GT)n dinucleotide repeat length polymorphism in the promoter of HMOX1 gene. In addition, –413A>T SNP was assessed by the specific TaqMan® SNP Genotyping Assay, and (GT)n polymorphism was determined by PCR product size analysis. We found that endometriosis is associated with an increased frequency of −413A(GT)31,32 haplotype (OR (95%CI) = 1.27 (1.01–1.60), p = 0.0381) and −413A(GT)31,32 homozygous genotype . These data suggest that endometriosis is associated with functional polymorphism of HMOX1 gene, and this gene may play a part in the pathogenesis of this disorder.Endometriosis is a common gynecological disorder characterized by the ectopic growth of endometrial-like tissue outside the uterine cavity. Etiopathogenesis of endometriosis is poorly understood; it is plausible, however, that the disease may be associated with oxidative stress related to local heme and iron metabolism. Therefore, the aim of the study was to reveal a possible association of endometriosis with a stress-inducible heme oxygenase 1 (HMOX1). For this purpose, 228 patients with clinically confirmed endometriosis and 415 control parous women from general Polish population were examined for functional –413A>T (rs2071746) single-nucleotide polymorphism (SNP) and ( Endometriosis is a common gynecological disorder affecting from 2% up to 22% (approximately 10%) women in reproductive age . EtiopatThere exist several theories of development of endometriosis ,5,6. HowHMOX1 gene located on chromosome 22q12 . However, the frequency of –413AA genotype was significantly higher in endometriosis group (OR (95%CI) = 1.48 (1.06–2.07), p = 0.0228). Endometriosis was also associated with a significantly decreased frequency of –413AT genotype (OR (95%CI) = 0.66 (0.48–0.92), p = 0.0132) (The distribution of  0.0132) .GT repeats in the promoter of HMOX1 gene in patients with endometriosis and control women is shown in GT)n alleles was uneven with many rare variants. When alleles with frequencies >5% were considered, two clearly different groups displaying 23 and 24 repeats as well as 31 and 32 repeats, respectively, could be distinguished. These two variant groups accounted for ca. 80% of all endometriosis and control cases, and therefore, only these groups were considered for specific analyses. They will be further referred to as (GT)23,24 and (GT)31,32 variants. The detailed results of distribution of (GT)n alleles and genotypes in relation to −413A>T SNP genotypes are given in Distribution of the number of GT)n genotype distribution was in Hardy–Weinberg equilibrium both among endometriosis patients (p = 0.09) and control subjects (p = 0.39). Distribution of (GT)23,24 and (GT)31,32 variants and their genotypes are shown in GT)23,24 in endometriosis and control groups was 30.8% and 31.6%, respectively, whereas the respective frequency of (GT)31,32 was 52.4% and 47.2%. The difference in distribution of (GT)23,24 in endometriosis and control group was not significant (OR (95%CI) = 0.96 (0.75–1.23), p = 0.777), whereas the difference in the frequency of (GT)31,32 had a borderline significance (OR (95%CI) = 1.23 (0.98–1.55), p = 0.0754). Analysis of distribution of (GT)23,24 and (GT)31,32 genotypes has revealed that endometriosis is associated with increased frequency of (GT)31,32 homozygotes (OR (95%CI) = 1.43 (1.01–2.05), p = 0.0480). There were no differences in distribution of (GT)23,24/(GT)31,32 heterozygotes and (GT)23,24 homozygotes in endometriosis group as compared with control n repeats are in linkage disequilibrium. In addition, –413A allele is associated with (GT)31,32 allele (r2 = 0.71), whereas –413T allele is associated with (GT)23,24 (r2 = 0.57).Haplotype analysis has revealed that HMOX1 haplotypes and their related genotypes is presented in 413A(GT)31,32 haplotype (OR (95%CI) = 1.27 (1.01–1.60), p = 0.0381). There was no difference in the frequency of –413T(GT)23,24 haplotype between endometriosis and control group (OR (95%) = 0.95 (0.67–1.35), p = 0.6954). Frequency of the other haplotypes was relatively low, and none of them showed any significant association with endometriosis (data not shown).Analysis of distribution of these two major 413A alleles in combination with (GT)31,32 variant is shown in 413A(GT)31,32/–413A(GT)31,32 homozygous genotype was significantly more frequent in women with endometriosis (OR (95%CI) = 1.51 (1.06–2.17), p = 0.0238). On the other hand, analysis of other combinations between –413AA homozygotes and other (GT)n variants, as well as (GT)31,32(GT)31,32 homozygotes and –413AT or –413TT did not reveal any difference between endometriosis and control group (HMOX1 genotypes including combinations of –413T and (GT)23,24 alleles were significantly associated with endometriosis (data not shown).Analysis of distribution of –ol group . None ofHMOX1 gene. The disease was found to be associated with an increased frequency of both –413AA and (GT)31,32(GT)31,32 homozygotes. In addition, we observed that the distribution of –413 HMOX genotypes among patients (but not controls) significantly departed from Hardy–Weinberg equilibrium (HWE). Since departure from HWE among cases has been proposed as an independent test detecting an association [HMOX locus is not neutral in endometriosis.The results of our study show that endometriosis is related to functional polymorphism in the promoter of ociation ,38, this413A(GT)31,32 haplotypes as well as –413A(GT)31,32/–413A(GT)31,32 genotypes. This indicates that disease may be related to homozygosity in either –413A or (GT)31,32 allele or may be linked to their combination. However, it was not possible to distinguish between the effect of –413AA or (GT)31,32(GT)31,32 homozygosity because of the very low frequency of subjects in whom these two alleles were not found together . Analysis of the frequency of HMOX1 (GT)n alleles has revealed two clearly different groups displaying 23–24 repeats or 31–32 (GT) repeats which account for ca. 80% of all studied subjects. This observation is also consistent with other reports on (GT)n allele distribution [GT)n alleles were classified into short or long and sometimes medium variants. However, criteria for such classification were arbitrary, and therefore, to avoid a bias related to a great diversity of (GT)n variants, we decided to limit our analysis only to specific long (GT)31,32 and short (GT)23,24 alleles.Distribution of –413A>T ulations ,29 and iribution . It shou413A HMOX1 homozygote is associated with a higher risk of women’s’ hypertension and coronary artery disease. In respect to the (GT)nHMOX1 polymorphism, our results are also similar to results reported for a variety of other diseases including some cardiovascular disorders and transplanted organ dysfunction [GT)n variants (n>30) were associated with increased susceptibility to the disease.Our present result in women with endometriosis is similar to the previous reports of Ono et al. ,29 who ffunction  as well function , where lHMOX1 promoter polymorphism still remains a matter of controversy. It is generally accepted that short (GT)n variants are associated with an increased HMOX1 expression and activity. This is based on both the biological observations of cell lines isolated from subjects carrying different (GT)n variants [HMOX1 promoter/luciferase assays [GT)n variants and did not take into consideration a possible influence of −413A>T SNP polymorphism and possibly other yet-unknown factors. In contrast to these reports, luciferase reporter assays performed by Ono et al. [A(GT)30-harboring HMOX1 promoter constructs was about six times higher than that of −413T(GT)23 ones. This strongly implies that the relationship between the length of (GT)n variants and prevalence of −413A>T alleles needs further elucidation.The biological significance of variants ,41 and ce assays ,43. It so et al.  have revHMOX1 may be stimulated by inflammatory cytokines as well as oxidative stress agents [Expression of s agents ,23,24 whs agents ,13,14,15s agents  which maThe specific role of HMOX1 in etiopathogenesis of endometriosis also remains to be established. There is a growing evidence that this enzyme may exert a variety of cytoprotective effects ,23,24. HHMOX1 promoter polymorphism in endometriosis also remains obscure. It would be of interest to correlate HMOX1 polymorphism with some clinical features of endometriosis such as stage of the disease, lesion type and their location. However, the patients’ cohort recruited in the present study consisted mostly (95%) of women with moderate/severe (III/IV) stage of the disease. Furthermore, 95% of patients had endometriotic ovarian cysts, and only 5% of them had peritoneal lesions. Such patients’ stratification made any statistical correlation analysis meaningless. Therefore, further studies on a group of patients including significantly more cases with minimal/mild stage of endometriosis and higher prevalence of only peritoneal lesions would be necessary.Clinical significance of HMOX1 gene promoter. This suggests that HMOX1 plays an important role in etiopathogenesis of this disorder, possibly by exerting a protective effect and facilitating survival of endometriotic cells in ectopic sites. This, however, needs further extensive studies.In summary, our present results show that endometriosis is associated with functional polymorphism of


Anti-TIF1γ antibodies are a class of myositis-specific antibodies (MSAs) and are closely associated with adult cancer-associated myositis (CAM). The heterogeneity in anti-TIF1γ+ myositis is poorly explored, and whether anti-TIF1γ+ patients will develop cancer or not is unknown at their first diagnosis. Here, we aimed to explore the subtypes of anti-TIF1γ+ myositis and construct machine learning classifiers to predict cancer in anti-TIF1γ+ patients based on clinical features.A cohort of 87 anti-TIF1γ+ patients were enrolled and followed up in Xiangya Hospital from June 2017 to June 2021. Sankey diagrams indicating temporal relationships between anti-TIF1γ+ myositis and cancer were plotted. Elastic net and random forest were used to select and rank the most important variables. Multidimensional scaling (MDS) plot and hierarchical cluster analysis were performed to identify subtypes of anti-TIF1γ+ myositis. The clinical characteristics were compared among subtypes of anti-TIF1γ+ patients. Machine learning classifiers were constructed to predict cancer in anti-TIF1γ+ myositis, the accuracy of which was evaluated by receiver operating characteristic (ROC) curves.Forty-seven (54.0%) anti-TIF1γ+ patients had cancer, 78.7% of which were diagnosed within 0.5 years of the myositis diagnosis. Fourteen variables contributing most to distinguishing cancer and non-cancer were selected and used for the calculation of the similarities (proximities) of samples and the construction of machine learning classifiers. The top 10 were disease duration, percentage of lymphocytes (L%), percentage of neutrophils (N%), neutrophil-to-lymphocyte ratio (NLR), sex, C-reactive protein (CRP), shawl sign, arthritis/arthralgia, V-neck sign, and anti-PM-Scl75 antibodies. Anti-TIF1γ+ myositis patients can be clearly separated into three clinical subtypes, which correspond to patients with low, intermediate, and high cancer risk, respectively. Machine learning classifiers  had good predictions for cancer in anti-TIF1γ+ myositis patients. In particular, the prediction accuracy of random forest was >90%, and decision tree highlighted disease duration, NLR, and CRP as critical clinical parameters for recognizing cancer patients.Anti-TIF1γ+ myositis can be separated into three distinct subtypes with low, intermediate, and high risk of cancer. Machine learning classifiers constructed with clinical characteristics have favorable performance in predicting cancer in anti-TIF1γ+ myositis, which can help physicians in choosing appropriate cancer screening programs. Idiopathic inflammatory myopathies  are a group of highly heterogeneous systemic autoimmune diseases. IIMs have five main subgroups—dermatomyositis (DM), polymyositis (PM), immune-mediated necrotizing myopathy (IMNM), sporadic inclusion body myositis (sIBM), and overlap myositis (including antisynthetase syndrome) , 2. An iAutoantibodies are important biomarkers in IIMs. Myositis-specific autoantibodies (MSAs) are specific to IIMs, while myositis-associated autoantibodies (MAAs) are associated with myositis overlap syndromes. MSAs are closely correlated with distinct disease phenotypes; for example, anti-MDA5 antibodies are associated with clinically amyopathic DM and rapidly progressive interstitial lung disease (RP-ILD) .Transcriptional intermediary factor 1γ (TIF1γ) is a protein belonging to the tripartite motif (TRIM) superfamily that plays diverse roles in transcriptional elongation, DNA repair, cell differentiation, mitosis, and embryonic development. Anti-TIF1γ antibodies were first referred to as anti-p155 in the serum of myositis patients, which immunoprecipitated a 155-kDa nuclear protein , 12. AntIn this study, we analyzed 44 clinical characteristics at baseline of 87 anti-TIF1γ+ adult myositis patients and their outcome of cancer during the follow-up time. Fourteen variables most important for distinguishing cancer and non-cancer were selected. Then, multidimensional scaling (MDS) plot and hierarchical cluster analysis divided anti-TIF1γ+ myositis into three clusters based on sample similarities calculated with these variables, which corresponded to patients with low, intermediate, and high cancer risk. Distinct clinical characteristics were found among clusters. In addition, machine learning classifiers for cancer were constructed and verified with excellent performances. Overall, our study highlighted a new strategy to manage anti-TIF1γ+ patients with machine learning algorithms to stratify and predict their cancer risk at their first visit at hospitals.This was a longitudinal study conducted at Xiangya Hospital in Hunan Province, south-central China, from June 2017 to June 2021. The inclusion criteria were a clinical diagnosis of myositis in adult patients according to either the 1975 Bohan/Peter  or 2017 Medical records of patients at the time of enrollment before treatment were reviewed, and data on 44 clinical variables .Machine learning algorithms were performed in R or Python. Elastic net was used to select the most important clinical variables out of all 44 clinical variables for the classification of anti-TIF1γ+ myositis with cancer or not. The importance of selected variables was evaluated by random forest with mean decrease Gini. The similarities (random forest proximities) of samples were calculated with the selected variables, based on which multidimensional scaling (MDS) plot and hierarchical cluster analysis were used to cluster and visualize these samples. Five machine learning classifiers  were also constructed with these selected clinical variables to predict cancer probability of anti-TIF1γ+ patients. Stratified 10-fold cross-validation was performed to estimate the accuracy of each model and optimize the models with hyperparameter tuning. Eighty-seven patients were split into a training set containing 70% of the observations and a test set containing the remaining 30%. The training set was used to build the classificatory models, and the testing set was used to evaluate the accuracy of the model. All steps were performed using Python V.3.9.1, scikit-learn V.0.24.2, NumPy V.1.19.5, and pandas V.1.2.1. ROC (receiver operating characteristic) curves were drawn with Matplotlib V.3.3.3.p <0.05 was considered statistically significant. Quantitative data were described as the means (standard deviations) or medians (interquartile ranges) according to data distribution and homoscedasticity. Qualitative data were described as frequencies (percentages). Accordingly, one-way ANOVA or Kruskal–Wallis tests (adjusted with Dunnett T3 or Dunn’s test) and Pearson chi-square test or Fisher’s exact test (adjusted with Bonferroni test) were performed to compare multiple groups. The heatmap was plotted by ggplot2 in R.The statistical analysis of comparisons among anti-TIF1γ+ myositis patients in the three clusters was performed in SPSS v.22 or GraphPad 8.0, and Anti-TIF1γ+ myositis patients enrolled in our study were mainly female (64.4%), with an average age of 57 years and a median disease duration of 6 months. Their most common manifestation was typical DM rashes (95.4%), followed by proximal weakness (56.3%), ILD (17.2%), and arthritis/arthralgia (11.5%). Two patients (2.3%) were complicated with another connective tissue disease—Sjogren’s syndrome.Forty-seven (54.0%) of our anti-TIF1γ+ patients in total met the diagnostic criteria of CAM. Their temporal relationships between the diagnosis of myositis and cancer are shown in ), the 87 anti-TIF1γ+ patients can be separated into three distinct clusters. The patient numbers in clusters 1, 2, and 3 were 28 (32.2%), 28 (32.2%), and 31(35.6%), respectively. Twenty-seven (96.4%) patients in cluster 2 developed cancer, which was almost the opposite of cluster 1 with only one (3.6%) cancer patient. Cluster 3 had 19 (61.3%) patients with cancer and were intermediate between clusters 1 and 2. Therefore, we inferred clusters 1, 2, and 3 corresponded to three stratifications with low, high, and intermediate cancer risk. The importance of the 14 selected clinical variables was ranked with random forest algorithm according to mean decrease Gini  of patients were calculated by these 14 variables. As shown in the MDS plot and hierarchical clustering tree (p < 0.001). Patients in cluster 2 had an older age (p = 0.020); a higher percentage of skin ulcers (p = 0.008); a shorter disease duration (p < 0.001); higher levels of CRP (p = 0.005), lactic dehydrogenase (LDH) (p < 0.001), creatine kinase (CK) (p = 0.005), N% (p = 0.013), and NLR (p < 0.001); and a lower level of L% (p < 0.001). Clusters 1 and 3 also had significant difference in disease duration (p < 0.001), LDH (p < 0.001), N% (p < 0.001), L% (p < 0.001), and NLR (p < 0.001). Interestingly, clusters 2 and 3 differed obviously in cancer types: 87.5% patients with breast cancer were in cluster 3 and 100.0% patients with nasopharyngeal carcinoma were in cluster 2 (p = 0.005 and p < 0.001). Ovarian cancer, peritoneal tumor, renal pelvis cancer, pancreatic cancer, pelvic tumor, and tonsil cancer were all developed in cluster 3. Conversely, bladder cancer, esophageal cancer, retroperitoneal cancer, and liver cancer were all found in cluster 2 .We then analyzed the clinical characteristics of anti-TIF1γ+ patients in the three clusters Table 1.), respectively.Early diagnosis of cancer in anti-TIF1γ+ myositis is crucial for improving prognosis, especially for patients with intermediate and high cancer risk in clusters 2 and 3. We constructed machine learning models with the selected 14 most important variables to predict anti-TIF1γ+ myositis with or without cancer. The ROC curves of machine learning models calculated using the training and testing sets are shown in In this study, anti-TIF1γ+ myositis is separated into three different clinical subtypes. Clusters 1, 2, and 3 correspond to patients with low, high, and intermediate risk of cancer, respectively. Anti-TIF1γ+ patients with low cancer risk have distinct clinical characteristics from those with high and intermediate cancer risk, which enables the construction of models to predict cancer in anti-TIF1γ+ patients. Indeed, machine learning classifiers  showed good classification in discriminating anti-TIF1γ+ patients with cancer or not. In particular, the prediction accuracy of random forest was >90%. Decision tree can conveniently stratify and manage anti-TIF1γ+ myositis patients with disease duration, NLR, and CRP in clinical practice. It is of great value because myositis patients who are positive for anti-TIF1γ antibodies and predicted to develop cancers by our models should undergo more careful and intensive tumor screening than those predicted with low cancer risk.Fourteen clinical variables were selected for model construction in our study. The top 10 variables ranked by importance were disease duration, L%, N%, NLR, sex, CRP, shawl sign, arthritis/arthralgia, V-neck sign, and anti-PM-Scl75 antibodies. The disease duration  was shorter in the cluster with intermediate and high cancer risks than in the cluster with low cancer risk. This may be related to the more severe disease state of patients in clusters with higher cancer risk, manifested as more patients with deteriorated general condition and increased CK levels. Anti-TIF1γ+ myositis patients in clusters with intermediate and high cancer risk had a higher N% and NLR but a lower L%. Neutrophil dysregulation is pathogenic in myositis, mainly through neutrophil extracellular traps (NETs) and the subset of low-density granulocytes (LDGs) . The undThere were no differences in anti-TIF1γ intensities  among clusters with different cancer risks. It is still controversial whether the levels of anti-TIF1γ antibodies correlate with the evolution of cancer –25. TestOur study showed that 78.7% 37/47) of cancers were diagnosed within 0.5 years of the myositis diagnosis. A time span of 3 years before and after myositis diagnosis is emphasized in the definition of CAM 7 of canc–8. In faThis is the first study identifying subtypes and predicting cancer in anti-TIF1γ+ myositis by machine learning algorithms, although several limitations exist. First, compared with anti-MDA5 (30%) and anti-JO1 (19%) antibodies, the positive rate of anti-TIF1γ antibodies (7%) among myositis patients is relatively low . Thus, mIn conclusion, anti-TIF1γ+ myositis can be divided into three distinct subtypes based on their clinical characteristics, which are corresponding to patients with low, intermediate, and high cancer risk. Machine learning models showed satisfactory accuracy in the prediction of cancer in anti-TIF1γ+ myositis patients. These findings suggested that the stratified management of anti-TIF1γ+ myositis patients is necessary to avoid excessive cancer screening examinations in patients with low cancer risk and make the best use of targeted cancer screening in patients predicted to develop cancer.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of the Institutional Review Board at Xiangya Hospital. The patients/participants provided their written informed consent to participate in this study.LZ, HZ, and HL designed the study. SX, BZ, CS, ZG, WP, LL, JiZ, QP, JuZ, JP, YZ, LZ, and LS collected the data. LZ and HZ analyzed the results and wrote the draft. HL revised the manuscript for content. All authors contributed to the article and approved the submission for publication.This work was supported by grants from the National Natural Science Foundation of China  and Hunan Provincial Natural Science Foundation (2019JJ40503).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Investigate the effect and mechanism of berberine on the small intestinal mucosa of non-steroidal anti-inflammatory drugs (NSAIDs) related small intestinal injury.Twenty-four SD rats were randomly divided into control group, model group and intervention group. The model group and intervention group were treated with diclofenac , a total of 4 days tube feeding, and the intervention group was treated with 50 mg/kg·d intragastric administration of berberine after 2 days. The control group was treated with 7.5 mg/kg·d, 2/d 0.9% saline tube feeding. Then we screened differential expression of colonic mucosal gene by the liquid chip technology.P < 0.05), HTR4, HTR1a, F2RL3, CALCA, NPY, CRHR2, IL1b, P2RX3, TPH1, HMOX1, TRPV1, VIP, F2RL1, SLC6A4, TFF2, AQP8 content were significantly increased (P < 0.05), NOS1 content decreased significantly (P < 0.05); Compared with the model group, macroscopic and histology score of the intervention group improved significantly (P < 0.05), and HTR4, F2RL3, NPY, CRHR2, IL1b, VIP, AQP8 content were significantly lower (P < 0.05), NOS1 content increased significantly (P < 0.05).Compared with the control group, macroscopic and histology score of the model group increased significantly (Berberine has a protective effect on NSAID-associated small intestinal injury, the mechanism may be that berberine decreases the expression of intestinal mucosa HTR4, F2RL3, NPY, CRHR2, IL1b, VIP, AQP8, and increases the expression of NOS1, that to reduce intestinal permeability and protect intestinal mucosal barrier. Non-steroidal anti-inflammatory drugs (NSAIDs) is a kind of anti-inflammatory drug that does not contain steroid structure, which is widely known that have effect on anti-inflammatory, anti-rheumatism, alleviating pain, pyretolysis and anticoagulated blood and so on. With the wild application of NSAIDs in rheumatoid arthritis, cardiovascular and cerebrovascular diseases, its adverse reactions are also exposed. These side effects involve the digestive system, blood system, skin connective tissue and respiratory system, among which the most common is the adverse reactions of the gastrointestinal tract . In currIt is reported that various damage features of the small intestine are common among long-term NSAIDs users: inflammation accounts for 60–70%, ulceration accounts for 30–40%, increased permeability is up to 70%, bleeding/anemia is about 30%, and malabsorption occupies 40–70%  so severA lot of research on the mechanism of NSAID-induced small intestinal injury and therapeutic method had been made through experiments and clinical capsule studies. And selective COX-2 inhibitors, prostaglandin derivatives, cytoprotective drugs, Phosphatidylcholine-NSAID, and probiotics all shown to have potential protective effects on NSAID-induced small intestinal injury. However, no new promising and standardized drugs had been developed for NSAID-associated small intestinal injury until recently . BerberiTherefore, our present study aims to screen differential expression of small intestinal mucosa gene by the liquid chip technology so as to build a gene regulatory network for NSAID- induced small intestinal disease. Meanwhile, investing the specific mechanism of berberine to prove it has effective therapeutic effects from the gene level.n = 8), the model group (n = 8) and the intervention group (n = 8). This study was reviewed and approved by the Medical ethics committee of Zhejiang Chinese Medical University. All sections of this report follow the ARRIVE Guidelines for reporting animal research.Twenty-four female Sprague Dawley (SD) rats , provided by Animal Experiment Center of Zhejiang Chinese Medicine University, without feeding drugs or used as experimental animals, were kept in room cages at 22–24 °C, humidity< 60%, noise <50db. Rats were fed with standard diet and distilled water. The rats were acclimatized for a week before the start of the experiment, and were divided randomly into three groups, the control group  into 0.9% saline. Referring to the long-term of human oral dose (150 mg/d), and converting into rats oral dose 7.5 mg/kg·d, 2/d, a total of 4 d tube feeding preparation model of intestinal injury in 8 SD rats.After 2d of diclofenac sodium tube feeding, the berberine  crushed dissolved in 0.9% saline, 50 mg/kg·d tube feeding to 8 SD rats for 2d.All rats were fasted for 24 h, and put to death by intraperitoneal injection of 1%pentobarbital sodium on 5th day. Opening the rats’ abdominal cavity cutting 2 cm bowel from the ileocecal valve 5 cm, taking intestinal mucosa macroscopic and histology evaluation.Anatomical lesion score from Wallace et al.  was usedEach intestinal segment was processed into paraffin. Preparing Serial paraffin sections (4 μm) and staining with hematoxylin–eosin and PAS for morphological examination. Chiu scale  was usedThe main procedure was as below: High-throughput sequencing was conducted on independent samples. Total ribonucleic acid (RNA) was extracted from duodenal tissues through TRIzol  one-step method. Then we did primer test and sample assay. After purification, RNA concentration was analyzed by Nanodrop  and quality testing was conducted by using BioAnalyzer . SuperScript II reverse transcriptase was used to synthesize complementary DNA(cDNA). Luminex 100TM was used to detect the gene expression of intestinal mucosa.−x ± SD, and was analysed by using one-factor analysis of variance. The data of nonnormal distribution was demonstrated by median and full range with using rank sum test, P < 0.05 showed that the difference had statistical significance.The data of normal distribution was demonstrated as P < 0.05). The lesion scores of small intestinal mucosal of berberine intervention group (2.57 ± 0.33) was significantly lower (P < 0.05) compared with the model group  had higher scores than the control group (0.86 ± 0.24). Compared with model group,the histological scores of berberine were lower (P < 0.05)  Fig. B. Model P < 0.05), and higher levels of HTR4, HTR1a, F2RL3, CALCA, NPY, CRHR2, IL1b, P2RX3, TPH1, HMOX1, TRPV1, VIP, F2RL1, SLC6A4, TFF2, AQP8(P < 0.05). After berberine intervention, HTR4, F2RL3, NPY, CRHR2, IL1b, VIP, AQP8 were significantly decreased (P < 0.05), and NOS1 were significantly increased (P < 0.05)  regular used NSAIDs and around 43 million adults (19.0%) took aspirin more than 3 months(> 3 times per week). Widespread use of NSAIDs has increased the damage to the digestive tract such as small intestine which worth our attention –26: HTR4Our experiments have confirmed that the pathogenesis of NSAID-induced small intestinal disease is related to intestinal motility abnormalities, increases permeability of the intestinal mucosa caused by neuroendocrine, inflammatory reactions and disorders of water metabolism. The 5-Hydroxytryptamine Receptor 4(HTR4) and 5-Hydroxytryptamine Receptor 1a(HTR1a) produce a sustained excitatory response by activating protein kinase A and binding to 5-Hydroxytryptamine (5-HT). In this experiment, the expression of HTR4 and HTR1a in the ileum mucosa increased, which suggested that NSAIDs mediated the intestinal nervous system to control the movement of intestinal chromaffin cells and increased the excitability of intestinal motility, and then caused intestinal motility dysfunction . As wellThe liquid chip technology is an easier and more cost-effective alternative to purchasing and running several real-time instruments in parallel. In conclusion, while testing clinical specimens, the Luminex assay shows clinical sensitivity and specificity comparable to real-time Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The assay can process large numbers of specimens at the same time, while significantly reducing the cost. Furthermore, the platform’s flexibility allows for the possibility of adding more targets and of easily changing primer sequences in order to detect more genes at the same time .Berberine, which is extracted from traditional Chinese medicine Rhizoma coptidis, is usually used to treat gastrointestinal disorders, due to its antimicrobial propertie . RecentlCoagulation factor II receptor-like 3 (F2RL3) encodes a member of the protease-activated receptor subfamily, also known as protease-activated receptor 4(PAR4),which takes partin platelet activation, intimal hyperplasia and inflammation . Some stOn the contrary, vasoactive intestinal peptide (VIP) has potent anti-inflammatory effects via downregulates TNF-α expression  and NF-ΚIn humans, AQP8 protein is found as a water selective channel  and is lIn summary, multiple genes involved in the pathogenesis of NSAID-induced small intestinal disease, associated with intestinal motility disorder, intestinal barrier damage. Chinese medicine berberine has a protective effect on NSAID-associated intestinal injury, the mechanism may be that berberine decreases the expression of intestinal mucosa HTR4, F2RL3, NPY, CRHR2, IL1b, VIP, AQP8, and increases the expression of NOS1 through alleviating inflammation, nerve endocrine regulation to reduce intestinal permeability and protect intestinal mucosal barrier.


It is important to determine the activation status of Rac and Cdc42 in cancer tissues for the prediction of metastasis and patient prognosis. However, it has been impossible to detect their spatial activation on formalin-fixed paraffin embedded (FFPE) surgical specimens thus far. Here, we established a novel detection technique for activated Rac/Cdc42 in human colon cancer FFPE tissues by using a p21-activated kinase (PAK)-Rac binding domain (RBD) detection probe fused with glutathione S-transferase (GST), designated GST-PAK-RBD, and novel rapid-immunohistochemistry (R-IHC) systems using noncontact alterating-current electric field mixing, although there is a technical limitation in that it may not distinguish between Rac members and Cdc42. In 50 cases of colon cancer, various activation patterns of Rac/Cdc42 were observed, which were designated plasma membrane, cytoplasm, mixed pattern, and polarized distribution. The activity was striking in the invasive fronts of tumors and significantly correlated with tumor invasion properties evaluated by TNM classification. Of note, in tissue microarray (TMA) samples, 29 of 33 cases demonstrated higher Rac1/Cdc42 activity in the tumor area than the corresponding normal mucosa. In addition, positive correlations were detected between Rac/Cdc42 activity and clinicopathological factors such as venous and lymphatic vessel invasion. These results suggest that understanding Rac and Cdc42 activations in cancer tissues would be valuable as an option for molecular therapy as personalized medicine. Although an understanding of tumor migration and invasion capabilities would be indispensable for predicting metastasis and patient prognosis, to date, only microscopic examination using H&E staining has been performed on surgical specimens of human cancer formalin-fixed paraffin embedded (FFPE) tissues2.Tumor invasion and metastasis are prognostic factors in various cancer patients. The potential of tumor cell migration and invasion seems to be individually distinct, as evaluated by the expression of epithelial-mesenchymal transition (EMT) markers such as Snail, Slug, N-cadherin, and Vimentin3.Rac and Cdc42 are key regulators that promote cell motility and are associated with tumor invasion and metastasis. Small GTPases comprise several families, including the Ras, Rho, and Rab families. The Rho family contains three well-characterized GTPases, RhoA, Rac1, and Cdc42, which cooperatively regulate the assembly and organization of the actin cytoskeleton. RhoA participates in the formation of stress fibers and focal adhesion complexes, Rac1 promotes the assembly of a meshwork of actin filaments to produce lamellipodia and membrane ruffles, and Cdc42 leads to the formation of filopodia and actin-rich surface protrusions5. Rac1/Cdc42-GTP bind to p21-activated kinase (PAK) and regulate various cellular functions, such as cytoskeleton dynamics and gene expression, leading to cell movement, migration, and proliferation8. Activated Rho family proteins contribute to promoting the metastasis of tumor cells by disrupting epithelial sheet organization, increasing cell motility, and promoting the degradation of the extracellular matrix9. Rac1b, a splice variant of Rac1 showing constitutively active GTPase, was preferentially expressed in patients with colon and breast cancer12. In addition, overexpression and gene amplification of PAK1 have been shown in various cancers, including breast, colon, ovarian, bladder, brain, and pancreatic cancers13, suggesting a pivotal role of active Rac1/Cdc42 in human carcinogenesis. Thus, it is important to evaluate the spatial activation of intrinsic Rac1/Cdc42 in tumor tissues.Small GTPases are activated by guanine nucleotide exchange factor (GEF) binding to GTP but inactivated by GTPase-activating protein (GAP). The GTP-bound form of small GTPases interacts with distinct effector molecules to execute their cellular function14. Classically, microwave ovens and ultrasound devices have been utilized for rapid IHC17. However, the quality of immunostaining was reduced, and the specimen was damaged by long-term ultrasonic irradiation18.The efficient detection method of small G-protein by using FFPE tissues has not yet been established due to the low binding frequency of the detection probe. In the present study, we developed a novel detection technique for activated Rac/Cdc42 in human cancer FFPE tissues using a recently developed rapid immunohistochemistry (R-IHC) device, in which the antigen–antibody reaction can be facilitated up to 1,200-fold by alternating current-based high-speed microaggitationIn this study, we attempted the R-IHC system to facilitate the binding frequency between endogenous Rac/Cdc42-GTP protein and GST-PAK-RBD probe without disturbing conformation of Rac- and Cdc42-GTP. We succeeded in detecting Rac/Cdc42 activation in human colon cancer FFPE tissues with various patterns, strikingly in the invasive front, of which activity was correlated with clinicopathological factors such as lymphatic vessel invasion, raising the value for predicting invasion and metastasis in various types of cancers; although it needs to be noted that the binding to and detection of other Rac members, such as Rac2 and Rac3 and/or Cdc42, cannot be excluded in patient-derived material.293T human embryonic kidney cells were cultured in Dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum . Two human colon cancer cell lines, HCT116 and WiDr cells, were cultured in McCoy’s 5A (Gibco®-Life Technologies) and RPMI 1640 (Nissui) medium containing 10% FBS, respectively.For the GST pull-down assay, immunofluorescence, and construction of the cell block, 293T cells were transiently transfected with pCXN2-Flag-Rac1 V12 or pCXN2-Flag-Rac1 N17 using Fugene HD transfection reagent .2, 10% glycerol, 1 mM PMSF). For the GST pull-down assay, 1 mg of protein lysate was incubated with 10 µg GST-PAK2 RBD (recombinant protein) with rotation for 30 min at 4 °C, followed by incubation with 20 µl glutathione Sepharose 4B beads  with additional rotation for 30 min at 4 °C. After brief centrifugation, the supernatant was discarded, and the precipitates were washed with lysis buffer three times. Final precipitates were suspended in 20 µl of 2 × SDS sample buffer (100 mM Tris–HCl (pH 6.8), 4% SDS, 10% β-mercaptoethanol, 20% glycerol, and 0.01% BPB), boiled for 5 min, and subjected to electrophoresis using a 12% SDS–PAGE gel. PVDF membrane-transferred separated proteins were immunoblotted with mouse monoclonal anti-Rac1 antibody (Ab) . For immunoprecipitation, 1 mg of protein lysate was incubated with anti-active Rac1 Ab  at 4 °C overnight. After brief centrifugation, the subsequent steps were performed as described above.293T cells with or without enforced Rac1 V12 or Rac1 N17 were lysed with lysis buffer (25 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% NP40, 10 mM MgClTo reveal activation of Rac1 in human colon cancer cell lines, HCT116 and WiDr cells were serum-starved overnight and stimulated with FBS for 30 min. The cell lysate was subjected to GST pull-down assay as described above.293T cells expressing Rac1 V12 were cultured on glass-based dishes  and fixed in 3% paraformaldehyde  in PBS for 15 min. The cells were permeabilized with 0.1% Triton X-100 for 4 min and blocked with 1% BSA for 20 min. To detect activated Rac1, the cells were treated with 2 µg/ml GST-PAK-RBD for 2 h at room temperature (RT), followed by anti-GST Ab overnight at 4 °C and Alexa Fluor 488-conjugated anti-mouse IgG  for 1 h at RT. In comparison, the fixed and permeabilized cells were stained with anti-active Rac1 Ab (New East) at 4 °C overnight. Images were acquired using a fluorescence microscope .2O2) for 5 min, GST-PAK-RBD for 5 min using an R-IHC machine, 1st Ab (α-GST Ab) at 37 °C for 30 min and for an additional 10 min at RT, polymer 2nd Ab for 30 min (Dako). During each step, specimens were washed with PBS containing 0.5% Tween 20. After coloring with diaminobenzidine  within 50 s, the tissues were nuclear stained (Mayer) for 30 s, dehydrated, penetrated, and mounted  tissues, optimal conditions of immunostaining were investigated using cell blocks of 293T cells with or without enforced Rac1 V12. 293T cells were transfected as described above and fixed in formalin, and cell blocks were constructed. The concentration of GST-PAK , dilution ratio of α-GST Ab , formalin-fixed duration , and requirement of antigen retrieval  were investigated. The established protocol was as follows: incubation at 37 °C for 10 min, deparaffinization for 10 min, endogenous peroxidase (3% H7) were subcutaneously injected into six- to eight-week-old female 10 nude mice, Balb/cA Jcl nu/nu  per cell type. After approximately one month, the mice were sacrificed by cervical fracture under isoflurane inhalation anesthesia, and the growing tumor tissues were resected. The tumors that grew to almost equal volume were randomly divided into two groups within the weight range group (3 mice/group) and fixed in formalin or the PAXgene Tissue system (Qiagen), which is a formalin-free system designed to improve the quality of molecular analysis without disturbing those of histopathological analysis. Rac and Cdc42 activation in the excised tumors was evaluated as described above. All animal experiments were conducted in accordance with the guidelines of the Hokkaido University Manual for Implementing Animal Experimentation, which was consistent with the ARRIVE Guidelines for the Care and Use of Laboratory Animals and for study design and approved by the Institutional Animal Care and Use Committee at Hokkaido University Graduate School of Medicine (Number 12-0092). All researchers who performed procedures using live animals were preapproved by the Animal Welfare Committee of Hokkaido University. For each animal experiment, four different investigators were involved as follows: two investigators  performed the surgical procedure and all steps for making tumor specimens and were aware of treatment group allocation . Two investigators  who were unaware of treatment evaluated the staining intensity and pattern of active Rac1/Cdc42.HCT116 and WiDr cells (1 × 10In tissue microarray (TMA) specimens from 33 patients with colon cancer (Stage 0–IV: 33 cases) diagnosed at the Department of Cancer Pathology, Hokkaido University Graduate School of Medicine between 2003 and 2015 with informed consent from participants, activated Rac and Cdc42 were immunostained, as shown in Supplementary Fig. P < 0.05 was considered statistically significant. All ethical issues related to human pathological specimens were discussed and approved by the Ethics Committee of Hokkaido University Graduate School of Medicine (Number 15-022).The colon cancer FFPE tissues of 50 patients diagnosed with colon cancer at the Department of Cancer Pathology, Hokkaido University Graduate School of Medicine between 2003 and 2015 with informed consent from participants are summarized in Supplementary Table P < 0.05 considered significant. The F test was used to investigate the equal or unequal variance of samples.Graphical data are presented as the mean and standard deviation (S.D.), and Student’s t test was used for comparisons, with In the process of tumor progression, the detection of active Rac/Cdc42 is useful for a better understanding of the migration and invasion abilities of tumor cells and the prediction of metastasis and patient prognosis. A GST pull-down assay using GST-PAK-RBD is available for the biochemical detection of active Rac and Cdc42. Recently, an anti-active Rac1 antibody (Ab) has been made commercially available. Here, we compared the detection sensitivity and specificity of active Rac1 between a GST pull-down assay and immunoprecipitation using an anti-active Rac1 Ab. In 293T cells, exogenous Rac1 V12, but not N17, was effectively detected in the GST pull-down assay, whereas immunoprecipitation using an anti-active Rac1 Ab failed . Meanwhile, in the remaining 4 cases , Rac/Cdc42 activity was reversed . Actual intensities of active Rac/Cdc42 in normal mucosa and tumor were as follows: 67.29 and 110.92 in all cases, 66.64 and 117.97 in 29 cases , 72.01 and 59.83 in 4 cases . Of note, positive correlations between Rac/Cdc42 activity and lymphatic vessel invasion, venous invasion, and lymph node metastasis were detected  samples from 33 patients with colon cancer were utilized. In 29 cases, Rac/Cdc42 activity was increased in the tumor region compared to normal mucosa  biosensors has been developed24. FRET technology enables the visualization of several intracellular events in living cells; Kiyokawa et al. demonstrated that Rac1 suppression at the apical membrane is essential for cyst structure maintenance using a FRET-based live imaging system25. Meanwhile, proficient skills to develop biosensors, uptake of FRET probes into living cells, and expensive equipment to detect FRET reactions are required. The R-IHC technique using a GST-PAK probe easily and rapidly enables the detection of activated Rac and Cdc42 in FFPE tissues, which contributes to realizing special activation of Rac/Cdc42 in cancer tissues. This technique can be applied to other small GTPases, such as Rho and Rap1, in addition to the Ras family, by proper combination with probes. Meanwhile, it should be noted that Baker and colleagues have conclusively shown that the active Rac1 antibody (used in this manuscript too) does not detect active Rac1 at all but seems to bind to vimentin26. This is supportive information for our results , leading to rapid application of cetuximab for personalized medicine. In addition, rapid immunohistochemistry (IHC) for Ki-67/MIB-1, CK (AE1/AE3), CK7, CK20, EMA, TTF-1, CD10, GFAP, CD20, and CD30 is enabled within 20 min using this device, which provides valuable information for intraoperative rapid pathological diagnosis29. Recently, the usefulness of this device has been reported in the fields of rapid dual in situ hybridization (RISH) targeting HER2, scoring of programmed death ligand 1 (PD-L1), and cell biology33. In the future, our newly developed method raises the possibility of providing valuable information in many fields, not only in cancer but also embryology, cell biology, pathobiology of various diseases, pathological diagnosis, and so on. We also found special activation of Rac and Cdc42 in the fundic gland (Supplemental Fig. The most direct method to measure the activities of small GTPases is P34. Thus, the detection of activated Rac1 in cancer tissues enables the prediction of the invasive property of tumor cells, forthcoming metastasis, and patient prognosis, thereby making it a useful biomarker.Of note, in TMA samples from 33 patients, we demonstrated positive correlations between Rac/Cdc42 activity of cancer cells and clinicopathological factors such as lymphatic vessel invasion Fig. b. Rac/CdIn this study, we newly found various activation patterns of Rac and Cdc42 in colon cancer FFPE tissues, designated as membrane, cytoplasm, mixed type of membrane and cytoplasm, and polarized patterns Fig. , which mIn conclusion, this is the first study to detect activated Rac and Cdc42 in human cancer FFPE tissues using the R-IHC system. Rac/Cdc42 activity was significantly higher in the tumor area than in the normal mucosa in colon cancer patients, especially in the invasive front, increasing its usefulness in the prediction of invasion and metastasis in various types of cancers. We expected that this technology would also provide valuable information for other small GTPases, such as Ras, which collectively leads to effective personalized medicine.Supplementary Information.


Visabron c (4–4) was selected froma minilibrary with conformational diversity based on its potency andselectivity in functional adhesion cellular assays. Visabron c (4–4)’s serum stability, pharmacokinetics,and therapeutic effects following ip injection were assessed in anexperimental autoimmune encephalomyelitis (EAE) animal model. Furthermore,visabron c (4–4)’s lack of toxic effectsin mice was verified by blood analysis, tissue pathology, immunogenicity,and “off-target” effects, indicating its significanttolerability and lack of immunogenicity. Visabron c (4–4) can be delivered systemically. The in vitro and in vivo data justify visabron c (4–4) as a safe alternative peptidomimetic lead compound/drugto monoclonal anti-α4 integrin antibodies, steroids, and otherimmunosuppressant drugs. Moreover, visabron c (4–4)design may pave the way for developing new therapies for a varietyof other inflammatory and/or autoimmune diseases.Integrins α4β1/α9β1 are important in thepathogenesis and progression of inflammatory and autoimmune diseasesby their roles in leukocyte activation and trafficking. Natalizumab,a monoclonal antibody selectively targeting α4β1 integrinand blocking leukocyte trafficking to the central nervous system,is an immunotherapy for multiple sclerosis (MS). However, due to itsadverse effects associated with chronic treatment, alternative strategiesusing small peptide mimetic inhibitors are being sought. In the presentstudy, we synthesized and characterized visabron  The cytoplasmic domains of the α4and α9 integrins share about 40% amino acid sequence identity.4 These integrins are important regulators of cytoskeletaldynamics in cell adhesion and migration. They are overexpressed inpathological states such as wounds, inflammation, autoimmune diseases,and cancer, making these receptors important therapeutic targets formany inflammatory diseases.5Integrins are a familyof cell surface receptors composed of noncovalentlylinked α and β subunits that regulate many biologicalfunctions in eukaryotic cells and play essential roles in regulatingcell adhesion and migration.6 However, the therapeutic efficacy of natalizumabis associated with adverse effects.7 Moreover,neutralizing antibodies toward natalizumab that cause loss of drugefficacy have been identified in about 6% of patients. The greatestrisk of natalizumab treatment is associated with the development ofprogressive multifocal leukoencephalopathy (PML), a rare but seriousviral infection (JC-virus) leading to inflammation and demyelination,resulting in severe disability or death.8 Because of the risks associated with natalizumab and its biosimilarantibodies, there are many reservations over their use in the clinicas a preferred treatment option. Although antibodies and small moleculeshave been developed to inhibit α4β1/α4β7 integrinswith potency and selectivity, there is an unmet clinical need forthe development of cheaper, effective α4β1/α4β7peptidomimetic antagonists with improved safety and pharmacokineticsfor the therapy of inflammatory and autoimmune diseases. Moreover,most of the known compounds are either dual antagonists of α4β1/α4β7integrins or are selective for α4β7, while very few compoundsare selective for α4β1. Peptide lead compounds containingthe amino acid motif LDTSL (Leu-Asp-Thr-Ser-Leu) or their truncatedversions such as Asp-Thr and Leu-Asp-Thr were found to bind α4β7and inhibit adhesion of leukocytes, expressing α4β7 tothe MAdCAM-1 ligand.3 A library of conformationallyrestricted end-to-end cyclic hexapeptides was synthesized, presentingthe pharmacophore Leu-Asp-Thr (LDT) sequence in different conformations,indicating potent and selective α4β7 integrin antagonismof MAdCAM-1 binding to the α4β7 integrin.9 Mannose-based peptidomimetics selectively blocking MAdCAM-1were also developed.10 In another approach,PTG-100 and its PN-943 analogue, an oral α4β7 integrinpeptide antagonist, were developed to treat inflammatory bowel disease.In addition, high-affinity, selective BIO-1211 inhibitors of α4β1,based on the Leu-Asp-Val (LDV) sequence11 and small, end-to-end cyclic Ile-Leu-Asp-Val (ILDV) peptides, werealso described.12Multiplesclerosis (MS) is a central nervous system (CNS) neurologicaldisorder generally viewed as having an autoimmune origin. Likewise,in other various inflammatory diseases, including asthma, rheumatoidarthritis, and inflammatory bowel disease (IBD), the binding of theleukocytes α4β1 integrin to VCAM-1 ligand expressed onendothelial cells initiates adhesion of the leukocyte to the vascularendothelium followed by extravasation into the CNS tissue, thus contributingto the inflammation and pathogenesis of the MS disease. Treatmentwith the monoclonal antibodies natalizumab (Tysabri) for therapy ofMS and vedolizumab (Entyvio) for IBD, which inhibit α4β1and α4β7, respectively, has been found to be efficientin the clinic for therapy of chronic relapsing inflammation of demyelinatingMS disorders and treatment of resistant Crohn’s disease.14 Thereafter, our strategy was to transform the linear peptide motifinto an active backbone-cyclic peptide with improved drug properties.Backbone cyclization (BC) was already proven a valuable tool in themethodological conversion of active regions of proteins to cyclicpeptidomimetic drug leads.16 The BC method is employedto introduce global constraints to peptides and active regions inproteins. It differs from other cyclization methods since it utilizesthe amide bond nitrogen to anchor the cyclizing bridge, thus maintainingthe active pharmacophore. It is based on the incorporation of N-alkylated, epsilon-functionalized amino acids into theactive sequence for cyclization. BC proved superior to other stabilizationmethods since the resultant peptides had defined structures that ledto better selectivity17 and improved pharmacologicalproperties.15 However, obtaining the desiredactive cyclic analogue based on a linear sequence is not a straightforwardprocess and, therefore, requires selection from designed librarieswith conformational diversity.15 In thisstudy, we describe the isolation of visabres disintegrin from vipervenom, identification of the TMLD motif selective for α4β1/α4β7integrin inhibition, and attempts for its cyclization to generatevisabron, a backbone cyclic peptide dual antagonist of α4β1(VLA-4)/ α9β1 integrin with druglike properties. The pharmacologicalprofile of visabron was achieved by in vitro functionalcell adhesion and selectivity assays, characterizations of its pharmacokinetic,and safety properties and measurement of its therapeutic effect in vivo in a multiple sclerosis–experimental autoimmuneencephalomyelitis mouse model.The present approachfor the development of antagonists for α4β1/α4β7integrins is based on the identification of novel amino acid sequencemotifs targeting α4β1/α4β7 integrins presentin snake venom disintegrins. These are a family of low-molecular-weight,cysteine-rich proteins that potently and selectively target and inhibitthe α4β1/α4β7 integrins.18 intothree protein fractions .Fraction Vd-III, which was less toxic, not hemorrhagic, and less contaminatedwith protease and phospholipase A2 activity but enrichedin α4-inhibitory activity (Tables S1 and S2), was further separated by a consecutive step of reversed-phaseHPLC using a linear gradient of increasing acetonitrile concentration. The α4-inhibitorydisintegrin fraction, named visabres, was identified by inhibitionof Jurkat cell adhesion to human recombinant VCAM-1 . Mass spectroscopy analyses of this fractionindicated it is enriched in disintegrins but still contaminated withother venom proteins (Table S3). Finalpurification of visabres was achieved by a consecutive, similar HPLCseparation resulting in a pure α4-disintegrin eluted as a singlepeak at an acetonitrile concentration of ∼43% . The purity of visabres was confirmedby SDS-PAGE in reduced and nonreduced conditions, indicating the dimericstructure of this molecule . The amino acid sequence of visabres (Table S4) was determined using a standard procedure, previously appliedfor other dimeric snake venom disintegrins.19 Alignment of the sequence of the α4/α9-integrin-bindingmotif in visabres subunit B, compared to other dimeric disintegrins. Visabres attenuated byabout 50% the clinical neurological score in EAE mice, upon i.p. injection,using a nontoxic, cumulative dose of 2.5 mg/kg (data not shown).The first stepof the isolation of visabres was based on the preparative fractionationof the venom by FPLC gel filtrationproteins S3. FinalTable S6, sequences 1 and 2 respectively), covering part of the sequenceof chain B of visabres (Table S4). It wasfound that the TMLD-containing linear peptide was important in therecognition and inhibition of the α4β1 integrin . Thereafter, by removingamino acids from the amino and carboxy terminals of this linear peptide,in order to generate a small peptide with druglike properties withmolecular weight around 1000 Da,20 we foundthat the linear pharmacophoric KRTMLDGL sequence is selective forinhibition of α4β1/α9β1 integrin,21 albeit of very low potency . Additional work indicated that replacingthreonine in position 41 to alanine, as found in some natural disintegrins, further indicating that the TMLD “hot spot” in theintegrin-binding loop, at the exact position as the RGD motif presentin other disintegrins,13 is important forthe integrin-binding affinity. In the past, we prepared differentcyclic peptides with cysteine bridges at different positions, in whichthe KRTMLDGL sequence was stabilized and folded. However, althoughsome of these peptides selectively inhibited α4β1 integrin-mediatedcell adhesion, they suffered from poor solubility and stability inaqueous solutions (data not shown). Therefore, the present rationalfor the synthesis of a visabres-derived backbone cyclic peptide namedvisbron was to replace the visabres arginine at position 40 by glycine) by forming a ringbetween the amide bond nitrogens of the two glycines at positions40 and 45. Thus, an N-alkylated glycine-building unit  replaced the arginine residue in visabronenabling backbone cyclization (n and m length),22 a minilibrary was prepared. Three backbone cyclic analogueswere synthesized by SPPS and purified by preparative HPLC with highyields, named visabron c (2–2), c (4–4), and c (6–6), with ring sizesof 23, 27, and 31 atoms, respectively and MS characterizations  ofthe synthesized backbone cyclic visabron peptides indicated 95–99%purity and molecular weights of 945, 1001, and 1057 Da, respectively(Table S7). The visabron peptides werecharacterized by very good aqueous solubility as also reflected bytheir cLogP, which is smaller than 5.0 (Table S8), a property important for a drug to be administrated orallyor by injection.23 Visabron is a polarpeptide with a strong positive charge  onone end, a property that is required for the α4β1/α9β1integrin-binding affinity, considering that substitution of lysine39 to arginine in Arg-visabron c (4–4) caused significant loss ofactivity (c (4–4) > visabron c(6–6) > visabron c (2–2) emphasized that the activity of the visabron cyclicpeptides was derived from a conformational effect. Therefore, we chosethe most active backbone cyclic peptide visabron c (4–4) as the lead structure for further pharmacological characterizations.At first,we began the lead peptide development by solid-phase peptide synthesis(SPPS) and screening for competition in adhesion binding to α4β1/α9β1compared to α1/α2/α5-β1 integrin of two linearpeptides of 31 and 27 amino acids  on Jurkat cell adhesion and migration through the humanumbilical vein endothelial cell (HUVEC) monolayer activated by theproinflammatory cytokine TNFα. Minimal spontaneous adhesion(8–12%) occurred, but this increased markedly (25–35%of added Jurkat cells) when the cells were activated with TNFα(c (2–2)and c (4–4) in the range of 1–10 μM(c (6–6), whilevisabres was very active and used as a positive control (c (4–4),similar to natalizumab and visabres, indicating its α4β1-mediated,leukocyte-trafficking-inhibitory activity (c (4–4)’s pharmacological action as a novelleukocyte trafficking inhibitor.Leukocytes migrate through vascular endothelium and theninto the connective tissue. As an ith TNFα2A. The a 1–10 μM2A but wa control 2A, insetactivity 2B,C. Thec (4–4) compared to natalizumab on mice with EAE, the bodyweight and clinical neurological score were tested daily for up to35 days. No neurological symptoms were observed during the entireperiod of 35 days in the nondisease, control group. The EAE diseasegroup began to show neurological deficit symptoms at 13–16days postimmunization, and the mean neurological score of mice withEAE increased rapidly, reaching the maximal level at 21 days postimmunization.From the onset of disease (13–16 days) to 35 days postimmunization,the increase in the neurological score in the visabron c (4–4) and natalizumab groups was significantly lower thanthat in the EAE group (c (4–4) and natalizumab groups (c (4–4) and natalizumab groups(c (4–4) and natalizumab groups were also significantlydecreased compared with those of the EAE/PBS group (c (4–4), in a dose-dependent fashion, induced a significanttherapeutic preventive effect in the mice with EAE, similar to natalizumabused as a positive control, in a range of 12.5 mg/kg until 300 mg/kg,as measured by the clinical neurological score at the onset of thedisease (c (4–4) and natalizumabon the degree of inflammatory cell infiltration in the spinal cordtissue was measured by cell counting on H&E stained slices. TheEAE-disease group showed a larger amount of inflammatory cell infiltrationthan the control group. Treatment with either visabron c (4–4) or natalizumab significantly reduced the inflammatorycell infiltration , being a dual antagonistof α4β1/α9β1 integrins, is also blocking thesepathways, enhancing the therapeutic effect on mice with EAE disease.To evaluate the therapeutic effect of visabron AE group 3A. The bb groups 3B. The lb groups3C and thBS group 3D. Visabdisease 3E.The eltration 3F, refleFigure S7). They were stable after 180min of incubation in rat plasma, while the half-life of the linearvisabron was only 7 min, indicating fast degradation of the linearpeptide by plasma enzymes. Following i.v. dose administration in rats,visabron c (4–4) exposure was with a meanplasma area under the curve (AUC) value of 46.6 + 2 μg·min/mL.The mean clearance (CL) value was 11.08 + 0.5 mL/min/kg, and the volumeof distribution was 0.39 + 0.03 L/kg. The mean half-life (t1/2) was 23.8 + 1.6 min (c (4–4) has minimal distribution into the tissues, which isin accord with the site of its pharmacologic activity, preventinglymphocyte extravasation from the blood vessels into the extravascularfluids, also indicating that it can undergo efficient eliminationby the clearing organs kidney and liver.The cyclic visabrons have high metabolicstability in rat plasma,relative to the linear parent peptides, due to their backbone cyclization, and subsequently in their pharmacokinetics.28Several toxin-derived peptides thathave become drugs were usedfor the management of diabetes, hypertension, chronic pain, and othermedical conditions.Figures S8 and S9, the relationship between the PK profile of visabron c (4–4) and its pharmacodynamics therapeutic effectsare indirect,29 taking time for development,and are not apparentlyrelated to plasma concentration, reminiscent of trafficking behaviorof helper T cells in response to methylprednisolone.30 In the EAE model experiment, visabron c (4–4) was administered i.p. every 48 h by five injections.Given that the peptide’s half-life is 24 min, it is assumedthat following 4 half-lives (approximately 96 min), visabron c (4–4) will be eliminated from the plasma. Therefore,it can be concluded that there is an “indirect pharmacodynamic(PD)” relationship since >48 h postadministration in theEAEexperiment the active peptide visabron c (4–4)no longer exists in the plasma, and yet significant therapeutic effectswere exhibited among mice treated with visabron c (4–4) at cumulative doses of 300, 90, and 25 mg/kg. Therefore,the PK/PD relationship of this cyclic peptide is complex and is atype of “indirect PD” and is predicted to be dependedon various biological parameters such as expression and activationlevels of the α4β1/α9β1 integrins, lymphocytesturnover, and their trafficking regulation and not solely by the plasmaconcentration level of visabron c (4–4). Tohighlight this indirect PD phenomenon it is important to note thatthe kinetics of the measured  response is muchslower than the elimination kinetics of the active cyclic peptidevisabron c (4–4) in the body.As can be seen from c (4–4) 24 h after i.v. injection in C57BL/6 malemice (Tables S9 and S10), without short-termadverse pathological effects on major organs, when administered eitheri.v. or per o.s., at a dose of 500 mg/kg for a period of 48 h (Table S11). The “off-target” effect may resultin adverse effects of the drug resulting in drug withdrawal. Therefore, in vitro pharmacological profiling is increasingly beingused earlier in the drug discovery process to identify undesirableoff-target activity profiles that could hinder or halt the developmentof a candidate drug.31 Eurofins Cerep Safetyscan in vitro analyses of visabron c (4–4), including 44 binding, enzymatic, and transporterspanel assays, provided data regarding the potential radioligand bindingcompetition by visabron c (4–4) on major physiologicaltargets, known to show a clear correlation with observed invivo toxic effects.31 We choseto investigate a concentration of 1 mM visabron c (4–4) which is about 200-fold higher than the IC50 for α4β1 integrin inhibition using “in cell”assay and assumed to be higher than the therapeutic window, in orderto predict visabron c (4–4) interactions attoxic overdoses. We set a threshold of 40% for binding competitionon all tested targets as summarized in c (4–4) did not interact with the majority of thephysiological targets, such as (a) cholinergic muscarinic receptors;(b) α and β adrenergic receptors subtypes; (c) cannabinoidreceptors; (d) GABA (benzodiazepine) receptors; (e) several dopaminergicand (f) serotonergic receptors; (g) glutamatergic and (h) histaminergicreceptors; (i) glucocorticoid and (j) androgen receptors; (k) vasopressinreceptor, and the most abundant (l) voltage-dependent sodium, potassium,and calcium channels, and (m) the typical catecholamine transportersfor norepinephrine, dopamine, and serotonin. However, at this highconcentration, but not at 100 μM, visabron c (4–4) competed with radioligand agonists of three G-proteincoupled pain receptors (GPCRs), cholecystokinin CCK1 (CCKA) (94.4%)and opiate receptors δ (DOP) (93%) and μ (MOP) (92%) (c (4–4) (data not shown).Cumulatively, these findings support the high selectivity of visabron c (4–4) on an α4β1/α9β1 integrintarget and indicate that at a very high concentration it may causeanalgesic effects by binding to opiate receptors and inhibiting cyclooxygenases,adverse effects that may be beneficial in multiple sclerosis patientsthat complain of pain. Safety screening in early drug discovery testedcompounds at a 10 μM concentration and assume that a high numberof off-target hits (up to 14) generally led to severe side effects,which later led to in vivo screening only occurringwith molecules having <7 off-targets.32 Application of this safety lead optimization screening strategyduring the present early stage of drug discovery led to the identificationof visabron c (4–4) with CCK and opioid receptorsoff-target binding at a very high, nonphysiological concentration.In the case of CCK1 there is sequence similarity between visabron c (4–4) and CCK133 ascan be seen in Figure S10, which can accountfor the binding of visabron c (4–4) to theCCK receptors. In the case of the opioids, visabron c (4–4) did not possess the main pharmacophore groups, namelytwo aromatic rings, an aromatic hydroxyl group, and a polar or chargednitrogen essential for binding to the opioid MOP and DOP receptors.34 Visabron c (4–4) containstwo out of four pharmacophores essential for opioid receptor bindingand activation (NH3+ of Lys39 andOH of Thr41); however, it does not contain the two aromaticrings unless we consider the side chains of Met42, Leu43, and Leu46 as aromatic isosteres.35 It may be hypothesized that at a very high concentration,Visabron c (4–4) will be a positive allostericmodulator of the opioid receptors, similar to several nonpeptidylsmall molecules.36 However, since at 100μM visabron c (4–4) did not interactwith any of the above receptors, it is less plausible that in vivo visabron c (4–4) will activateopioid receptors. Nevertheless, to ensure risk mitigation, this possibilityneeds to be confirmed by future experiments to get a full pharmacologicalprofile of visabron c (4–4)’s effecton CCK and opioid receptors to safely exclude the possibility of potentialside effects.Single-dose toxicity evaluationby hematocrit cell counting and biochemicalanalysis indicated acute tolerability of 500 mg/kg of visabron P) (92%) 5, and moP) (92%) 6. Howevec (4–4) was investigated on apanel of 20 tyrosine protein kinases of immune cells that were reportedto be involved in leukocyte trafficking, immunosuppression, and/orinduction of inflammation.38 The measurements ofthe activity of cytoplasmic and receptor recombinant tyrosine proteinkinases were based on the level of chelation-enhanced fluorescencethat is directly proportional to the amount of phosphorylated, real-timesensors consisting of sulfonamido-oxine (Sox) chromophore linked toa peptide or protein substrate.39 At aconcentration of 1 mM, but not 0.1 mM, visabron c (4–4) inhibited from 25% to 40% Abl, EGFR, JAK3, LynA, andC-Raf   2 was sol40 In contrastto natalizumab, visabron c (2–2) alone ortogether with Freund‘s complete adjuvant did not induce antibodyproduction in a BALB/c mice model, as evident from the lack of a significantreaction in ELISA by comparison to the preimmune serum . Visabron c (4–4) was synthesized, characterized, and pharmacologicallyidentified as a selective backbone cyclic peptide antagonist of VCAM-1/α4β1/α9β1 integrins, blocking leukocyte migration in vitro, and conferring therapy in MS-EAE in vivo mice model. Visabron c (4–4) is proposedas an alternative backbone cyclic peptide drug to monoclonal anti-α4integrin due to its safety properties, lack of immunogenicity, andlow price of production. Visabron c (4–4)can be delivered systemically and represent a safe alternative tosteroids and immunosuppressant drugs for therapy of autoimmune inflammatorydiseases. We are currently conducting structural modifications ofVisabron c (4–4) to generate a prodrug fororal application. Additional preclinical ex vivo and in vivo experimentation is necessary to confirm whethervisabron c (4–4) possesses a full safety liabilitybefore conducting first-in-human studies.In conclusion, the use of backbonecyclization to constrain theTMLD peptide motif in defined conformational space has led to theselection of visabron H-1,2,3-triazolopyridinium-3-oxidehexafluorophosphate (HATU), 1-hydroxy-7-azabenzotriazole (HOAt), 9-fluorenylmethyloxycarbonyl-Nα-protected amino acids (Fmoc-Nα-AA-OH), and N-(9-fluorenylmethoxycarbonyloxy)succinimide(Fmoc-OSu) were purchased form Chem-Impex International, Inc. . Fmoc-Rink-amide methylbenzhydrylamine (MBHA) resin  was purchased from Iris Biotech GmbH . 1,2-Diaminoethane, 1,4-diaminobutane, 1,6-diaminohexane,2,4,6-trimethylpyridine (collidine), bis(trichloromethyl) carbonate(BTC), tetrakis(triphenylphosphine) palladium(0), diethyl ether, bromoaceticacid, acetic anhydride (Ac2O), piperidine, trifluoroaceticacid (TFA), diisopropylethylamine (DIPEA), methanol (MeOH), triethylamine(Et3N), triisopropylsilane (TIS), dibromomethane (DBM),dimethyl sulfoxide (DMSO), and other organic materials were purchasedfrom Across Organics N.V. . Organic solvents for solid-phasepeptide synthesis (SPPS) and for high-performance liquid chromatography(HPLC) including N-methyl-2-pyrrolidone (NMP), dichloromethane(DCM), N,N-dimethylformamide (DMF),and acetonitrile (ACN) were purchased from J.T. Baker (NJ). CellTrackerGreen 5-chloromethylfluorescein diacetate (CMFDA) was purchased fromInvitrogen/Molecular Probes . Human recombinant tumornecrosis factor-α (TNFα), phorbol 12-myristate 13-acetate(PMA), and Complete Freund’s Adjuvant were purchased from Sigma. Other chemicals and reagents were of analyticalgrade. Pertussis toxin was obtained from List Biological Laboratories,Campbell, CA.1-[Bis(dimethylamino)methylene]-1Collagen IV(from bovine placentavilli) was purchased from Chemicon Temecula, CA, and Collagen I (fromrat tail) was purchased from BD Biosciences . RecombinantHuman VCAM-1 was purchased from PeproTech Co. , andrecombinant human VCAM-1/CD106 Fc and MAdCAM-1 Fc chimeras, as wellas recombinant human integrin α4β1 or α4β7were obtained from R&D Systems Co . Human fibronectinand laminin were purchased from Sigma-Aldrich Israel Ltd. .Vipera lebetina obtuse.42 The disintegrin anti-α1β1 viperistatinand the C-type lectin protein anti-α2β1 vixapatin wereisolated from the venom of Vipera xantina palestinae as previously described.44The clinical-grademonoclonal antibodies natalyzumab (Tysabri) anti-α4β1produced by Biogen Co.  and vedolizumab (Entyvio) anti-α4β7,produced by Takeda Co. , were a kind gift from the pharmacyof the Hadassah Hebrew University Medical Center. The anti-integrinα9β1 monoclonal antibody [Y9A2] was purchased from AbcamCo. Cambridge, UK (ab27947), and the polyclonal and monoclonal antibodiesagainst α4β1 and α4β7 were purchased fromLifeSpan BioSciences Inc.  and ThermoFischer ScientificCo. , respectively. The anti-α4 disintegrin VLO5was purified from the venom of V. lebetina obtusa was purchasedfrom Latoxan Serpentarium . The venomof the Israeli Vipera daboia was purchased from SISCo.  which collected and maintained the snakes undergood laboratory practice (GLP) conditions, according to the requirementsof the Israeli Ministry of Health for antiserum production.The venom of All of the reactions that were performedon solid support utilized Fmoc chemistry for the Nα protection.The reactions were shaken using a Bigger Bill orbital shaker. Preactivationtubes were stirred over a vortex. Excluding the cleavage procedure,all other reactions on the solid support were performed under basicconditions of pH 8–9. The process of the reactions was monitoredby HPLC/MS following a “small cleavage” procedure. Thesynthesis via solid support was performed in a vessel equipped witha sintered glass bottom. Fmoc Rink Amide methylbenzhydrylamine (FmocRink Amide-MBHA) resin (loading capacity 0.66 mmol/g resin) was thesolid support for this synthesis. The equivalents of all reagentsused in SPPS were calculated with respect to the resin loading capacityand weight. The volume of the solvents in all of the reactions wasfixed to 30 mL to maintain a constant concentration of reagents. Aftercleavage from the resin, the crude final peptides were dissolved inTDW/ACN 1:1 mixture, lyophilized, and analyzed by HPLC/MS.α-AA-OH (3equiv) and HOAt (3 equiv) in NMP was prepared and cooledto 0 °C. HATU (3 equiv) was then added for preactivation of theamino acid prior to reaction with the peptidyl-resin, and the solutionwas shaken at 0 °C for 3 min. The preactivated solution was addedto the peptidyl-resin, and the mixture was shaken for 60 min. Theprocedure was repeated twice. The resin beads were washed with 30mL NMP (4 × 2 min) and DCM (2 × 2 min).A solution of Fmoc-NFor the removal of the Fmoc protectinggroup, a solution of 20% piperidine in NMP was added to the peptidyl-resin.The reaction was performed in a vessel shaken at room temperaturefor 30 min. The reaction was repeated once using a fresh solutionof 20% piperidine in NMP. At the end of the second cycle, the peptidyl-resinbeads were washed with 30 mL of NMP (4 × 2 min) and DCM (2 ×2 min).N-methylmorpholine(2.5%), and DCM (92.5%), and the mixture was shaken for 5 min, afterwhich tetrakis(triphenylphosphine)palladium(0) Pd(PPh3)4 (0.1 equiv)was added. The reaction mixture was shaken vigorously in the darkand under argon for 3 h, after which the peptidyl-resin beads werewashed with a 30 mL solution of 0.5% v/v DIPEA in DMF (5 × 2min), 0.5% sodium diethyldithiocarbamate trihydrate salt in DMF (5× 2 min), and DCM (5 × 2 min).For the removal of the Alloc protectinggroup, the peptidyl-resin was added to a saturated argon solutioncomposed of acetic acid (5%), On-resin urea cyclization was carriedout by adding a solution of BTC (0.33 equiv) and DIPEA (10 equiv)in DCM to the peptidyl-resin and the mixture was shaken for 2 h, afterwhich the resin beads were washed with 30 mL of DCM (4 × 2 min),and DMF (2 × 2 min).In this method,simultaneous removalof the synthesized peptides from the solid support together with theremoval of the acid-labile, side chain protecting groups was performedby the following procedure: 30 mL of precooled solution (at 0 °C)composed of TFA (95%), TDW (2.5%), and TIS (2.5%) was added to driedand desiccated 2.1 g peptide-resin beads. The reaction mixture waskept standing at 0 °C for 30 min after which it was shaken for150 min at room temperature. The TFA solution containing the cleavedpeptide was then separated from the resin beads via filtration, andthe TFA was partially evaporated by a stream of nitrogen. Cold diethylether was added to the remaining volume of TFA, and the mixture wascentrifuged to separate scavengers and other hydrophobic impuritiesfrom the precipitated peptide. Diethyl ether was then removed fromthe precipitate by decanting. The cycle of precipitation, centrifugation,and decanting was repeated three times. The precipitate was dissolvedin 10 mL of ACN/TDW (1:1) (relative to 2.1 g peptide-resin beads),and the solution was lyophilized overnight prior to purification viapreparative HPLC. The lyophilized crude product was obtained as fluffywhite solid.The crude peptides were dissolved in a TDW/ACN 1:1mixture, filteredthrough a 0.45 μm PTFE filters, and injected in 5–10mL volumes to a reversed-phase preparative HPLC column of Vydac . The analysis utilized a Merck-HitachiL-6200A pump and L-7400 variable wavelength detector recording at220 nm at room temperature. The gradient of the mobile phase consistedof A: TDW (0.1% v/v TFA) and B: ACN (0.085% v/v TFA). First, the columnwas equilibrated for 5 min at 95% A, and then a linear gradient wasapplied from 5 to 40 min to reach 95% B. The mobile phase remainedfor 5 min at 95% B for column equilibration. The gradient was returnedback to the starting conditions  within 5 min and keptat this point for additional 5 min for column equilibration. The flowrate of the mobile phase was 9 mL/min. The collected fractions wereanalyzed by MS and lyophilized, and samples were injected into ananalytical HPLC column to determine the degree of purity.All samples were dissolved in a TDW/ACN1:1 mixture, filtered through a 0.45 μm PTFE filter, and injectedinto a reversed-phase analytical HPLC column of Vydac . The analysis utilized a Merck-Hitachi L-7100pump and L-7400 variable wavelength detector recording at 220 nm atroom temperature. The gradient of the mobile phase consisted of (A)TDW (0.1% v/v TFA) and (B) ACN (0.085% v/v TFA). First, the columnwas equilibrated for 5 min at 95% A, and then a linear gradient wasapplied from 5 to 20 min to reach 95% B. The mobile phase remainedfor 5 min at 95% B for column equilibration. The gradient was returnedback to the starting conditions  within 5 min and keptat this point for additional 5 min for column equilibration. The flowrate of the mobile phase was 1 mL/min. The collected fractions werefurther analyzed by MS.Mass spectrawere acquired ona LCQ Fleet Ion Trap mass spectrometer (Thermo Scientific) utilizingelectrospray ionization. For HRMS analyses, the spectra were recordedon an Agilent 6550 iFunnel Q-TOF LC/MS system.Recombinanthuman VCAM-1 and MadCAM-1 , were immobilized on the wells of a 96-well ELISAplate (Dynatech Immulon) by overnight incubation at 4 °C. Afterwashing with HBSST  the plates were blockedwith 5% BSA in HBSST by incubation at 37 °C for 1 h. The plateswere washed with HBSST, and recombinant human integrin α4β1or α4β7 (3 mg/mL) was added in the presence or absenceof competitor cyclic peptides, disintegrins, or monoclonal anti-integrinantibodies dissolved in HBSS containing 1% BSA. The plates were thereafterincubated at 30 °C for 3 h, as time-binding had reached a plateau,and then washed 3 times with HBSST. Bound integrins were quantitatedusing primary anti-integrin polyclonal and monoclonal antibodies ata concentration of 0.5 mg/mL in HBSST containing 1% BSA, and incubatedat 37 °C for 1 h. The plates were washed 3 times with HBSST andsecondary antibodies- alkaline phosphatase (AP) conjugated (SigmaCo.) were added at dilution 1:1000 and incubated at 37 °C for1 h. Thereafter, the plates were washed 3 times with HBSST and twicewith HBSS and the alkaline phosphatase substrate 4-nitrophenyl phosphatewas added to the wells for 30 min. The plates were read using TecanSpectrophotometer at 405 nm. The level of nonspecific binding wasmeasured in each experiment by determining the level of binding towells coated with BSA alone. Control experiments indicated that theinteraction of ligands to integrins was specific, since the bindingwas significantly and equally, inhibited by EDTA, selective disintegrins,and by antifunctional monoclonal antibodies directed against eitherthe α4β1 or α4β7.46 or with the empty vector pcDNAneoI  bycalcium phosphate precipitation. Transfected cells were maintainedin Dulbecco’s modified Eagle’s medium (DMEM) supplementedwith 10% fetal calf serum and the neomycin analog G-418 (1 mg/mL). The cell lines expressed high surface levelsof integrins as determined by flow cytometry using the respectivemonoclonal antibodies.K562 cells transfectedwith α1and α2 integrins were originally provided by Dr.M. Hemler . Κ562 humanimmortalized myelogenous leukemia cell line, Jurkat immortalized humanT-lymphocyte cell line, Ramos ,SW480 epithelial human adenocarcinoma cell line, and U-937 cells withmonocytic-type phenotype that differentiate to macrophages followingPMA stimulation were purchased from ATCC . Human umbilicalvein endothelial (HUVE) cells were purchased from Clonetics , grown in endothelial cell growth medium containing 2% fetal bovineserum, human recombinant epidermal growth factor (10 ng/mL), gentamycin(50 mg/mL), amphotericin B (50 ng/mL), bovine brain extract (12 mg/mL),and hydrocortisone (1 mg/mL), and used between passage 4 and 8. α4and α9- and mock-transfected SW480 and glioma cells were generatedby transfection with full-length α9 expression plasmid pcDNAIneoa947 with minormodifications. The day before the experiment, each well was coatedwith VCAM-1 (3 mg/mL) immobilized on the 96-well plate in PBS by overnightincubation at 4 °C. Thereafter, nonspecific binding was blockedby incubating the wells with 1% (w/v) bovine serum albumin (BSA) inHank’s Balanced Salt Solution (HBSS) containing 5 mM MgCl2 at room temperature for 1 h before use. The cells were labeledby incubation with 12.5 μM 5-chloromethylfluorescein diacetate(CMFDA) in HBSS without 1% BSA at 37 °C for 30 min. The labeledcells were then centrifuged at 1000 rpm and washed twice with HBSScontaining 1% BSA to remove excess CMFDA. Labeled cells (about 1 ×105 cells/well) were plated on each well in the presenceor absence of visabrons and incubated at 37 °C for 60 min. Unboundcells were removed by washing the wells three times with 1% (w/v)BSA in HBSS, and bound cells were lysed by the addition of 0.5% TritonX-100 (diluted in DDW). The fluorescence in each well was quantifiedwith a SPECTRAFluor Plus plate reader (Tecan), at λex = 485 nm and λem = 530 nm. To determine the numberof adhered cells from the fluorescence values, a standard curve wasgenerated by serial dilution of known numbers of CMFDA-labeled cells.Cell adhesionassay was carriedout as previously describedtrans-Endothelialleukocyte migration was assessed as follows: HUVEC cells were platedonto collagen type IV (5 μg/mL) coated polycarbonate inserts in serum-containing endothelial cellgrowth medium and allowed to grow to confluence over 72 h. Twenty-fourhours before assays, the upper chambers were washed twice with serum-freemedium, and a new medium with or without 3 ng/mL TNFα was added.Immediately prior to the addition of the cells, the upper chamberswere washed twice with serum-free DMEM, and the medium in the lowerchamber was replaced with 500 μL of serum-free DMEM. Jurkatcells labeled with CMFDA for 1 h. Thereafter, the cells were suspendedin DMEM at a density of 5 × 104 cells in a volumeof 300 μL, preincubated in the presence or absence of visabronsfor 1 h at room temperature, and applied on the layer of HUVEC. Insetswere placed into a 24-well plate containing 700 μL of DMEM with2% FBS used as a chemoattractant. After 1, 2, and 24 h at 37 °Cin 5% CO2, nonadherent cells in the upper chamber wereremoved. Medium including migrating cells from the lower chamber wascollected, the lower chamber was rinsed several times to collect allof the Jurkat cells that had transmigrated, and the absence of additionaladherent cells was confirmed microscopically. The medium and all washeswere pooled, and the fluorescence in each well was quantified witha SPECTRAFluor Plus plate reader at λex = 485 nmand λem = 530 nm. To determine the number of adheredcells from the fluorescence values, a standard curve was generated,by serial dilution of known numbers of CMFDA-labeled cells. The experimentswere carried out in sixplicate and repeated three times.35–55 peptide in salineand an equal volume of 5 mg/mL Complete Freund’s Adjuvant (CFA)containing 1 mg/mL heat-killed M. tuberculosis  . The emulsification was made fromequal parts of oil and liquid portions (1:1) in two syringes connectedto each other with Leur lock, transferred to an insulin syringe, and0.2 mL was injected to the right flank of each mouse. On the day ofimmunization, pertussis toxin (200 ng/mL) was injected i.p. at a volumedose of 0.2 mL/mouse. The injection of the pertussis toxin (i.p) wasrepeated after 48 h to boost the immune recognition reaction. Fromday four, natalizumab and Visabron c (4–4)were injected i.p. each other day, five times, and the weight, theneurological score and the disease incidence of the mice were monitoreduntil 35 days after immunization. EAE scoring system: mice were observeddaily for the appearance of neurological symptoms, which were scoredas follows: 0, asymptomatic; 1, partial loss of tail tonicity; 1.5,limp tail; 2, hind limb weakness (right reflex); 3, ataxia; 4, earlyparalysis; 5, full paralysis; and 6, moribund or dead. The diseaseincidence was calculated as a percentage of the ratio of the numberof new diseased mice per number of living mice at each time-point.In experiments to estimate cell infiltration in the spinal cord at35 days postimmunization, the mice were euthanized by cervical dislocationand lumbar spinal cords were collected. The mice were perfused withnormal saline and 4% paraformaldehyde through the blood circulationand the spinal cord was fixed with 4% paraformaldehyde for 24 h, embeddedin paraffin, and cut into 5 μm thick sections. Hematoxylin-Eosinstaining (H&E) was performed. To assess the degree of inflammatorycell infiltration, in each mouse, three histological sections wereexamined. The inflammation score was calculated based on the degreeof infiltration of the inflammatory cells: 0, no infiltrating cells;1, a small amount of infiltrating cells; 2, inflammatory infiltratingtissue around blood vessels; 3, extensive perivascular scar infiltration.All animal experimentswere conducted under the guidelines and supervision of the HebrewUniversity Ethical Committee, which approved the methods employedin this project (Permit No. MD-18-15643-5). C57Bl/6 female mice, 8weeks old, obtained from the Harlan animal breeding center were usedin the study and were maintained in the SPF unit of the Faculty ofMedicine. EAE was induced by s.c. injection of 0.2 mL/mouse emulsioncontaining 200 μg of MOGc (2–2) (150 mg/kg), 0.25 mL Visabron c (2–2) (150 mg/kg) + 0.25 mL of Freund‘s complete adjuvant,and 0.25 mL natalizumab (18 mg/kg). After 14 days of the first inoculation,the mice received a booster with 0.5 mL of the preparations mentionedabove. To verify the presence of antibodies against visabron c (2–2) or natalizumab, the serum collected frommice tail blood was tested 40 days after the first inoculation byindirect ELISA using visabron c (2–2) or natalizumabantigens immobilized to the plate .Then the plates were washed three times with PBS containing 0.05%Tween 20 (T20) and incubated for 1 h at 37 °C with 5% nonfatdry milk in PBS-T20 . Serum samples (n = 18) were diluted at 1:100 in BB, added to the plate,and incubated for 1 h at 37 °C. The plates were then washed threetimes with PBS–T20. Antimouse whole molecule IgG-peroxidaseconjugated (Sigma) was added at a dilution of 1:1500 in BB, and incubatedfor 1 h at 37 °C. After three washes with PBS–T20, substrateand chromogenic reagent  were added and incubated for 5 min. The reaction wasstopped with 12.5% H2SO4, and absorbance wasmeasured at 490 nm. The results were considered significant when theabsorbance was at least two times higher than that obtained with thepreimmune serum.BALB/c male mice, aged 60 days(22 gr) were divided into three groups each containing six mice. Themice were immunized intraperitoneal with 0.5 mL of Visabron Supporting Information.Pharmacokineticswas performed asdetailed in the c (4–4) by iv injection in a volume of 0.2 mL/mouseat a dose of 10 mg/mice (about 500 mg/kg), and another group of fivemice (7688–92) received per-os visabron c (4–4)at a dose of 10 mg/mice, dissolved in a nanoemulsifying drug deliverysystem,48 in a bolus of 0.4 mL/mouse. After48 h of exposure, the animals were sacrificed and the organs wereharvested for the detection of possible toxicological lesions in theframework of a safety assessment. Then the organs were fixed in 4%formaldehyde, trimmed in a standard position per organ, and transferredto embedding cassettes. Paraffin blocks were sectioned at approximately3–5 μm thickness. The sections were applied on a glassslide and stained with hematoxylin and eosin (H&E). Pictures weretaken on a microscope (Olympus BX60) at magnifications of X4 and X10using the microscope’s camera (Olympus DP73). An experiencedpathologist (Dr. Loeb Emanuel) examined the slides. Microscopicallyfindings were classified with standard pathological nomenclature andthe severity of the findings was graded on a scale of minimal, mild,or severe. Grades of severity for microscopic findings were subjective;minimal was the least extent discernible and severe was the greatestextent possible.Eight-week-oldC57Bl/6 mice were obtained from Envigo animal breeding center in Israel.All experiments were approved by the Institutional Animal Care andUse Committee, and performed according to OECD guidelines for chemicaltesting. One group of five mice (7683–87) received visabron c (4–4) was tested at 1 and 0.1 mM.Compound binding was calculated as the percentage of inhibition ofthe binding of a radioactively labeled ligand specific for each target.The compound enzyme inhibitory effect was calculated as the percentageof inhibition of control enzyme activity. Results showing inhibitionhigher than 40% were considered to represent significant effects ofthe tested compound. Results showing inhibition lower than 25% asobtained with 0.1 mM Visabron c (4–4) werenot considered significant and were mostly attributable to the variabilityof the signal around the control level. In each experiment, the respectivereference compound was tested concurrently with visabron c (4–4), and the data were compared with historical valuesdetermined at Eurofins. The experiment was statistically acceptedin accordance with the Eurofins validation standard operating procedure.Screening ofpotentially significant off-target effects to binding and enzyme targetswas performed via SafetyScreen44 offered by Eurofins Cerep-Panlabs.Visabron Supporting Information.Kinase activitywas measured using the PhosphoSenstechnology  as detailed in the p < 0.05.Each experiment was performedin triplicates.Unless otherwise stated, one-way ANOVA was performed using IBM SPSSsoftware. In case of significance, Bonferroni posthoc analysis wasperformed. The results were considered significant when