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 $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.

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.

Single-stage nipple-areolar complex reconstruction technique, outcomes, and patient satisfaction.

INTRODUCTION: Nipple-areolar reconstruction (NAR) is the final phase of breast reconstruction and is associated with increased patient satisfaction. Nipple-areolar reconstruction is typically performed in 2 separate stages, which include nipple reconstruction and tattooing of the nipple-areolar complex (NAC). Previous studies have demonstrated that increased duration of the reconstruction is associated with decreased patient satisfaction. Because a 2-stage reconstruction prolongs the reconstructive process, we introduce a simple and novel method of single-stage NAR (SS NAR), which combines the use of local flaps for nipple reconstruction and medical tattooing of the NAC in 1 session and delivers predictable outcomes with high patient satisfaction. METHODS: A retrospective chart review of patients who underwent SS NAR at our institution during the period from September 2010 to May 2012 was performed. Patient demographics, complications, outcomes, and overall patient satisfaction were assessed. A modified questionnaire (Likert scale) was used to assess patient satisfaction of nipple size, color, shape, and projection. RESULTS: Twenty-nine SS NARs were performed in 18 patients: 7 unilateral and 11 bilateral. Mean age was 45 years (range, 34-60 years). No major complications were identified. Mean length of follow-up was 10 months (range, 2-22 months). A 17% complication rate was observed: 14% (4/29) had irregular dye uptake of the areola, and 3% (1/29) had dehiscence from silicone guard pressure on the incision. Two patients underwent revisions: one patient underwent additional tattooing, and the other required flap readvancement and implant downsizing secondary to the wound dehiscence. We obtained a 70% survey response rate with 100% of responders who reported that they were "very satisfied" with NAC in each dimension. CONCLUSIONS: Our study demonstrates that SS NAR is a safe procedure with reproducible, excellent clinical results and very low complication rates or need for revisions. This method is cost-effective, convenient for the patient, and shortens patient recovery time with high patient satisfaction.

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.

I'm clear, you're clear, we're all clear: improving consultation communication skills in undergraduate medical education.

Requesting and providing consultations are daily occurrences in most teaching hospitals. With increased attention on transitions of care in light of the recent scrutiny of duty hours, consultations and other interphysician interactions, such as handoffs, are becoming increasingly important. As modern medicine increases in complexity, the skill of communicating with medical colleagues throughout the continuum of care becomes more challenging. Like many of the other skills acquired by medical students, consultation communication is often learned by casual observation and through trial and error. Without formal training, however, miscommunications will continue to occur, nearly ensuring that medical errors happen. Interphysician communication skills, therefore, need to be emphasized in undergraduate and graduate medical education instead of being left to happenstance or hit-or-miss practice. In this article, the authors review two models for understanding and teaching the consultation process--5Cs and PIQUED--both of which were developed for specific subsets of learners. They then combine the two to create a consultation model that may be more widely applied.

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.

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.

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.

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.

Regional gap junction inhibition increases defibrillation thresholds.

It is clear that ischemia inhibits successful defibrillation by altering regional electro-physiology. However, the exact mechanisms are unclear. This study investigated whether regional gap junction inhibition increases biphasic shock defibrillation thresholds (DFT). Sixteen swine were instrumented with a mid-left anterior descending (LAD) perfusion catheter for regional infusion of 0.5 mM/h heptanol (n = 8) or saline (n = 8). DFT values and effective refractory periods (ERP) at five myocardial sites were determined. Regional conduction velocity (CV) was determined in an LAD drug-perfused and nondrug-perfused region in an additional seven swine. Regional heptanol infusion increased 50% DFT values by 33% (P = 0.01) and slowed CV by 42-59% (P < 0.01) but did not affect ERP. Regional heptanol also increased CV dispersion by approximately 270% (P < 0.05) but did not change ERP dispersion. Regional placebo did not alter any of these parameters. Furthermore, regional heptanol infusion induced spontaneous ventricular fibrillation in eight of eight animals. Increasing spatial conduction velocity dispersion by impairing regional gap junction conductance increased DFT values. Dispersion in conduction velocity slowing during regional ischemia may be an important determinant of defibrillation efficacy.

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.