Alex Chin

Strain-Dependent Induction of Enterocyte Apoptosis by Giardia lamblia Disrupts Epithelial Barrier Function in a Caspase-3-Dependent Manner

ABSTRACT We recently demonstrated that Giardia lamblia rearranges cytoskeletal proteins and reduces transepithelial electrical resistance. The effect of G. lamblia on enterocyte apoptosis is unknown, and a possible link between microbially induced enterocyte apoptosis and altered epithelial permeability has yet to be established. The aim of this study was to assess whether G. lamblia induces enterocyte apoptosis in duodenal epithelial monolayers and whether this effect increases epithelial permeability. Monolayers of nontransformed human duodenal epithelial cells were incubated with sonicated or live G. lamblia trophozoites (NF, S2, WB, or PB strains) for 8, 24, and 48 h. Cell cultures were assessed for apoptosis by Hoechst fluorescence staining, enzyme-linked immunosorbent assay for apoptotic nucleosomes, and electron microscopy. In separate experiments, monolayers were pretreated with or without 120 μM caspase-3 inhibitor (Z-DEVD-FMK) for 1 h and were assessed for production of apoptotic nucleosomes, tight junctional integrity (with fluorescent ZO-1 staining followed by confocal laser microscopy), and transepithelial permeability for fluorescein isothiocyanate-dextran. G. lamblia strains NF and S2, but not strains WB or PB, induced enterocyte apoptosis within the monolayers, and this effect was inhibited by Z-DEVD-FMK pretreatment. Using the G. lamblia NF isolate, additional experiments investigated the possible link between enterocyte apoptosis and altered epithelial permeability. G. lamblia NF disrupted tight junctional ZO-1 and increased epithelial permeability, but these effects were also prevented by pretreatment with the caspase-3 inhibitor. These findings indicate that strain-dependent induction of enterocyte apoptosis may contribute to the pathogenesis of giardiasis. This effect is responsible for a loss of epithelial barrier function by disrupting tight junctional ZO-1 and increasing permeability in a caspase-3-dependent manner.

PAR2 activation alters colonic paracellular permeability in mice via IFN-γ-dependent and -independent pathways

Activation of colonic proteinase‐activated receptor‐2 (PAR2) caused inflammation and increased mucosal permeability in mouse colon. The present study was aimed at characterizing the possible links between these two phenomena. We evaluated the effects of intracolonic infusion of PAR2‐activating peptide, SLIGRL, on colonic paracellular permeability and inflammation at two different doses, 5 and 100 μg per mouse, in an attempt to discriminate between both PAR2‐mediated effects. We further investigated the possible involvement of interferon γ (IFN‐γ) and calmodulin‐dependent activation of myosin light chain kinase (MLCK), and alterations of zonula occludens‐1 (ZO‐1) localization in PAR2‐induced responses. Thus, at the lower dose, SLIGRL increased colonic permeability without causing inflammation. Western blotting showed phosphorylation of mucosal myosin light chain (MLC) expression after both doses of SLIGRL. Moreover, while the MLCK inhibitor, ML‐7, abolished the permeability effects of the low dose of SLIGRL, it only partially inhibited that of the high dose. In IFN‐γ‐deficient mice (B6 ifng−/−), the increases in permeability were similar for both doses of SLIGRL and prevented by ML‐7. In addition, MLCK immunoprecipitation revealed an increase of calmodulin binding to MLCK in the mucosa of mice treated with either dose of SLIGRL. Finally, we have shown that direct activation of PAR2 on enterocytes is responsible for increased permeability and ZO‐1 disruption. Moreover, SLIGRL at a dose that does not produce inflammation increases permeability via calmodulin activation, which binds and activates MLCK. The resulting tight junction opening does not depend upon IFN‐γ secretion, while the increased permeability in response to the high dose of PAR2 agonist involves IFN‐γ secretion.

Proteinase-activated receptor 1 activation induces epithelial apoptosis and increases intestinal permeability.

Proteinase-activated receptor 1 (PAR1)-mediated inflammation remains poorly understood. Here we characterize previously unrecognized effects of PAR1-induced apoptosis signaling, which contributes to epithelial barrier dysfunction. Incubation of epithelial cells with PAR1 agonists induced apoptosis and increased epithelial permeability in a caspase-3-dependent manner. Similarly, studies in vivo demonstrated that intracolonic infusion with PAR1 agonists increased colonic permeability in mice, and that this effect was abolished by pretreatment with a caspase-3 inhibitor. PAR1 agonists induced tight junctional zonula-occludens 1 disruption and apoptotic nuclear condensation. Investigation into signaling pathways showed that these effects were dependent on caspase-3, tyrosine kinase, and myosin light chain kinase. Conversely, the Src kinase inhibitor PP1 augmented zonula-occludens 1 injury and nuclear condensation induced by PAR1 agonists. These results support a role for proteinases and PARs in intestinal disease and provide new directions for possible therapeutic applications of PAR1 antagonists.

Infection of human and bovine epithelial cells with Cryptosporidium andersoni induces apoptosis and disrupts tight junctional ZO-1: effects of epidermal growth factor

The effects of Cryptosporidium andersoni on human or bovine epithelia are poorly defined. Epidermal growth factor inhibits colonisation of the gastrointestinal epithelium with bacteria and the enteric protozoan parasite Giardia lamblia. This study characterised whether C. andersoni infects human or bovine epithelial cells in vitro, assessed its impact on apoptosis and tight junctional Zonula-Occludens-1, and determined whether these effects may be altered by epidermal growth factor. Monolayers of human colonic CaCo(2) cells, SCBN (non-malignant small intestinal epithelial cells), and Madin Darby bovine kidney epithelial cell lines (MDBK and NBL-1) were grown to confluency in Dulbeccos Modified Eagle Medium. Monolayers were assigned to one of three experimental groups-(1) control: exposed to culture medium alone; (2) untreated: exposed to 10(3) live C. andersoni oocysts or (3) epidermal growth factor-treated: apically pre-treated with recombinant human epidermal growth factor and then exposed to Cryptosporidium. Oocyst viability, infection with Cryptosporidium, apoptosis, and integrity of tight junctional Zonula-Occludens-1 were assessed. In addition, live Cryptosporidium oocysts were incubated with epidermal growth factor to assess whether epidermal growth factor had cryptosporicidial activity. Cryptosporidium andersoni oocysts infected all human and bovine monolayers, increased nuclear fragmentation, and disrupted Zonula-Occludens-1. Apical epidermal growth factor significantly reduced infection with C. andersoni in all cell lines and inhibited the Cryptosporidium-induced apoptosis and disruption of Zonula-Occludens-1. Epidermal growth factor did not affect oocyst viability.

Tilmicosin Induces Apoptosis in Bovine Peripheral Neutrophils in the Presence or in the Absence of Pasteurella haemolytica and Promotes Neutrophil Phagocytosis by Macrophages

ABSTRACT Pathogen virulence factors and inflammation are responsible for tissue injury associated with respiratory failure in bacterial pneumonia, as seen in the bovine lung infected with Pasteurella haemolytica. Tilmicosin is a macrolide antibiotic used for the treatment of bovine bacterial pneumonia. Recent evidence suggests that tilmicosin-induced neutrophil apoptosis may have anti-inflammatory effects. Using bovine leukocytes, we sought to define whether liveP. haemolytica affected tilmicosin-induced neutrophil apoptosis, assessed the proapoptotic effects of tilmicosin in comparison with other drugs, and characterized its impact on phagocytic uptake of neutrophils by macrophages. Induction of apoptosis in the presence or absence of P. haemolytica was assessed by using an enzyme-linked immunosorbent assay for apoptotic nucleosomes. In addition, fluorescent annexin-V staining identified externalized phosphatidylserine in neutrophils treated with tilmicosin, penicillin, ceftiofur, oxytetracycline, or dexamethasone. Neutrophil membrane integrity was assessed by using propidium iodide and trypan blue exclusion. As phagocytic clearance of apoptotic neutrophils by macrophages contributes to the resolution of inflammation, phagocytosis of tilmicosin-treated neutrophils by esterase-positive cultured bovine macrophages was assessed with light microscopy and transmission electron microscopy. Unlike bovine neutrophils treated with penicillin, ceftiofur, oxytetracycline, or dexamethasone, neutrophils exposed to tilmicosin became apoptotic, regardless of the presence or absence ofP. haemolytica. Tilmicosin-treated apoptotic neutrophils were phagocytosed at a significantly greater rate by bovine macrophages than were control neutrophils. In conclusion, tilmicosin-induced neutrophil apoptosis occurs regardless of the presence or absence of live P. haemolytica, exhibits at least some degree of drug specificity, and promotes phagocytic clearance of the dying inflammatory cells.

Simulation of repetitive diagnostic blood loss and onset of iatrogenic anemia in critical care patients with a mathematical model

Anemia is prevalent among critical care patients and is attributed to pathologic and iatrogenic processes and bleeding. The extent that diagnostic blood loss contributes to anemia among adult critical care patients is controversial and multi-factorial. The aim of this study is to describe an erythrokinetic model that integrates rates of phlebotomy, erythropoiesis and red cell senescence with patient characteristics to predict the onset of iatrogenic anemia in an objective manner. Using sex-specific parameters, the model predicts that adults with (average body weight and average blood volume), initial hemoglobin concentration at mid-reference interval, active erythropoiesis and losing 53 mL of blood per day by phlebotomy would require 40-70 days to reach 70 g/L of hemoglobin. To mimic critical care patients with low initial hemoglobin and suppressed erythropoiesis, the influence of daily blood loss and total blood volume was predicted. Simultaneous lack of erythropoiesis, initial hemoglobin concentrations at the lower limit of the reference interval (110 g/L), low body weight and increased phlebotomy accelerated onset of ~70 g/L hemoglobin transfusion threshold to 9-14 days. This computer simulation depicts the extent that adult critical care patients with anemia risk factors could benefit from conservative test ordering practices and subsequent reduced diagnostic phlebotomy.

Defining contralateral adrenal suppression in primary aldosteronism: implications for diagnosis and outcome

Unilateral primary aldosteronism (PA) should have a contralaterally normal and therefore suppressed adrenal zona glomerulosa. However, there is no validated definition of adrenal suppression. We created two biochemical hypotheses of adrenal suppression based upon measurements taken during adrenal vein sampling (AVS) to determine whether either proved useful for interpretation of AVS or prediction of hypertension outcome in operated cases.

Unadjusted Plasma Renin Activity as a “First-Look” Test to Decide Upon Further Investigations for Primary Aldosteronism

The authors sought to define the 95th percentile of plasma renin activity (PRA) in a sample of patients with confirmed primary aldosteronism (PA) prior to adjustment of medications as a practical “first‐look” test to identify those with very low ultimate likelihood of having PA. The aldosterone to renin ratio (ARR) was measured without adjustment of antihypertensive medications, with further workup as appropriate. Two groups were defined: patients with surgically “confirmed PA” (n=58) and patients with “high‐probability PA” (n=59), defined as having any of the following: computed tomography–confirmed adrenal adenoma plus lateralizing adrenal vein sampling (AVS) without surgery, high ARR and hypokalemia but nonlateralizing AVS, or ARR more than four times the upper limit of normal. The PRA 95th percentile was 1.0 ng/mL/h. All outliers had hypokalemia and two had adrenal adenomas. There was no difference between the confirmed and high probability groups. In the absence of highly suspicious clinical features, patients with unadjusted PRA >1.0 ng/mL/h do not warrant further investigation for PA.

Implementation of an intervention to reduce population-based screening for vitamin D deficiency: a cross-sectional study

BACKGROUND We describe the implementation of an intervention in Alberta in support of the Choosing Wisely Canada recommendation against population screening for vitamin D deficiency (as determined by serum total 25-hydroxyvitamin D testing). We hypothesized that the introduction of a specialized requisition for vitamin D testing would reduce the annual number of vitamin D tests performed. METHODS We performed a cross-sectional observational study that included all vitamin D tests ordered in Alberta between Apr. 1, 2015, and Mar. 31, 2016. There were no exclusion criteria. A special requisition for ordering vitamin D tests in Alberta was introduced on Apr. 1, 2015. Using an interrupted time series model, we compared predicted versus observed vitamin D test volumes for the 12-month period following the introduction of the new requisition. The sole outcome measure was the monthly change in volume of vitamin D testing. In addition, we calculated any cost savings as a result of reduced testing. RESULTS Over the first 12 months of the intervention, there was a reduction in the number of tests ordered from a predicted 342 477 tests to 29 525 tests (91.4% reduction). This decrease represented a direct spending decrease of Can

Resolution of Spurious Immunonephelometric IgG Subclass Measurement Discrepancies by LC-MS/MS

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