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The key function of innate immunity is to sense danger signals and initiate effective responses as a defense mechanism against pathogens. Simultaneously, effector responses must be regulated to avoid excessive inflammation with resulting tissue damage. microRNAs (miRNAs), are small endogenous molecules, that has recently gained attention as important regulatory elements in the human inflammation cascade. The control over host miRNA expression may represent a previously uncharacterized molecular strategy exploited by pathogens to mitigate innate host cell responses.

Vibrio cholerae is a Gram-negative bacterium that colonizes the human small intestine and causes life-threatening secretory diarrhea, essentially mediated by cholera toxin (CT). It is considered a non-invasive pathogen and does not cause clinical inflammation. Still, cholera is associated with inflammatory changes of the small intestine. Furthermore, CT-negative strains of V. cholerae cause gastroenteritis and are associated with extra-intestinal manifestations, suggesting that other virulence factors than CT are also involved in the pathogenesis.

The innate immune response to V. cholerae is poorly investigated and the potential role of miRNA in cholera had not been studied before. Therefore, this thesis explores the role of intestinal epithelial cells in response to V. cholerae infection with a focus on regulatory miRNA as a potential contributor to the pathogenesis. The in vivo material was small intestinal biopsies from patients suffering from V. cholerae infection. As an in vitro model for V. cholerae attack on intestinal epithelium, we used tight monolayers of T84 cells infected with V. cholerae and their released factors. We analyzed changes in levels of cytokines, immunomodulatory microRNA and their target genes.

We showed that miRNA-146a and miRNA-155 reached significantly elevated levels in the intestinal mucosa at acute stages of disease in V. cholerae infected patients and declined to normal levels at the convalescent stage. Low-grade inflammation was identified at the acute stage of V. cholerae infection, which correlated with elevated levels of regulatory miRNA. Furthermore, outer membrane vesicles (OMVs) released by the bacteria were shown to induce miR-146a and live bacteria induced miR-155 in intestinal epithelial cells. In addition, OMVs decreased epithelial permeability and caused mRNA suppression of pro-inflammatory cytokines, including immune cell attractant IL-8 and CLL20, and the inflammasome markers IL-1b and IL-18. These results propose that V. cholerae regulates the host expression of miRNA during infection and may set the threshold for activation of the intestinal epithelium.

Moreover, we showed that V. cholerae also harbors inflammatory-inducing capabilities, by secreting a pore-forming toxin, Vibrio cholerae cytolysin (VCC). By using genetically modified strains as well as soluble protein challenge experiments, VCC was found solely responsible for the increased epithelial permeability and induction of several pro-inflammatory cytokines in intestinal epithelial cells. In contrast to OMVs, VCC displayed strong upregulation of the pro-inflammatory cytokines IL-8, TNF-a, CCL20 and IL-1b and IRAK2, a key signaling molecule in the IL-1 inflammasome pathway. This suggest that VCC is an important virulence factor in the V. cholerae pathogenesis, particularly in CT-negative strains. Furthermore, we showed that the bacterium could control the inflammatory actions of VCC by secreting the PrtV protease, which degraded VCC and consequently abolished inflammation.  

In summary, we showed that V. cholerae harbors immunomodulating capabilities, both at the gene level, through induction of host regulatory miRNA, and at the protein level, through secretion of VCC and PrtV. These strategies may be relevant for V. cholerae to promote survival in the gut and cause successful infections in the human host.

Background. Emotional intelligence (EI), the ability to understand and regulate one’s and other’s emotions, has been linked to academic and clinical performance and stress management, making it an essential skill to develop during medical school. Nevertheless, uncertainty remains about the impact of medical education on EI, its association with sociodemographic factors, and the potential moderating role of gender. Therefore, this study aimed to explore levels of global EI among Swedish medical students based on their completed semesters while analyzing the potential moderator role of gender and identifying potential EI differences associated with age, gender, prior education, work experience, and previous experience working in a leadership position.

Methods: The participants were medical students in semesters 1, 3, 5, 7, 9, and 11 at a Swedish University. Participants answered the self-report Trait Emotional Intelligence Questionnaire - Short Form (TEIQue-SF) and demographic questions. For each participant, the mean global trait EI was calculated (range 1–7), and differences were compared based on semesters and sociodemographic factors. In addition, we investigated the relationship between semester and EI scores with gender as a moderator.

Results: Of the 663 invited medical students, 429 (65%) responded, including 269 women (62.7%), 157 men (36.6%), and 3 identifying as others (0.7%). The participants had a mean global trait EI score of 5.33. Final-year students demonstrated significantly higher global trait EI scores than first-year students, and gender did not have a moderating effect across semesters. Furthermore, students in the age group 25–29 years showed higher EI scores compared to those in the age group 21–24 years, while there were no significant differences in EI scores for older students (≥ 30 years) compared to other age groups. Higher EI scores were also positively associated with previous work-and leadership experiences. Gender and previous education did not significantly impact EI scores.

Conclusions. Our findings suggest that higher EI scores are associated with semesters of medical education, age, and previous work and leadership experience. Future longitudinal studies are needed to identify factors that could improve EI among medical students to design curricular activities aimed at supporting the EI of the next generation of physicians.

The small intestinal epithelium of Vibrio cholerae infected patients expresses the immunomodulatory microRNAs miR-146a and miR-155 at acute stage of disease. V. cholerae release outer membrane vesicles (OMVs) that serve as vehicles for translocation of virulence factors including V. cholerae cytolysin (VCC). The aim was to investigate whether OMVs, with and/or without VCC-cargo could be responsible for induction of microRNAs in intestinal epithelial cells and thereby contribute to immunomodulation. Polarized tight monolayers of T84 cells were challenged with OMVs of wildtype and a VCC deletion mutant of the non-O1/non-O139 (NOVC) V. cholerae strain V:5/04 and with soluble VCC. OMVs, with and without VCC-cargo, caused significantly increased levels of miR-146a. Increase was seen already after 2 hours challenge with OMVs and persisted after 12 hours. Challenge with soluble VCC caused significant increases in interleukin-8 (IL-8), tumour necrosis factor-α (TNF-α), CCL20, IL-1β, and IRAK2 mRNA levels while challenge with OMVs did not cause increases in expression levels of any of these mRNAs. These results suggest that V. cholerae bacteria release OMVs that induce miR-146a in order to pave the way for colonization by reducing the strength of an epithelial innate immune defence reaction and also preventing inflammation in the mucosa that factors like VCC can evoke.

The potential immunomodulatory role of microRNAs in small intestine of patients with acute watery diarrhea caused by Vibrio cholerae O1 or enterotoxigenic Escherichia coli (ETEC) infection was investigated. Duodenal biopsies were obtained from study-participants at the acute (day 2) and convalescent (day 21) stages of disease, and from healthy individuals. Levels of miR-146a, miR-155 and miR-375 and target gene (IRAK1, TRAF6, CARD10) and 11 cytokine mRNAs were determined by qRT-PCR. The cellular source of microRNAs in biopsies was analyzed by in situ hybridization. The ability of V. cholerae bacteria and their secreted products to cause changes in microRNA- and mRNA levels in polarized tight monolayers of intestinal epithelial cells was investigated. miR-146a and miR-155 were expressed at significantly elevated levels at acute stage of V. cholerae infection and declined to normal at convalescent stage (P<0.009 versus controls; P = 0.03 versus convalescent stage, pairwise). Both microRNAs were mainly expressed in the epithelium. Only marginal down-regulation of target genes IRAK1 and CARD10 was seen and a weak cytokine-profile was identified in the acute infected mucosa. No elevation of microRNA levels was seen in ETEC infection. Challenge of tight monolayers with the wild type V. cholerae O1 strain C6706 and clinical isolates from two study-participants, caused significant increase in miR-155 and miR-146a by the strain C6706 (P<0.01). One clinical isolate caused reduction in IRAK1 levels (P<0.05) and none of the strains induced inflammatory cytokines. In contrast, secreted factors from these strains caused markedly increased levels of IL-8, IL-1β, and CARD10 (P<0.001), without inducing microRNA expression. Thus, miR-146a and miR-155 are expressed in the duodenal epithelium at the acute stage of cholera. The inducer is probably the V. cholerae bacterium. By inducing microRNAs the bacterium can limit the innate immune response of the host, including inflammation evoked by its own secreted factors, thereby decreasing the risk of being eliminated.

Background: Concern over medical students' well-being is a global issue, with studies showing high psychological distress rates. Impostor Phenomenon (IP), i.e., underestimating one's abilities, attributing success to external factors, and feeling like a fraud, has been implicated as one reason behind these troubling findings. Meanwhile, resilience has been suggested to protect against psychological distress. This study aimed to investigate the prevalence of IP and its association with resilience among undergraduate medical students.

Methods: The Clance Impostor Phenomenon Scale (CIPS), the Brief Resilience Scale (BRS), and sociodemographic questions were completed by 457 medical students registered in their 2-10th semester at a Swedish university. Of the respondents, 62.6% identified as women, 36.1% as men, and 1.3% as others.

Results: The prevalence of IP was 58.4% (defined as CIPS score ≥ 62). According to the CIPS scoring guidelines, 10.3% of participants had low IP, 29.5% moderate, 41.6% frequent, and 18.6% intense IP. Of all participants, almost 90% experienced at least moderate and 60.2% frequent to intense IP. Women had significantly higher CIPS scores and lower BRS scores than men. In contrast, neither attending semester nor age group significantly impacted CIPS scores. Finally, there was a moderate inverse correlation between the level of resilience and the prevalence of IP.

Conclusions: Our findings suggest that the majority of Swedish medical students feels like an impostor, and of these students, most do so frequently. Furthermore, IP is more prevalent among female students - who also show lower levels of resilience. Moreover, our results indicated that IP could hinder achieving higher resilience. Future longitudinal studies should investigate how gender norms may contribute to IP feelings and explore the potential advantages of counteracting IP and strengthening resilience among medical students. However, targeted interventions addressing individual students' IP and resilience are insufficient. There is also a need to address structural aspects of the educational environment, such as gender stereotypes, that may contribute to IP.

We suggest that VCC is capable of causing an inflammatory response characterized by increased permeability and production of IL-8 and TNF-alpha in tight monolayers. Pure VCC at a concentration of 160 ng/ml caused an inflammatory response that reached the magnitude of that caused by Vibrio-secreted factors, while higher concentrations caused epithelial cell death. The inflammatory response was totally abolished by treatment with PrtV. The findings suggest that low doses of VCC initiate a local immune defense reaction while high doses lead to intestinal epithelial lesions. Furthermore, VCC is indeed a substrate for PrtV and PrtV seems to execute an environment-dependent modulation of the activity of VCC that may be the cause of V. cholerae reactogenicity.