Exploring the relationship between anal fistula and colorectal cancer based on Mendelian randomization and bioinformatics.

The association between anal fistula patients and colorectal cancer, as well as the potential pathophysiological mechanisms, remains unclear. To explore the relationship between anal fistula and colorectal cancer and its potential mechanisms. Analysis of GEO and TCGA databases. Disease-related genes were also referenced from Coremine Medical, GeneCard and OMIM. Core hub genes were identified through protein-protein interaction analysis by intersecting differentially expressed genes from the datasets with disease data. On one hand, a prognostic model was developed using genes and its prognostic role was validated. On the other hand, the optimal diagnostic genes were selected through machine learning. Mendelian randomization (MR) analysis was conducted to explore the potential causal link between anal fistula and colorectal cancer. Thirteen core genes were identified (TMEM121B, PDGFRA, MID2, WNT10B, HOXD13, BARX1, SIX2, MMP1, SNAL1, CDKN2A, ITGB3, TIMP1, CALB2). Functional enrichment analysis revealed that the intersecting genes between anal fistula and colorectal cancer were associated with extracellular matrix components, signalling pathways, cell growth, protein modification, as well as important roles in cellular activities, tissue and organ development, and biological function maintenance. These genes were also involved in pathways related to Wnt signalling and colorectal cancer development. Prognostic analysis and immune infiltration analysis indicated a close relationship between core hub genes and the prognosis and immune infiltration in colorectal cancer. Machine learning showed that core genes played an essential role in the diagnostic differentiation of colorectal cancer. MR results suggested no causal relationship between anal fistula and colorectal cancer. This study identified shared core genes between anal fistula and colorectal cancer, involved in various pathways related to tumour development. These genes play crucial roles in prognosis and diagnosis.

Polymorphisms of the TNF Gene and Three Susceptibility Loci Are Associated with Crohn's Disease and Perianal Fistula Crohn's Disease: A Study among the Han Population from South China.

BACKGROUND Although 90 susceptibility loci of Crohn's disease (CD) have been confirmed in the Asian population, susceptibility genes for perianal fistula of CD (pCD) in this population remain unknown. This study explored susceptibility genes for CD and pCD in the Han population from South China. MATERIAL AND METHODS In total, 490 patients diagnosed with CD between July 2012 and June 2016 at the Sixth Affiliated Hospital of Sun Yat-sen University were included and divided into the CD group (n=240) and the pCD group (n=250). The healthy control group was composed of 260 volunteers. Peripheral blood samples were taken, and single nucleotide polymorphism (SNP) locus sequencing was used to screen for susceptibility loci. SNPs were sequenced using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. RESULTS Nine SNPs in TNFSF1 on chromosome 9 were associated with CD. Among them, the rs6478106 locus is a risk locus for CD. The distribution frequency of the T allele of the rs6478106 SNP was significantly different between cases and controls (32.49% versus 18.27%, P<0.001). Rs72553867, located in the IRGM gene on chromosome 5, rs4409764, located in the NKX2-3 gene on chromosome 10, and rs3731772, located in the AOX1 gene on chromosome 2, were susceptibility factors for pCD. Nine SNPs located in TNFSF15 on chromosome 9 were related to CD in Han individuals from Southern China. CONCLUSIONS The rs6478106 T allele is associated with the risk of CD in the investigated population. SNPs rs72553867 (IRGM gene), rs4409764 (NKX2-3 gene), and rs3731772 (AOX1 gene) increase the risk of pCD.

Mutations of MYH14 are associated to anorectal malformations with recto-perineal fistulas in a small subset of Chinese population.

BACKGROUND: Anorectal malformations (ARMs) are among the most commonly congenital abnormalities of distal hindgut development, ranging from anal stenosis to anal atresia with or without fistulas and persistent cloaca. The etiology remains elusive for most ARM cases and the majority of genetic studies on ARMs were based on a candidate gene approach. MATERIALS AND METHODS: In all eight family members of a non-consanguineous Chinese family, we performed whole-exome sequencing. Subsequently, exome sequencing of MYH14 in 72 unrelated probands with ARMs was performed. The accurate distribution of non-muscle myosin II heavy chain (NMHC II) was investigated by immunohistochemistry in serial sagittal sections of E11.5-13.5 mouse cloacal regions. RESULTS: A homozygous mutation in MYH14 was identified in the two siblings of family 1. Compound heterozygous MYH14 changes were identified in an unrelated individual. Immunohistochemical analysis suggest stronger NMHC IIC localization in the epithelium of the murine embryonic cloaca, urorectal septum and hindgut compared with another two NMHC II isoforms. CONCLUSION: This is the first identification of mutations in MYH14 as a cause of ARMs. The stronger localization of NMHC IIC in E11.5-13.5 mouse cloacal regions further supports the role of MYH14 in anorectal development.

Prognostic factors affecting outcomes in fistulating perianal Crohn's disease: a systematic review.

BACKGROUND: One in three patients with Crohn's disease will develop a perianal fistulae, and one third of these will achieve long-term healing or closure. A barrier to conducting well-designed clinical trials for these patients is a lack of understanding of prognostic factors. This systematic review sets out to identify factors associated with prognosis of perianal Crohn's fistulae. METHODS: This review was registered on the PROSPERO database (CRD42016050316) and conducted in line with PRISMA guidelines along a predefined protocol. English-language studies assessing baseline factors related to outcomes of fistulae treatment in adult patients were included. Searches were performed on MEDLINE and Embase databases. Screening of abstracts and full texts for eligibility was performed prior to extraction of data into predesigned forms. Bias was assessed using the QUIPS tool. RESULTS: Searches identified 997 papers. Following removal of duplicates and secondary searches, 923 were screened for inclusion. Forty-seven papers were reviewed at full-text level and 13, 2 of which were randomised trials, were included in the final qualitative review. Two studies reported distribution of Crohn's disease as a prognostic factor for healing. Two studies found that CARD15 mutations decreased response of fistulae to antibiotics. Complexity of fistulae anatomy was implicated in prognosis by 4 studies. CONCLUSIONS: This systematic review has identified potential prognostic markers, including genetic factors and disease behaviour. We cannot, however, draw robust conclusions from this heterogeneous group of studies; therefore, we recommend that a prospective cohort study of well-characterised patients with Crohn's perianal fistulae is undertaken.

Multimodal single-cell analyses reveal mechanisms of perianal fistula in diverse patients with Crohn's disease.

BACKGROUND: Crohn's disease complicated by perianal fistulae is more prevalent and severe in patients of African ancestry. METHODS: We profiled single cells from diverse patients with Crohn's disease with perianal fistula from colorectal mucosa and fistulous tracts. Immunofluorescence was performed to validate predicted cell-cell interactions. Unstimulated monocytes were chronically cultured in diverse cohorts. A subset was analyzed by single-nucleus RNA + ATAC sequencing. FINDINGS: Fistulous tract cells from complete proctectomies demonstrated enrichment of myeloid cells compared to paired rectal tissues. Ligand-receptor analysis highlights myeloid-stromal cross-talk and cellular senescence, with cellular co-localization validated by immunofluorescence. Chitinase-3 like-protein-1 (CHI3L1) is a top upregulated gene in stromal cells from fistulae expressing both destructive and fibrotic gene signatures. Monocyte cultures from patients of African ancestry and controls demonstrated differences in CHI3L1 and oncostatin M (OSM) expression upon differentiation compared to individuals of European ancestry. Activating protein-1 footprints are present in ATAC-seq peaks in stress response genes, including CHI3L1 and OSM; genome-wide chromatin accessibility including JUN footprints was observed, consistent with reported mechanisms of inflammatory memory. Regulon analyses confirm known cell-specific transcription factor regulation and implicate novel ones in fibroblast subsets. All pseudo-bulked clusters demonstrate enrichment of genetic loci, establishing multicellular contributions. In the most significant African American Crohn's genetic locus, upstream of prostaglandin E receptor 4, lymphoid-predominant ATAC-seq peaks were observed, with predicted RORC footprints. CONCLUSIONS: Population differences in myeloid-stromal cross-talk implicate fibrotic and destructive fibroblasts, senescence, epigenetic memory, and cell-specific enhancers in perianal fistula pathogenesis. The transcriptomic and epigenetic data provided here may guide optimization of promising mesenchymal stem cell therapies for perianal fistula. FUNDING: This work was supported by grants U01DK062422, U24DK062429, and R01DK123758.

Study on the effect and mechanism of Yanghe decoction Huacai on tissue repair ofsyndrome after anal fistula surgery.

OBJECTIVE: To observe the clinical efficacy and safety of Yanghe decoction Huacai for the repair of Yin syndrome wounds with slow-healing after anal fistula surgery. METHODS: A total of 120 patients with slow-healing negative wounds with after low-grade anal fistula surgery who met the inclusion criteria were divided into a treatment group and a control group based on a random number table method, with 60 patients in the treatment group and 60 patients in the control group. The treatment group was given Yanghe decoction Huacai in combination with routine treatment; the control group was only given routine treatment, in which the wound surface was disinfected with iodine, and then covered with sterile gauze. The course of treatment in both groups was 10 d. After treatment, the wound secretion score, wound granulation tissue score, the expression levels of basic fibroblast growth factor (bFGF), transforming growth factor ß1 (TGF-ß1), and epidermal growth factor (EGF) in the wound, wound healing time and clinical efficacy were compared. RESULTS: There was no significant difference in age or gender between the two groups (P > 0.05). On the 10th and 15th days after the surgery, the wound secretion scores were higher in the treatment group than in the control group (P < 0.01). Comparing the two groups at the 10th and 15th day after surgery, the granulation tissue growth scores in the treatment group were better than the in control group (P < 0.01). On the 10th and 15th day after operation, the expression levels of bFGF, TGF-ß1 and EGF factors in the treatment group were stronger than those in the control group. The healing time of the wounds in the treatment group was significantly shorter than in the control group (P < 0.01). The clinical efficacy of the two groups after treatment was compared, and the overall efficacy of the treatment group was significantly higher than that of the control group (P < 0.01). CONCLUSIONS: Yanghe decoction Huacai have significant efficacy in the treatment of slow-healing wounds with Yin syndrome after anal fistula surgery. It improves wound secretions, promotes the growth of wound granulation tissue, and shortens wound healing time. Its mechanism of action may be related to the control of wound inflammation. It is related to increasing the expression of bFGF, TGF-ß1 and EGF in wound tissue, and promoting wound angiogenesis and fibroblast proliferation.

Mutation in TAGAP is protective of anal sepsis in ileocolic Crohn's disease.

BACKGROUND: Anal complications of Crohn's disease range from painless skin tags to debilitating fistulas that are imperfectly treated with tumor necrosis factor antagonists. The recent discovery of more than 190 single-nucleotide polymorphisms associated with Crohn's disease offers the opportunity to genetically define the severity of anal disease in Crohn's disease and possibly predict prognosis and anti-tumor necrosis factor response. OBJECTIVES: This study aimed to identify single nucleotide polymorphisms associated with anal disease generally, septic anal disease specifically and the responsivity to anti-tumor necrosis factor treatment. DESIGN: All patients with ileocolonic Crohn's disease were identified from our IBD registry. One hundred ninety-six Crohn's disease-related single-nucleotide polymorphisms were analyzed by the use of a custom microarray chip. Patients' response to anti-tumor necrosis factor treatment was then assessed. RESULTS: One hundred sixteen patients with ileocolonic Crohn's disease were identified and assigned to septic anal disease (abscesses/fistulas, n = 35), benign anal disease (skin tags/fissures/isolated pain, n = 17), and no anal disease (n = 64) cohorts. Single-nucleotide polymorphism rs212388 negatively correlated with the presence of anal disease overall and septic disease specifically. The presence of the non-wild-type allele 'G' was protective of anal sepsis with homo- and heterozygotes having a 75% chance of no anal disease (p = 0.0001). The homozygous wild-type group had the highest risk of septic disease and included 3 of 4 patients requiring diverting ileostomies. Twenty-four patients were treated with anti-tumor necrosis factors. Nine had a beneficial response (assessed at >6 months); however, no single-nucleotide polymorphism correlated with anti-tumor necrosis factor response. Rs212388 is associated with the TAGAP molecule involved in T-cell activation. CONCLUSIONS: Rs212388 most significantly correlated with the presence and severity of anal disease in ileocolonic Crohn's disease. A single copy of the risk allele was protective, whereas wild-type homozygotes had the highest risk of septic disease and stoma creation. In this select group, no single-nucleotide polymorphism was predictive of anti-tumor necrosis factor response. Mutations in TAGAP may predict a more benign form and course of anal disease in Crohn's disease.

RNA Sequencing Reveals the Wound Repair Mechanism of Cuyuxunxi Prescription in Surgical Patients with Anal Fistulas.

BACKGROUND: Anal fistula is one of the most common colorectal and perirectal diseases in the world. Cuyuxunxi (CYXX) prescription is an efficient herbal fumigant used to promote the surgical wound healing of anal fistulas. OBJECTIVES: This study aimed to explore the underlying molecular mechanism of CYXX prescription on surgical wound healing of anal fistulas. METHODS: Ten patients with anal fistula were randomized into a control group or treatment group. The wound surface of patients in the control group was rinsed by normal saline, while that in the treatment group was rinsed by CYXX prescription. The wound tissues of patients with anal fistulas seven days after the surgery were collected for hematoxylin-eosin (HE) staining and RNA sequencing. The expressions of differentially expressed genes (DEGs) were validated by real-time quantitative PCR (RT-qPCR). RESULTS: HE staining showed that CYXX treatment reduced the infiltration of inflammatory cells. A total of 472 DEGs, including 141 up-regulated genes and 331 down-regulated genes, were identified. These genes were significantly related to skin development, xenobiotic stimulus, and inflammation. In addition, the consistency rate of RT-qPCR and sequencing results was 83.33%, which showed a high relative reliability of the sequencing results. CONCLUSION: CYXX prescription could improve epidermis repair and reduce inflammatory responses.

CARD15 mutations and perianal fistulating Crohn's disease: correlation and predictive value of antibiotic response.

BACKGROUND: CARD15 mutations alter bowel immunity and increase susceptibility to Crohn's disease (CD). However, the relation between these mutations and Crohn's perianal fistulas has not been fully clarified. AIMS: To assess whether CARD15 mutations are associated with risk of developing Crohn's perianal fistulas and whether these mutations are predictors of the response of perianal fistulas to antibiotics. METHODS: CARD15 mutations were investigated in 203 consecutive CD patients. Presence/absence of history of perianal fistula was recorded. Patients with history of perianal fistula were divided into two groups (with/without CARD15 mutations), and response to antibiotics was evaluated in both groups. RESULTS: Of the 203 patients, 60 (29.6%) showed at least one CARD15 mutation and 55 (27.1%) had history of perianal fistula. History of perianal fistula was identified in 13 (21.7%) patients with mutations and in 42 (29.4%) patients without mutations (P = 0.260). Mean age at diagnosis of first perianal fistula was similar in patients with/without CARD15 mutations (28.7 ± 9.8 versus 29.7 ± 10.1 years, P = 0.758). Average time between disease onset and diagnosis of first perianal fistula was also similar in the two groups (4.6 ± 5.1 versus 5.0 ± 5.9 years, P = 0.816). Response of perianal fistulas to antibiotics (metronidazole alone or combined with ciprofloxacin) was significantly higher in patients without CARD15 mutations (7.7% versus 40.5%, P = 0.041). CONCLUSIONS: In CD, CARD15 mutations are not associated with risk of developing perianal fistulas or with time of their outbreak. Nevertheless, patients with perianal fistulas and CARD15 mutations showed worse response to antibiotics.

Molecular differences of adipose-derived mesenchymal stem cells between non-responders and responders in treatment of transphincteric perianal fistulas.

BACKGROUND: Injection of autologous adipose tissue (AT) has recently been demonstrated to be an effective and safe treatment for anal fistulas. AT mesenchymal stem cells (AT-MSCs) mediate the healing process, but the relationship between molecular characteristics of AT-MSCs of the injected AT and fistula healing has not been adequately studied. Thus we aimed to characterize the molecular and functional properties of AT-MSCs isolated from autologous AT injected as a treatment of cryptogenic high transsphincteric perianal fistulas and correlate these findings to the healing process. METHODS: 27 patients (age 45 ± 2 years) diagnosed with perianal fistula were enrolled in the study and treated with autologous AT injected around the anal fistula tract. AT-MSCs were isolated for cellular and molecular analyses. The fistula healing was evaluated by MRI scanning after 6 months of treatment. AT-MSC phenotype was compared between responders and non-responders with respect to fistula healing. RESULTS: 52% of all patients exhibited clinical healing of the fistulas as evaluated 6 months after last injection. Cultured AT-MSCs in the responder group had a lower short-term proliferation rate and higher osteoblast differentiation potential compared to non-responder AT-MSCs. On the other hand, adipocyte differentiation potential of AT-MSCs was higher in non-responder group. Interestingly, AT-MSCs of responders exhibited lower expression of inflammatory and senescence associated genes such as IL1B, NFKB, CDKN2A, TPB3,TGFB1. CONCLUSION: Our data suggest that cellular quality of the injected AT-MSCs including cell proliferation, differentiation capacity and secretion of proinflammatory molecules may provide a possible mechanism underlying fistula healing. Furthermore, these biomarkers may be useful to predict a positive fistula healing outcome. TRIAL REGISTRATION: NTC04834609, Registered 6 April 2021. https://clinicaltrials.gov/ct2/show/NCT04834609.

Importance of early detection of infantile inflammatory bowel disease with defective IL-10 pathway: A case report.

RATIONALE: Infantile inflammatory bowel disease (IBD) is an extremely rare subgroup of IBD that includes patients whose age of onset is younger than 2 years old. These patients can have more surgical interventions, and a severe and refractory disease course with higher rates of conventional treatment failure. Monogenic defects play an important role in this subgroup of IBD, and identification of the underlying defect can guide the therapeutic approach. PATIENT CONCERNS: In 2007, a 4-month-old girl from a nonconsanguineous family presenting with anal fistula, chronic diarrhea, and failure to thrive. She underwent multiple surgical repairs but continued to have persistent colitis and perianal fistulas. DIAGNOSIS: Crohn's disease was confirmed by endoscopic and histologic finding. INTERVENTION: Conventional pediatric IBD therapy including multiple surgical interventions and antitumor necrosis factor alpha agents were applied. OUTCOMES: The patient did not respond to conventional pediatric IBD therapy. Interleukin-10 (IL-10) receptor mutation was discovered by whole-exome sequencing and defective IL-10 signaling was proved by functional test of IL-10 signaling pathway by the age of 12. The patient is currently awaiting hematopoietic stem cell transplantation. LESSONS: Early detection of underlying genetic causes of patients with infantile-IBD is crucial, since it may prevent patients from undergoing unnecessary surgeries and adverse effects from ineffective medical therapies. Moreover, infantile-IBD patients with complex perianal disease, intractable early onset enterocolitis and extraintestinal manifestations including oral ulcers and skin folliculitis, should undergo genetic and functional testing for IL-10 pathway defect.

Immunopathological characteristics of cryptoglandular and Crohn's anorectal fistulas.

Anorectal fistulas are quite common proctologic disorders. They can be either of cryptoglandular origin or can be associated with Crohn's disease and chronic ileocolitis. Mechanical obstruction and local infections are prime causes of this pathological condition. Genetic predisposition and inadequate immune response with overproduction of pro-inflammatory cytokines appear prominently in the course of Crohn's disease. Interferon-gamma, a Th1 type cytokine, reflecting the engagement of cellular immune mechanisms, is the first to be produced in the intestinal mucosa. The inflammatory process in the colon mucosa induced by the abundant microbial flora is sustained and turned chronic by the gradual elevation of the local TNF-a and regulatory cytokines levels (interleukin-10, transforming growth factor-beta). The number of activated local memory T cells CD4+CD45RBl0 increases significantly. The regulatory CD4+CD25+ T lymphocytes producing interleukin-10 increase also trying to counterbalance the cytokine reaction. The chronic inflammatory infiltrates of the colon mucosa are represented by lymphocytes, plasma cells, macrophages. The long-term activation of macrophages by the released interferon-gamma leads to tissue damage and potentiation of angiogenesis--a risk factor for carcinoma development. Management of anorectal abscesses and fistulas is complex aiming to alleviate the symptoms, prevent relapses, reduce the risk of sphincter damage and improve quality of life. The main approach (surgery) should be combined with antimicrobial infection control and immunomodulation by intravenous or local administration of anti-TNF-alpha antibodies.

Genetic risk profiling and prediction of disease course in Crohn's disease patients.

BACKGROUND & AIMS: Clinical presentation at diagnosis and disease course of Crohn's disease (CD) are heterogeneous and variable over time. Early introduction of immunomodulators and/or biologicals might be justified in patients at risk for disease progression, so it is important to identify these patients as soon as possible. We examined the influence of recently discovered CD-associated susceptibility loci on changes in disease behavior and evaluated whether a genetic risk model for disease progression could be generated. METHODS: Complete medical data were available for 875 CD patients (median follow-up time, 14 years; interquartile range, 7-22). Fifty CD-associated polymorphisms were genotyped. Kaplan-Meier survival analyses, multiple logistic regression, and generalized multifactor dimensionality reduction analyses (GMDR) were performed, correcting for follow-up time. RESULTS: Homozygosity for the rs1363670 G-allele in a gene encoding a hypothetical protein near the IL12B gene was independently associated with stricturing disease behavior (odds ratio [OR], 5.48; 95% confidence interval [CI], 1.60-18.83; P = .007) and with shorter time to strictures (P = .01), especially in patients with ileal involvement (P = .0002). Male patients carrying at least one rs12704036 T-allele in a gene desert had the shortest time to non-perianal fistula (P < .0001). The presence of a C-allele at the CDKAL1 single nucleotide polymorphism rs6908425 and the absence of NOD2 variants were independently associated with development of perianal fistula (OR, 8.86; 95% CI, 1.13-69.78; P = .04 and OR, 0.56; 95% CI, 0.38-0.83; P = .004, respectively), particularly when colonic involvement and active smoking were present. CONCLUSIONS: CD-associated polymorphisms play a role in disease progression and might be useful in identifying patients who could benefit from an early top-down treatment approach.

Polymorphism of the IRGM gene might predispose to fistulizing behavior in Crohn's disease.

OBJECTIVES: Recently, genome-wide association analyses have identified single nucleotide polymorphisms in the IRGM gene (rs1000113 and rs4958847) as strong candidate susceptibility factors for Crohn's disease (CD). The aim of our study was to test whether these variants are associated with inflammatory bowel disease (IBD) in adult- and childhood-onset Italian patients. METHODS: Allele and genotype frequencies of rs1000113 and rs4958847 were determined in 823 CD (265 younger than 19 years at diagnosis), 353 ulcerative colitis (UC) (130 younger than 19 years at diagnosis), and 578 controls. Genotype distributions were examined both within IBD clinical sub-phenotypes and CARD15 genotypes. RESULTS: rs1000113 and rs4958847 were both associated with adult-onset (P=2 x 10(-4); P=2.5 x 10(-3), respectively) and childhood-onset (P=4 x 10(-4); P=8 x 10(-3), respectively) CD cohorts. Similarly, the genotype frequencies remained significantly different for both variants (adult rs1000113, P=1 x 10(-4); rs4958847, P=1 x 10(-3); pediatric rs1000113, P=2.3 x 10(-4); rs4958847, P=9.6 x 10(-3)). At logistic regression, the rs4958847 polymorphism was associated with fistulizing behavior (P=0.037, OR=1.54, CI=1.02-2.31) and perianal fistulas (P=0.045, OR=1.55, CI=1.01-2.38). Conversely, no association with UC and sub-phenotypes was shown. CONCLUSIONS: We replicated the previously reported associations between CD and rs1000113 and rs4958847, confirming that IRGM is a susceptibility locus only for CD, either adult- or early-onset in the Italian population; furthermore, we have also shown its influence on specific clinical features (fistulizing disease).

NOD2/CARD15 gene variants are linked to failure of antibiotic treatment in perianal fistulating Crohn's disease.

OBJECTIVES: The Crohn's disease (CD) susceptibility gene, nucleotide-binding oligomerizetion domain 2 (NOD2)/caspase recruitment domain 15 (CARD15), is linked to the innate immune response associated with altered epithelial bacterial defense. Its relevance in antibiotic therapy of perianal fistulating CD remains elusive. The aim of the study was to explore systematically the association between NOD2/CARD15 variants and clinical response of perianal fistulas in patients using antibiotic therapy. METHODS: Fifty-two patients (median age 36 yr) with draining perianal fistulas were treated with ciprofloxacin (N = 49) or metronidazole (N = 3) for a median duration of 7 wk. Complete response was defined as the absence of any draining fistula despite gentle finger compression. Genotyping for NOD2/CARD15 variants and human beta (beta)-defensin 2 (HBD-2) copies was performed by 5' nuclease assays (Applied Biosystems, Foster City, CA). The examiners and laboratory investigators were blinded. The Fisher exact test and Wilcoxon signed rank test were used for statistical analysis. RESULTS: Ciprofloxacin was discontinued in one patient due to diarrhea after 2 wk. Complete fistula response was observed in 13 of 39 patients with NOD2/CARD15 wild-type (33.3%) compared with none in patients carrying NOD2/CARD15 variants (0%, P= 0.02). The median number of HBD-2 gene copies between responders and partial/nonresponders was similar. CONCLUSIONS: The study result suggests a substantial contribution of NOD2/CARD15 to the antibiotic treatment outcome of perianal fistulating CD. NOD2/CARD15 variants may predispose to an altered intestinal microflora in perianal fistulas that is less responsive to antibiotic treatment.

Root extractive from Daphne genkwa benefits in wound healing of anal fistula through up-regulation of collagen genes in human skin fibroblasts.

Wound healing is the main problem in the therapy of anal fistula (AF). Daphne genkwa root has been traditionally used as an agent to soak sutures in operation of AF patients, but its function in wound healing remains largely unclear. The aim of the present study was to illuminate mechanisms of D. genkwa root treatment on AF. In the present study, 60 AF patients after surgery were randomly divided into two groups, external applied with or without the D. genkwa extractive. Wound healing times were compared and granulation tissues were collected. In vitro, we constructed damaged human skin fibroblasts (HSFs) with the treatment of TNF-α (10 µg/ml). Cell Count Kit-8 (CCK-8) and flow cytometry analysis were used to determine the effects of D. genkwa root extractive on cell viability, cell cycle and apoptosis of damaged HSFs. Furthermore, protein levels of TGF-ß, COL1A1, COL3A1, Timp-1, matrix metalloproteinase (MMP)-3 (MMP-3) and MEK/ERK signalling pathways were investigated both in vivo and in vitro Results showed that D. genkwa root extractive greatly shortens the wound healing time in AF patients. In granulation tissues and HSFs, treatment with the extractive significantly elevated the expressions of COL1A1, COL3A1, Timp-1, c-fos and Cyclin D1, while reduced the expression of MMP-3 Further detection presented that MEK/ERK signalling was activated after the stimulation of extractive in HSFs. Our study demonstrated that extractive from D. genkwa root could effectively improve wound healing in patients with AF via the up-regulation of fibroblast proliferation and expressions of COL1A1 and COL3A1.

[Detection and analysis of the characteristic expression of microRNAs of anal fistula patients].

OBJECTIVE: To detect and analyze the characteristic miRNAs profile of anal fistula and explore their possible target genes and potential clinical significance. METHODS: The anal mucosa close to the hemorrhoids were collected from three patients undergoing fistulectomy and hemorrhoidectomy (fistula group) as well as three patients receiving only hemorroidectomy(hemorrhoids group), matching with fistula group in age, gender and body weight. miRNA microarray was used to compare the expression of 1 285 human miRNAs of the anal mucosa between two groups. Cluster analysis was adopted to analyze the accumulation of the differentially expressed miRNAs(P<0.05, fold≥2.0 or ≤0.5) and their target genes were predicted with 10 softwares such as DIANAmT, miRanda, miRDB, miRWalk etc. Comprehensive scoring was performed to identify genes with highest predictive score. Gene ontology (GO) concentration technique was used to analyze the target gene-associated biological process. Immunohistochemistry was used to examine protein expression of genes with the highest score. RESULTS: Among 1285 miRNAs in fistula group, 13 miRNAs were differentially expressed with those in hemorrhoid group, including 2 of up-regulation and 11 of down-regulation. Paired t test showed that in fistula group, miRNA-3609 up-regulation was 5.98 folds(P=0.0231) and miR-181a-2-3p down-regulation was 0.13 folds(P=0.0067) compared to those in hemorrhoid group, which had the greatest differential expression. Cluster analysis suggested that up-regulated miR-3609 and miR-6086 had similar change trend in both groups. Among 11 down-regulated miRNAs, miR-125bp-1-3p and miR-548q had similar expression and other 9 miRNAs had similar expression as well, including miR-1185-1-3p, miR-532-3p, miR-1233-5p, miR-769-5p, miR-149-5p, miR-99b-3p, miR-141-3p, miR-138-5p, and miR-181a-2-3p. Target gene prediction analysis of above 13 genes showed that 7 miRNAs(53.8%) were eligible to predict their potential target genes, yielding totally 104 possible target genes. The rest of 6 miRNAs(46.2%) failed to predict any target gene. The highest score in prediction of target gene was chitinase 1(ChIT1) and its corresponding differential miRNA was miR-769-5p(r=-0.94286, P=0.0167). Gene ontology analysis showed that the most associated biological process related with these 104 target genes was keratinization, immune response and signal transduction. Immunohistochemistry revealed ChiT1 expression of anal mucosa in fistula group was significantly higher compared to hemorrhoid group(P<0.01). CONCLUSIONS: There is a characteristic miRNAs profile in anal fistula patients, which may play a role in the occurrence and development of anal fistula.

Results of the Fifth Scientific Workshop of the ECCO (II): Pathophysiology of Perianal Fistulizing Disease.

The fifth scientific workshop of the European Crohn's and Colitis Organization (ECCO) focused on the relevance of fistulas to the disease course of patients with Crohn's disease (CD). The objectives were to reach a better understanding of the pathophysiological mechanisms underlying the formation of CD fistulas; to identify future topics in fistula research that could provide insights into pathogenesis; to develop novel therapeutic approaches; and to review current therapeutic strategies (with clarification of existing approaches to prevention, diagnosis and treatment). The results of the workshop are presented in two separate manuscripts. This manuscript describes current state-of-the-art knowledge about fistula pathogenesis, including the roles of epithelial-to-mesenchymal transition and cytokine matrix remodelling enzymes, and highlights the common association between fistulas and stenosis in CD. The review also considers the possible roles that genetic predisposition and intestinal microbiota play in fistula development. Finally, it proposes future directions and needs for fistula research that might substantially increase our understanding of this complex condition and help unravel novel therapeutic strategies and specific targets for treatment. Overall, it aims to highlight unanswered questions in fistula research and to provide a framework for future research work.

The NOD2 Single Nucleotide Polymorphism rs72796353 (IVS4+10 A>C) Is a Predictor for Perianal Fistulas in Patients with Crohn's Disease in the Absence of Other NOD2 Mutations.

BACKGROUND: A previous study suggested an association of the single nucleotide polymorphism (SNP) rs72796353 (IVS4+10 A>C) in the NOD2 gene with susceptibility to Crohn's disease (CD). However, this finding has not been confirmed. Given that NOD2 variants still represent the most important predictors for CD susceptibility and phenotype, we evaluated the association of rs72796353 with inflammatory bowel disease (IBD) susceptibility and the IBD phenotype. METHODOLOGY: Genomic DNA from 2256 Caucasians, including 1073 CD patients, 464 patients with ulcerative colitis (UC), and 719 healthy controls, was genotyped for the NOD2 SNP rs72796353 and the three main CD-associated NOD2 mutations rs2066844, rs2066845, and rs2066847. Subsequently, IBD association and genotype-phenotype analyses were conducted. RESULTS: In contrast to the strong associations of the NOD2 SNPs rs2066844 (p=3.51 x 10(-3)), rs2066845 (p=1.54 x 10(-2)), and rs2066847 (p=1.61 x 10(-20)) with CD susceptibility, no significant association of rs72796353 with CD or UC susceptibility was found. However, in CD patients without the three main CD-associated NOD2 mutations, rs72796353 was significantly associated with the development of perianal fistulas (p=2.78 x 10(-7), OR 5.27, [95% CI 2.75-10.12] vs. NOD2 wild-type carriers). CONCLUSION/SIGNIFICANCE: Currently, this study represents the largest genotype-phenotype analysis of the impact of the NOD2 variant rs72796353 on the disease phenotype in IBD. Our data demonstrate that in CD patients the IVS4+10 A>C variant is strongly associated with the development of perianal fistulas. This association is particularly pronounced in patients who are not carriers of the three main CD-associated NOD2 mutations, suggesting rs72796353 as additional genetic marker for the CD disease behaviour.