Jennifer M. Loeb

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

ABSTRACT 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 [35S]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 [35S]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.

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.

HIV Viral RNA Extraction in Wax Immiscible Filtration Assisted by Surface Tension (IFAST) Devices

The monitoring of viral load is critical for proper management of antiretroviral therapy for HIV-positive patients. Unfortunately, in the developing world, significant economic and geographical barriers exist, limiting access to this test. The complexity of current viral load assays makes them expensive and their access limited to advanced facilities. We attempted to address these limitations by replacing conventional RNA extraction, one of the essential processes in viral load quantitation, with a simplified technique known as immiscible filtration assisted by surface tension (IFAST). Furthermore, these devices were produced via the embossing of wax, enabling local populations to produce and dispose of their own devices with minimal training or infrastructure, potentially reducing the total assay cost. In addition, IFAST can be used to reduce cold chain dependence during transportation. Viral RNA extracted from raw samples stored at 37°C for 1 week exhibited nearly complete degradation. However, IFAST-purified RNA could be stored at 37°C for 1 week without significant loss. These data suggest that RNA isolated at the point of care (eg, in a rural clinic) via IFAST could be shipped to a central laboratory for quantitative RT-PCR without a cold chain. Using this technology, we have demonstrated accurate and repeatable measurements of viral load on samples with as low as 50 copies per milliliter of sample.

Activation of the κ-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.

Nucleic Acid Sample Preparation using Spontaneous Biphasic Plug Flow

Nucleic acid (NA) extraction and purification has become a common technique in both research and clinical laboratories. Current methods require repetitive wash steps with a pipet that are laborious and time-consuming, making the procedure inefficient for clinical settings. We present here a simple technique that relies on spontaneous biphasic plug flow inside a capillary to achieve sample preparation. By filling the sample with oil, aqueous contaminants were displaced from the captured NA without pipetting wash buffers or use of external force and equipment. mRNA from mammalian cell culture was purified, and polymerase chain reaction (PCR) amplification showed similar threshold cycle values as those obtained from a commercially available kit. Human immunodeficiency virus (HIV) viral-like particles were spiked into serum and a 5-fold increase in viral RNA extraction yield was achieved compared to the conventional wash method. In addition, viral RNA was successfully purified from human whole blood, and a limit of detection of approximately 14 copies of RNA extracted per sample was determined. The results demonstrate the utility of the current technique for nucleic acid purification for clinical purposes, and the overall approach provides a potential method to implement nucleic acid testing in low-resource settings.

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.

Using Exclusion-Based Sample Preparation (ESP) to Reduce Viral Load Assay Cost

Viral load (VL) measurements are critical to the proper management of HIV in developing countries. However, access to VL assays is limited by the high cost and complexity of existing assays. While there is a need for low cost VL assays, performance must not be compromised. Thus, new assays must be validated on metrics of limit of detection (LOD), accuracy, and dynamic range. Patient plasma samples from the Joint Clinical Research Centre in Uganda were de-identified and measured using both an existing VL assay (Abbott RealTime HIV-1) and our assay, which combines low cost reagents with a simplified method of RNA isolation termed Exclusion-Based Sample Preparation (ESP).71 patient samples with VLs ranging from <40 to >3,000,000 copies/mL were used to compare the two methods. We demonstrated equivalent LOD (~50 copies/mL) and high accuracy (average difference between methods of 0.08 log, R2 = 0.97). Using expenditures from this trial, we estimate that the cost of the reagents and consumables for this assay to be approximately

Regional gap junction inhibition increases defibrillation thresholds

5 USD. As cost is a significant barrier to implementation of VL testing, we anticipate that our assay will enhance access to this critical monitoring test in developing countries.