involved ethical committees and Swissethics . All procedures performed in this study involving human participants were in accordance with the ethical standards of the research committee and with the 1964 Helsiniki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study.The study population comprised 373 patients of whom 319 (85.5%) were female and 54 (14.5%) male. The median (Interquartile Range (IQR)) age at time of first clinical manifestation of SLE was 31.0 (21.6–43.9) years, the median age at the time of blood sampling 43.1 (32.2–54.3) years, the median age at SLICC index assessment 44.8 (34.6–57.5) years, and the median SLE duration at the time of damage assessment since first clinical manifestation was 9.5 (5.2–18.1) years. Of all patients 74.5% were Caucasian, 10% African, 9.7% Asian, 5.1% Native Americans and 0,3% Pacific Islanders. At the time of the study visit 44.2% had a SLEDAI score of 6 or more (median (IQR) SLEDAI score 4 (2–9)) and 53.4% had a PGA equal to 1 or higher.The basic demographic and clinical characteristics of all patients are summarized in Table The median (IQR) MBL level was 1131 (335–2344) ng/ml. 129 patients (34.6%) showed levels below 500 ng/ml and 175 patients (46.9%) below 1000 ng/ml  and is, therefore, comparable considering the close relationship between systemic sclerosis and SLE.Distribution data of MBL deficiency from healthy Swiss controls were not available. However, likewise to our study population, the frequency of MBL deficiency below 500 ng/ml in SLE patients of our study cohort (median (IQR) 1130 (340–2340) ng/ml, 34.6%) was similar (p = 0.22) to the frequency observed in Swiss scleroderma patients (median (IQR) 1290 (330–2300) ng/ml, 30.3%) as described previouslyFurthermore, we investigated the potential association of erythrocyte sedimentation rate (ESR) as marker of inflammation with MBL levels. We observed a very weak correlation between MBL plasma concentration and the corresponding ESR  as well as the daily prednisolone (PDN) dose , respectively.50, we saw a weak association between patients with an elevated ESR and a MBL deficiency below 1000 ng/ml (p = 0.047). However, this association was not found (p = 0.084) when using a 500 ng/ml cut-off. Further, we saw no such association between MBL deficiency and steroid intake, but as expected patients with a higher ESR were more likely to receive higher doses of PDN as part of the treatment .In addition, using a commonly used pathologic ESR cut-off level of 28 mm/hIn total, 62 patients 16.6%) had at least one CV manifestation, of these patients 77.4% were Caucasians, 11.3% African, 6.5% Asians and 4.8% Native Americans. 20 patients (5.3%) had more than one CVD manifestation, leading to a total of 93 distinctive CVDs that were documented in the study population. Separated into subcategories, 10.2% of the patients had a CVI (38/373), 7.8% a CHD (20/373), 4.3% a MCI (16/373), 2.4% a PAD (9/373) and 1% (1/373) a documented mesenteric insufficiency. . Similarly, MBL levels below 1000 ng/ml were not associated with an increased rate of CVD . In addition, MBL deficiency was not associated with the occurrence of any subcategory of CVD.Furthermore, MBL levels were similarly distributed in CVD versus no-CVD patients (median (IQR) 1180 339–2344) ng/ml vs. 955 (306–2389) ng/ml, p = 0.5, Fig. 39–2344 nSimilarly, the distribution of MBL levels in subgroup analyses . Likewise, ethnicity was not associated with the occurrence of CVD.2 and 42.4% had at least smoked once in their life. In addition, 43.7% of our patients had APL+.With regard to common CVD risk factors, more than half of the patients (52.3%) had hypercholesterolemia, 41.5% had arterial hypertension, 6,7% were diabetic, more than one-third (35.2%) were overweight with a BMI ≥ 25 kg/mAs shown in Table The ESR median in patients with CVD was 18.5 mm/h vs. 16.0 mm/h in patients without CVD (p = 0.3). ESR or the dose of PDN were not associated with the incidence of cardiovascular events in the present study (data not shown).Adjusting the risk of CVD for MBL levels, potential and traditional CV risk factors, revealed a significant association of CVD with male sex (p = 0.05), age (p < 0.001), disease duration (p < 0.001), APL+ (p < 0.001), hypertension (p < 0.001), hypercholesterolemia (p < 0.001) and history of smoking (p = 0.02) in the univariate analysis, while the association with MBL levels p = 0.34), disease activity , BMI (p = 0.58) and DM (p = 0.12) did not reach statistical significance. The final multiple logistic regression model including significant variables in the univariate analysis  revealed an independent significant association between CVD in SLE patients with age (p < 0.001), disease duration (p < 0.001), hypertension (p < 0.005) and APL+ (p < 0.003), while the association with male sex (p = 0.49) and smoking (p = 0.26) did not reach significance anymore  but not with ischemic heart disease57, and Jönsen et al. could show that classical risk factors  were relatively more important for the development of CVD than MBL deficiency58.However, Calvo-Alen Our study was designed to elucidate whether MBL deficiency, based on the resulting blood concentrations, is also associated with an increased incidence of CVD in SLE patients. To address this question, we measured MBL plasma levels and investigated their potential association with clinical CV manifestation and with the main CV risk factors.Our results suggest that MBL deficiency is not a determinant of CVD in SLE patients, independent of other risk factors.58. With regard to the variability of MBL levels within one genotype29, MBL plasma levels seem to be more discriminative in terms of immunological functionality36. Second, we focused on all CVD out of the 12 organ systems that are recorded by the SLICC damage index, including CVI, CHD, MCI PAD and mesenteric insufficiency. Garred et al.59 were the first to describe a higher frequency of thrombotic disease in SLE patients with MBL low-producing genotypes and this analysis was limited to arterial thrombosis in a later study by ∅hlenschlaeger et al.55, while Calvo-Alen et al.57 restricted their analysis to CVI. Of interest, Jönsen et al.58 and Calvo-Alen et al.57 used a similar outcome as in our study, allowing a better direct comparison between the studies. While the latter one showed an association with CVI, the first one, similarly to our study, rather described traditional CV risk factors to be primarily associated with CVD.In part, our findings are in contrast to data reported by previous studies. These differences can be explained by a number of factors. First, our study focused on the determination of plasma MBL concentrations and not the genotypeset al.58 our study population had a considerably shorter follow-up time (in years) (median (range) 14 (0–49) vs. median (IQR) 9.5 (5.2–18.1)) but a somewhat higher age (in years) of the patients (median (range) 40 (10–83) vs. median (IQR) 44.8 (34.6–57.5)).In direct comparison with the study by Jönsen et al.55, Jönsen et al.58 and Font et al.54 but slightly less patients than the study by Calvo et al.57 Although in our study MBL deficiency was numerically more common in the group of SLE patients with CVD than in the Non-CVD group (40.3% vs. 33.4% below 500 ng/ml and 50.0% vs. 46.3% below 1000 ng/ml), this difference was not significant, which could be due to the relatively small number of events in total. In this context, it is of interest to note that patients in the study by ∅hlenschlaeger et al.55 developed an arterial thrombosis in 26%, a frequency 10% higher than in our study population. Likewise, Calvo-Alen et al.57, Jönsen et al.58 and Font et al.54 showed a considerably higher rate of CV events, which might be related to the younger age of our SLE patients and the relatively short follow-up time in our study. As a consequence, in spite of the relatively large number of SLE patient, our study might be limited in power due to its comparatively small number of CVD in total.Another parameter that needs to be considered is the limited number of patients and, as a consequence, the limited number of patients with CVD investigated in previous studies. In comparison, our study evaluated a larger study population than the studies by ∅hlenschlaeger A further limitation that needs to be mentioned is that the damage assessment was carried out using the SLICC index and not with a specific focus on CVD at the time of data collection. Last, risk stratification analyses could not be adjusted to the thyroid function, but, according to our database records, the vast majority of our patients had no evidence of thyroid disease.In balance with these limitations, the strength of our study is the large cohort size of SLE patients being prospectively included in the Swiss SLE Cohort Study (SSCS). The study population included both genders in a typical distribution as observed in patients with SLE and was shown to have typical patient characteristics. In addition, we focused on the determination of plasma MBL concentrations which maybe best reflect functionality. MBL levels in our patients were found to be stable over time and not related to SLE disease activity.In this setting, we could not find a significant association between MBL deficiency and the occurrence of CVD in SLE patients. However, large prospective studies with long follow-ups would be required to definitely exclude a role of MBL in SLE-associated CVD.Supplementary information


Heterobothrium okamotoi, which leads to huge economic losses in aquaculture of the tiger pufferfish Takifugu rubripes. Then, using a simulation study, we tested the possibility of simultaneous improvement of parasite resistance, assessed by parasite counts on host fish (HC), and standard length (SL). Each trait showed moderate heritability . The predictive abilities of genomic prediction among 12 models, including genomic Best Linear Unbiased Predictor (GBLUP), Bayesian regressions, and machine learning procedures, were also moderate for both transformed HC (0.248‒0.344) and SL (0.340‒0.481). These results confirmed the feasibility of GS for this trait. Although an undesirable genetic correlation was suggested between transformed HC and SL (rg = 0.228), the simulation study suggested the desired gains index can help achieve simultaneous genetic improvements in both traits.Parasite resistance traits in aquaculture species often have moderate heritability, indicating the potential for genetic improvements by selective breeding. However, parasite resistance is often synonymous with an undesirable negative correlation with body size. In this study, we first tested the feasibility of genomic selection (GS) on resistance to heterobothriosis, caused by the monogenean parasite  In particular, pedigree-based breeding methods have contributed to aquaculture development by improving economically important traits, as seen in the salmonids and tilapias4. However, pedigree-based methods have innate drawbacks where it is assumed estimated breeding values (EBVs) of target traits for candidate individuals are the average breeding values of parents, ignoring Mendelian segregation within families5. Thus, pedigree-based methods can not differentiate EBVs among full sibs and large-scale pedigrees were needed to evaluate breeding values. On the other hand, molecular markers can be effective in handling Mendelian sampling by capturing genetic variance at DNA levels, i.e., full sibs could have different EBVs. By harnessing whole-genome high-density markers and advanced regression methods, Meuwissen et al.6 proposed genomic selection (GS) to estimate the genomic estimated breeding values (GEBVs) of selection candidates. Thanks to the recent advances in genotyping by sequencing technologies, it is now affordable to genotype genome-wide single nucleotide polymorphisms (SNPs) for GS aquaculture breeding programs7. As expected, the greater performance of GS over the pedigree-based method in prediction and inbreeding control has been demonstrated by empirical studies using cultured fish populations9.Selective breeding is potentially able to boost aquaculture efficiency, now the fastest-growing food production industryTakifugu rubripes is a delicacy in Japan and is one of the most valuable marine fish species in Japanese aquaculture, ranking fourth in production value among cultured finfish10. To fulfill the growing demand for this species, selective breeding will be a practical approach to boost farming efficiency; however, tiger pufferfish aquaculture has not yet fully applied this technology12. Apart from growth-related traits, disease resistance should be highly valued in the breeding program, as disease outbreaks easily hamper the aquaculture industry. For instance, heterobothriosis, the gill disease caused by a monogenean parasite Heterobothrium okamotoi, severely threatens tiger pufferfish productivity and welfare13. The most severe infectious occurs at early phases of production, just after transfer from land-based hatcheries to sea cages15. These naïve juveniles are afflicted by the parasite, persistently present at oceanic aquaculture sites, resulting in retarded growth and high mortality rate16. While the mechanisms of host immune system response to H. okamotoi are still unclear18, host resistance to heterobothriosis is considered polygenic19 and it is difficult to apply marker-assisted selection, which has worked well in infectious pancreatic necrosis resistant Atlantic salmon20. Recently, the potential of GS for disease resistance has been demonstrated in farmed populations of Atlantic salmon 22, rainbow trout (Oncorhynchus mykiss)9, European sea bass (Dicentrarchus labrax)23, and gilthead seabream (Sparus aurata)24. As most of the disease resistance traits have moderate or high heritability in fish species25, GS can also be applied to facilitate heterobothriosis resistance in the tiger pufferfish.The tiger pufferfish 26. For example, the breeding program which improves the resistance to sea lice possibly diminishes growth-related traits in farmed Atlantic salmon27. Likewise, improving resistance to H. okamotoi may negatively affect growth-related traits in the tiger pufferfish11. Thus, simultaneous genetic improvement of resistance to heterobothriosis and body size is most desirable for aquaculture of the tiger pufferfish, although complicated by traits with antagonistic genetic correlations. One of the conventional methods for multiple-trait improvement is the linear selection index (LSI) method developed by Smith and Hazel29. Net genetic merit  of each animal is calculated from each target trait and used for ranking breeding candidates. To maximize the selection response, a general LSI is computed by a linear combination of phenotypes or EBVs and the corresponding coefficients. Extensive LSI methods have been proposed30, as determined by the method of coefficient calculation. For instance, the desired gain selection index allows breeders to restrict traits according to the expected change of genetic gain values of traits31. In the era of GS, those LSI methods can be directly applied to compute the linear genomic selection index (LGSI), which showed higher efficiency in both simulation and real data, compared to pedigree-based LSI32. Although LGSI showed great advantages, successful applications of LGSI still largely depend on the accurate estimation of GEBVs and genetic parameters33, which are sensitive to many factors, including the genetic architecture of target traits, population structure, genotyping technologies, etc.36. Consequently, an LGSI method might have different performances in different cases. Therefore, it is essential to find the optimal strategy incorporating LGSI and examine its performance in each breeding program. The GS breeding simulator will be a practical tool that approximates the real genetic progress by sophisticated modeling of the meiosis and GS procedure at the DNA level37. Further, as regards selection targeting disease resistance traits in aquaculture, a recent simulation study of acute hepatopancreatic necrosis disease (AHPND) in shrimp (Litopenaeus vannamei) showed that GS was superior to pedigree-based methods38. Therefore, with the assistance of simulation, the breeding strategies incorporating LGSI are expected to greatly accelerate the simultaneous genetic improvement of disease resistance and growth-related traits.Selecting one quantitative trait may improve or diminish others due to the genetic pleiotropy and/or linkage disequilibriumIn this study, we tested the possibility of GS to improve heterobothriosis resistance of the tiger pufferfish and designed a GS breeding strategy that could improve the resistance trait concurrent with growth-related traits. We initiated artificial infection on cultured tiger pufferfish obtained from wild parents, applied genome-wide association studies (GWAS), and genetic parameter estimation to survey the genetic architecture of target traits. We then examined the possibility of genomic prediction (GP) for both traits by applying 12 different prediction models. Finally, we investigated the optimal breeding strategy incorporating LGSI using a simulation study by comparing six breeding scenarios.p = 3.79 × 10–6, alpha level = 0.05) while SL approximated a normal distribution . Therefore, we applied a square-root transformation on (HC + 1), approximating a normal distribution . Transformed HC was used in the following genetic analysis. Weak but significant phenotypic correlation was observed between HC and SL .We produced test fishes by artificially crossing 11 wild males and 10 wild females, and subjected 240 4-month-old individuals to an artificial infection for 37 days. Heterobothriosis resistance was evaluated by counting the number of parasites attached to the branchial cavity walls (HC), and the standard length (cm) was measured on each fish (SL). The phenotypic mean was 15.85 (± 9.15 S.D.) for HC and 9.83 (± 0.78 S.D.) for SL Fig. . As the 39. The MiSeq sequencing generated an average of 174,870  raw reads per fish. After the quality-trimming step, the mean number of reads for each fish was 161,426  with the mean read length of 124 bp. The survived reads were mapped onto a reference fugu genome (FUGU5/fr3) for SNP calling. Following the quality filtration of SNPs, 6718 putative SNPs were yielded. Missing SNPs were imputed using LinkImputeR40. At this imputation step, 11 SNPs were discarded and 6707 imputed SNPs were called for each individual with the imputation accuracy of 0.888.We genotyped genome-wide SNPs of each individual using AmpliSeqt-SNE analysis41 based on SNP data . With the same model, the strength of the genetic correlation between the transformed HC and SL was also estimated. We detected a moderate antagonistic genetic correlation (rg = 0.228), where large individuals were suffering from higher parasitic loads. This genetic correlation could be due to the phenotypic correlation, although phenotypic correlation between HC and SL was weak as described above. Therefore, we tested correlation between GEBV for each trait using a univariate linear model (i.e. GBLUP); to predict GEBV for HC, SL was included as the covariate to minimize non-genetic effects from SL. If genetic correlation exists between the two phenotypes, the GEBVs would also show a correlation. We found positive correlation . This supports genetic correlation between the two traits.To investigate the extent of genetic effects on the phenotypic variation, heritability was estimated by a multivariate linear mixed model. Moderate heritability was observed for each trait (transformed HC: 10 (0.05/6707)), SNPs with relatively high association were found in the chromosome 1, 6 and 9 for HC (– log10(p) = 3.48, 3.46 and 3.95, respectively) and in the chromosome 8 and 12 for SL (– log10(p) = 3.58 and 3.51, respectively).GWAS was applied to detect loci highly associated with transformed HC and SL Fig. . Althougr between the GEBVs and observed phenotypes by means of a tenfold cross-validation scheme. Predictive abilities for transformed HC ranged from 0.248 to 0.344 under 12 models , support vector machine regression with a linear kernel (SVR-linear), SVR with a poly kernel (SVR-poly), SVR with a radial basis function kernel (SVR-rbf), feedforward neural networks (FNN), and multi-task feedforward neural networks (multi-task FNN). We compared predictive ability defined as Pearson’s rg = 0.228), using simulation studies. We simulated six scenarios each different in selection schemes, i.e. random mating (RAND), GS on HC only (GSHC), GS on SL only (GSSL), selection applying Smith-Hazel index29  and desired gains index31 (SDGI), for 10 generations with 50 replications . For SDGI, d was set as  for HC and SL, so that SL can be improved preferentially while HC can be reduced by 30% after 10 generations (− 3 * 10/100 = − 30%). To compare the two selection index methods, we also ran an additional scenario (S2SHI) based on Smith-Hazel index, where the economic weight for each trait was set the same as the designed weights of SDGI . As expected, only SDGI could improve the two traits simultaneously, where true breeding values (TBVs) of parasite load (HC) decreased while SL increased in each generation .In this study, we tested the possibility of GS for genetic improvements in heterobothriosis resistance of the tiger pufferfish from empirical data and conducted a simulation study to design a GS breeding strategy that could improve the resistance trait concurrent with growth-related traits. Overall, our results suggest GS for the parasite resistance trait is feasible (predictive ability = 0.248‒0.344) and breeding strategy incorporating the DGI method can simultaneously improve both HC and SL, even though an unfavorable antagonistic genetic correlation was suggested  and SL  were obtained, indicating selective breeding for those traits is feasible. The estimated heritability was comparable to those estimated for resistance against sea lice in Atlantic salmon (h2 = 0.22 to 0.33 with 35 k SNPs)22 and bacterial cold water disease resistance  in farmed rainbow trout (h2 = 0.33 with 35 k SNPs)9. This suggested our small SNP panel could successfully capture the genetic variance for HC in the tiger pufferfish. In this study, we could not detect significant SNPs from GWAS. Even with the small SNP panel and small sample size, strong effect SNPs (the sex-determining SNP) could be detected in a cultured population of the tiger pufferfish39. Therefore, our GWAS result suggests the parasitic resistance is controlled by a large number of quantitative trait locus (QTL) with small or moderate effects, and marker-assisted selection is not feasible. This result is consistent with the previous QTL analysis using the interspecies hybrid system of pufferfishes19. Although the effect was not significant, genes neighboring the SNP with highest – log10(p) values on chromosome 9  deserve further investigation, because the genomic region including this site was reported to have a small QTL effect on host specificity of H. okamotoi19.With 6707 SNP makers, moderate estimated heritability of transformed HC  were similar (0.303‒0.312) and scarcely higher than the GBLUP model (0.307 ± 0.018) for HC. This suggests that these linear models did not greatly differ regarding the predictive ability and the assumptions of the prior distribution of genetic effects have a limited impact on this trait. Bayes RKHS showed slightly better performance in HC compared to these linear models. For SVR-poly and SVR-rbf models, relatively low abilities for HC were observed, however, high abilities were found for SL. Since the default hyperparameters were used in the SVR models, hyperparameter tuning may aid achievement of better performance for HC as in the case of the previous study43. The architectures of FNN and multi-task FNN were tuned to achieve high predictive ability of GS for HC, however, the same architecture was applied to calculate the predictive ability of GS for SL. As expected, these models resulted in high predictive ability for HC but low for SL. This indicates that a deep learning model is task-specific and high accuracy can be obtained with careful optimization as described previously44. However, a great improvement in predictive ability was not achieved by FNN methods compared to GBLUP and Bayesian models even with the model complexity.The predictive abilities for HC estimated under 12 models were moderate (0.248‒0.344), and within the range observed for other disease resistance traits examined in other fish species45, especially when target traits show a negative correlation. On the other hand, DGI, a variation of the selection index methods, allows selection with restrictions on multiple traits via the desired gains vector (d). In this study, the d vector was set with intending to reduce HC by 30% during 10 generations while maximizing SL. The desired gains vector (d) can be further optimized by comparing simulation scenarios with various d to achieve the self-defined breeding goal. Unfavorable genetic correlation between body size and disease resistance is commonly observed in aquaculture species, e.g. vibriosis in Atlantic cod46, bacterial cold water disease in rainbow trout47, and piscirickettsiosis in coho salmon48. Therefore, it is expected that DGI or the similar LGSI method can be widely applied for the simultaneous improvement of disease resistance trait and growth-related traits, which are the primary targets of most breeding programs.Our simulation study showed the availability of DGI for simultaneous genetic improvement in HC and SL even when the unfavorable antagonistic genetic correlation was assumed. The two scenarios incorporating the Smith-Hazel index showed the undesired consequences, where the average TBV for both SL and HC increased . This happened because the breeding scheme only selected the individuals with the top LGSI values, but the high LGSI calculated by the Smith-Hazel index method does not guarantee the selected individuals are superior in both of the traitsIn summary, the availability of GS for HC and SL was confirmed in this study. Moderate heritability for both traits suggests the genetic return from GS is high. GBLUP and Bayesian linear regression models showed similar predictive abilities for these traits. Although an unfavorable antagonistic genetic correlation was suggested between the two traits, the GS breeding strategy incorporating DGI can be a solution for the simultaneous genetic improvement.12 with minor modification. In brief, females were anesthetized with 200 mg/l of 2-phenoxyethanol and then ripened by injection of 150 µg/kg of luteinizing hormone-releasing hormone . Gametes were stripped from each individual and fertilized per male–female pair . Fertilized eggs of each maternal half-sib family were mixed and kept in a hatching jar. After hatching, each maternal half-sib was kept in a holding tank for 1 month and then all families were mixed and cultured in a three-ton communal tank. Rearing and feeding conditions were set as previously described10. At 4 months age, 240 fish were randomly collected and subjected to the artificial challenge test.The empirical experiments were performed in the Fisheries Laboratory, University of Tokyo . All samples (n = 240) were generated by a full-factorial mating among 10 wild males and 11 wild females, which were commercially caught from Wakasa Bay . For the mating, artificial fertilization was applied following the previous study49. A day before the infection, fish were equally distributed into three identical one-ton experimental tanks  supplied with H. okamotoi-free fresh seawater (UV treated and filtered). Meanwhile, eggs of H. okamotoi were collected from tanks containing infected fish and kept in a glass jar containing fresh seawater until infection. Hatching was induced by physical stimulation (shaking at 140 rpm for 10 min) and the density of oncomiracidia, the free-living larvae of H. okamotoi, in the suspensions was determined under the microscope just before the infection. At infection, the water depth of experimental tanks was adjusted to 15 cm, and approximately 4000 oncomiracidia was introduced into each tank. At 3 h post-exposure, fish were transferred into three, newly-setup one-ton holding tanks and reared for 32 days, when H. okamotoi reaches maturation and moves to the branchial cavity walls (BCW)13. At the 32-day mark, fish were euthanized, measured for SL and the BCWs dissected from both sides. For each fish, the caudal fin was clipped and kept in 600 µl TNE8U buffer50 (10 mM Tris–HCl (pH 7.5), 125 mM NaCl, 10 mM EDTA, 1% SDS, 8 M urea) at room temperature to extract genomic DNA for genotyping. Collected BCWs tissues were kept in 10% formalin until counting the number of parasites. The parasites attached to the whole BCWs were counted under a stereo microscope. The host resistance against H. okamotoi is assessed by parasite count on the entire BCWs (HC).Artificial infection was done following previous studies39. In short, 3187 genome-wide target regions were amplified by the first PCR with the custom AmpliSeq primer pools. After the adapter ligation and purification steps, PCR products were barcoded by a second PCR with 8-base index oligo sequences 53. Reads with mapping quality values (MAPQ) less than 10 were removed by samtools (v1.7-2)54. Genotype calling of each sample was done using GATK-4.1.6.055HaplotypeCaller with the following options: –output-mode EMIT_ALL_CONFIDENT_SITES -ERC GVCF –stand-call-conf 30. Obtained gVCF files were combined using GATK CombineGVCFs and then joint genotyping was performed using GATK GenotypeGVCFs. Variant filtering was done using vcftools (v0.1.5)56 with the following parameters: –min-meanDP 15 –max-meanDP 500 –max-missing 0.7 –hwe 0.05 –minDP 10 –remove-indels. The missing values of genotypes were imputed by LinkImputeR-1.2.140. At first, SNPs which did not fulfill the maximum missingness per SNP and sample of 0.9 were filtered out, and then the missing genotypes were imputed. All samples were retained but 11 out of 6718 SNPs were discarded. Subsequently, the imputation accuracy was accessed with numbermasked option (set as 500). PLINK (v1.0.7)57–recodeA option was used to generate the allele coding matrix from the imputed VCF files.Genomic DNA was extracted using a Gentra Puregene Tissue Kit  following the manufacture’s instruction and applied for AmpliSeq genotyping as previously describedt-distributed stochastic neighbor embedding (t-SNE)58. At first, t-SNE transforms the genomic data into conditional probabilities that represent pairwise similarity in the high-dimensional space. Then, transformed data were applied to a heavy-tailed Student’s t-distribution that measures pairwise similarities of corresponding samples in the low-dimensional embedding space. Finally, t-SNE minimized the sum of the Kullback–Leibler divergence between those two probability distributions (Kullback–Leibler divergence is the measure of the difference between two probability distributions). The t-SNE analysis was implemented in sklearn.manifold.TSNE class of Python/scikit-learn-0.20.3. The perplexity was set as 20 and default values were used for the other parameters.The population structure was analyzed with a nonlinear dimension reduction technique, i (i = 1 for transformed HC and 2 for SL); A.mat function in R/sommer-4.0.160 with the default settings; mmer function in R/sommer-4.0.1 to solve the equations. The heritability .To investigate the associated markers with transformed HC and SL, GWAS was performed based on the linear mixed model:63. Samples were randomly and equally divided into ten subsets: one for testing and the remaining for training. The phenotypic values of the test set were masked, and the regression model was trained using the training set. GEBVs of the test set were predicted and predictive ability was calculated as the correlation (Pearson's r) between GEBVs and observed values of the test set. This step was repeated with rotating the test sets among the ten subsets, and the average of Pearson's r was obtained. This cross-validation process was repeated 10 times to obtain the mean and the standard error of the mean (S.E.) for the predictive abilities. Transformed HC instead of the original phenotype was used in GBLUP and Bayesian models. To generate the consistent random state for sampling among 12 models, we fixed seeds for random sampling among the models.Predictive abilities were obtained under 12 regression models described below. The tenfold cross-validation scheme was applied for the ability calculation following the procedure described by Hosoya et al.The same model as the Eq.  was used65 are expressed as follows:, X, i; i. following a specific prior distribution. Bayes A assumes a scaled-t distribution for the prior while Bayes B assumes a mixture of gaussian distribution and a point mass at zero. The prior of Bayes C is a mixture of scaled-t distribution and a point of mass at zero. The prior of Bayes Ridge and Bayes LASSO is a normal distribution and a double exponential distribution, respectively. These models were solved using R/BGLR-1.0.866 with nIter = 10,000 and burnIn = 2000.The models of Bayes A, B, C, Ridge, and LASSO67 structured as:Bayesian RKHS is a Bayesian approach of semi-parametric regressionε-deviation from all observed phenotypes while balancing the model complexity and prediction error69. The method of Lagrange multipliers is used to solve the optimization problem, and the derived approximate function follows:sklearn.svm.SVR function in Python/scikit-learn-0.20.370. The gamma parameter was set to ‘auto’ and the default setting was used for the other parameters.The SVR method can be viewed as a convex optimization problem that finds a function from genotypes to phenotypes at most 71. Neural cells were modeled by non-linear functions (or activation function) and the network was mimicked by the chain structure. Our FNN had one input layer, two hidden layers, and a regression output. The number of input units was 6707, equivalent to the number of SNPs. The first hidden layer has 200 hidden units and the second 20. The rectified linear unit was used as an activation function in hidden layers. FNN was trained by minimizing the loss function, that is, the mean squared error in this case:n is the sample size of the training group; Feedforward neural networks (FNN), inspired by the biological neural network, can model genotype–phenotype regression73. The model has one input layer, two hidden layers, one main regression output, and one auxiliary regression output. The first hidden layer has 200 units, which is a sharing layer for both tasks. Both outputs have separated second hidden layers that differ in the number of the hidden units (20 for the main trait and 100 units for the auxiliary trait). For the estimation of HC, the main regression output is for HC and auxiliary regression output is for SL. The model setting of the main regression and auxiliary regression output was reversed for SL estimation. The activation function and the loss function were the same as the FNN model described above. FNN and multi-task FNN were implemented in Python/keras-2.4.3 package74 with tensorflow-gpu-2.2.1 backend75. FNN used "Adam" optimizer, and multi-task FNN used "RMSprop" optimizer, both with the default parameters. Both models were trained by model.fit method in Python/keras with the parameters as epochs = 30, batch_size = 128, and others followed the default. Many combinations of model architecture, loss function, activate the function, and optimizer was arbitrarily chosen and tested to find the models here that have a high accuracy of GP for HC.Multi-task deep neural network (Multi-task FNN) is based on an assumption where HC and SL share underlying genetic architecture to some extent. These models can improve the accuracy of estimation of the main output using the related auxiliary task as an inductive bias to the main task in reproducing kernel Hilbert space76. The tested scenarios were named for the selection scheme applied: random mating (RAND), GS on HC only (GSHC), GS on SL only (GSSL), Smith-Hazel index (S1SHI and S2SHI), and Desired gains index (SDGI). The workflow of the simulation study is illustrated in Fig. SHI, S2SHI and SDGI was constructed as:SHI and S2SHI was computed as:SHI. varP function of R/AlphaSimR, and mmer function of R/sommer-4.0.1 following the same procedure for the estimation of genetic correlation except that original HC value was used. On the other hand, DGI was computed as:SHI while DGI was assigned to SHI.To investigate the breeding strategy that can improve SL and HC simultaneously, six scenarios different in recurrent selection schemes were simulated for ten generations with 50 replicates using R/AlphaSimR-0.11.0 packageAll experiments done in this study were approved by and carried out in accordance with the IACUC  of the Graduate School of Agricultural and Life Sciences, University of Tokyo (P17-161). All methods were performed in accordance with the IACUC guidelines and regulations.Supplementary Information.


By contrast, there was an increase in the number of Treg cells and the levels of the Treg-related cytokines IL-10 and Foxp3. MFXD and butyrate increased the levels of ZO-1 in the colon. This study indicated MFXD exerts therapeutic effects against AR, possibly by regulating the gut microbial composition and Th17/Treg balance.Mahuang Fuzi Xixin Decoction (MFXD), a Chinese traditional herbal formulation, has been used to treat allergic rhinitis (AR) in China for centuries. However, the mechanism underlying its effect on AR is unclear. This study investigated the mechanism underlying the therapeutic effects of MFXD on AR. Ovalbumin-induced AR rat models were established, which were then treated with MFXD for 14 days. Symptom scores of AR were calculated. The structure of the gut microbiota was analyzed by 16S rRNA gene sequencing and qPCR. Short-chain fatty acid (SCFA) content in rat stool and serum was determined by GC-MS. Inflammatory and immunological responses were assessed by histopathology, ELISA, flow cytometry, and western blotting. Our study demonstrated that MFXD reduced the symptom scores of AR and serum IgE and histamine levels. MFXD treatment restored the diversity of the gut microbiota: it increased the abundance of Firmicutes and Bacteroidetes and decreased the abundance of Proteobacteria and Cyanobacteria. MFXD treatment also increased SCFA content, including that of acetate, propionate, and butyrate. Additionally, MFXD administration downregulated the number of Th17 cells and the levels of the Th17-related cytokines IL-17 and ROR Allergic rhinitis (AR), an immunoglobulin E- (IgE-) mediated respiratory allergic disease, is characterized by nasal itching, congestion, and sneezing . The incShanghan Lun, is a traditional polyherbal mixture composed of Mahuang , Fuzi , and Xixin (the root and rhizome of Asarum heterotropoides f. mandshuricum (Maxim) Kitag.). Mahuang, Fuzi, and Xixin have anti-inflammatory effects [Mahuang Fuzi Xixin Decoction (MFXD), which is described in Zhongjing Zhang's  effects , 6. MFXD effects . In prev effects . Moreove effects . Recent  effects . As MFXDGut microbiota is considered beneficial because it provides protection from pathogens, nutrition, metabolic benefits, and immune system support . HoweverIn the present study, we investigated the effect of MFXD on gut microbial composition and Th17/Treg balance and further examined the therapeutic mechanisms of MFXD. This study might provide a new insight into the immunomodulatory effects of MFXD on AR.β, and IL-23 ELISA kits were purchased from Cusabio Biotech Co., Ltd. . Rat peripheral blood lymphocyte separation medium kit was purchased from Solarbio . Rat-FITC-anti-CD4, Rat-Cy3-anti-CD25, Rat-APC-anti-Foxp3, and Rat-Alexa Flour-488 IL-17 were purchased from eBioscience . Anti-rat ZO-1 antibody was purchased from Abcam . The other chemical reagents used were of analytical grade.Mahuang, Fuzi, and Xixin decocting pieces were obtained from Kangmei Pharmaceutical Co., Ltd. . The voucher specimens (No. 160350561) were deposited in our laboratory. Ovalbumin (OVA) was purchased from Sigma . Aluminum hydroxide was purchased from Damao Chemical Reagent Factory . IgE and histamine (HIS) ELISA kits were purchased from Nanjing Jiancheng Bioengineering Institute . Rat IL-10, IL-17, IL-1MFXD consisted of Mahuang, Fuzi, and Xixin at a ratio of 2 : 3 : 1. Mahuang was immersed in distilled water  for 30 min and boiled for 20 min. Next, Fuzi and Xixin were added to the suspension, which was then simmered for another 90 min. Filtrates were concentrated to 1.52 g/mL by rotary evaporation.ad libitum and acclimated for one week. The study was approved by the Institutional Animal Care and Use Committee of Southern Medical University (No. L2018130).Specific pathogen-free male Wistar rats (160 ± 20 g) were obtained from the Experimental Animal Center of Southern Medical University  and housed in a specific pathogen-free animal laboratory with a relative humidity of 60% and a temperature of 25°C under a 12 h light/dark cycle. All rats were provided specific pathogen-free food and water μL of 5% OVA solution by a micropipette into each nostril once daily for 7 days. Establishment of AR models was confirmed by observing the behaviors of rats and detecting serum IgE level.The animal experiment was conducted by referencing Ren's design with minor modification . The whon = 6). AR rats were randomly divided into four groups (n = 6): the AR model (sterile water), MFXD (7.6 g/kg), loratadine (1 mg/kg), and sodium butyrate groups (200 mg/kg). The dose of MFXD in the study is equivalent to 84 g, recorded by Shanghan Lun, according to the conversion by body surface area. The treatments were orally administered once daily from day 22 to day 35. The rats were intranasally dripped every other day to maintain nasal sensitization during treatment.Normal rats were used as the control group  and Periodic Acid-Schiff (PAS). Histopathological changes were assessed and photographed by an optical photomicroscope.Nasal mucosa was fixed with 4% paraformaldehyde, dehydrated, and embedded in paraffin. The tissues were cut into 4 Colonic tissue sections were dewaxed, rehydrated, and retrieved antigen. After washing with phosphate-buffered saline (PBS), the sections were incubated with anti-rat ZO-1 antibody at 4°C overnight, followed by incubation with secondary antibodies at 37°C for 60 min. After washing with PBS, staining was coupled three times, and 30-diaminobenzidine was added for color development. All slices were counterstained with hematoxylin at 25°C for 1 min. Microscopic images were obtained with a light microscope.β and IL-23 in lung tissues were detected using ELISA kits, according to the manufacturers' instructions.The levels of IgE, HIS, IL-10, and IL-17 in serum and IL-1Total genomic DNA in stool samples was extracted using E.Z.N.A. Stool DNA Kit. The V4 region of the eukaryotic ribosomal RNA gene was amplified by PCR using the primers 341F (CCTACGGGNGGCWGCAG) and 806R (GGACTACHVGGGTATCTAAT). Amplicons were extracted from 2% agarose gels, purified by AxyPrep DNA Gel Extraction Kit, and quantified by QuantiFluor-ST. The concentration and purity of DNA were measured with a spectrophotometer.16S rRNA high-throughput sequencing was performed on an Illumina MiSeq2500 commercial platform. Purified amplicons were pooled in equimolar concentrations and paired-end sequenced (2 × 250) on an Illumina platform according to the standard protocols. Reads were assembled by FLASH (version 1.2.11) and filtered by QIIME (version 1.9.1). After quality filtering and chimera removal, clean data in each sample were clustered into operational taxonomic units (OTUs) with 97% similarity identity using UPARSE pipeline. The phylogenetic affiliation of each 16S rRNA gene sequence was assigned by RDP classifier. Bioinformatics analysis will be carried out with the obtained sequencing data.Enterococcus spp., Butyricicoccus pullicaecorum, and Lactobacillus group belong to Firmicutes. The Bacteroides fragilis group belongs to Bacteroidetes. qPCR assays were carried out in a 96-well optical plate on a LightCycler® 480 Real-Time PCR System . All assays were performed twice. The reaction mixtures consisted of TaKaRa Premix Taq (10 μL), template DNA (2 μL), 10 μM forward primer (0.4 μL), 10 μM reverse primer (0.4 μL), and ddH2O (7.2 μL). The copy number of target DNA was measured by serially diluting standards running on the same plate. Bacterial quantity was presented as log10 bacteria per gram of stool.Based on the sequencing data, the bacteria selected for qPCR are well-known bacteria in the intestines. μL), and ether (1 mL) were added into a 4 mL EP tube and vortex-mixed. The mixture was then centrifuged at 4000 rpm and 4°C for 20 min. The serum samples (100 μL) were added methanol (200 μL), mixed, and centrifuged at 10000 rpm and 4°C for 10 min. The supernatant was analyzed by GC-MS to determine SCFA content.Stool samples were diluted in water, ultrasonically mixed, and centrifuged at 15000 rpm and 4°C for 10 min. The supernatant (0.5 mL), anhydrous sodium sulfate (0.3 g), 50% sulfate acid  were subjected to 10% SDS-PAGE and then transferred onto PVDF membranes. The membranes were then blocked in 5% nonfat milk solution, incubated with the appropriate concentration of primary antibodies against RORγt or Foxp3 overnight at 4°C, and then incubated with secondary antibody. Finally, protein bands were visualized using an enhanced chemiluminescence detection kit and quantified by optical density using the ImageJ software. The results are expressed as a ratio to β-actin.For Foxp3 and RORpost hoc test. Statistical significance was accepted at P values of less than 0.05.All data are presented as the mean ± SD. Statistical analyses were performed using one-way analysis of variance, followed by Tukey's P < 0.05), but treatment with MFXD markedly decreased these scores . MFXD treatment significantly increased the levels of SCFAs in both the feces and serum. Acetic, propionic, and butyric acids showed the same trend and returned to normal levels after MFXD treatment . However, after treatment with MFXD, changes in the levels of IL-10 and IL-17 were noticeably reversed , but promoted the expression of Foxp3, compared with that in the AR group (P < 0.01).Because the differentiation of CD4+ T cells to Th17 or Treg cells was mainly regulated by nuclear transcription factors, we further assessed changes in the expression of Foxp3 and RORblotting . In agreShanghan Lun, the experimental dose was determined by the conversion of body surface area. These measures ensured the safety of MFXD and avoided the occurrence of side effects.Although the therapeutic effect of MFXD on AR has been confirmed in clinical research, its mechanism of action remains elusive. In the present study, we showed that MFXD improved AR symptoms in rats and that this effect was associated with the regulation of gut microbiota structure and the balance of Th17/Treg cells. Herbal medicines can have a risk of adverse effects if not properly used . The toxα-diversity indicators of gut microbiota. The Chao index is used to estimate the species richness information of samples. The Shannon index reflects the diversity of species as a comprehensive reflection of species structure. The larger the value, the higher the diversity of gut microbiota. In present study, we noted that the Chao and Shannon indexes were significantly decreased in the AR group. This was consistent with epidemiological observations that low gut bacterial diversity during infancy augments the risk of allergic diseases [α-diversity, indicating that MFXD increased the richness and evenness of gut microbiota. Moreover, PCA and PCoA showed that the AR group was distinguished from the control group, suggesting that AR altered gut microbial composition. MFXD improved this pathological change due to AR to the normal condition. These results suggested that MFXD as a treatment of AR altered the structure of gut microbiota.AR is a common respiratory allergic disease, in which gut microbiota has been known as a key factor . Chao andiseases . HoweverEscherichia coli and Bacteroides fragilis. Tregs can be induced by Lactobacillus and Streptococcus strains in the intestinal lamina propria [Lachnospiraceae and Ruminococcaceae and an increase in the Enterobacteriaceae which are correlated with the Treg/Th17 imbalance [Lactobacillus group and Ruminococcaceae and decreased the levels of Bacteroides fragilis and Enterococcus spp., which indicated MFXD modulates the abundance of gut microbiota related to the differentiation of Th17 and Treg cells. Moreover, MFXD markedly increased the relative abundance of Anaerotruncus, Butyricicoccus, Lachnospiraceae, and Ruminococcaceae, which are bacteria capable of fermenting carbohydrates, degrading oligosaccharides, and producing SCFAs [In the present study, MFXD increased the abundance of Firmicutes, Bacteroidetes, and Actinobacteria and decreased that of Proteobacteria and Cyanobacteria at the phylum levels, suggesting that MFXD regulated the composition of gut microbiota. Our results implied that MFXD modulates gut microbiota which may benefit in regulating Th17/Treg balance and improving the AR. Gut microbiota is necessary for the expansion and differentiation of intraintestinal and systemic immune cells, which regulates the host immune. For example, Th17 cells are produced by colonization of  propria . A previmbalance . In our ng SCFAs . This isng SCFAs , 29. Theβ and are expanded and stabilized further by IL-23 [β and IL-23 in the lung could be decreased by the MFXD. Our results speculated that MFXD reversed the balance of Th17/Treg which may relate to the inhibition of Th17 differentiation.SCFAs mediate the communication between gut microbiota and the host immune system , regulatby IL-23 , which iγt and Foxp3 are transcription factors of Th17 and Treg, respectively, which impact their differentiation. Thus, we studied the expression of RORγt and Foxp3 in the colon. The data showed that the expression of RORγt and Foxp3 increased and decreased in AR rats, respectively, consistent with the result of a previous study [RORus study , implyinOur study showed that MFXD treatment improved the structure of gut microbiota, increased fecal SCFA content, and restored the balance of Th17/Treg, thereby protecting rats from AR. These findings have advanced our understanding of the mechanism underlying effect of MFXD on AR in terms of gut microbiota and immune regulation.


Intensive care unit (ICU) delirium is a frequent secondary neurological complication in critically ill patients undergoing prolonged mechanical ventilation. Quantitative pupillometry is an emerging modality for the neuromonitoring of primary acute brain injury, but its potential utility in patients at risk of ICU delirium is unknown.This was an observational cohort study of medical-surgical ICU patients, without acute or known primary brain injury, who underwent sedation and mechanical ventilation for at least 48 h. Starting at day 3, automated infrared pupillometry—blinded to ICU caregivers—was used for repeated measurement of the pupillary function, including quantitative pupillary light reflex  and constriction velocity . The relationship between delirium, using the CAM-ICU score, and quantitative pupillary variables was examined.p < 0.05). After adjusting for the SOFA score and the cumulative dose of analgesia and sedation, lower q-PLR was associated with an increased risk of ICU delirium .A total of 59/100 patients had ICU delirium, diagnosed at a median 8 (5–13) days from admission. Compared to non-delirious patients, subjects with ICU delirium had lower values of q-PLR (25 [19–31] vs. 20 [15–28] %) and CV (2.5 [1.7–2.8] vs. 1.7 [1.4–2.4] mm/s) at day 3, and at all additional time-points tested (Sustained abnormalities of quantitative pupillary variables at the early ICU phase correlate with delirium and precede clinical diagnosis by a median 5 days. These findings suggest a potential utility of quantitative pupillometry in sedated mechanically ventilated ICU patients at high risk of delirium. Delirium is a common neurological complication of intensive care unit (ICU), particularly in patients requiring prolonged mechanical ventilation and sedation. Delirium pathophysiology is multifactorial, involving abnormalities of microcirculatory and endothelial function, neurotransmitter imbalance, increased cytokine release and activation of neuroinflammation , 2. The The pupillary light reflex (PLR) is regulated by the cholinergic system, which mediates pupillary constriction to light stimulation ; therefoComposite prediction scores . The study was approved by the ethical committee of the Lausanne University (project-ID 2016-01923), and a waiver of consent was granted because non-invasive pupillometry is standard care. The study conforms with the STROBE guidelines for the report of observational studies.This was a prospective observational cohort study conducted from December 2016 to March 2018 at the Department of Adult Intensive Care Medicine, Centre Hospitalier Universitaire Vaudois (CHUV), University Hospital and University of Lausanne, Switzerland. Subjects were medical-surgical critically ill patients requiring sedation and mechanical ventilation for at least 48 h, at high risk (about 50%) of ICU delirium . ExclusiManagement of analgesia and sedation was based on a written institutional algorithm, in line with current recommendations . SedatioAn automated infrared pupillometer  was used for repeated measurements of quantitative PLR  and constriction velocity . Normative values for the q-PLR range between 30 and 40%, and for the CV between 1.5 and 2.2 mm/s; low values are defined as a q-PLR < 20% and a CV < 1 mm/s, respectively .Pupillary measurements were conducted on both eyes by an experienced clinician or nurse, who was not involved in patient care, and were performed during the day in stable conditions of ambient light. Measurements were performed twice daily (in the morning and the afternoon), starting at day 3 from mechanical ventilation, and were repeated at days 4 and 5, up to a maximum of day 7. At each time-point, the average values of q-PLR and CV from both eyes were retained for the analysis. All pupillometry variables were blinded to clinicians and nurses involved in patient care.As soon as the Richmond Agitation-Sedation Score was equal or greater than − 2, delirium was assessed twice daily until discharge from the ICU using the CAM-ICU. Patients were considered as having delirium when they had at least one positive CAM-ICU during their ICU stay. The duration of coma was calculated as the number of days spent with a motor Glasgow Coma Scale < 6 from ICU admission.Demographic and clinical variables included age, gender, medical versus surgical ICU admission, admission APACHE II score, daily SOFA score, sepsis diagnosis (according to the Sepsis-3 definition ), cumulap < 0.05. Statistical analysis was conducted with R 3.5.1 and JMP-14®. The statistical analysis was performed by an independent statistician (JP).Data are presented as median and interquartile range (IQR), except when otherwise stated. Univariate comparisons between the two main outcome groups (delirium vs. no-delirium) were analyzed using the non-parametric Wilcoxon test. A multivariable stepwise logistic regression model was performed by entering the day 3 q-PLR as the variable of interest, and the day 3 SOFA score, the cumulative fentanyl dose, and the cumulative dose of sedatives as pre-specified co-variates. Statistical significance was set at A total of 100 patients were studied. Patient demographics are summarized in Table As shown in Table p < 0.05 for comparisons, Table n = 30), q-PLR remained significantly lower in patients with delirium .Compared to non-delirious subjects, patients with ICU delirium had lower values of q-PLR and CV, at all time-points tested, starting at day 3 from mechanical ventilation, and up to day 5   Table .Table 4p > 0.2).A model combining SOFA score and q-PLR at day 3 yielded an area under the ROC curve of 0.71 for delirium prediction. Of note, independent associations between delirium and q-PLR remained significant at days 4 and 5. No statistically significant interaction was found between q-PLR and the dose of midazolam and fentanyl, at any time-point tested between day 3 and 5 , in which there are currently no monitoring tools for early delirium detection . The delThe study was single-center and utilized a convenience sample size, without formal sample size calculation, thereby implying a potential risk of biases. The cohort was selected to be representative of a high-risk ICU delirium population, undergoing mechanically ventilation for at least 48 h or more, i.e., a setting where neuromonitoring may be of greatest potential utility. However, pupillometry was not started early on ICU admission in all patients expected to be on mechanical ventilation for at least 48 h, but rather was restricted to patients who were actually still mechanically ventilated after 48 h. It therefore remains to be investigated whether very early pupillometry assessment may provide even earlier evidence for risk of delirium. The duration of the delirium was not available in all patients, which is an additional limitation.Neuroimaging was not systematically performed, but we excluded all patients admitted for a primary acute brain injury or with a previous known neurological disease thereby limiting as much as possible intrinsic brain factors that may potentially alter pupillometry assessment , 43. FurAutomated infrared pupillometry revealed a strong association between lower pupillary light constriction and ICU delirium, irrespective of baseline injury severity and cumulative analgesia and sedation dose. Importantly, alterations in quantitative pupillary constriction variables occurred early and preceded delirium diagnosis by a median 5 days. The findings of this study provide novel insights into ICU delirium pathophysiology and suggest a potential clinical utility of quantitative pupillometry for the neuromonitoring of sedated mechanically ventilated patients at high risk for ICU delirium.


Emerging evidence showed promising effects of vitamin D on headaches characteristics. Thus, it seems there is still a need for more researches to clarify the mechanisms by which this vitamin exerts anti-migraine effects.The present study was conducted as a 16-week randomized double-blind placebo-controlled trial on 80 episodic migraine patients allocated in 2 parallel groups each consisted of 40 patients who received vitamin D 2000 IU/d or placebo. At baseline and after the intervention completion, headache diaries and migraine disability assessment questionnaire (MIDAS) were used to assess migraine related variables in patients. Also, interictal serum concentration of calcitonin gene-related peptide (CGRP) (as the dominant mediator of migraine pain pathogenesis) was evaluated using ELISA method.P value: 0.016). Moreover, after controlling for baseline levels, and other variables using ANCOVA, CGRP level was appeared to be significantly lower following vitamin D supplementation (153.26 (133.03–173.49) ng/L) than the patients in the placebo arm (188.35 (167.15–209.54) ng/L) .The mean (SD) of age in the vitamin D and placebo groups was 37 (8) and 38 (12) years, respectively. ANCOVA test adjusted for baseline values, and confounders showed vitamin D supplementation resulted in a significant improvement in MIDAS score after 12 weeks in the intervention group (21.49 (16.22–26.77)) compared to placebo (31.16 (25.51–36.82) According to the current findings, vitamin D supplementation in episodic migraineurs, particularly in those with migraine with aura, may potentially improve migraine headache characteristics and disability probably through attenuating CGRP levels. Therefore, these results could provide a new insight into anti-nociceptive effects of vitamin D; however, more studies are required to confirm our findings.IRCT20151128025267N6.The trial is registered in the Iranian registry of clinical trials (IRCT) at 11 July 2018, with IRCT code:  According to the report of global burden of disease (GBD) 2016, migraine headache has been established as the leading disability cause in the globe among under 50 years old . MigrainTwo main categories of migraine include episodic migraine (having < 15 headache days per month) and chronic migraine  , 7.Both genetics and environment are supposed to be related to migraine development. Some dietary factors, misuse of caffeine, hormonal fluctuations, high stress level, increased weight, smoking, and suffering from tension type headache or medication overuse headache are among the suggested factors affecting migraine susceptibility and or chronification .There is no established mechanism to explain migraine pathogenesis, although there have been much efforts directed in this field. The current evidence suggest a variety of mechanisms which are thought to be involved in pathophysiology of migraine including cortical spreading depression (CSD), trigeminovascular pathway activation (a pathway for nociceptive information conduction), neuro-inflammation as well as dysfunction in vascular system . Also, dCurrent evidence highlighted a need for compounds that suppress the neuro-inflammatory state in migraine as novel effective medications in its treatment , 10, 11.The details on study design and data collection has been described in our previous publication . BrieflyThe current randomized clinical trial (RCT) was performed in Tehran, Iran from July 2018 to July 2019. Totally, 98 migraine patients who were referred to the tertiary headache clinic of Sina University Hospital, were interviewed for the current trial. Inclusion criteria were diagnosis of episodic migraine (EM) according to headache characteristics and the International Classification of Headache Disorders, 3rd edition (ICHDIII criteria) , age betAfter initial examination by the study expert headache-specialist neurologists, 32 subjects were excluded due to exclusion criteria as illustrated in Fig. At the first visit, all demographic and socioeconomic information including age, sex, marital status, educational status, and job, headache onset years, having migraine with/without aura, familial history of migraine and smoking status, number and type of medications, and data on past medical history of patients were collected through face to face interview by an expert researcher.Anthropometric data including weight, and height were also evaluated at study entry and the end of the trial as follow: body weight was assessed to the nearest 0.5 kg and height to the nearest 0.1 cm applying a Seca 755 dial column medical scale and standard stadiometer, respectively. BMI was calculated as weight in kilograms divided by height in square meters.Patients in the intervention group received 1 pearl containing 2000 IU of vitamin D/d in addition to routine medications prescribed by our study neurologists. The patients in the placebo group were given 1 placebo pearl which was indistinguishable from vitamin D pearls in taste and appearance. Throughout the trial, the subjects were allowed to consume their usual acute/ prophylactic drugs, though they were asked not to change their medications during the trial. Both vitamin D and placebo pearls were produced by Zahravi Pharmaceutical Company, Tabriz, Iran.All participants received a 30-days headache diary to fill out the migraine characteristics  . BrieflyIn addition, at the end of investigation we asked the participants to roughly rate their overall perception of headaches improvement following the current intervention using a six-point Likert scale, ranging from 1 (less than 10%) to 6 (more than 70%) .At baseline and after the intervention completion, MIDAS questionnaire was used to assess migraine related disability in patients. The questionnaire includes seven questions that examine the levels of impact of headaches on work, home, social and leisure activities over the past 3 months. Questions 1 through 5 of this questionnaire assess the migraine-related decline in performance. The questionnaire was validated previously in Iran .At the first visit and the end of the trial, 5 ml blood samples were obtained from all the patients to assess serum concentration of 25OHD and CGRP. Blood samples were centrifuged at 4 °C for 10 min and serum samples were kept at − 80 °C until laboratory analysis. Vitamin D levels in the form of serum 25OHD (S25OHD) were evaluated using chemiluminescence immunoassays (CLIA) method. Serum level of CGRP was evaluated using enzyme linked immunosorbent assay (ELIZA) method and Crystal Day kit, E1061 Hu, 96 tests . The interval of time from the last attack of headache to the day of blood samples collection was also recorded.Compliance was indicated using tablet count after 12-week of intervention. A studied participant was considered compliant if ≥70% of the tablets had been used throughout the trial. During study follow-up, the compliance was additionally stimulated by weekly telephone calls and reminding the participants to use the supplements. Also, after 12-week trial, S25OHD status was evaluated in order to assess the compliance of the subjects in the intervention arm. Also, patients were asked to report any minor or major adverse effects which were likely to be associated with supplementation. The subjects were also asked to rate their satisfaction with participation in the study using a five-point Likert scale ranging from 1 (very dissatisfied) to 5 (very satisfied) .According to the below formula, it was assumed that by estimating 40 samples in each group with an average of 8 headache day/month (standard deviation (SD): 4 days), aiming to detect at least 4 days reduction in headache days, 90% statistical power will be achieved  .\documeP < 0.05 level. All statistical analyses were performed using SPSS program version 19 .Means (SD) were used to describe the quantitative variables and frequencies  were reported for categorical variables. Within and between groups comparisons were made using paired sample t-test or either independent t test or Chi square test, respectively. Further, one-way analysis of variance (ANOVA) was used to compare between groups when aiming to assess variables changes before and after supplementation with vitamin D or placebo in episodic migraine patients with/without aura. In order to analysis the effects of intervention on headache characteristics, MIDAS score, and serum CGRP levels, analysis of covariance (ANCOVA) adjusted for baseline values and confounders was applied. In the case of using ANCOVA, the adjusted means and 95% confidence intervals (95% CI) were also reported. Pearson correlation test was used to determine the correlation between changes in S25OHD and outcome variables. The figures was prepared by GraphPad Prism 5.0 . Significance level was defined as n = 40, 80.0% women) or placebo group . Four subjects in the placebo group and 2 patients in the intervention arm were lost to follow-up. The reasons are described in Fig. Figure Table P value = 0.017); however, after 12 weeks of study, serum levels of this vitamin significantly increased in the vitamin D group  compared to no significant changes observed in the placebo group (from 33.70 to 33.95 ng/ml) .Mean baseline S25OHD levels among patients received placebo was significantly higher than those in the vitamin D group (P = 0.024).The medications consumption of both studied groups at baseline and after the intervention consisted of abortive and prophylactic drugs are demonstrated in Fig. P value< 0.001), whereas there were no significant changes observed within the placebo group when comparing to the baseline data. Following the trial, ANCOVA test adjusted for baseline values and confounders demonstrated that patients in vitamin D group had significantly lower headache days (4.71 day/month) in comparison with placebo group (6.43 day/month) .Detailed results of present RCT on headache characteristics have been reported previously . PatientP value from ANCOVA test = 0.016)). Within group comparisons revealed this score significantly reduced in vitamin D group (mean changes: − 10.38 (12.29); P value< 0.001), while there were no significant changes noted within the placebo group . Vitamin D supplementation resulted in a significant improvement in MIDAS score after 12 weeks in the intervention group (21.49 (16.22–26.77)) compared to placebo (31.16 (25.51–36.82) P value≤0.002). Interestingly, migraineurs with aura who received vitamin D supplements had the highest reduction of mean frequency of headache days per month (− 4.58) and MIDAS score (− 16.92) as compared to other studied groups   Table .Table 3n = 7) and 10% (n = 4) of patients in the vitamin D group reported 50–70% and > 70% overall improvement in their headaches, respectively; while majority of patients in control group reported < 20% improvement   Fig. .Fig. 3PP value = 0.063). Within group comparisons showed CGRP levels significantly reduced in the vitamin D group compare to baseline (mean changes: − 27.31 (59.69); P value = 0.006), while it slightly increased in the placebo group (mean changes: 12.57 ± 23.85); P value = 0.004). After the trial, the results of independent t test showed no significant differences in mean CGRP concentration of the two studied group; however, after controlling for baseline CGRP levels, age, sex, BMI change, the years having headache, and time from last attack using ANCOVA, CGRP level was appeared to be significantly lower following vitamin D supplementation among patients in the intervention group (153.26 (133.03–173.49) ng/L) than the patients in the placebo arm (188.35 (167.15–209.54) ng/L ).At baseline, mean time from the last attack to the day of blood samples collection was reported to be about 4 days in both vitamin D and placebo groups. At the end of trial, mean days since the last attack was estimated as 5 days in vitamin D group and 4 days in placebo group. The changes in serum concentrations of CGRP in two studied groups before and after supplementation with vitamin D or placebo are demonstrated in Table r = − 0.201, P value = 0.094), MIDAS score , and CGRP levels  throughout the intervention .No statistically significant differences were observed between the studied groups with regard to the total number of patients experienced adverse effects over the trial. Minor gastrointestinal complaints were the most reported adverse effects among both groups with 5 patients (12.5%) in vitamin D group and 8 patients (22.2%) in controls. None of the studied subjects reported any serious adverse reactions, and the interventions overly were well-tolerated. The median score of patients’ satisfaction with the trial was higher among vitamin D receiving patients (median = 3) than placebo groups (median = 2) ) and beta-CGRP (which is mainly present in enteric nervous system), only differ in 3 amino acid constituents . The senThe CGRP concentration is detected to be augmented in migraineurs especially during head pain periods , 36. TheTo our knowledge this is the first investigation in migraineurs that assessed the levels of CGRP following vitamin D supplementation. According to available evidence, the mechanisms through which vitamin D influence CGRP synthesis and or release have not been clearly defined yet. There are a number of studies in which the effects of vitamin D or its derivatives on CGRP concentration were evaluated in animal models or cell cultures in different conditions; however, the findings have been inconclusive –43 due, In the present research, it was found that patients in the intervention group had significantly lower S25OHD levels, and on the other hand, a slightly higher serum CGRP levels at baseline; however, supplementation with the vitamin resulted in significant increment of S25OHD and reduction of CGRP, though we did not find any dose-response correlation throughout the trial between either of headache related parameters  and S25OHD. Therefore, it is likely that the protective effects of vitamin D in ameliorating migraine related features and CGRP levels reduction may be attributed to its anti-nociceptive properties and some indirect pathways. It is worth noting that NO, might elevate CGRP and SP synthesis/secretion release that ultimately may result in triggering nociceptive neurons and inflammation specially in trigeminovascular system , 39, 44.The strengths of this study are the prospective- double blind, placebo- controlled design, using a safe daily dosage of vitamin D as a somewhat inexpensive and available over-the-counter supplement, exploring CGRP levels as the dominant indicator of migraine pathogenesis, enrolling the study participant following examinations by expert headache-specialist neurologists, and confirming episodic migraine diagnosis based on the most recent ICHD criteria (ICHDIII). In spite of these strengths, there were a number of limitations. First of all, due to ethical considerations and according to migraine trials guidelines , it was According to the current findings, vitamin D supplementation may potentially improve migraine headache, which has been established as the leading cause of disability in < 50 years, probably through attenuating CGRP levels. Therefore, daily administration of vitamin D 2000 IU as an adjuvant therapy could be considered a well-tolerated and effective agent in improvement of migraine characteristics and disability level particularly among those patients who have migraine with aura. This could provide a new insight into anti-nociceptive effects of vitamin D. However, to confirm current promising findings, large sample size RCTs with longer duration of follow-up are required especially in patients with chronic migraine. Also, well-designed animal and/or clinical studies addressing the mechanisms through which vitamin D may influence migraine pathogenesis, are of crucial importance.