Development and Implementation of a Pediatric Nursing Emergency Behavioral Health Assessment Tool.

INTRODUCTION: The national pediatric mental and behavioral health crisis dramatically increased emergency department mental and behavioral health visits and changed emergency nursing practice. Acuity assessment determines patient severity level and supports appropriate resources and interventions. There are no established nursing tools that assess pediatric mental or behavioral health acuity in the emergency department setting. Our goal was to develop and implement the novel pediatric emergency nurse Emergency Behavioral Health Acuity Assessment Tool. METHODS: This quality-improvement project used the plan, do, study, act model to design/refine the Emergency Behavioral Health Acuity Assessment Tool and a non-experimental descriptive design to assess outcomes. The setting was a 47-bed urban level 1 pediatric trauma center with more than 60,000 annual visits. The team designed the tool using published evidence, emergency nurse feedback, and expert opinion. The tool objectively captured patient acuity and suggested acuity-specific nursing interventions. Project outcomes included acuity, length-of-stay, restraint use, and patient/staff injuries. Analyses included descriptive statistics and correlations. RESULTS: With over 3000 annual mental/behavioral-related visits, the emergency department had an average daily census of 23 mental and behavioral health patients. Implementation occurred in August 2021. The Emergency Behavioral Health Acuity Assessment Tool dashboard provided the number of patients, patient location, and acuity. Length-of-stay did not change; however, patient restraint use and patient/staff injuries declined. Number of restraints positively correlated with moderate acuity levels (r = 0.472, P = 0.036). DISCUSSION: For emergency nurses, the Emergency Behavioral Health Acuity Assessment Tool provided an objective measure of patient acuity. Targeted interventions can improve the care of this population.

Chronic unilateral arm lymphedema correlates with increased intima-media thickness in the brachial artery.

Drainage of the arterial wall via adventitial lymphatic vessels has been shown to play a pivotal role for vessel wall homeostasis. Also, retrograde cholesterol transport is ensured via this route, but no studies exist to demonstrate that lymphatic stasis would represent a mechanism to initiate atherosclerotic lesion formation in human arteries. To test this hypothesis, we embarked on a simple clinical experiment, assessing wall thickness in limb arteries with lymphedema after surgical intervention, with the contralateral limb serving as control. Using ultrasound imaging, the differential thickness was assessed separately for the three arterial wall layers. The potential of disease progression by lymphostasis was addressed by depiction of longitudinal results according to the time after lymph dissection.

Std fimbriae-fucose interaction increases Salmonella-induced intestinal inflammation and prolongs colonization.

Expression of ABO and Lewis histo-blood group antigens by the gastrointestinal epithelium is governed by an α-1,2-fucosyltransferase enzyme encoded by the Fut2 gene. Alterations in mucin glycosylation have been associated with susceptibility to various bacterial and viral infections. Salmonella enterica serovar Typhimurium is a food-borne pathogen and a major cause of gastroenteritis. In order to determine the role of Fut2-dependent glycans in Salmonella-triggered intestinal inflammation, Fut2+/+ and Fut2-/- mice were orally infected with S. Typhimurium and bacterial colonization and intestinal inflammation were analyzed. Bacterial load in the intestine of Fut2-/- mice was significantly lower compared to Fut2+/+ mice. Analysis of histopathological changes revealed significantly lower levels of intestinal inflammation in Fut2-/- mice compared to Fut2+/+ mice and measurement of lipocalin-2 level in feces corroborated histopathological findings. Salmonella express fimbriae that assist in adherence of bacteria to host cells thereby facilitating their invasion. The std fimbrial operon of S. Typhimurium encodes the π-class Std fimbriae which bind terminal α(1,2)-fucose residues. An isogenic mutant of S. Typhimurium lacking Std fimbriae colonized Fut2+/+ and Fut2-/- mice to similar levels and resulted in similar intestinal inflammation. In vitro adhesion assays revealed that bacteria possessing Std fimbriae adhered significantly more to fucosylated cell lines or primary epithelial cells in comparison to cells lacking α(1,2)-fucose. Overall, these results indicate that Salmonella-triggered intestinal inflammation and colonization are dependent on Std-fucose interaction.

TLR-9 and IL-15 Synergy Promotes the In Vitro Clonal Expansion of Chronic Lymphocytic Leukemia B Cells.

Clinical progression of B cell chronic lymphocytic leukemia (B-CLL) reflects the clone's Ag receptor (BCR) and involves stroma-dependent B-CLL growth within lymphoid tissue. Uniformly elevated expression of TLR-9, occasional MYD88 mutations, and BCR specificity for DNA or Ags physically linked to DNA together suggest that TLR-9 signaling is important in driving B-CLL growth in patients. Nevertheless, reports of apoptosis after B-CLL exposure to CpG oligodeoxynucleotide (ODN) raised questions about a central role for TLR-9. Because normal memory B cells proliferate vigorously to ODN+IL-15, a cytokine found in stromal cells of bone marrow, lymph nodes, and spleen, we examined whether this was true for B-CLL cells. Through a CFSE-based assay for quantitatively monitoring in vitro clonal proliferation/survival, we show that IL-15 precludes TLR-9-induced apoptosis and permits significant B-CLL clonal expansion regardless of the clone's BCR mutation status. A robust response to ODN+IL-15 was positively linked to presence of chromosomal anomalies (trisomy-12 or ataxia telangiectasia mutated anomaly + del13q14) and negatively linked to a very high proportion of CD38(+) cells within the blood-derived B-CLL population. Furthermore, a clone's intrinsic potential for in vitro growth correlated directly with doubling time in blood, in the case of B-CLL with Ig H chain V region-unmutated BCR and <30% CD38(+) cells in blood. Finally, in vitro high-proliferator status was statistically linked to diminished patient survival. These findings, together with immunohistochemical evidence of apoptotic cells and IL-15-producing cells proximal to B-CLL pseudofollicles in patient spleens, suggest that collaborative ODN and IL-15 signaling may promote in vivo B-CLL growth.

Ex vivo perfusion of the isolated rat small intestine as a novel model of Salmonella enteritis.

Using an ex vivo perfused rat small intestinal model, we examined pathological changes to the tissue, inflammation induction, as well as dynamic changes to smooth muscle activity, metabolic competence, and luminal fluid accumulation during short-term infection with the enteropathogenic bacteria Salmonella enterica serovar Typhimurium and Yersinia enterocolitica. Although few effects were seen upon Yersinia infection, this system accurately modeled key aspects associated with Salmonella enteritis. Our results confirmed the importance of the Salmonella Pathogenicity Island 1 (SPI1)-encoded type 3 secretion system (T3SS) in pathology, tissue invasion, inflammation induction, and fluid secretion. Novel physiological consequences of Salmonella infection of the small intestine were also identified, namely, SPI-1-dependent vasoconstriction and SPI-1-independent reduction in the digestive and absorptive functions of the epithelium. Importantly, this is the first small animal model that allows for the study of Salmonella-induced fluid secretion. Another major advantage of this model is that one can specifically determine the contribution of resident cell populations. Accordingly, we can conclude that recruited cell populations were not involved in the pathological damage, inflammation induction, fluid accumulation, nutrient absorption deficiency, and vasoconstriction observed. Although fluid loss induced by Salmonella infection is hypothesized to be due to damage caused by recruited neutrophils, our data suggest that bacterial invasion and inflammation induction in resident cell populations are sufficient for fluid loss into the lumen. In summary, this model is a novel and useful tool that allows for detailed examination of the early physiopathological effects of Salmonella infection on the small intestine.

Randomized phase III trial of APF530 versus palonosetron in the prevention of chemotherapy-induced nausea and vomiting in a subset of patients with breast cancer receiving moderately or highly emetogenic chemotherapy.

BACKGROUND: APF530 provides controlled, sustained-release granisetron for preventing acute (0-24 h) and delayed (24-120 h) chemotherapy-induced nausea and vomiting (CINV). In a phase III trial, APF530 was noninferior to palonosetron in preventing acute CINV following single-dose moderately (MEC) or highly emetogenic chemotherapy (HEC) and delayed CINV in MEC (MEC and HEC defined by Hesketh criteria). This exploratory subanalysis was conducted in the breast cancer subpopulation. METHODS: Patients were randomized to subcutaneous APF530 250 or 500 mg (granisetron 5 or 10 mg) or intravenous palonosetron 0.25 mg during cycle 1. Palonosetron patients were randomized to APF530 for cycles 2 to 4. The primary efficacy end point was complete response (CR, no emesis or rescue medication) in cycle 1. RESULTS: Among breast cancer patients (n = 423 MEC, n = 185 HEC), > 70 % received anthracycline-containing regimens in each emetogenicity subgroup. There were no significant between-group differences in CRs in cycle 1 for acute (APF530 250 mg: MEC 71 %, HEC 77 %; 500 mg: MEC 73 %, HEC 73 %; palonosetron: MEC 68 %, HEC 66 %) and delayed (APF530 250 mg: MEC 46 %, HEC 58 %; 500 mg: MEC 48 %, HEC 63 %; palonosetron: MEC 52 %, HEC 52 %) CINV. There were no significant differences in within-cycle CRs between APF530 doses for acute and delayed CINV in MEC or HEC in cycles 2 to 4; CRs trended higher in later cycles, with no notable differences in adverse events between breast cancer and overall populations. CONCLUSIONS: APF530 effectively prevented acute and delayed CINV over 4 chemotherapy cycles in breast cancer patients receiving MEC or HEC. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT00343460 (June 22, 2006).

Slow-release granisetron (APF530) versus palonosetron for chemotherapy-induced nausea/vomiting: analysis by American Society of Clinical Oncology emetogenicity criteria.

BACKGROUND: APF530 is a novel sustained-release formulation of granisetron. In a Phase III trial, APF530 500 mg was noninferior to palonosetron 0.25 mg in preventing acute chemotherapy-induced nausea and vomiting (CINV) after moderately (MEC) or highly emetogenic chemotherapy (HEC) and delayed CINV after MEC, but not superior in preventing delayed CINV after HEC. Emetogenicity was classified by Hesketh criteria; this reanalysis uses newer American Society of Clinical Oncology criteria. METHODS: Complete responses (no emesis or rescue medication) after cycle one were reanalyzed after reclassification of MEC and HEC by American Society of Clinical Oncology criteria. RESULTS: APF530 maintained noninferiority to palonosetron. CONCLUSION: Single-dose APF530 is a promising alternative to palonosetron for preventing acute and delayed CINV after MEC or HEC. The Clinicaltrials.gov identifier for this study is NCT00343460.

Comparison of an extended-release formulation of granisetron (APF530) versus palonosetron for the prevention of chemotherapy-induced nausea and vomiting associated with moderately or highly emetogenic chemotherapy: results of a prospective, randomized, double-blind, noninferiority phase 3 trial.

PURPOSE: Subcutaneous APF530 provides controlled sustained release of granisetron to prevent acute (0-24 h) and delayed (24-120 h) chemotherapy-induced nausea and vomiting (CINV). This randomized, double-blind phase 3 trial compared APF530 and palonosetron in preventing acute and delayed CINV after moderately (MEC) or highly emetogenic chemotherapy (HEC). METHODS: Patients receiving single-day MEC or HEC received single-dose APF530 250 or 500 mg subcutaneously (SC) (granisetron 5 or 10 mg) or intravenous palonosetron 0.25 mg. Primary objectives were to establish APF530 noninferiority to palonosetron for preventing acute CINV following MEC or HEC and delayed CINV following MEC and to determine APF530 superiority to palonosetron for preventing delayed CINV following HEC. The primary efficacy end point was complete response (CR [using CI difference for APF530-palonosetron]). A lower confidence bound greater than -15 % indicated noninferiority. RESULTS: In the modified intent-to-treat population (MEC = 634; HEC = 707), both APF530 doses were noninferior to palonosetron in preventing acute CINV after MEC (CRs 74.8 % [-9.8, 9.3] and 76.9 % [-7.5, 11.4], respectively, vs. 75.0 % palonosetron) and after HEC (CRs 77.7 % [-11.5, 5.5] and 81.3 % [-7.7, 8.7], respectively, vs. 80.7 % palonosetron). APF530 500 mg was noninferior to palonosetron in preventing delayed CINV after MEC (CR 58.5 % [-9.5, 12.1] vs. 57.2 % palonosetron) but not superior in preventing delayed CINV after HEC. Adverse events were generally mild and unrelated to treatment, the most common (excluding injection-site reactions) being constipation. CONCLUSIONS: A single subcutaneous APF530 injection offers a convenient alternative to palonosetron for preventing acute and delayed CINV after MEC or HEC.

Cytotoxic necrotizing factor-Y boosts Yersinia effector translocation by activating Rac protein.

Pathogenic Yersinia spp. translocate the effectors YopT, YopE, and YopO/YpkA into target cells to inactivate Rho family GTP-binding proteins and block immune responses. Some Yersinia spp. also secrete the Rho protein activator cytotoxic necrotizing factor-Y (CNF-Y), but it has been unclear how the bacteria may benefit from Rho protein activation. We show here that CNF-Y increases Yop translocation in Yersinia enterocolitica-infected cells up to 5-fold. CNF-Y strongly activated RhoA and also delayed in time Rac1 and Cdc42, but when individually expressed, constitutively active mutants of Rac1, but not of RhoA, increased Yop translocation. Consistently, knock-out or knockdown of Rac1 but not of RhoA, -B, or -C inhibited Yersinia effector translocation in CNF-Y-treated and control cells. Activation or knockdown of Cdc42 also affected Yop translocation but much less efficiently than Rac. The increase in Yop translocation induced by CNF-Y was essentially independent of the presence of YopE, YopT, or YopO in the infecting Yersinia strain, indicating that none of the Yops reported to inhibit translocation could reverse the CNF-Y effect. In summary, the CNF-Y activity of Yersinia strongly enhances Yop translocation through activation of Rac.

Resonance IR: a coherent multidimensional analogue of resonance Raman.

This work demonstrates the use of triply resonant sum frequency (TRSF) spectroscopy as a "resonance IR" analogue to resonance Raman spectroscopy. TRSF is a four-wave-mixing process where three lasers with independent frequencies interact coherently with a sample to generate an output at their triple summation frequency. The first two lasers are in the infrared and result in two vibrational excitations, while the third laser is visible and induces a two-quantum anti-Stokes resonance Raman transition. The signal intensity grows when the laser frequencies are all in resonance with coupled vibrational and electronic states. The method therefore provides electronic enhancement of IR-active vibrational modes. These modes may be buried beneath solvent in the IR spectrum and also be Raman-inactive and therefore inaccessible by other techniques. The method is presented on the centrosymmetric complex copper phthalocyanine tetrasulfonate. In this study, the two vibrational frequencies were scanned across ring-breathing modes, while the visible frequency was left in resonance with the copper phthalocyanine tetrasulfonate Q band, resulting in a two-dimensional infrared plot that also reveals coupling between vibrational states. TRSF has the potential to be a very useful probe of structurally similar biological motifs such as hemes, as well as synthetic transition-metal complexes.

Reported experiences with Medicaid managed care models among parents of children.

The proportion of children enrolled in Medicaid managed care arrangements has grown significantly over the past decade. Yet, few studies have attempted to assess differences in parental reports and ratings of care for children enrolled in different types of Medicaid managed care. We examine parental reports and ratings of care to explore whether and how patient and parent experiences vary by child health status and managed care plan type, including provider-sponsored specialized plans serving only children. Parents of children in a Florida Medicaid demonstration project in two counties over 3 years were surveyed using Consumer Assessment of Health Providers and Systems surveys (n = 2,741-11,067). Ordered logistic regression models with interaction terms were used to assess relationships between plan type, presence of chronic condition, and measures of patient experience. Parents of children enrolled in provider-sponsored plans that focus on pediatrics were more likely to provide a positive rating for their doctor, health plan, and specialty care compared to parents of children in an health maintenance organization (HMO). Parents of children with a chronic condition were less likely than parents of children without a chronic condition to provide a favorable rating of overall health care, their doctor, or health plan. The interaction term that assessed whether patient experience by plan type was impacted by the child's health status was not statistically significant. Parents of Medicaid children may prefer provider-sponsored arrangements over HMOs. Findings can inform the future development of other integrated models of care involving provider-sponsored arrangements, such as pediatric Accountable Care Organizations and Patient-Centered Medical Homes.

Study protocol for a randomised open-label clinical trial examining the safety and efficacy of the Android Artificial Pancreas System (AAPS) with advanced bolus-free features in adults with type 1 diabetes: the 'CLOSE IT' (Closed Loop Open SourcE In Type 1 diabetes) trial.

INTRODUCTION: Multiple automated insulin delivery (AID) systems have become commercially available following randomised controlled trials demonstrating benefits in people with type 1 diabetes (T1D). However, their real-world utility may be undermined by user-associated burdens, including the need to carbohydrate count and deliver manual insulin boluses. There is an important need for a 'fully automated closed loop' (FCL) AID system, without manual mealtime boluses. The (Closed Loop Open SourcE In Type 1 diabetes) trial is a randomised trial comparing an FCL AID system to the same system used as a hybrid closed loop (HCL) in people with T1D, in an outpatient setting over an extended time frame. METHODS AND ANALYSIS: Randomised, open-label, parallel, non-inferiority trial comparing the Android Artificial Pancreas System (AAPS) AID algorithm used as FCL to the same algorithm used as HCL. Seventy-five participants aged 18-70 will be randomised (1:1) to one of two treatment arms for 12 weeks: (a) FCL-participants will be advised not to bolus for meals and (b) HCL-participants will use the AAPS AID algorithm as HCL with announced meals. The primary outcome is the percentage of time in target sensor glucose range (3.9-10.0 mmol/L). Secondary outcomes include other glycaemic metrics, safety, psychosocial factors, platform performance and user dietary factors. Twenty FCL arm participants will participate in a 4-week extension phase comparing glycaemic and dietary outcomes using NovoRapid (insulin aspart) to Fiasp (insulin aspart and niacinamide). ETHICS AND DISSEMINATION: Approvals are by the Alfred Health Ethics Committee (615/22) (Australia) and Health and Disability Ethics Committees (2022 FULL 13832) (New Zealand). Each participant will provide written informed consent. Data protection and confidentiality will be ensured. Study results will be disseminated by publications, conferences and patient advocacy groups. TRIAL REGISTRATION NUMBERS: ACTRN12622001400752 and ACTRN12622001401741.

Fully coherent triple sum frequency spectroscopy of a benzene Fermi resonance.

In this paper we present a new multiresonant coherent multidimensional spectroscopy (CMDS) technique employing a pathway that is both fully coherent and necessarily unique. This technique is based on a Triple Sum Frequency (TSF) coherence pathway with three excitation pulses having frequencies ω1, ω2, and ω3 and the phase matching condition k→1 + k→2 + k→3. Two-dimensional spectra are created by independently tuning the ω1 and ω2 pulses across vibrational resonances while monitoring the intensity of a visible output beam created by a Raman transition induced by the ω3 pulse. Two-dimensional plots of the coherent dynamics are created by independently scanning the τ21 and τ31 delay times between the different frequency excitation pulses over all time orderings. TSF CMDS separates fundamental and overtone/combination band states uniquely onto the ω1 and ω2 axes when τ21 ≠ 0. TSF is valuable in its ability to probe states of complementary parity to those seen in Doubly Vibrationally Enhanced Four-Wave Mixing (DOVE-FWM), the other fully coherent mixed electronic/vibrational CMDS method. This capability is demonstrated through the use of neat benzene as a model system, where the center of inversion imposes strict parity selection rules.

Triply resonant sum frequency spectroscopy: combining advantages of resonance Raman and 2D-IR.

This article describes the new multidimensional spectroscopy technique triply resonant sum frequency spectroscopy, a four-wave mixing technique sharing advantages of both 2D-IR and resonance Raman experiments. In this technique, lasers with three independent frequencies interact coherently within a sample and generate an output frequency at their triple summation. The output intensity depends on coupled electronic and vibrational resonances in the sample. We use an organic dye as a model system to demonstrate fully resonant, fully coherent multidimensional spectroscopy using two independently tunable mid-infrared vibrational interactions and one visible electronic interaction. When the pulses are time ordered, the method has a single coherence pathway, eliminating interference between pathways. Fundamental vibrational transitions appear on one axis and overtones and combinations bands on the other, allowing anharmonicities of the modes to be determined easily and conveying molecular coupling information. The experiments demonstrate coupling between seven vibrational ring modes and an electronic state, the resolution of a Fermi resonance, detection of low concentrations, elimination of excitation pulse scattering and fluorescence, background suppression of solvent and co-solutes, and observation of coherence dephasing dynamics. The electronic resonance enhancements used in this methodology are similar to the enhancements responsible for resonance Raman spectroscopy and can be considered resonance 2D-IR spectroscopy.

Systematic review and meta-analysis of the effect of fecal microbiota transplantation on behavior in animals.

The bi-directional interaction between gut microbiota and the central nervous system has been coined the gut microbiota-brain axis. Fecal microbiota transplantation (FMT) is the administration of a solution of fecal matter from a donor into the intestinal tract of a recipient. Preclinical FMT experiments are essential to prove causality in the context of the gut microbiota-brain axis. In this systematic review, we assess the body of evidence related to the ability of FMT to modulate an animal's behavior. Accordingly, we provide a detailed summary of the use of FMT in behavior-related animal studies, an extensive risk of bias analysis, and a meta-analysis of the overall effect of FMT on behavioral outcome measures in 64 studies, representing 4889 animals. The resulting meta-analysis revealed FMT was effective at changing animal behavior, thereby substantiating evidence for the gut microbiota-brain axis. However, our study also highlights an urgent need for methodological safeguards within this research field to reduce the risk of bias and improve the internal validity of future studies.

Salmonella enterica Infection of Human and Mouse Colon Organoid-Derived Monolayers.

Intestinal epithelial organoids reflect the morphology and function of an in vivo epithelial barrier. The composition of epithelial cell types reflects the cellular composition of the original tissue (small or large intestine) and organoids can be grown from different species. Thus, intestinal organoids constitute an ideal model to investigate infections of different hosts with enteric pathogens. In this chapter, we will focus on Salmonella infection of human and mouse colonoids grown in a 2D monolayer on permeable filter supports.

Bacterial pathogenesis: exploiting cellular adherence.

Cell adhesion molecules, such as integrins, cadherins, the immunoglobulin superfamily of cell adhesion molecules and selectins, play important structural roles and are involved in various signal transduction processes. As an initial step in the infectious process, many bacterial pathogens adhere to cell adhesion molecules as a means of exploiting the underlying signaling pathways, entering into host cells or establishing extracellular persistence. Often, bacteria are able to bind to cell adhesion molecules by mimicking or acting in place of host cell receptors or their ligands. Recent studies have contributed to our understanding of bacterial adherence mechanisms and the consequences of receptor engagement; they have also highlighted alternative functions of cell adhesion molecules.

Measuring endogenous corticosterone in laboratory mice - a mapping review, meta-analysis, and open source database.

Evaluating stress in laboratory animals is a key principle in animal welfare. Measuring corticosterone is a common method to assess stress in laboratory mice. There are, however, numerous methods to measure glucocorticoids with differences in sample matrix (e.g., plasma, urine) and quantification techniques (e.g., enzyme immunoassay or radioimmunoassay). Here, the authors present a mapping review and a searchable database, giving a complete overview of all studies mea­suring endogenous corticosterone in mice up to February 2018. For each study, information was recorded regarding mouse strain and sex; corticosterone sample matrix and quantification technique; and whether the study covered the research theme animal welfare, neuroscience, stress, inflammation, or pain (the themes of specific interest in our con­sortium). Using all database entries for the year 2012, an exploratory meta-regression was performed to determine the effect of predictors on basal corticosterone concentrations. Seventy-five studies were included using the predictors sex, time-since-lights-on, sample matrix, quantification technique, age of the mice, and type of control. Sex, time-since-lights-on, and type of control significantly affected basal corticosterone concentrations. The resulting database can be used, inter alia, for preventing unnecessary duplication of experiments, identifying knowledge gaps, and standardizing or heterogenizing methodologies. These results will help plan more efficient and valid experiments in the future and can answer new questions in silico using meta-analyses.

Virulence is positively selected by transmission success between mammalian hosts.

Virulence, defined as damage to the host, is a trait of pathogens that evolutionary theory suggests benefits the pathogen in the "struggle for existence". Pathogens employ virulence mechanisms that contribute to disease. Central to the evolution of virulence of the infectious agents causing an array of bacterial disease is the evolutionary acquisition of type III secretion, a macromolecular complex that creates a syringe-like apparatus extending from the bacterial cytosol to the eukaryotic cytosol and delivers secreted bacterial virulence factors (effectors) into host cells. In this work, we quantify the contribution of virulence determinants to the evolutionary success of a pathogen. Using a natural pathogen of mice, we show that virulence factors provide a selective advantage by enhancing transmission between hosts. Virulence factors that have a major contribution to disease were absolutely required for transmission of the pathogen to naive hosts. Virulence-factor mutants with more subtle defects in pathogenesis had quantifiable roles in the time required to transmit the pathogen between mice. Virulence-factor mutants were also found to lose in competition with wild-type bacteria when iteratively transmitted from infected to uninfected mice. These results directly demonstrate that virulence is selected via the fitness advantage it provides to the host-to-host cycle of pathogenic species.

CD14 and ALPK1 Affect Expression of Tight Junction Components and Proinflammatory Mediators upon Bacterial Stimulation in a Colonic 3D Organoid Model.

Cd14 and Alpk1 both encode pathogen recognition receptors and are known candidate genes for affecting severity in inflammatory bowel diseases. CD14 acts as a coreceptor for bacterial lipopolysaccharide (LPS), while ALPK1 senses ADP-D-glycero-beta-D-manno-heptose, a metabolic intermediate of LPS biosynthesis. Intestinal barrier integrity can be influenced by CD14, whereas to date, the role of ALPK1 in maintaining barrier function remains unknown. We used colon-derived 3D organoids, first characterised for growth, proliferation, stem cell markers, and expression of tight junction (TJ) components using qPCR and immunohistochemistry. They showed characteristic crypt stem cells, apical shedding of dead cells, and TJ formation. Afterwards, organoids of different genotypes (WT, Il10 -/-, Cd14 -/-, and Alpk1 -/-) were then stimulated with either LPS or Escherichia coli Nissle 1917 (EcN). Gene expression and protein levels of cytokines and TJ components were analysed. WT organoids increased expression of Tnfα and tight junction components. Cd14 -/- organoids expressed significantly less Tnfα and Ocln after LPS stimulation than WT organoids but reacted similarly to WT organoids after EcN stimulation. In contrast, compared to WT, Alpk1 -/- organoids showed decreased expression of different TJ and cytokine genes in response to EcN but not LPS. However, Western blotting revealed an effect of ALPK1 on TJ protein levels. These findings demonstrate that Cd14, but not Alpk1, alters the response to LPS stimulation in colonic epithelial cells, whereas Alpk1 is involved in the response upon bacterial challenge.