Redefining the role of biomarkers in heart failure trials: expert consensus document.

Heart failure is a growing cardiovascular disease with significant epidemiological, clinical, and societal implications and represents a high unmet need. Strong efforts are currently underway by academic and industrial researchers to develop novel treatments for heart failure. Biomarkers play an important role in patient selection and monitoring in drug trials and in clinical management. The present review gives an overview of the role of available molecular, imaging, and device-derived digital biomarkers in heart failure drug development and highlights capabilities and limitations of biomarker use in this context.

The role of L. monocytogenes serotype 4b gtcA in gastrointestinal listeriosis in A/J mice.

Serotype 4b strains of Listeria monocytogenes have been responsible for most large outbreaks of listeriosis. In L. monocytogenes serotype 4b, gtcA and gltA have been implicated in serotype-specific glycosylation of the teichoic acid of the cell wall with galactose and glucose. In this study, we investigated the impact of mutations in gltA (resulting in absence of glucose on teichoic acid) and gtcA (resulting in absence of galactose, and markedly reduced glucose on teichoic acid) on virulence following intragastric infection of anesthetized A/J mice. The gltA mutant was not impaired in virulence in this model. In contrast, testing of gtcA mutants constructed in two different strains showed that the mutants were recovered in lower numbers than their respective parent strains from the spleen, liver, ceca, and gall bladders of intragastrically inoculated mice. Genetic complementation of the gtcA mutation partially restored gastrointestinal virulence. When mice were inoculated intravenously, the gtcA mutants were also recovered in lower numbers from the liver (for both mutant strains) and the spleen (for one mutant strain) than their respective parental strains. The mutants were also evaluated for invasion and intracellular multiplication in the Caco-2 human intestinal epithelial cell line. Inactivation of gltA did not affect invasion or intracellular growth of the bacteria. In contrast, gtcA mutants showed decreased invasion, but normal multiplication in Caco-2 cells. Overall, these data demonstrate a role for gtcA in the pathogenesis of gastrointestinal listeriosis in mice, and suggest that diminished ability of gtcA mutants to invade intestinal epithelial cells may be partly responsible for decreased gastrointestinal virulence.

Lipopolysaccharide increases cell surface P-glycoprotein that exhibits diminished activity in intestinal epithelial cells.

Increasingly, it is recognized that commensal microflora regulate epithelial cell processes through the dynamic interaction of pathogen-associated molecular patterns and host pattern recognition receptors such as Toll-like receptor 4 (TLR4). We therefore investigated the effects of bacterial lipopolysaccharide (LPS) on intestinal P-glycoprotein (P-gp) expression and function. Human SW480 (P-gp+/TLR4+) and Caco-2 (P-gp+/TLR4-) cells were treated with medium control or LPS (100 ng/ml) for 24 h prior to study. P-gp function was assessed by measuring the intracellular concentration of rhodamine 123 (Rh123). To confirm P-gp-specific effects, breast cancer resistance protein (BCRP/ABCG2) and multidrug resistance-associated protein 2 (MRP-2/ABCC2) were also analyzed. Treatment of SW480 cells with LPS led to diminished P-gp activity, which could be prevented with polymyxin B (control: 207+/-16 versus LPS: 402+/-22 versus LPS+polymyxin B: 238+/-26 pmoles Rh123/mg protein, p<0.05 control versus LPS). These effects could be blocked by using polymyxin B and were not seen in the P-gp+/TLR4--Caco-2 cell line (control: 771+/-28 versus LPS: 775+/-59 pmoles Rh123/mg protein). Total cellular levels of P-gp did not change in LPS-treated SW480 cells; however, a significant increase in cell surface P-gp was detected. No change in activity, total protein, or apically located MRP-2 was detected following LPS treatment. Sequence analysis confirmed wild-type status of SW480 cells. These data suggest that activation of TLR4 in intestinal epithelial cells leads to an increase in plasma membrane P-gp that demonstrates a diminished capacity to transport substrate.

The poloxamer P85 is protective against Listeria monocytogenes invasion.

Listeria monocytogenes remains an important foodborne pathogen, and strategies designed to decrease the susceptibility of selected patient populations to foodborne pathogens are therefore desirable. Our objective was to determine if the poloxamer P85 was protective against L. monocytogenes infection. Caco-2 cells were treated with 0.1% (w/v) P85 and challenged with 10(7) L. monocytogenes EGD for 1 hour. A standard gentamicin protection assay was performed to determine invasion differences between the experimental groups. Effects of P85 on the pathogen were studied by measuring bacterial growth and ATP concentrations. In a murine model of listeriosis, FVB mice were administered 150 mg/kg P85 or vehicle control 45 minutes prior to intragastric inoculation of 10(7) L. monocytogenes. Dissemination of the pathogen from the gastrointestinal tract to the liver and spleen was determined 24 hours after bacterial challenge. Pretreatment of Caco-2 cells with P85 significantly decreased L. monocytogenes invasion compared to controls. Repletion of ATP reversed the protective effects of P85. No changes in bacterial ATP or growth profile were detected in P85-treated bacteria. Administration of P85 to mice prior to infection led to decreased dissemination to the liver and spleen compared to vehicle-treated mice. P85 is protective against L. monocytogenes infection when administered prior to bacterial challenge. Modulation of host ATP levels appears to be crucial for the protective effects of P85.

Endomorphin-1 alters interleukin-8 secretion in Caco-2 cells via a receptor mediated process.

Administration of opioids that bind to the classical mu opioid receptor has been shown to lead to unintended alterations in immune function. Traditionally, altered immune function has been investigated with circulating immune cells. Effects of mu agonists on intestinal epithelial immune function have not been described. Since the oral route of administration is frequently employed with opiates, we determined if the mu receptor specific agonist endomorphin-1 altered interleukin-8 (IL-8) production by Caco-2 cells. Using RT-PCR and immunocytochemistry, Caco-2 cells were found to constitutively express (mu) mu opioid receptors. Activation of the mu receptor by endomorphin-1 (1 and 10 microM) resulted in significant increases in IL-8 when Caco-2 cells were stimulated with IL-1beta. Increased IL-8 secretion due to endomorphin-1 could be blocked by pre-incubating cells with the mu receptor antagonist, beta-funaltrexamine. These results indicate that the intestinal epithelial IL-8 response can be altered by a muopioid receptor mediated mechanism.

Activation of the kappa-opioid receptor in Caco-2 cells decreases interleukin-8 secretion.

The immunomodulatory effects of kappa-opioid agonists at the intestinal epithelial level are not well characterized. In the present study, we determined that Caco-2 cells express the kappa-opioid receptor and its activation by trans-(+/-)-3,4-dichloro-N-methyl-N[2-(1-pyrolidinyl)cyclohexyl]benzeneacetamide methanesulfonate (U-50488) leads to decreased interleukin-8 secretion in the presence of interleukin-1beta. These effects were detected over a wide range (10 nM-50 microM) of U-50488 concentrations and were reversible using the kappa-opioid receptor antagonist nor-binaltorphimine. Our data suggest that activation of kappa-opioid receptors on Caco-2 cells decreases interleukin-8 secretion and thus may alter the chemotactic stimulus at the epithelial level.

Tachycardia-induced heart failure does not alter myocardial P-glycoprotein expression.

STUDY OBJECTIVE: To determine the effects of tachycardia-induced heart failure on myocardial P-glycoprotein (P-gp) expression. DESIGN: Nonblinded, parallel, sham-controlled, animal model study. SETTING: University laboratory. ANIMALS: Thirty mongrel dogs. INTERVENTION: Heart failure was induced by rapid ventricular pacing over 4 weeks; sham procedures were performed for the control group. MEASUREMENTS AND MAIN RESULTS: Myocardial biopsies were taken from the left ventricular lateral wall and prepared for P-gp quantification by laser-induced fluorescence. The relative amount of P-gp messenger RNA (mRNA) was assessed by reverse transcriptase polymerase chain reaction. Rapid ventricular pacing produced heart failure and reduced the area ejection fraction from 48% +/- 6% to 21% +/- 6% (p<0.05 vs baseline). However, heart failure did not alter the quantity of myocardial P-gp (0.20 +/- 0.02 microg/ml for the control group vs 0.23 +/- 0.02 microg/ml for the intervention group, p=0.4). Furthermore, heart failure did not alter P-gp expression significantly. CONCLUSION: Myocardial P-gp does not change in response to tachycardia-induced heart failure. Thus, there is a low likelihood for P-gp-related drug resistance during a syndrome similar to tachycardia-induced heart failure.

The effects of thermal injury on transcellular permeability and intestinal P-glycoprotein in rats.

This study was designed to assess intestinal drug transport via transcellular absorption and intestinal P-glycoprotein content following thermal injury in rats using propranolol as a marker substrate. Male, Sprague Dawley rats (n=30) underwent either a 30% total body surface area full thickness burn or sham treatment. Twenty-four hours later, animals were anesthetized, underwent laparotomy and the proximal jejunum was cannulated. The jejunal segment was perfused with buffer containing [3H] propranolol. Following euthanasia, jejunal tissue was harvested for Western immunoblotting of P-glycoprotein and villin, and immunohistochemical analysis of P-glycoprotein. Dramatic structural changes in jejunal integrity were observed following thermal injury; however, no significant differences in the absorption characteristics of propranolol following thermal injury were observed. Mean effective permeability of propranolol was 5.67+/-1.79 and 5.85+/-1.67cm/sx10(-5) for burn and sham groups, respectively (P>0.05). P-glycoprotein and villin content in the jejunum were significantly decreased in burn animals. The transcellular transport of propranolol is unaffected 24h following thermal injury in rats, despite alterations in intestinal P-glycoprotein content. The decrease in P-glycoprotein and villin content in thermally injured animals may reflect loss of mature enterocytes at the villus tips.

A P60 mutant of Listeria monocytogenes is impaired in its ability to cause infection in intragastrically inoculated mice.

A spontaneous P60 mutant of Listeria monocytogenes was less able to cause systemic infection in A/J mice, following intragastric inoculation, than the parental wild type strain (SLCC 5764, serotype 1/2a). Significantly fewer CFU were recovered from internal organs (spleen, liver, gall bladder) and from the cecum of mice inoculated intragastrically with the P60 mutant than mice inoculated with wild type L. monocytogenes. The P60 mutant also exhibited a diminished ability to invade and multiply within Caco-2 intestinal epithelial cells. These findings indicate that P60 is required for maximal virulence of L. monocytogenes in the gastrointestinal tract of mice.

Lactobacillus casei alters hPEPT1-mediated glycylsarcosine uptake in Caco-2 cells.

Augmentation of the normal flora of the gastrointestinal tract with probiotic bacteria is currently under investigation as a therapeutic tool for several diseases. However, it is unknown whether probiotic bacteria such as Lactobacillus casei alter the expression and function of intestinal transport proteins such as hPEPT1. The effects of 24 and 48 h incubation of Caco-2 cells with 10(8)/L L. casei on the hPEPT1-mediated uptake rate of 20 micro mol/L [(3)H]glycylsarcosine were examined. Dipeptide uptake did not differ from the control at 24 h (15.9 +/- 2.4 vs. 11.5 +/- 1.4 cm.s(-1).mg protein(-1)); however, a significant increase in uptake occurred after 48 h of L. casei treatment (23.7 +/- 1.5 vs. 12.0 +/- 1.9 cm.s(-1).mg protein(-1); P = 0.005). hPEPT1 involvement was confirmed in experiments using excess substrate. Increased uptake of [(3)H]glycylsarcosine appeared to be the result of the direct interaction of the bacteria with Caco-2 cells because conditioned medium had no effect on dipeptide uptake. hPEPT1 mRNA levels did not differ at any time point. These results show that prolonged incubation of Caco-2 cells leads to increased hPEPT1 activity and that this occurs by a mechanism distinct from increased gene expression.

Intestinal P glycoprotein acts as a natural defense mechanism against Listeria monocytogenes.

Mechanisms by which the intestinal epithelium resists invasion by food-borne pathogens such as Listeria monocytogenes are an evolving area of research. Intestinal P glycoprotein is well known to limit the absorption of xenobiotics and is believed to act as a cytotoxic defense mechanism. The aim of this study was to determine if intestinal P glycoprotein is involved in host defense against L. monocytogenes. Caco-2 cells and a P-glycoprotein-overexpressing subclone (Caco-2/MDR) were employed in addition to mdr1a(-/-) mice and wild-type controls. In vitro invasion assays and in vivo experiments were employed to measure bacterial invasion and dissemination. In addition, L. monocytogenes proteins were labeled with [(35)S]methionine, and the transepithelial transport across Caco-2 monolayers was characterized in both directions. Overexpression of P glycoprotein in Caco-2/MDR cells led to increased resistance to L. monocytogenes invasion, whereas P-glycoprotein inhibition led to increased invasion. Flux of [(35)S]methionine-labeled L. monocytogenes proteins was significantly greater in the basolateral-to-apical direction than in the apical-to-basolateral direction, indicating dependence on an apically located efflux transporter. Moreover, inhibiting P glycoprotein reduced the basolateral-to-apical flux of the proteins. Early dissemination of L. monocytogenes from the gastrointestinal tract was significantly greater in the mdr1a(-/-) mice than in wild-type controls. Expression and function of intestinal P glycoprotein is an important determinant in resistance to early invasion of L. monocytogenes.

Sodium pentobarbital anesthesia transiently enhances the severity of infection following intragastric, but not intravenous, inoculation of Listeria monocytogenes in mice.

In the present study we observed that mice anesthetized with sodium pentobarbital were far more susceptible to gastrointestinal challenge with Listeria monocytogenes than were unanaesthetized mice. The effect of pentobarbital anesthesia was transient (gone within 2 h) and did not alter the severity of infection following i.v. challenge with L. monocytogenes. Treatment with pharmacological inhibitors of gastric acidity (i.e. cimetidine and omeprazole), or intestinal motility (loperamide), did not duplicate the effect of pentobarbital on gastrointestinal listeriosis. These findings suggest that sodium pentobarbital anesthesia causes a short-lived but striking diminution in resistance to gastrointestinal listeriosis in mice, via an undefined mechanism.

Human jejunal permeability of cyclosporin A: influence of surfactants on P-glycoprotein efflux in Caco-2 cells.

PURPOSE: The purpose of this work was to determine the jejunal permeability of cyclosporin A (CsA) in humans and whether formulation variables modulate the effects of P-glycoprotein (P-gp) on the permeability of CsA in Caco-2 cells. METHODS: A solution containing CsA, phenylalanine, propranolol, polyethyleneglycol (PEG) 400, and PEG 4000 was perfused through a 10-cm jejunal segment in 12 subjects. Caco-2 transport studies were performed using previously reported methodology. RESULTS: The mean Peff (+/- SD) of CsA in humans was 1.65 (0.53). The mean permeabilities for phenylalanine, propranolol, and PEG 400 were 4.54 (2.39), 2.90 (1.28), and 0.83 (0.51) x 10(-4) cm/s, respectively. The presence of surfactants significantly decreased the permeabilities of CsA in both directions in Caco-2 cells. CONCLUSIONS: The results suggest that the effects of surfactants via micellar solubilization and inhibition of P-gp efflux on CsA transport in Caco-2 cells are significant. CsA can rightly be classified as a low solubility-high permeability Class II BCS drug and its highly variable absorption from Sandimmune oral formulations is the result of poor dissolution characteristics.