RhoB expression associated with chemotherapy response and prognosis in colorectal cancer.

PURPOSE: To examine the role of RhoB expression in relation to chemotherapy response, clinical outcomes and associated signaling pathways in colorectal cancer patients. MATERIALS AND METHODS: The study included 5 colon cancer cell lines, zebrafish embryos and 260 colorectal cancer patients treated with 5-fluorouracil (5-FU) and oxaliplatin (OXL). The methods consisted of CRISPR/Cas9, reactive oxygen species (ROS), caspase-3 activity, autophagy flux, in-silico RNA sequencing and immunohistochemistry. Gene expression analysis and pathway analysis were conducted using RNA-seq data. RESULTS: All cancer lines tested, including SW480, SW480-KO13 (RhoB knockout), SW480-KO55 (RhoB knockout), HCT116 and HCT116-OE (RhoB overexpressed), exhibited cytotoxicity to 5-FU and OXL. RhoB knockout cell lines demonstrated significantly reduced migration compared to the control cell lines. Furthermore, RhoB played a role in caspase-3-dependent apoptosis, regulation of ROS production and autophagic flux. The mRNA sequencing data indicated lower expression levels of oncogenes in RhoB knockout cell lines. The zebrafish model bearing SW480-KO showed a light trend toward tumor regression. RhoB expression by immunohistochemistry in patients was increased from normal mucosa to tumor samples. In patients who received chemotherapy, high RhoB expression was related to worse survival compared to low RhoB expression. Furthermore, the molecular docking analysis revealed that OXL had a higher binding affinity for RhoB than 5-FU, with a binding affinity of -7.8 kcal/mol and HADDOCK predicted molecular interactions between RhoB and caspase 3 protein. Gene-set enrichment analysis supported these findings, showing that enrichment of DNA damage response pathway and p53 signaling in RhoB overexpression treatment group, while the RhoB knockout treatment group exhibited enrichment in the negative regulation pathway of cell migration. CONCLUSION: RhoB was negatively associated with chemotherapy response and survival in colorectal cancers. Therefore, RhoB inhibition may enhance chemotherapeutic responses and patient survival.

Autoimmune and Metabolic Diseases and the Risk of Early-Onset Colorectal Cancer, a Nationwide Nested Case-Control Study.

Incidence of early-onset (<50 years) colorectal cancer (EOCRC) is increasing in developed countries. The aim was to investigate autoimmune and metabolic conditions as risk factors for EOCRC. In a nationwide nested case-control study, we included all EOCRC cases in Sweden diagnosed during 2007-2016, together with controls, matched for birth year, sex, and county. Information on exposure of autoimmune or metabolic disease was collected from the National Patient Register and Prescribed Drugs Registry. Hazard ratios (HR) as measures of the association between EOCRC and the exposures were estimated using conditional logistic regression. In total, 2626 EOCRC patients and 15,756 controls were included. A history of metabolic disease nearly doubled the incidence hazard of EOCRC (HR 1.82, 95% CI 1.66-1.99). A sixfold increased incidence hazard of EOCRC (HR 5.98, 95% CI 4.78-7.48) was seen in those with inflammatory bowel disease (IBD), but the risk increment decreased in presence of concomitant metabolic disease (HR 3.65, 95% CI 2.57-5.19). Non-IBD autoimmune disease was not statistically significantly associated with EOCRC. IBD and metabolic disease are risk factors for EOCRC and should be considered in screening guidelines.

CD45RA+CD62L- ILCs in human tissues represent a quiescent local reservoir for the generation of differentiated ILCs.

Innate lymphoid cells (ILCs) are highly plastic and predominantly mucosal tissue-resident cells that contribute to both homeostasis and inflammation depending on the microenvironment. The discovery of naïve-like ILCs suggests an ILC differentiation process that is akin to naïve T cell differentiation. Delineating the mechanisms that underlie ILC differentiation in tissues is crucial for understanding ILC biology in health and disease. Here, we showed that tonsillar ILCs expressing CD45RA lacked proliferative activity, indicative of cellular quiescence. CD62L distinguished two subsets of CD45RA+ ILCs. CD45RA+CD62L+ ILCs (CD62L+ ILCs) resembled circulating naïve ILCs because they lacked the transcriptional, metabolic, epigenetic, and cytokine production signatures of differentiated ILCs. CD45RA+CD62L- ILCs (CD62L- ILCs) were epigenetically similar to CD62L+ ILCs but showed a transcriptional, metabolic, and cytokine production signature that was more akin to differentiated ILCs. CD62L+ and CD62L- ILCs contained uni- and multipotent precursors of ILC1s/NK cells and ILC3s. Differentiation of CD62L+ and CD62L- ILCs led to metabolic reprogramming including up-regulation of genes associated with glycolysis, which was needed for their effector functions after differentiation. CD62L- ILCs with preferential differentiation capacity toward IL-22-producing ILC3s accumulated in the inflamed mucosa of patients with inflammatory bowel disease. These data suggested distinct differentiation potential of CD62L+ and CD62L- ILCs between tissue microenvironments and identified that manipulation of these cells is a possible approach to restore tissue-immune homeostasis.

Hereditary evaluation and genetic counselling in young individuals with colorectal cancer in a population-based cohort.

AIM: Early-onset colorectal cancer should raise suspicions of a hereditary colorectal cancer (CRC) syndrome, including Lynch syndrome (LS) and Familial Adenomatous Polyposis (FAP). Collection of family history and genetic counselling (GC) is mandatory but previous studies have revealed low awareness of hereditary CRC among clinicians why there has been an incentive to implement universal LS screening. In this population-based cohort study, we aimed to observe the uptake of GC in the Swedish South-Eastern medical care region for young CRC patients and to investigate the frequency of patients diagnosed with LS. METHODS: Patients below 50 years of age diagnosed with CRC between 2008 and 2017 were identified from the national Swedish Colorectal Cancer Registry. Medical records were reviewed regarding family history, co-morbidity and referral for GC, with a follow-up time of at least three years. RESULTS: The analysis included 278 patients with 287 tumours, 108 (38%) located in rectum and 179 (62%) in colon. One hundred sixteen (42%) individuals were referred to the Regional Clinical Genetics service, whereof 74 (27%) underwent complete investigation. Thirteen (18%) patients were identified with a mutation, eleven (15%) had LS and two (3%) FAP. The remaining 61 (82%), without proven mutation, were considered as familial CRC. Younger age correlated with a higher chance of referral for GC. CONCLUSION: The study found that only a minority of young CRC patients underwent genetic counselling, contrary to clinical guidelines. Hereditary CRC is therefore probably underdiagnosed even among young individuals.

Increased Numbers of Enteric Glial Cells in the Peyer's Patches and Enhanced Intestinal Permeability by Glial Cell Mediators in Patients with Ileal Crohn's Disease.

Enteric glial cells (EGC) are known to regulate gastrointestinal functions; however, their role in Crohn's disease (CD) is elusive. Microscopic erosions over the ileal Peyer's patches are early signs of CD. The aim of this work was to assess the localization of EGC in the follicle and interfollicular region of the Peyer's patches and in the lamina propria and study the effects of EGC mediators on barrier function in CD patients and non-inflammatory bowel disease (non-IBD) controls. EGC markers, glial fibrillary acidic protein (GFAP), and S100 calcium-binding protein ß (S100ß) were quantified by immunofluorescence and Western blotting. Both markers showed significantly more EGC in the Peyer's patches and lamina propria of CD patients compared to the non-IBD controls. In CD patients there were significantly more EGC in Peyer's patches compared to lamina propria, while the opposite pattern was seen in controls. Barrier function studies using Ussing chambers showed increased paracellular permeability by EGC mediators in CD patients, whereas permeability decreased by the mediators in controls. We show the accumulation of EGC in Peyer's patches of CD patients. Moreover, EGC mediators induced barrier dysfunction in CD patients. Thus, EGC might have harmful impacts on ongoing inflammation and contribute to the pathophysiology of the disease.

Antagonism of Adherent Invasive E. coli LF82 With Human α-defensin 5 in the Follicle-associated Epithelium of Patients With Ileal Crohn's Disease.

BACKGROUND: The first visible signs of Crohn's disease (CD) are microscopic erosions over the follicle-associated epithelium (FAE). The aim of the study was to investigate the effects of human α-defensin 5 (HD5) on adherent-invasive Escherichia coli LF82 translocation and HD5 secretion after LF82 exposure in an in vitro model of human FAE and in human FAE ex vivo. METHODS: An in vitro FAE-model was set up by the coculture of Raji B cells and Caco-2-cl1 cells. Ileal FAE from patients with CD and controls were mounted in Ussing chambers. The effect of HD5 on LF82 translocation was studied by LF82 exposure to the cells or tissues with or without incubation with HD5. The HD5 secretion was measured in human FAE exposed to LF82 or Salmonella typhimurium. The HD5 levels were evaluated by immunofluorescence, immunoblotting, and ELISA. RESULTS: There was an increased LF82 translocation across the FAE-model compared with Caco-2-cl1 (P < 0.05). Incubation of cell/tissues with HD5 before LF82 exposure reduced bacterial passage in both models. Human FAE showed increased LF82 translocation in CD compared with controls and attenuated passage after incubation with sublethal HD5 in both CD and controls (P < 0.05). LF82 exposure resulted in a lower HD5 secretion in CD FAE compared with controls (P < 0.05), whereas Salmonella exposure caused equal secretion on CD and controls. There were significantly lower HD5 levels in CD tissues compared with controls. CONCLUSIONS: Sublethal HD5 reduces the ability of LF82 to translocate through FAE. The HD5 is secreted less in CD in response to LF82, despite a normal response to Salmonella. This further implicates the integrated role of antimicrobial factors and barrier function in CD pathogenesis.

MTDH genetic variants in colorectal cancer patients.

The colorectal carcinogenesis is a complex process encompassing genetic alterations. The oncoprotein AEG-1, encoded by the MTDH gene, was shown previously to be involved in colorectal cancer (CRC). The aim of this study was to determine the frequency and the spectrum of MTDH variants in tumor tissue, and their relationship to clinicopathological variables in CRC patients. The study included tumors from 356 unselected CRC patients. Mutation analysis of the MTDH gene, including coding region and adjacent intronic sequences, was performed by direct DNA sequencing. The corresponding normal colorectal tissue was analyzed in the carriers of exonic variant to confirm germline or somatic origin. We detected 42 intronic variants, where 25 were novel. Furthermore, we found 8 exonic variants of which four, one missense (c.977C > G-germline) and three frameshift mutations (c.533delA-somatic, c.1340dupA-unknown origin, c.1731delA-unknown origin), were novel. In silico prediction analyses suggested four deleterious variants (c.232G > T, c.533delA, c.1340dupA, and c.1731delA). There were no correlations between the MTDH variants and tumor stage, differentiation or patient survival. We described several novel exonic and intronic variants of the MTDH gene. The detection of likely pathogenic truncating mutations and alterations in functional protein domains indicate their clinical significance, although none of the variants had prognostic potential.

Appendectomy versus antibiotic treatment in acute appendicitis. a prospective multicenter randomized controlled trial.

BACKGROUND: Appendectomy has been the treatment for acute appendicitis for over 120 years. Antibiotic treatment has occasionally been used in small uncontrolled studies, instead of operation, but this alternative has never before been tried in a multicenter randomized trial. PATIENTS AND METHODS: Male patients, 18-50 years of age, admitted to six different hospitals in Sweden between 1996 and 1999 were enrolled in the study. No women were enrolled by decision of the local ethics committee. If appendectomy was planned, patients were asked to participate, and those who agreed were randomized either to surgery or to antibiotic therapy. Patients randomized to surgery were operated on with open surgery or laparoscopically. Those randomized to antibiotic therapy were treated intravenously for 2 days, followed by oral treatment for 10 days. If symptoms did not resolve within 24 hours, an appendectomy was performed. Participants were monitored at the end of 1 week, 6 weeks, and 1 year. RESULTS: During the study period 252 men participated, 124 in the surgery group and 128 in the antibiotic group. The frequency of appendicitis was 97% in the surgery group and 5% had a perforated appendix. The complication rate was 14% in the surgery group. In the antibiotic group 86% improved without surgery; 18 patients were operated on within 24 hours, and the diagnosis of acute appendicitis was confirmed in all but one patient, and he was suffering from terminal ileitis. There were seven patients (5%) with a perforated appendix in this group. The rate of recurrence of symptoms of appendicitis among the 111 patients treated with antibiotics was 14% during the 1-year follow-up. CONCLUSIONS: Acute non-perforated appendicitis can be treated successfully with antibiotics. However, there is a risk of recurrence in cases of acute appendicitis, and this risk should be compared with the risk of complications after appendectomy.

Choice of anesthesia and risk of reoperation for recurrence in groin hernia repair.

OBJECTIVE: To analyze the relative risk of reoperation for recurrence using 3 anesthetic alternatives, general anesthesia (GA), regional (spinal-, epidural-) anesthesia (RA), and local anesthesia (LA), and to study time trends for various anesthetic and operative methods, as well as other risk factors regarding reoperation for recurrence. BACKGROUND: The method of anesthesia used for hernia repair is generally assumed not to affect the long-term outcome. The few studies on the topic have rendered conflicting results. METHODS: Data from the Swedish Hernia Register was used. Relative risk was first estimated using univariate analysis for assumed risk variables and then selecting variables with the highest or lowest univariate risk for multivariate analysis. RESULTS: From 1992 through 2001, 59,823 hernia repairs were recorded. Despite the fact that univariate analysis showed a somewhat lower risk for reoperation in the LA group, the multivariate analysis showed that LA was associated with a significantly increased risk for reoperation in primary but not in recurrent hernia repair. The Lichtenstein technique carried a significantly lower reoperation risk than any other method of operation. CONCLUSIONS: LA was associated with a higher risk of reoperation for recurrence after primary hernia repair. The use of mesh techniques has increased considerably, and among these the Lichtenstein repair was associated with a significantly lower risk for reoperation than any other repair.

Recurrence and pain three years after groin hernia repair. Validation of postal questionnaire and selective physical examination as a method of follow-up.

OBJECTIVES: To evaluate recurrence rate and chronic groin pain three years after hernia repair and to validate a postal questionnaire with selective physical examination as a method of follow-up. DESIGN: Prospective cohort study. SETTING: County hospital, Sweden. PATIENTS: Prospective data were retrieved from the Swedish Hernia Register for patients aged 15-80 years at the time of groin hernia repair, operated on during 1994. INTERVENTIONS: Three years after operation patients were mailed a three-item questionnaire and invited to have a physical examination. Those examined answered a detailed questionnaire about pain and functional impairment. When appropriate an extended physical examination was undertaken to find out the probable cause of the pain. MAIN OUTCOME MEASURES: Recurrence, pain, and functional impairment. RESULTS: 272 hernias were repaired in 264 patients. 24 patients had died and 16 had a recurrence before the follow-up examination. After a median observation time of 44 months, 218 patients with 223 repairs (96%) were examined. Depending on the definition of recurrence and completeness of physical examination (selective or all patients) the recurrence rate varied between 10% (25/239) and 15% (35/239) including recurrences diagnosed before follow-up. 40 patients (18%) reported groin pain at follow-up, which was considered to be caused by a previous hernia repair in 34 (15%), 12 of whom (5%) had moderate or severe pain. Postoperative complications were associated with an increased risk of chronic pain, whereas type of hernia and use of mesh had no influence. CONCLUSIONS: The incidence of recurrence and chronic pain after hernia repair requires continuous audit in non-specialised units. Participation in a register and follow-up by a three-item questionnaire and selective physical examination provides a solid basis for quality control.