Peer-led pediatric resuscitation training: effects on self-efficacy and skill performance.

BACKGROUND: Peer-led basic life support training in medical school may be an effective and valued way of teaching medical students, yet no research has been conducted to evaluate the effect on the self-efficacy of medical students. High self-efficacy stimulates healthcare professionals to initiate and continue basic life support despite challenges. METHODS: A randomized controlled trial, in which medical students received pediatric basic life support (PBLS) training, provided by either near-peer instructors or expert instructors. The students were randomly assigned to the near-peer instructor group (n = 105) or expert instructor group (n = 108). All students received two hours of PBLS training in groups of approximately 15 students. Directly after this training, self-efficacy was assessed with a newly developed questionnaire, based on a validated scoring tool. A week after each training session, students performed a practical PBLS exam and completed another questionnaire to evaluate skill performance and self-efficacy, respectively. RESULTS: Students trained by near-peers scored significantly higher on self-efficacy regarding all aspects of PBLS. Theoretical education and instructor feedback were equally valued in both groups. The scores for the practical PBLS exam and the percentage of students passing the exam were similar in both groups. CONCLUSIONS: Our findings point towards the fact that near-peer-trained medical students can develop a higher level of PBLS-related self-efficacy than expert-trained students, with comparable PBLS skills in both training groups. The exact relationship between peer teaching and self-efficacy and between self-efficacy and the quality of real-life pediatric resuscitation should be further explored. TRIAL REGISTRATION: ISRCTN, ISRCTN69038759 . Registered December 12th, 2019 - Retrospectively registered.

Distribution of human colonic dendritic cells and macrophages.

To define the phenotype of intestinal dendritic cells and macrophages, resected colonic specimens were used to obtain lamina propria cell suspensions by EDTA treatment, then enzymatic digestion. The phenotype of dendritic cell-enriched suspensions was compared with that of macrophage-enriched populations by immunocytochemistry using the avidin-biotin-peroxidase (ABC) system and immunoelectron microscopy. Dendritic cells expressed HLA-DR (L243) and HLA-DQ-associated (RFD) antigens and CD68 in a perinuclear distribution. Staining for S100 was weak or absent. Macrophages also expressed HLA markers (L243 and RFD1) and CD68. The 25F9 antigen was expressed strongly, whilst CD14 was absent from cells isolated from noninflamed tissues. To determine their anatomic distribution, immunohistochemistry was performed using single- and double-labelling techniques (ABC +or- alkaline phosphatase anti-alkaline phosphatase method). Mutually exclusive subsets of 25F9+ and S100+ cells were seen: 25F9+ macrophages were concentrated in a band immediately beneath the luminal epithelium; S100+/HLA-DR+ dendritic cells formed a reticular network throughout the lamina propria and beneath the basement membrane of the crypts. This distribution suggests that macrophages may help regulate intestinal responses by acting as the first line of defence against the entry of luminal antigens. A breach of the macrophage 'barrier' by invading antigens may necessitate the recruitment of T cell responses by immunostimulatory dendritic cells.

Evidence for a CD14+ population of monocytes in inflammatory bowel disease mucosa--implications for pathogenesis.

Lipopolysaccharide (LPS) is abundant in the intestinal lumen. CD14 is the receptor for the LPS-LPS binding protein complex, and its presence on mononuclear phagocytes allows cell activation by pg/ml concentrations of LPS. We have shown that the recently recruited blood monocyte in inflammatory bowel disease mucosa is CD14+. This study examined the expression of CD14 on macrophages in inflamed (n = 13) and uninflamed (n = 7) intestine by immunohistochemistry, and on disaggregated lamina propria mononuclear cells (12 from inflamed, 17 from uninflamed intestine) and peripheral blood mononuclear cells (n = 26) by flow cytometry, using a panel of three MoAbs directed against CD14. Immunohistochemistry revealed that 3.7% of macrophages in uninflamed intestine were CD14+, while 25.1% of macrophages in active inflammatory bowel disease expressed CD14 (P < 0.02). Flow cytometry demonstrated that CD14 expression by macrophages from Crohn's disease and ulcerative colitis was augmented significantly (P = 0.02 and P = 0.01, respectively) compared with uninflamed intestine, with a discrete population of macrophages in inflammatory bowel disease, not present in normal intestine, which strongly expressed CD14. The characteristically high levels of CD14 on blood monocytes were unaffected by the presence of intestinal inflammation. Given the exposure of lamina propria cells to LPS present in the lumen of the terminal ileum and colon, the increased numbers of CD14+ macrophages in inflammatory bowel disease may result in greatly increased production of inflammatory mediators, thereby suggesting a mechanism for the perpetuation of mucosal inflammation.

Dendritic cells, the major antigen-presenting cells of the human colonic lamina propria.

Induction of T-cell responses requires the recognition of antigen in association with class II major histocompatibility complex (MHC) antigens on specialized antigen-presenting cells. It was previously demonstrated that dendritic cells were the major antigen-presenting cell in the mouse intestinal lamina propria whilst macrophages were shown to be suppressive. The aim of this study was to compare the antigen-presenting cell activity of human colonic dendritic cells with macrophages. Colonic mucosa was removed from 46 specimens resected for cancer and other non-malignant conditions and lamina propria cell suspensions obtained by EDTA treatment followed by enzymatic digestion. Lamina propria cell suspensions, depleted of macrophages by adherence to insolubilized human immunoglobulin and carbonyl iron phagocytosis, were enriched for dendritic cells by density gradient centrifugation. Yields represented 0.9% (range 0.7-1.4%) of the starting cell number and the degree of enrichment was 30-50%. Immunocytochemistry demonstrated high levels of class II MHC antigen expression, but low levels or absent expression of macrophage and other markers. The ultrastructural features of the low-density cell fraction were typical of dendritic cells with cytoplasmic extensions or veils and the absence of phagocytic vesicles. Populations of cells enriched for macrophages were obtained by harvesting the human immunoglobulin-adherent cells. These cells were > 70% positive for macrophage markers using immunocytochemistry. The ability of lamina propria cells to induce primary T-cell activation was assayed using allogeneic peripheral blood T cells as responders in the mixed leucocyte reaction (MLR). When antigen-presenting activity was assessed using the MLR, the stimulatory activity was present in the dendritic cell-enriched fraction, with little activity present in the macrophage fraction. These data indicate that dendritic cells, not macrophages, are the major cell population capable of generating a mixed leucocyte reaction in the human colonic lamina propria.

Cell associated urokinase activity and colonic epithelial cells in health and disease.

It is not known if urokinase-type plasminogen activator (uPA) is associated with normal colonic epithelial cells. The aims of this study were to determine if normal colonic epithelial cells have uPA activity and whether this is concentrated at the cell membrane. In addition, the contribution of colonic epithelial cell associated uPA activity to disease related pertubations of mucosal uPA activity were examined. A highly enriched population of colonic epithelial cells was isolated from resected colon or biopsy specimens by an enzymatic technique. uPA activity was measured in cell homogenates by a specific and sensitive colorimetric method and expressed relative to cellular DNA. In two experiments subcellular fractionation of colonic epithelial cells was performed by nitrogen cavitation followed by ultracentrifugation over a linear sucrose gradient. The fractions collected were analysed for uPA and organelle-specific enzyme activities. Normal colonic epithelial cells have cell associated uPA activity (mean (SEM) 5.6 (1.1) IU/mg, n = 18). This colocalised with fractions enriched for leucine-beta-naphthylamidase and 5'-nucleotidase, markers of plasma membrane. uPA activities in epithelial cells from cancerous colons (9.8 (3.1) n = 7) or from mucosa affected by inflammatory bowel disease (3.8 (0.7) n = 15) were not significantly different from normal (paired t test), while that in epithelial cells from greatly inflamed mucosa was similar to that from autologous normal or mildly inflamed areas (4.4 (1.2) v 5.9 (3.6), n = 9). Thus normal colonic epithelial cells have cell associated uPA activity which is concentrated on the plasma membranes, suggesting the presence of uPA receptors. Increased mucosal levels of uPA previously reported in patients with inflammatory bowel disease are not due to increased colonic epithelial cell associated uPA.

Isolation of colonic crypts that maintain structural and metabolic viability in vitro.

The aim of this study was to develop a method by which colonic epithelial cells can be isolated from resected mucosa or colonoscopic biopsy specimens and viability maintained in the short term. The principles of the technique are to digest the lamina propria from the epithelium with Dispase and collagenase, to disrupt the epithelium by trituration, and to purify the epithelial cells by seiving and differential sedimentation. Whole and partial crypts were isolated with consistently high purity of 93.5% +/- 1.2% (excluding red cells). Structural integrity was confirmed by light and electron microscopy, exclusion of trypan blue, minimal leakage of lactic dehydrogenase over 5 h (4.1% +/- 1.7%), and 51Cr leakage of less than 2% per hour over 16 h. Functional integrity was supported by continued deoxyribonucleic acid synthesis [( 3H]thymidine uptake) over 16 h and the formation of epithelial monolayer cultures on plastic. Thus, this simple method yields a highly enriched cell population that maintains high viability in vitro for at least 16 h. Such cells may be useful for the study of the biology of colonic epithelial cells.

Lysis of colonic epithelial cells by allogeneic mononuclear and lymphokine activated killer cells derived from peripheral blood and intestinal mucosa: evidence against a pathogenic role in inflammatory bowel disease.

A sensitive 4 h 51Cr-release cytotoxicity assay has been developed using as targets colonic epithelial cells obtained by Dispase-collagenase digestion of resected mucosa or colonoscopic biopsies. Peripheral blood mononuclear cells (MNC) from most healthy donors showed low, but significant levels of cytotoxicity for normal epithelial cell target cells of 8.7 (4.4) % (mean (SD] and similar levels were found in 14 ulcerative colitis (6.5 (4.4) %) and 16 Crohn's disease (6.2 (5.2) %) patients. Neither drug therapy nor disease activity influenced the results. The sensitivity of colonic epithelial cells isolated from inflamed and histologically normal mucosa to lysis by peripheral blood MNC from a single donor was not affected by the underlying disease. Anti-epithelial cell activity did not correlate with anti-K562 activity and the cytotoxic cell was plastic non-adherent and Leu-11b-. None of 15 MNC populations isolated from mucosa of normal, tumour bearing, or chronically inflamed intestine exhibited significant lysis of colonic epithelial cells despite killing of K562 target cells in 10. Lymphokine activated killer (LAK) cells, generated by interleukin-2 stimulation in vitro of nine intestinal and seven peripheral blood MNC populations, exhibited high levels of lysis of K562 cells but, on every occasion, failed to lyse colonic epithelial cells. These data indicate that spontaneously cytotoxic or LAK cells are unlikely to play a role in the generation of colonic epithelial cell injury by direct cytotoxicity in inflammatory bowel disease.

Ulcerative colitis--a disease characterised by the abnormal colonic epithelial cell?

The leakiness of the cell membranes of colonic epithelial cells isolated by the collagenase/Dispase technique from normal or diseased colons was assessed in a 4 h 51Cr release assay. Cells from normal, adenoma bearing or cancer bearing colons showed 51Cr release of 8% or less in almost all of 46 cell populations tested. In contrast, cells from mucosa affected by ulcerative colitis [11.9 (4.3%) n = 23] or Crohn's disease [8.4 (2.7%) n = 18] released significantly more 51Cr than the non-inflamed groups. Values are expressed as mean (SD). Overall, release values were greater in ulcerative colitis than Crohn's disease (p less than 0.01). In Crohn's disease, cells obtained from histologically inflamed mucosa released significantly more 51Cr [9.7 (2.5%) n = 11] than those from non-inflamed mucosa [6.4 (1.5%) n = 7, p less than 0.02] whereas, in ulcerative colitis, abnormal release values were found in 8 of 13 cell populations isolated from mucosa showing no histological evidence of active disease. In five patients with distal ulcerative colitis, cells from mucosa not apparently involved demonstrated normal 51Cr release in four of five studies despite abnormal release from cells from involved mucosa suggesting that a diffuse abnormality of the colonic epithelial cell is not usually present. These data indicate that chronic mucosal inflammation per se is associated with abnormalities of the colonic epithelial cell but that, in ulcerative colitis, the abnormality remains in many patients with quiescent disease. Identification of the local factors responsible for such an abnormality may contribute to an understanding of the pathogenesis of ulcerative colitis.

On the stimulation of respiration by alpha-adrenergic agonists in perfused rat liver.

Interactions between phenylephrine-induced oxygen consumption, lactate and pyruvate output, and urea and glucose production were examined in perfused livers from fed or 48-h-fasted rats. Within 2 min of phenylephrine infusion, oxygen consumption in perfused livers was increased by approximately 40%. Increases in oxygen consumption induced by phenylephrine were essentially abolished in the presence of carboxyatractyloside, whereas those induced by dinitrophenol were still evident. Phenylephrine-induced increases in oxygen consumption were accompanied by enhanced rates of gluconeogenesis and ureogenesis in livers from fed or 48-h-fasted animals. These data indicate that phenylephrine-induced increases in respiration in perfused rat liver may result from an enhanced rate of mitochondrial oxidative phosphorylation in response to an increased cellular energy requirement.

The stimulation of tricarboxylic acid-cycle flux by alpha-adrenergic agonists in perfused rat liver.

Output of 14CO2 from 1-14C-labelled glutamate, 2-oxoglutarate or octanoate and from 4-methyl-2-oxo[2-14C]pentanoate was increased by more than 100% after infusion of phenylephrine into perfused livers of fed rats. Infusion of ethanol or sorbitol raised 3-hydroxybutyrate/acetoacetate ratios and decreased the output of 14CO2. Increases in 14CO2 output induced by phenylephrine were observed in the presence or absence of ethanol or sorbitol and were accompanied by elevated 3-hydroxybutyrate/acetoacetate ratios under all conditions examined. Phenylephrine had no effect on total tissue ATP/ADP ratios in livers from fed or starved rats. The data suggest that phenylephrine-induced increases in tricarboxylic acid-cycle flux do not arise from lowered matrix NADH/NAD+ or ATP/ADP ratios.

Effect of depolarizing concentrations of potassium on calcium uptake and metabolism in rat liver.

Exposure of perfused livers of fed rats to 60 mM K+ induces rapid responses in the Ca2+-sensitive metabolic events, glycogenolysis, cytoplasmic and mitochondrial NADH/NAD ratios and octanoate oxidation. All increase within 45 s of K+ addition. Metabolic responses were not observed following K+ addition to livers perfused in the absence of added Ca2+. Movements of Ca2+ into the liver were suggested from experiments in which 45Ca2+ uptake was measured. The Ca2+ antagonists verapamil, diltiazem and Ni2+ essentially abolished changes to tissue metabolism and Ca2+ fluxes induced by K+ addition. K+-induced changes were consistent with Ca2+ channel activation.

An assessment of the calcium content of rat liver mitochondria in vivo.

The effect of systematically altering the isolation conditions on the total calcium content of mitochondria isolated from perfused rat liver was examined. We showed that, under most isolation conditions, significant redistributions of mitochondrial calcium occurred resulting in up to 5-fold changes of the total calcium content. Mitochondrial Ca2+ flux inhibitors such as Ruthenium Red and nupercaine were only partially effective in inhibiting such redistributions. We present evidence indicating that the total calcium content of rat liver mitochondria in situ may approximate 2 nmol X (mg of protein)-1.