Surface-enhanced Raman spectroscopy analysis reveals biochemical difference in urine of patients with perianal fistula.

BACKGROUND & AIMS: Perianal fistulising Crohn's disease (PFCD) is different from the characteristics and outcomes of traditional non-inflammatory bowel disease (IBD) anal fistulas. The presence of perianal disease was a poor prognostic indicator for Crohn's disease (CD) patients and PFCD patients were more likely to bear an increased risk of recurrence. However, the effective and accurate diagnosis methods to early distinguish PFCD from simple perianal fistula were still scarce. The purpose of this study is to develop a non-invasive detecting approach to predict CD in patients with perianal fistulas. METHODS: Data on patients with anal fistulizing disease were collected from July 2020 to September 2020 in two IBD centers. Urine samples from PFCD and simple perianal fistula patients were investigated by surface-enhanced Raman spectroscopy (SERS). Principal component analysis (PCA)-support vector machine (SVM) was utilized to establish classification models to distinguish PFCD from simple perianal fistula. RESULTS: After a case-matched 1:1 selection by age and gender, 110 patients were included in the study. By analyzing the average SERS spectra of PFCD and simple perianal fistula patients, it revealed that there were significant differences in intensities at 11 Raman peaks. The established PCA-SVM model distinguished PFCD from simple perianal fistula with a sensitivity of 71.43%, specificity 80.00% and accuracy 75.71% in the leave-one-patient-out cross-validation. The accuracy of the model in validation cohort was 77.5%. CONCLUSIONS: Investigation of urine samples by SERS helps clinicians to predict Crohn's disease from perianal fistulas, which make patients achieve benefit from a more individualized treatment strategy.

Non-invasive diagnosis of Crohn's disease based on SERS combined with PCA-SVM.

Crohn's disease (CD) is an idiopathic chronic inflammatory bowel disease without a cure. Most of the CD patients are firstly diagnosed by invasive endoscopy, and clinical and pathological examinations are further required to confirm the diagnosis. Hence, the development of a non-invasive, rapid and accurate diagnosis method for CD patients is essential. In this study, urine samples from 95 CD patients (including 58 active CD (aCD) patients and 37 inactive CD (iCD) patients) and 48 healthy controls (HC) were investigated by surface-enhanced Raman spectroscopy (SERS). The statistical analysis of the three groups (i.e., CD/HC, aCD/HC and iCD/HC) was performed on the measured data. Principal component analysis (PCA)-support vector machine (SVM) and PCA-linear discriminant analysis (LDA) were then employed to establish classification models to distinguish between patients and HC. For the average SERS spectra of patients and HC, the Raman peaks belonging to lipids, proteins and nucleic acids were stronger in patients than those in HC. It showed that the classification accuracy of CD/HC based on PCA-SVM was higher than that of PCA-LDA (82.5% vs. 69.9%). And the classification accuracy of aCD/HC based on PCA-SVM was higher than that of iCD/HC (86.8% vs. 76.5%). The classification model we established distinguished between aCD and HC with 86.2% sensitivity and 87.5% specificity. It indicates that the metabolic change of patients could be identified by measuring urine with SERS, and aCD and HC could be distinguished more effectively. Our findings are helpful for clinicians to diagnose CD patients and monitor the progress and recurrence of the disease.

Increased AOC1 Expression Promotes Cancer Progression in Colorectal Cancer.

BACKGROUND: Amine oxidase copper containing 1 (AOC1) is a gene whose biological function in colorectal cancer (CRC) has not been elucidated. Therefore, the purpose of this study was to investigate the clinical significance of AOC1 expression in CRC and its biological function in CRC cell lines. MATERIALS AND METHODS: AOC1 expression levels were examined in paired CRC and peritumoral tissues, and distant liver metastatic tissues were examined using quantitative real-time PCR, western blotting, and immunohistochemistry staining. The log-rank test and Cox regression model were used to analyze the relationship between AOC1 expression and prognosis. Proliferation assays (Cell Counting Kit-8 and colony formation assays), migration assays (Transwell and wound healing assays) and xenograft tumor formation in nude mice were performed to assess the biological role of AOC1 in CRC cells. RESULTS: AOC1 expression significantly increased in human CRC tissues, especially in liver metastases, and was associated with a worse prognosis. In addition, AOC1 had higher expression in tumor organoids than in normal organoids, suggesting that it was highly expressed in the tumor epithelium. Functional analysis demonstrated that AOC1 knockdown inhibited the proliferation and migration of CRC cells by inducing EMT in vitro. Xenograft tumor formation in nude mice showed that knockdown of AOC1 inhibited the tumor xenografts growth in vivo. CONCLUSION: High expression of AOC1 was significantly associated with worse clinical outcomes, was an independent risk factor for poor prognosis, and promoted aggressive CRC cell phenotypes. AOC1 is expected to become a novel biomarker for predicting the prognosis of patients with CRC and an effective therapeutic target in clinical practice.

Surgical repair of rectovaginal fistulas: predictors of fistula closure.

INTRODUCTION AND HYPOTHESIS: We report the clinical outcome of surgical repair for rectovaginal fistula (RVF) carried out by one operative team. We also investigate the predictive factors for fistula healing. METHODS: A retrospective cohort of 63 patients underwent local surgical repair of RVF during January 2008 and December 2017 by one operative group. The clinical features of the patients were reviewed. The association between fistula closure and diverse clinical parameters, including operative method, fistula location, prior repair, and diverting stoma, was analyzed. RESULTS: Sixty-three consecutive patients underwent 80 local surgical repairs by our surgical team. Forty-five patients eventually healed after an average of 1.22 procedures. The overall success rate per procedure was 71.2%, whereas the closure rate of the first operation was 55.5% (n = 35). The etiology of the fistula did not impact on the success rate of surgical repair. The history of prior repair predicted a lower success rate on both overall procedure (RR = 0.59, 95% CI 0.41-0.85, p = 0.008) and the first repair in our institution (RR = 0.50, 95% CI 0.31-0.80, p = 0.003). There was no difference in closure rate between the stoma group and the non-stoma group. Nevertheless, among the 15 patients who underwent more than one operation in our center, a diverting stoma seemed to be necessary (10 patients healed in the stoma group and none of the patients healed in the non-stoma group, p = 0.02). CONCLUSIONS: History of prior surgical repair is a risk factor for failure. Diverting stoma did not increase the overall closure rate, but it seemed to be necessary for patients in whom the first operation failed.

Combination of CDX2 expression and T stage improves prognostic prediction of colorectal cancer.

OBJECTIVE: Prognostic prediction of colorectal cancer (CRC) remains challenging because of its heterogeneity. Aberrant expression of caudal-type homeobox transcription factor 2 (CDX2) is strongly correlated with the prognosis of CRC. METHODS: Tissue samples of patients with CRC who underwent surgery in Xinhua Hospital (Shanghai, China) from January 2010 to January 2013 were collected. CDX2 expression was semiquantitatively evaluated via immunohistochemistry. RESULTS: In total, 138 patients were enrolled in this study from a prospectively maintained institutional cancer database. The median follow-up duration was 57.5 months (interquartile range, 17.0-71.0 months). In the Cox proportional hazards model, low CDX2 expression combined with stage T4 CRC was significantly the worst prognostic factor for disease-free survival (hazard ratio = 7.020, 95% confidence interval = 3.922-12.564) and overall survival (hazard ratio = 5.176, 95% CI = 3.237-10.091). In the Kaplan-Meier survival analysis, patients with low CDX2 expression and stage T4 CRC showed significantly worse disease-free survival and overall survival than those with low CDX2 expression alone. CONCLUSION: CDX2 expression combined with the T stage was more accurate for predicting the prognosis of CRC. Determining the prognosis of CRC using more than one variable is valuable in developing appropriate treatment and follow-up strategies.

Reduced Risk of Inflammatory Bowel Disease-associated Colorectal Neoplasia with Use of Thiopurines: a Systematic Review and Meta-analysis.

BACKGROUND AND AIMS: The association between thiopurines and colorectal neoplasia risk remains controversial in inflammatory bowel disease [IBD] patients. We performed a systematic review and meta-analysis examining this association. METHODS: A comprehensive search of the PubMed, EMBASE and Cochrane Library databases was performed to identify relevant literature. Random-effects models were applied to calculate the pooled odds ratio [OR] and relative risk [RR] with corresponding 95% confidence intervals [CIs] among case-control and cohort studies. RESULTS: Eleven cohort and 16 case-control studies involving 95397 patients were included in this study. Overall, the use of thiopurines was associated with a reduced risk of colorectal neoplasia both in case-control [OR = 0.49, 95% CI: 0.34-0.70] and cohort studies [RR = 0.96, 95% CI: 0.94-0.98]. Moreover, a protective effect of thiopurines against advanced neoplasia [high-grade dysplasia and cancer] [OR = 0.51, 95% CI: 0.31-0.84 for case-control studies; RR = 0.96, 95% CI: 0.94-0.98 for cohort studies] and colorectal cancer [CRC] [OR = 0.56, 95% CI: 0.34-0.93 for case-control studies; RR = 0.96, 95% CI: 0.94-0.98 for cohort studies] was also observed. Furthermore, when the analysis was conducted on patients at a high risk for colorectal neoplasia, the chemopreventive effect was confirmed in patients with long disease duration [> 8 years] but not in those with extensive colitis or primary sclerosing cholangitis. CONCLUSIONS: This study demonstrated that thiopurine use was associated with a reduced risk of colorectal neoplasia, advanced neoplasia and CRC in IBD patients, especially those with long disease duration [> 8 years].

Long-term quality of life associated with early surgical complications in patients with ulcerative colitis after ileal pouch-anal anastomosis: A single-center retrospective study.

BACKGROUND: Ileal pouch-anal anastomosis (IPAA) is recommended for patients with ulcerative colitis (UC) in terms of surgical treatment. Measuring surgical complications of IPAA and long-term quality of life (QOL) are important to achieve an acceptable risk/benefit ratio for patients with UC. MATERIALS AND METHODS: Patients with UC who underwent total proctocolectomy (TPC) with IPAA from February 2008 to July 2016 at our institute were included. Early surgical complications were defined as mechanical/infectious events within one month after IPAA. Assessment of QOL was performed using the Cleveland Global Quality of Life instrument (CGQL), with 50% improvement as a cut-off value. Demographic and clinical variables were compared with univariable analysis and step-wise logistic regression models were also performed. RESULTS: A total of 58 eligible patients had a median follow-up time of 78.5 months [interquartile range (IQR), 34.4-92.8] from February2008 to March 2017, including 25 cases (43.1%) developed early surgical complications. Age at pouch surgery and excessive blood loss were risk factors associated with early surgical complications (p < 0.05). In multivariate analysis, older age at surgery [odds ratio (OR), 1.05; 95% confidence interval (CI), 1.01-1.1] and significant blood loss (≧400 ml) (OR, 4.31; 95% CI, 1.21-16.87) were contributing factors for developing early surgical complications. The CGQL score was significantly increased after IPAA (0.728 ± 0.151 vs. 0.429 ± 0.173, p < 0.001). Early surgical complications (OR, 5.55; 95%CI, 1.44-21.37), older age at surgery (OR, 1.06; 95% CI, 1.01-1.12) and use of immunomodulatory (OR, 17.50; 95% CI, 1.52-201.39) were associated with poor long-term QOL. CONCLUSION: The study demonstrated that early surgical complications might contribute to develop a poor CGQL score, suggesting intentional control of risk factors associated with early surgical complications should be taken into consideration for patients with UC for pouch surgery.

Phosphorylase kinase ß affects colorectal cancer cell growth and represents a novel prognostic biomarker.

PURPOSE: To study the expression and intracellular localization of phosphorylase kinase ß (PHKß) protein in colorectal cancers (CRCs), analyze its correlation with clinicopathological features and prognosis, and study the biological roles and mechanism-of-action of PHKß in CRC cell lines. METHODS: Quantitative polymerase chain reaction (qPCR) and western blot assays were performed to compare the expressions of PHKß mRNA and protein in CRC tissues and matched normal mucosa. Tissue microarrays and immunohistochemical staining were performed to detect the expression and intracellular location of PHKß protein and analyze its correlation with the clinicopathological characteristics and prognosis in CRC patients. Proliferation, cell cycle, wound healing, and xenograft models were used to elucidate the potential role of PHKß in vitro and in vivo. RESULTS: PHKß mRNA and protein were found to be overexpressed in CRC tissue compared to the levels in normal mucosa. Positive expression of PHKß was significantly correlated with TNM stage and distal metastasis, and elevated expression of PHKß was an independent prognostic factor in patients with CRC. PHKß knockdown impaired proliferation of CRC in vitro and in vivo and induced cell cycle arrest. CONCLUSIONS: PHKß affects CRC cell growth and represents a novel prognostic biomarker.

Polymorphism of the methylenetetrahydrofolate reductase gene association with homocysteine and ischemic stroke in type 2 diabetes.

BACKGROUND: Ischemic stroke is a frequent heterogeneous multifactorial disease. A number of genetic mutations and environmental factors have been implicated. A polymorphism in the gene for methylenetetrahydrofolate reductase (MTHFR) has been reported to be associated with hyperhomocysteinemia a risk for atherosclerotic vascular diseases. AIM: A cross-sectional study was performed to determine the relationship between the gene polymorphism for MTHFR and ischemic stroke in type 2 diabetes mellitus. MATERIALS AND METHODS: Of the 215 unrelated patients with type 2 diabetes mellitus recruited, 119 patients had ischemic stroke, Control group included 142 healthy subjects. The genotype of the subjects for the C677T polymorphism of MTHFR was analyzed by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) followed by HinfI digestion. Plasma total homocysteine (Hcy) levels were measured using high-performance liquid chromatography (HPLC) with fluorescence detection. RESULTS: The genotype distribution did not differ between the control subjects and type 2 diabetic patients (P > 0.05). Plasma homocysteine levels were markedly higher in diabetic patients with TT genotype than those with CC or CT genotype (P > 0.05). Ischemic stroke was more frequently observed in type 2 diabetic patients with the TT genotype than in those with the CT and CC genotype (odds ratio = 4.04, 95% CI = 1.95-8.34, P = 0.0036). Logistic regression analysis revealed that the C677T mutation of MTHFR gene was independently associated with ischemic stroke in type 2 diabetes. CONCLUSION: MTHFR C677T gene polymorphism associated with a predisposition to hyperhomocysteinemia could constitute a useful predictive marker for ischemic stroke in type 2 diabetic Chinese patients.

Methylenetetrahydrofolate reductase polymorphism associated with susceptibility to coronary heart disease in Chinese type 2 diabetic patients.

OBJECTIVE: Epidemiological studies have identified hyperhomocyst(e)inemia as an independent risk factor for atherosclerosis. The C677T variant of the methylenetetrahydrofolate reductase (MTHFR) gene, one of the key enzymes catalyzing remethylation of homocysteine, might play a role in the development of coronary heart disease (CHD). In this study, we examined the distribution of the MTHFR genotypes in the Chinese population and the association between the C677T variant and CHD in Chinese type 2 diabetic patients. METHODS: Two hundred and twenty-eight unrelated patients with type 2 diabetes mellitus (126 with coronary heart disease) and 114 healthy control subjects were recruited. The MTHFR genotype was analyzed by PCR followed by HinfI digestion. Plasma total homocysteine levels were measured using high-performance liquid chromatography (HPLC) with fluorescence detection. RESULTS: In 114 healthy control subjects, the frequency of the mutant T allele was 38.0%, comparable to that of a Hong Kong (Chinese) population. The genotype distribution did not differ between control subjects and type 2 diabetic patients (chi(2) = 3.67, P > 0.05). Genotypic analysis revealed that type 2 diabetic patients with CHD displayed a greater prevalence of T allele (45.2%) than type 2 diabetic patients without CHD (30.4%) (chi(2) = 8.72, P < 0.005). The odds ratio for CHD in type 2 diabetic patients in presence of T allele was 1.89 (CI 95%, 1.24-2.88). The MTHFR genotype were different between diabetic patients with and without CHD (chi(2) = 11.98, P < 0.005). Moreover, plasma homocysteine levels were markedly higher in individuals with TT genotype than those with CC or CT genotype or CC plus CT genotype. CONCLUSIONS: The C677T mutation of MTHFR gene is common in the Chinese population. MTHFR C677T gene polymorphism associated with a predisposition to increased plasma homocysteine levels could constitute a useful predictive marker for CHD in Chinese type 2 diabetic patients.

Genetic polymorphism of methylenetetrahydrofolate reductase as a risk factor for diabetic nephropathy in Chinese type 2 diabetic patients.

OBJECTIVE: Genetic predisposition has been implicated in diabetic nephropathy (DN). The C677T variant of the methylenetetrahydrofolate reductase (MTHFR) gene, one of the key enzymes catalyzing remethylation of homocysteine, may play a role in the development of not only vascular disease but also diabetic microangiopathies. In this study, we examined the distribution of the MTHFR genotypes in the Chinese population and the association between the C677T variant and diabetic nephropathy. METHODS: 220 unrelated patients with type 2 diabetes mellitus and 130 controls were recruited. The MTHFR genotype was analyzed by PCR followed by HinfI digestion. Plasma total homocysteine levels were measured using high-performance liquid chromatography (HPLC) with fluorescence detection. RESULTS: In 130 healthy control subjects, the frequency of the mutant T allele was 30.0%, comparable to that of a Hong Kong (Chinese) population. The distribution of the three genotypes was as follows: TT genotype, 16.9%; CT genotype, 26.2%; and CC genotype, 56.9%. This genotype distribution did not differ between control subjects and type 2 diabetic patients in which 19.1% were TT, 34.5% were CT and 46.4% were CC (2=3.85, P>0.05). The frequency of the mutant T allele was 42.3% in diabetic patients with nephropathy (n=124) versus 28.6% in those without nephropathy (n=96). The genotype frequencies were TT, 21.0%; CT, 42.7%; CC, 36.3% in diabetic patients with nephropathy versus TT, 16.7%; CT, 23.9%; CC, 59.4% in those without nephropathy. The MTHFR genotype and allele frequencies were different between diabetic patients with and without nephropathy (chi2=12.27, P<0.005; chi2=8.77, P<0.005, respectively). Moreover, plasma homocysteine levels were markedly higher in individuals with TT genotype than those with CC or CT genotype. CONCLUSIONS: The C677T mutation of MTHFR gene is common in the Chinese population. MTHFR C677T gene polymorphism associated with a predisposition to increased plasma homocysteine levels may represent a genetic risk factor for diabetic nephropathy in Chinese type 2 diabetic patients.

The relationship between MTHFR gene polymorphisms, plasma homocysteine levels and diabetic retinopathy in type 2 diabetes mellitus.

OBJECTIVE: To evaluate the role of methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms and plasma homocysteine levels in patients with type 2 diabetes mellitus and diabetic retinopathy (DR). METHODS: Total of 208 patients with type 2 diabetes mellitus and 57 controls were recruited into the study. MTHFR genetic C677T polymorphisms were determined by PCR-RFLP. Plasma total homocysteine levels were measured using high-performance liquid chromatography (HPLC) with fluorescence detection. RESULTS: The frequencies of MTHFR TT homogeneous type, CT heterogeneous type and allele T (28.18%, 41.82%, 49.09%) were significantly higher in the type 2 diabetes mellitus with diabetic retinopathy group than those without retinopathy (18.37%, 29.59%, 33.16%) and those of controls (17.54%, 28.07%, 31.58%). The presence of the T allele appeared to have a strong association with the development of diabetic retinopathy. The odds ratio was 1.94 with a 95% confidence interval of 1.31 - 2.88. Moreover, plasma homocysteine levels were remarkably higher in patients with TT or CT genotype than in patients with the CC genotype. CONCLUSION: MTHFR gene C677T mutation associated with a predisposition to increased plasma homocysteine levels may be considered as a genetic risk factor for diabetic microangiopathy (such as DR) in Chinese patients with type 2 diabetes mellitus.

[The relationship of methylenetetrahydrofolate reductase gene polymorphism and plasma homocysteine levels in type 2 diabetes mellitus patients with diabetic retinopathy].

OBJECTIVE: To evaluate the role of methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms and plasma homocysteine levels in Chinese patients with type 2 diabetes mellitus and diabetic retinopathy (DR). METHODS: MTHFR genetic C677T polymorphisms were determined by PCR-restriction fragment length polymorphism. Total plasma homocysteine levels were measured using high-performance liquid chromatography (HPLC) with fluorescence detection. RESULTS: The frequencies of MTHFR T homogenetic type and CT heterogenetic type and allele T (28.18%, 41.82%, 49.09%) in type 2 diabetic patients with diabetic retinopathy were significantly higher than those in diabetic patients without retinopathy (18.37%,29.59%,33.16%) or the normal controls (17.54%, 28.07%, 31.58%). Howerver, there were no significant differences in the frequency of MTHFR genotype and allele between the type 2 diabetic patients without retinopathy and the normal controls. The presence of T allele appeared to have a strong association with the development of diabetic retinopathy. The odds ratio was 1.94 and the 95% confidence interval was 1.31-2.88. Moreover, the plasma homocysteine levels in patients with TT or CT genotype were markedly higher than those in patients with CC genotype. CONCLUSION: MTHFR gene C677T mutation associated with a predisposition to increase of plasma homocysteine may represent a genetic risk factor for diabetic retinopathy in Chinese type 2 diabetes mellitus.

[Association of polymorphism in neurogenic differentiation factor 1 gene with type 2 diabetes].

OBJECTIVE: To evaluate the role of polymorphism in the neurogenic differentiation factor 1(Neuro D) gene in Chinese patients with type 2 diabetes mellitus. METHODS: The genotypes of codon 45 variant (GCC-->ACC) in the Neuro D gene were determined by mismatch PCR-restriction fragment length polymorphism assay in 448 Chinese, including 124 subjects with normal glucose tolerance and 324 patients with type 2 diabetes mellitus. The diabetic patients were divided into two groups cutting off with the age of 40 at onset. RESULTS: No homozygote of the Ala45Thr variant was found in these subjects. The frequencies of AT heterozygous type were significantly higher in early-onset type 2 diabetic group than those in the control group and in the late-onset type 2 diabetic group (chi(2)=7.85, P=0.005; chi(2)=8.81, P=0.003). The frequencies of Thr45 allele in the early-onset type 2 diabetic group were significantly different from those of the control group (13.4% vs 5.2%, chi(2)=7.15, P=0.008) and the late-onset type 2 diabetic group (13.4% vs 5.8%, chi(2)=8.13, P=0.004). The presence of Thr45 allele was shown to have an association with early-onset type 2 diabetes (OR=2.52, 95% CI: 1.42-4.49). Furthermore, the subjects carrying the variant appeared to have lower serum concentration of C-peptide in diabetic group. However, the frequencies of polymorphism genotypes of Neuro D gene showed no difference between the late-onset type 2 diabetic group and the control group. CONCLUSION: The genetic polymorphism in the Neuro D is associated with the development of early-onset type 2 diabetes. The presence of Thr45 allele may represent a risk factor for early-onset type 2 diabetes among Chinese.