Simple sample processing enhances malaria rapid diagnostic test performance.

Lateral flow immunochromatographic rapid diagnostic tests (RDTs) are the primary form of medical diagnostic used for malaria in underdeveloped nations. Unfortunately, many of these tests do not detect asymptomatic malaria carriers. In order for eradication of the disease to be achieved, this problem must be solved. In this study, we demonstrate enhancement in the performance of six RDT brands when a simple sample-processing step is added to the front of the diagnostic process. Greater than a 4-fold RDT signal enhancement was observed as a result of the sample processing step. This lowered the limit of detection for RDT brands to submicroscopic parasitemias. For the best performing RDTs the limits of detection were found to be as low as 3 parasites per µL. Finally, through individual donor samples, the correlations between donor source, WHO panel detection scores and RDT signal intensities were explored.

Quadruplex priming amplification for the detection of mRNA from surrogate patient samples.

Simple and rapid methods for detecting mRNA biomarkers from patient samples are valuable in settings with limited access to laboratory resources. In this report, we describe the development and evaluation of a self-contained assay to extract and quantify mRNA biomarkers from complex samples using a novel nucleic acid-based molecular sensor called quadruplex priming amplification (QPA). QPA is a simple and robust isothermal nucleic acid amplification method that exploits the stability of the G-quadruplex nucleotide structure to drive spontaneous strand melting from a specific DNA template sequence. Quantification of mRNA was enabled by integrating QPA with a magnetic bead-based extraction method using an mRNA-QPA interface reagent. The assay was found to maintain >90% of the maximum signal over a 4 °C range of operational temperatures (64-68 °C). QPA had a dynamic range spanning four orders of magnitude, with a limit of detection of ~20 pM template molecules using a highly controlled heating and optical system and a limit of detection of ~250 pM using a less optimal water bath and plate reader. These results demonstrate that this integrated approach has potential as a simple and effective mRNA biomarker extraction and detection assay for use in limited resource settings.

Bronchial bifurcations and respiratory mass transport.

A new transport mechanism explains the importance of the shape of bronchial bifurcations in the transfer of gases and particles between the atmosphere and the alveoli. Photographs of flow visualization experiments illustrate the effect in models of bronchial branching. The mechanism provides a means of nondiffusional transport that helps to explain normal respiratory exchange of gases as well as successful ventilation with very low tidal volumes, as in some lung diseases and in the high-frequency panting of dogs.

Mitosis enhances transgene expression of plasmid delivered by cationic liposomes.

A critical requirement of gene therapy is expression of the delivered transgene. Transgene expression is facilitated by access to the transcription mechanism found primarily in the nucleus. Factors modulating the interactions between intracellular plasmid and nuclear access are not well understood. In this study, the effect of mitosis on transgene expression was examined by quantitative flow cytometry. Transfection of HeLa cells synchronized at late G1 phase or G2/M phase was performed using a liposomal vector containing 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and dioleoyl-phosphatidylethanolamine (DOPE) (1:1 mol/mol). Cell samples were transfected and subsequently maintained in G1 phase for various durations to modulate the time between plasmid entry and mitosis. The plasmid contains the sequence for a mutated green fluorescent protein (GFP(S65T)) that was used to examine transgene expression. Ethidium monoazide-labeled plasmid was employed to examine the association of plasmid with the cell membrane. The percentage of cells expressing GFP(S65T) increased sharply as the synchronized cell population passed through M phase, suggesting that an event associated with mitosis is essential for transgene expression. Expression levels of the transgene then declined 18 h after mitosis irrespective of transfection strategy. All transfection strategies resulted in the same maximum percentage of GFP(S65T) positive cells (40%) and average GFP(S65T) expression level (3.14x106 molecules per positive cell). Association of plasmid with the cell membrane at late G1 phase was 1.5-fold of that at G2/M phase. These data are evidence for control of transgene expression triggered by events associated with cell cycle.

Glucose-induced increase in paracellular permeability and disruption of beta-receptor signaling in retinal endothelium.

PURPOSE: To examine the effects of high glucose concentrations on retinal endothelial permeability in an in vitro model of the retinal microvasculature. METHODS: The permeability of the endothelial barrier to small solutes was measured in a chromatographic cell column consisting of bovine retinal endothelial cells cultured on porous fibronectin-coated microcarriers. In each cell column, permeability changes were evaluated by comparing the treatment permeability response over time with the initial baseline permeability. Short-term (2-hour) barrier effects of glucose were examined by measuring permeability at 15-minute intervals after an increase in perfusate concentration from baseline (5.5 mM) to high (25 mM) glucose. Long-term (to 57 days) effects were tested by addition of 25 mM glucose to microcarrier cultures. The effect of glucose on beta-receptor signaling was tested by measuring its effect on the permeability decrease produced by 1 microM isoproterenol. RESULTS: An increase from 5.5 mM to 25 mM glucose concentration did not change retinal endothelial cell monolayer permeability (n=6) during 2 hours. However, an increase in monolayer permeability was observed after 19 days (n=8) in the 25-mM glucose culture. Paralleling this time course, short-term exposure to 25 mM glucose did not prevent a decrease in permeability triggered by the beta-receptor agonist isoproterenol. However, the permeability effect of the agonist was blocked by long-term culture in 25 mM glucose. Permeability of retinal endothelial monolayers cultured in 5.5 mM glucose and treated with 1 microM isoproterenol decreased significantly to 0.71+/-0.06 of baseline (n=4; mean+/-SEM). However, permeability did not change in parallel cell columns made from microcarriers cultured in 25 mM glucose (0.97+/-0.2 of baseline permeability; n=4; mean+/-SEM). CONCLUSIONS: High-glucose culture decreases the retinal endothelial barrier and blocks the response to beta-adrenergic receptors. This model may prove valuable in exploring other hypotheses of increased permeability associated with diabetic retinopathy or other retinal diseases that break down the retinal vascular barrier.

Partial characterization of the human retinal endothelial cell tight and adherens junction complexes.

PURPOSE: In diabetic retinopathy and macular edema, the blood-retinal barrier fails to function properly, and there is transvascular leakage of proteins and solutes. The tight junction protein occludin and the adherens junction protein cadherin-5 have been shown to be critical to maintaining the endothelial barrier and regulating paracellular transport of large vessel endothelia. However, the expression and distribution of these junction proteins in the retinal endothelium is not well characterized. METHODS: Human and bovine retinal endothelial cells were isolated as described previously. Western blot analysis and flow cytometry techniques were used to assay for the presence of occludin, zonula occludens-1 (ZO-1), cadherin-5, and beta-catenin. The subcellular localization of the proteins was visualized by immunohistochemistry performed on cultured human retinal endothelial cells and cryosections of bovine retina. RESULTS: Western blot analysis and flow cytometry techniques found occludin, ZO-1, cadherin-5, and beta-catenin in cultured human retinal endothelial cells. Immunofluorescence staining of cultured retinal endothelial cells and cryosections of bovine retina showed junctional localization of occludin, ZO-1, cadherin-5, and beta-catenin. CONCLUSIONS: This report demonstrates the expression of occludin and cadherin-5 in retinal endothelial cells and their localization to sites of cell-cell contact. Expression of their respective regulatory proteins, ZO-1 and beta-catenin, at sites of cell-cell contact suggests that occludin and cadherin-5 play a role in maintaining the retinal endothelial barrier.

Convective exchange between the nose and the atmosphere.

It is generally accepted that there is little rebreathing of gas exhaled through the nose. A detailed physical model system has been used to quantify and identify the mechanisms responsible for this phenomenon. By the use of a cast of the upper respiratory tract and oscillating flows with a Reynolds number of 500 and nondimensional frequency of 1.6, corresponding to quiet tidal breathing through the nose, dye dilution measurements indicated an efficiency of tidal exchange of 0.95. Flow visualization studies performed to trace the expiratory flow, as well as the streamlines during steady inspiratory flow, support the hypothesis that the high efficiency of exchange is due to radical differences in the velocity fields between inspiratory and expiratory phases of this oscillatory flow. These findings confirm that convective gas exchange between the nose and the atmosphere is highly efficient; however, the underlying mechanism responsible for this exchange also maximizes the exposure of the respiratory system to aerosols contained in the ambient atmosphere.

Adenosine decreases permeability of in vitro endothelial monolayers.

The addition of adenosine to perfusates flowing through an in vitro cell-column model of the vasculature decreases the permeability of cell column fetal bovine aortic endothelial monolayers. At 10(-4) M adenosine, cell monolayer permeability to the paracellular tracers polyethylene glycol (mol wt 900) and cyanocobalamin (mol wt 1,355) is significantly decreased within 15 min. In continuous treatments with adenosine for up to 30 min, the permeability reduction is maintained, and removal of adenosine returns permeability to baseline levels within 15 min. The effect of adenosine is concentration dependent, with a 5% reduction in permeability with 10(-6) M adenosine, a 21% reduction with 10(-5) M adenosine, and a 37% reduction with 10(-4) M adenosine. The effects of adenosine are not blocked by the adenosine transport inhibitor dipyridamole. Permeability is significantly reduced by the A2-specific adenosine analogue 5'-(N-cyclopropyl)-carboxamidoadenosine but not by the A1-specific adenosine analogue N6-(L-2-phenylisopropyl)adenosine. In addition, the permeability decrease is blocked by the A2-receptor antagonist 8-phenyltheophylline (10(-5)M). We conclude that adenosine decreases the permeability of bovine fetal aortic endothelial monolayers via endothelial A2-purinoceptors.

Use of scaling theory to relate measurements of lung endothelial barrier permeability.

This work examined the relationships between lung microvascular permeability-surface area products (PS) for small solutes in animals of different size and for columns of endothelial-covered microcarrier beads. We assembled PS data (humans, sheep, lambs, and rabbits) for labeled sucrose, mannitol, urea, 1,2-propanediol, 1,3-propanediol, and 1,4-butanediol. In addition, PS for cell columns using sucrose, mannitol, and sodium fluorescein were evaluated. A new mathematical model for the analysis of cell columns that accounts for transit time variations was derived and compared with models neglecting this variation. Allometric relationships between PS and body weight or exchange surface (S) were examined. Permeability relative to diffusivity (P/D) correlated inversely with S for all animals. In addition, P/D for the cell columns fell near this regression line. The results suggest either that permeability for hydrophilic tracers is higher for smaller animals or that the indicator-dilution measurement is a fractal process dependent on scale. Furthermore, the P/D-S correlations may help relate cell column experiments to animal studies.

Perilla ketone increases endothelial cell monolayer permeability in vitro.

Perilla ketone (PK) is a potent lung toxin that causes increased microvascular permeability pulmonary edema in grazing animals. Because the mechanism of action of PK is not know, we investigated whether PK directly affects endothelial cells. Bovine aortic endothelial cells were grown to confluence on Cytodex-3 microcarrier beads and placed in a chromatographic cell column. Monolayer permeability was evaluated from the elution profiles of three optical tracers: blue dextran (2 x 10(6) mol wt), sodium fluorescein (NaF, 342 mol wt), and cyanocobalamin (B12, 1,355 mol wt). Perfusion with 1.2 mM PK increased permeability within 15 min to NaF and B12 by 51 +/- 6 and 54 +/- 11%, respectively. Permeability returned to baseline after PK removal. These in vitro results suggest that PK produces a rapid and reversible increase in endothelial permeability directly. Staining of fixed cells with rhodamine-phalloidin revealed a major disruption of actin microfilaments after PK treatment. Because previous reports suggested that PK may be activated via cytochrome P-450, we attempted to block this using the cytochrome P-450 inhibitor ketoconazole. Ketoconazole alone did not significantly affect permeability, and the combination of PK and ketoconazole resulted in permeability increases similar to those measured for PK alone. This suggests that PK may not require cytochrome P-450 to increase vascular permeability.

Effect of perfusate hematocrit on urea permeability-surface area in isolated dog lung.

Seven dog lower left lung lobes were statically inflated and perfused at a constant rate for each lobe with a perfusate in which the hematocrit was altered over a wide range. The permeability-surface area of urea was calculated from multiple indicator dilution curves using two separate injectates for each hematocrit level. One injectate contained only 125I-albumin as the vascular reference tracer and the other contained both 51Cr-erythrocytes and 125I-albumin as the vascular reference tracers; both contained [14C]urea as the permeating tracer. The results strongly indicate that the phenomenon of "erythrocyte trapping" of urea does not affect the calculation of urea permeability-surface area product provided the appropriate albumin-erythrocyte composite reference tracer is utilized in its calculation.

Chromatographic demonstration of reversible changes in endothelial permeability.

This report describes a new in vitro method for measuring the diffusional permeability of an endothelial monolayer and its use in investigating the modulation of permeability by various agents, e.g., isoproterenol, propranolol, dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP), and cytochalasin D. To determine permeability, tracers of different molecular weights were applied simultaneously on a chromatography column containing confluent endothelial cells cultured on porous microcarrier beads. The Sangren-Sheppard model was used to determine the permeability of the endothelial monolayer from the tracer elution profiles. For six radiolabeled tracers the mean (+/- SD) permeabilities (cm/s x 10(-5)) in order of increasing tracer molecular weight were [3H]water, 82.0 +/- 28.8; [14C]urea, 49.5 +/- 9.5; [14C]mannitol, 13.3 +/- 4.7; [14C]-sucrose, 14.1 +/- 2.5; [3H]polyethylene glycol (900 mol wt), 4.80 +/- 1.61; and [3H]polyethylene glycol (4,000 mol wt), 1.97 +/- 1.01. These permeabilities deviate less from in vivo values than those obtained in other in vitro systems and are 10 times higher than in vivo estimates. The values were reproducible for up to the 4 h tested. Modulation of endothelial monolayer permeability was studied in a separate series of experiments. The beta-adrenergic agonist isoproterenol (10(-6) M) decreased the permeability to mannitol by 36% and to polyethylene glycol (900 mol wt) by 49%; in both instances the decrease in permeability was reversed by propranolol. Propranolol alone had no effect. Dibutyryl cAMP (10(-3) M) decreased the permeability to mannitol by 40% and to polyethylene glycol by 47%; permeability returned to base line when dibutyryl cAMP was removed. Cytochalasin D (1 microgram/ml) increased permeability by 350% for mannitol and 380% for polyethylene glycol; the permeability change was reversed after removal of cytochalasin D. The results indicate that cell-column chromatography is a powerful method that can be used to characterize the permeability of endothelial monolayers and to investigate permeability changes produced by various agents.

Permeability characteristics of cultured endothelial cell monolayers.

The purpose of this study was to characterize the permeability characteristics of an in vitro endothelial cell monolayer system and relate this information to available in vivo data. We cultured bovine fetal aortic endothelial cells on fibronectin-coated polycarbonate filters and confirmed that our system was similar to others in the literature with regard to morphological appearance, transendothelial electrical resistance, and the permeability coefficient for albumin. We then compared our system with in vivo endothelium by studying the movement of neutral and negatively charged radiolabeled dextran tracers across the monolayer and by using electron microscopy to follow the pathways taken by native ferritin. There were a number of differences. The permeability of our monolayer was 10-100 times greater than seen in intact endothelium, there was no evidence of "restricted" diffusion or charge selectivity, and ferritin was able to move freely into the subendothelial space. The reason for these differences appeared to be small (0.5-2.0 micron) gaps between 5 and 10% of the endothelial cells. Although the current use of cultured endothelial cells on porous supports may provide useful information about the interaction of macromolecules with the endothelium, there appear to be differences in the transendothelial permeability characteristics of these models and in vivo blood vessels.

Passage of uncharged dextrans from blood to lung lymph in awake sheep.

To examine how molecular size alone influences the passage of macromolecules from the pulmonary microcirculation into lymph collected from the caudal mediastinal lymph node of the sheep, we infused polydisperse uncharged [3H]dextrans intravenously at a constant rate over a period of 7.5 h in nine awake sheep with lung lymph fistulas. Lymph and plasma were collected during hours 5.5-7.5 of the infusions, and the [3H]dextrans were separated by molecular sieve chromatography into fractions that ranged from 1.6 to 8.4 nm in effective molecular (Stokes-Einstein) radius. Lymph-to-plasma (L/P) ratios for [3H]dextrans were near 1.0 at 1.6-nm radius, decreased with increasing molecular size, and approached zero at radii above 5.0 nm. We confirmed that these L/P ratios represented steady-state values by extending the duration of the infusion to approximately 30 h in two of the nine sheep and finding that the L/P ratios remained unchanged. These results were consistent with molecular sieving through a homoporous membrane with cylindrical pores of 5.0-nm radius. We also found that the L/P ratio for albumin [0.76 +/- 0.13 (SE)] in five of the same sheep was much higher than that for the [3H]dextran fraction of the same effective molecular radius [0.11 +/- 0.02 (SE)]. These results suggest that the movement of macromolecules from the pulmonary microcirculation into pulmonary lymph collected from the caudal mediastinal node of the sheep is influenced by both molecular size and molecular charge and that, compared with uncharged dextrans, the steady-state passage of anionic endogenous proteins from plasma to lymph is enhanced.

Reduced expression of the adherens junction protein cadherin-5 in a diabetic retina.

PURPOSE: Transvascular leakage occurs in diabetic retinopathy. The tight junction proteins occludin and zonula occludens-1 (ZO-1) and adherens junction protein cadherin-5 are critical to the maintenance of endothelial barrier. We report a comparison of junction protein expression in the normal and diabetic retina. METHOD: Case report. Postmortem retinal cryosections were prepared from the left eye of a 73-year-old woman with diabetic retinopathy. Cryosections were immunostained for cadherin-5, occludin, and ZO-1 and compared with retinal cryosections from the right eye of a 72-year-old man with no progression of retinal disease. RESULTS: Immunofluorescence showed positive retinal vessel staining for occludin and ZO-1 in both eyes and cadherin-5 in the normal eye but reduced cadherin-5 staining in the retinal vessels of the diabetic eye. CONCLUSION: Increases in transvascular leakage observed in diabetic retinal vasculature may be associated with reduction in the expression of the critical adherens junction protein, cadherin-5.

The role of cadherin endocytosis in endothelial barrier regulation: involvement of protein kinase C and actin-cadherin interactions.

We have previously reported that exposure of endothelial monolayers to low (0.12 mM) extracellular calcium significantly decreased the endothelial solute barrier, and that this effect was reversed by restoring 'normal' (1.2 mM) calcium (1). This effect was shown to be dependent on cadherins, however the molecular mechanisms through which barrier was altered by low calcium were not characterized. Here we investigated the mechanism of increased endothelial permeability produced by low calcium exposure. Endothelial permeability was significantly increased by exposure to low (0.12 mM) calcium; this effect was attenuated by pre-treatment with the protein kinase C (PKC) inhibitor, staurosporine (2 x 10(-7) M) for 30 min. Cell border retraction and gap formation produced by low calcium was also prevented by staurosporine. Treatment of monolayers with 0.12 mM calcium also stimulated the endocytosis of endothelial cadherins. This low calcium mediated cadherin endocytosis was also prevented by pretreatment with staurosporine. Low calcium mediated endocytosis was also prevented by the actin filament toxin, cytochalasin D (1 ug/ml, 30 min). We conclude that the mechanism of low calcium mediated loss of endothelial barrier function is mediated in part by a PKC dependent endocytosis of endothelial cadherins, which may involve interactions with the actin cytoskeleton. Physiological regulation of the in vivo endothelial barrier may also involve PKC dependent-actin mediated endocytosis of cadherin junctional elements.

Platelets and a platelet-released factor enhance endothelial barrier.

The role of platelets in the maintenance of endothelial barrier is examined in an in vitro model of the microvasculature. Human platelets (6,000/microliters) perfused through a cell column of endothelial-covered microcarriers decrease paracellular permeability of sodium fluorescein (mol wt 342) to 63% of baseline values. This effect is reversible and a second application and removal of platelets produces a similar response. This effect occurs within 5 min and reverses within 10 min after platelet removal. The reduction in permeability is not due to mechanical obstruction of endothelial junctions, since the number of recirculating platelets is not reduced and releasate from unstimulated 2-h platelet incubations also decreases permeability. Releasate from platelets stimulated with 0.1 U/ml of thrombin for 15 min have the same permeability reducing effect. In this system, the platelet factors serotonin (10(-3) M) and ADP (10(-4) M) have no effect on permeability. However, the platelet factors adenosine (10(-4) M), ATP (10(-5) M), and beta-agonists decrease permeability. None of these appear to account for platelet permeability activity, since activity is not blocked by agents directed against these mediators (adenosine deaminase, apyrase, 8-phenyltheophylline, or propranolol). The active factor(s) is stable at -20 degrees C, heat stable, sensitive to trypsin, and has an apparent molecular weight > 100. We conclude that unstimulated platelets release a factor(s) that enhances endothelial barrier in vitro and may be important in maintenance of the normal in vivo barrier.

Role of cadherins 5 and 13 in the aortic endothelial barrier.

We investigated the role of the cadherins 5 and 13 in the solute barrier formed by aortic endothelial cells in vitro. In confluent monolayers of bovine aortic endothelial cells, immunofluorescence with antibodies to the external domain of cadherin 5 (Mab 9H7) or to cadherin 13 (Mab Ec6C10) found staining for both cadherins at endothelial cell borders. Western blotting with an antibody to the characteristic cadherin cytoplasmic tail or with an antibody to the extracellular domain of cadherin 5 revealed a single 125 kD protein band. A second larger band was found at 130 kD with the anti-cadherin 13 Mab which was not recognized by an antibody to the cadherin cytoplasmic tail. A calcium switch strategy was used to investigate the involvement of these cadherins in the endothelial barrier. Changes in the permeability of small solutes in an endothelial cell column produced by a decrease in calcium concentration followed by a return to normal calcium, with or without antibody, were recorded. We found that anti-cadherin 5 IgG (10 micrograms/ml) interfered with the reforming of interendothelial junctions after restoration of calcium at every time point tested for a total of 45 min after restoration of calcium. The anti-cadherin 13 IgG (10 micrograms/ml) did not block reforming of the endothelial barrier in a similar manner. The presence of this antibody delayed only by 15 min the restoration of the normal barrier. Without calcium switch, addition of either monoclonal antibody (10 micrograms/ml) to the endothelial cell column had no effect on solute permeability. These results suggest that cadherin 5 in bovine aortic endothelial cells has a major functional role in forming the calcium-sensitive endothelial junction in vitro and may play an important role in the normal structure and function of the in vivo barrier.

Convective exchange in oscillatory flow through bronchial-tree models.

An important and previously unappreciated longitudinal convective-transport mechanism has been shown to operate in tube-bifurcation models geometrically and dynamically similar to upper and lower regions of the bronchial tree. The mechanism depends on the differences in shape that exist between inspiratory and expiratory velocity profiles in laminar and turbulent flow within the bronchial tubes and provides new and deeper physical insight into the flow of gases, aerosol particles, and heat in the airways. By use of dimensional analysis to relate the flows in the physical models to those in the real bronchial tree, the mechanism is shown to be important in normal breathing of gases and aerosols and is likely to be important also in high-frequency ventilation.

An N-cadherin-like protein contributes to solute barrier maintenance in cultured endothelium.

We investigated the role of cadherins in the solute barrier maintained by endothelial cells in vitro. Cell-column chromatographic measurement of endothelial barrier showed that reducing normal extracellular calcium from 1.2 to 0.12 mM increased endothelial permeability to 250% of baseline after 20 min. Restoring normal calcium restored the barrier within 15 min which remained stable for at least 60 min. We used sulfo-NHS-biotin and anti-cadherin antibodies to characterize endothelial proteins with possible roles in the maintenance of endothelial barrier. The non-specific probe sulfo-NHS-biotin identified at least ten endothelial cell surface proteins, with greatest labelling occurring at molecular weights of 125 and 145 kD. Six proteins, including the 125 and 145 kD proteins, associated with the cytoskeleton. Western blotting for the presence of classical cadherins containing the conserved cytoplasmic sequence CDPTAPPYDSLLVFDYEG detected two bands at 145 and 125 kD which associated with the cytoskeleton. Western blotting with an antibody, which recognizes FHLRAHAVDINGNQV, an extracellular homotypic binding region of N-cadherin, detects three bands. Of these three, one protein had a molecular weight of 125 kD and was associated with the cytoskeleton. Immunofluorescence with both N-cadherin and anti-peptide 1 antibodies found staining at endothelial cell borders. The utility of a newly developed cell-column calcium switch assay was tested by verifying the functional role of the previously described epithelial cadherin, uvomorulin, in epithelial barrier. We then applied this method to endothelial cell columns and found the N-cadherin antibody interfered with the reforming of interendothelial junctions. These results suggest that, as in epithelial cells, cadherins in bovine endothelial cells have a functional role in forming the calcium sensitive endothelial junction and may play an important role in the formation of normal barrier.