Identification and analysis of chemokine-related and NETosis-related genes in acute pancreatitis to develop a predictive model.

Background: Chemokines and NETosis are significant contributors to the inflammatory response, yet there still needs to be a more comprehensive understanding regarding the specific molecular characteristics and interactions of NETosis and chemokines in the context of acute pancreatitis (AP) and severe AP (SAP). Methods: To address this gap, the mRNA expression profile dataset GSE194331 was utilized for analysis, comprising 87 AP samples (77 non-SAP and 10 SAP) and 32 healthy control samples. Enrichment analyses were conducted for differentially expressed chemokine-related genes (DECRGs) and NETosis-related genes (DENRGs). Three machine-learning algorithms were used for the identification of signature genes, which were subsequently utilized in the development and validation of nomogram diagnostic models for the prediction of AP and SAP. Furthermore, single-gene Gene Set Enrichment Analysis (GSEA) and Gene Set Variation Analysis (GSVA) were performed. Lastly, an interaction network for the identified signature genes was constructed. Results: We identified 12 DECRGs and 7 DENRGs, and enrichment analyses indicated they were primarily enriched in cytokine-cytokine receptor interaction, chemokine signaling pathway, TNF signaling pathway, and T cell receptor signaling pathway. Moreover, these machine learning algorithms finally recognized three signature genes (S100A8, AIF1, and IL18). Utilizing the identified signature genes, we developed nomogram models with high predictive accuracy for AP and differentiation of SAP from non-SAP, as demonstrated by area under the curve (AUC) values of 0.968 (95% CI 0.937-0.990) and 0.862 (95% CI 0.742-0.955), respectively, in receiver operating characteristic (ROC) curve analysis. Subsequent single-gene GESA and GSVA indicated a significant positive correlation between these signature genes and the proteasome complex. At the same time, a negative association was observed with the Th1 and Th2 cell differentiation signaling pathways. Conclusion: We have identified three genes (S100A8, AIF1, and IL18) related to chemokines and NETosis, and have developed accurate diagnostic models that might provide a novel method for diagnosing AP and differentiating between severe and non-severe cases.

Novel Prognostic Biomarkers in Gastric Cancer: CGB5, MKNK2, and PAPPA2.

INTRODUCTION: Gastric cancer is one of the most common malignant tumors of the digestive tract. However, there are no adequate prognostic markers available for this disease. The present study used bioinformatics to identify prognostic markers for gastric cancer that would guide the clinical diagnosis and treatment of this disease. MATERIALS AND METHODS: Gene expression data and clinical information of gastric cancer patients along with the gene expression data of 30 healthy samples were downloaded from the TCGA database. The initial screening was performed using the WGCNA method combined with the analysis of differentially expressed genes, which was followed by univariate analysis, multivariate COX regression analysis, and Lasso regression analysis for screening the candidate genes and constructing a prognostic model for gastric cancer. Subsequently, immune cell typing was performed using CIBERSORT to analyze the expression of immune cells in each sample. Finally, we performed laboratory validation of the results of our analyses using immunohistochemical analysis. RESULTS: After five screenings, it was revealed that only three genes fulfilled all the screening requirements. The survival curves generated by the prognostic model revealed that the survival rate of the patients in the high-risk group was significantly lower compared to the patients in the low-risk group (P-value < 0.001). The immune cell component analysis revealed that the three genes were differentially associated with the corresponding immune cells (P-value < 0.05). The results of immunohistochemistry also support our analysis. CONCLUSION: CGB5, MKNK2, and PAPPA2 may be used as novel prognostic biomarkers for gastric cancer.

Investigation of gastric microbiota in patients with coronary atherosclerosis disease infected by Helicobacter pylori.

Gastric microbiota may be involved in the pathogenicity of Helicobacter pylori(Hp). In the present study, 30 male patients with coronary atherosclerosis disease (CAD) infected with Hp and 30 healthy male volunteers with Hp infection as the control group were detectedby macrogenomic sequencing for gastric microbiota. According to the diversity of gastric microbiota, the CAD group was further divided into two subgroups: CAD treatment (CAD-T) and CAD fellow-up (CAD-F). Shannon index of CAD-T was significantly lower than that of the control group and CAD-F (P<0.05), Simpson index was significantly higher than that of the control group and CAD-F (P<0.05), and there was no statistical difference between CAD-T and the control group and CAD-F patients in Chao1 and ACE index (P>0.05). There is a difference in the dominant flora between the CAD group and the control group. After Hp eradication, Shannon index of gastric microbiota increased, Simpson index decreased, and there was statistical difference before and after Hp eradication in CAD-T group (P<0.05). There was no significant difference in Chao1 and ACE index between before and after Hp eradication (P>0.05). There is a significant difference in the dominant flora before and after eradication in CAD-T group. There were significant differences in clinical manifestations, endoscopic manifestations and pathological results among the three groups (P<0.05). The diversity of gastric microbiota is closely related to the pathogenicity of Hp,, regardless of dominant flora.

Hepatic veno-occlusive disease development in the hematopoietic stem cell transplantation patients: incidence and associated risk factors, a meta-analysis.

BACKGROUND: Now there are no efficient prophylactic or treatment strategies for hepatic veno-occlusive disease (VOD). Therefore, it is critical to early identify patients at high risk of VOD. AIM: To analyze the risk factors of VOD in the hematopoietic stem cell transplantation (HSCT) patients. METHODS: A comprehensive search of the population was conducted. RESULTS: Twenty-one studies with 27 679 HSCT patients were eligible. The incidence of VOD was 15% [95% confidence interval (CI) 13-17%]. The following were the risk factors for VOD: mismatched HLA [odds ratio (OR) 2.34, 95% CI 1.20-4.57, P = 0.01], history of liver disease (OR 2.72, 95% CI 2.03-3.64, P < 0.00001), elevated AST before transplant (OR 2.49, 95% CI 1.49-4.15, P = 0.0005), months from diagnosis to HSCT > 12 months (OR 1.76, 95% CI 1.15-2.69, P = 0.009), previous radiation (OR 1.86, 95% CI 1.49-2.31, P < 0.00001), busulphan (OR 3.69, 95% CI 2.58-5.29, P < 0.00001) and MTX (OR 1.81, 95% CI 1.22-2.69, P = 0.003). There were no significant differences for VOD presentation in the patients with regards to sex, number of HSCT, Karnofsky score <90%, unrelated donor, autologous HSCT, CYA and heparin prophylaxis. CONCLUSION: Mismatched HLA, liver disease (history of liver disease, elevated AST), months from diagnosis to HSCT >12 months, previous radiation and use of hepatotoxic drugs (BU and MTX) are the independent risk factors for VOD in the HSCT patients.

An improved method for the isolation and culture of rat pancreatic ductal epithelial cells.

BACKGROUND: This aim of this study was to explore a novel method that can be used to isolate and culture rat pancreatic ductal epithelial cells. METHODS: Retrograde injection of indigo carmine solution into the bile duct of rats revealed the main pancreatic duct, which was isolated using the naked eye (without using a stereomicroscope). The main pancreatic duct was sequentially digested with three enzymes, and the digested cells were cultured in RPMI-1640 medium containing 10-15% fetal bovine serum. After 72 h, the primary pancreatic ductal epithelial cells started to adhere to the wall. The cells reached 70-80% confluence after approximately 7 days and were subsequently digested with 0.25% trypsin and subcultured. Cells of the second and fourth passages were harvested. Cytokeratin (CK)-7 and CK-19 protein expressions in the cells and pancreatic tissue were detected by Western blot analysis. q-PCR was employed to examine CK-7, CK-19, somatostatin, amylase, insulin, and glucagon mRNA expression in the cells and pancreatic tissue after the main pancreatic duct was removed. RESULTS: The rats had one or two main pancreatic ducts meeting the bile ducts at a right or an acute angle. Rat pancreatic ductal epithelial cells isolated by this method grew well and showed a cobblestone-like appearance via microscopy. Western blot analysis showed that both the second and fourth passages of pancreatic ductal epithelial cells expressed CK-7 and CK-19 protein. The q-PCR results showed the expression of CK-7 and CK-19 genes in the second and fourth passages of pancreatic ductal epithelial cells, while the somatostatin, amylase, insulin, and glucagon genes were not expressed. The pancreatic tissue harvested after the removal of the main pancreatic duct did not express CK-7 or CK-19, while the somatostatin, amylase, insulin, and glucagon genes were expressed. CONCLUSIONS: The preliminary results show that this method can be applied to successfully isolate and culture rat pancreatic ductal epithelial cells.

Optimal timing of contrast-enhanced computed tomography in an evaluation of severe acute pancreatitis-associated complications.

Dynamic contrast-enhanced computed tomography (CECT) has been used previously to evaluate severe acute pancreatitis (SAP)-associated complications. However, optimal time points of CECT have not yet been established. The present study aimed to determine optimal timings for CECT to be undertaken for patients with SAP. The results of CECT from 309 patients with SAP, who were classified into either infected or non-infected SAP groups, were retrospectively analyzed. The severity and alterations in the periods within 72 h to >4 weeks of SAP onset were also assessed. In the analysis of the disease severity and changes, acute peripancreatic fluid collection was detected, where the number of areas increased within 1 week of SAP onset but decreased within 4 weeks and longer. However, no significant differences were observed between the infected and non-infected groups. The acute necrotic collection (ANC) areas were ≤30% of the area of the pancreas, with significantly more ANC areas and pancreatic necrosis in the infected SAP group compared with the non-infected SAP group at a time interval of >4 weeks. The exudation of pleural effusion (PE) was elevated within 1 week, but decreased within 2 weeks and longer. The difference in the alteration of the exudation of PE was not statistically different between the two groups. In conclusion, the results suggest that the period between 72 h and 1 week of SAP onset is optimal timing of CECT to assess SAP-associated complications, particularly for infected SAP patients.

Effect of adjuvant therapy with electroacupuncture on bone turnover markers and interleukin 17 in patients with rheumatoid arthritis.

OBJECTIVE: To evaluate the effect of electroacupuncture as an adjuvant treatment with first-line medications on bone metabolism biomarkers and interleukin-17 (IL-17) in the peripheral blood of patients with rheumatoid arthritis (RA). METHODS: Sixty RA patients were randomized into three groups. The control group was treated with methotrexate plus leflunomide (MTX+LEF), the acupuncture group was treated with simple needling plus MTX + LEF, and the patients in the electroacupuncture (EA) group were treated with EA plus MTX + LEF. EA or acupuncture was applied every other day for a total of 10 times over a treatment period of 8 weeks. RESULTS: In all three treatment groups, serum levels of the bone metabolism markers PICP, N-MID, and B-ALP were elevated and the concentrations of the inflammatory markers ß-CTx, IL-17, CRP, and TRACP-5b were reduced after treatment. These differences were significant for the EA group but not the other groups (P < 0.05). CONCLUSION: EA could effectively reduce the suffering and improve the quality of life of RA patients. It is a promising adjuvant therapy for enhancing the effectiveness of clinical therapeutics.

Role of Wnt/ß-catenin pathway agonist SKL2001 in Caerulein-induced acute pancreatitis.

The goal of this study was to clarify the protective role of the Wnt/ß-catenin pathway agonist SKL2001 in a rat model of Caerulein-induced acute pancreatitis. AR42J cells and rats were divided into 4 groups: control, Caerulein, SKL2001 + Caerulein, and SKL2001 + control. Cell apoptosis was examined using flow cytometry. Hematoxylin-eosin staining was performed to observe pathological changes in pancreatic and small intestinal tissues. Inflammatory cytokines were detected by enzyme-linked immunosorbent assay (ELISA), while genes related to the Wnt/ß-catenin pathway were quantified using quantitative real-time PCR. In vitro results showed that Caerulein promoted cell necrosis, inhibited the Wnt/ß-catenin pathway, and increased the level of inflammatory cytokines. However, SKL2001 reduced cell necrosis and inflammatory cytokines and activated the Wnt/ß-catenin pathway. Additionally, in vivo results demonstrated the accumulation of fluid (i.e., edema), hemorrhage, inflammation and necrosis of the pancreatic acini occurred 6 h after the final Caerulein induction, with the damage reaching a maximal level 12 h after the final Caerulein induction; meanwhile, the Wnt/ß-catenin pathway was evidently inhibited with an enhanced level of inflammatory cytokines. The aforementioned damage was further aggravated 12 h later. Nevertheless, the pancreatic and small intestinal tissue damages were alleviated in Caerulein-induced rats treated with SKL2001. In conclusion, activation of the Wnt/ß-catenin pathway could inhibit Caerulein-induced cell apoptosis and inflammatory cytokine release, thus improving pancreatic and intestinal damage in rats with acute pancreatitis.

Diagnosis of Genus Helicobacter through a hemi-nested PCR assay of 16S rRNA.

The present study aimed to establish a genus-specific PCR-based assay to detect helicobacters using 16S rRNA gene as the target template. We designed the hemi-nested primers based on sequences of 16S rRNA gene of 34 types of Helicobacter species. The inclusivity, sensitivity, and specificity of the PCR assay using these primers were examined in three different models, comprising feces simulated samples, BLAB/c mice infection model and clinic patients samples. The detection sensitivity of Helicobacter pylori, Helicobacter hepaticus and Helicobacter bilis strains from feces simulated samples was all 102 CFU/ml. We successfully detected H. hepaticus and H. bilis in the liver, cecum and feces of experimentally infected mice. H. pylori was successfully detected in the feces samples from 3 patients infected with H. pylori while not in the feces samples from 3 healthy human. However, the C97/C05-C97/C98 PCR assay detected H. pylori in the 2 positive samples. Due to the PCR assay's excellent inclusivity, high sensitivity and specificity it may be used to detect the presence of Helicobacters.

[Construction and identification of mouse wildtype-syndecan-1 and unshedding-syndecan-1 expressing vector].

AIM: To construct the expressing vector pcDNA3.0- wildtype-syndecan-1(WT-sdc1) and pcDNA3.0-unshedding-syndecan-1(uS-sdc1), and to explore the expression in vitro. METHODS: (1)The mouse WT-sdc1 DNA was successfully amplified by PCR and then cloned into pcDNA3.0. uS-sdc1 was construct by Gene splicing by overlap extension- PCR on the basis of WT-sdc1. The two vectors confirmed by DNA sequencing. (2)there are 4 groups in our research: control group, pcDNA3.0 transfected group, WT-sdc1 transfected group and uS-sdc1 transfected group. each vecter was transfected into IEC-6 cells by Lipofectamine(TM);2000. RT-PCR, Western blot and Dot blot were performed to detect the expression of syndecan-1 before and after stimulation of phorbol 12-myristate 13-acetate (PMA) for 15 min. RESULTS: The vector WT-sdc1 and uS-sdc1 were successfully constructed although an non-sense mutation was in uS-sdc1. Compared to control and pcDNA3.0 transfected groups, WT-sdc1 and uS-sdc1 groups showed a significant increase in the expression of syndecan-1 in both mRNA and protein levels. In response to the stimulation of PMA, the expression of syndecan-1 was down-regulated at the protein levels but not mRNA levels. CONCLUSION: The WT-sdc1 and uS-sdc1 are successfully constructed, which lays the foundation for further studying of syndecan-1 in gastrointestinal inflammation.

[Cloning and bioinformatics analysis of recombinant methyl-accepting chemotaxis signal transduction protein of Helicobacter hepaticus].

OBJECTIVE: To clone the gene encoding methyl-accepting chemotaxis signal transduction protein (MCSTP) of Helicobacter hepaticus and analyze the gene structures using bioinformatics methods. METHODS: With the specific primer of Helicobacter hepaticus MCSTP c1977, MCSTP gene was amplified by PCR from the genomic DNA of Helicobacter hepaticus and ligated to the prokaryotic expression vector pET22b(+). After sequencing, the sequence homology and structural feature of MCSTP gene were analyzed by bioinformatics method. RESULTS: A 99% similarity was identified between MCSTP gene cloned and its counterpart in standard Helicobacter hepaticus strain ATCC51449 genome DNA published by GenBank, with only a replacement of A by T at 1160 bp. A low homology was found in the MCSTP genes between Helicobacter hepaticus, Campylobacter jejuni and Helicobacter pylori by bioinformatics analysis, suggesting the specificity of MCSTP gene in Helicobacter hepaticus among the microbes. CONCLUSION: The prokaryotic expression plasmid pET22b(+)/MCSTP is constructed successfully, and the bioinformatics analysis provided evidences and clues for further study of the biological functions and pathogenic mechanism of MCSTP.