Apical Clearance Research Articles

Polarized SCAR and the Arp2/3 complex regulate apical cortical remodeling in asymmetrically dividing neuroblasts

Although the formin-nucleated actomyosin cortex has been shown to drive the changes in cell shape that accompany animal cell division in both symmetric and asymmetric cell divisions, the mitotic role of cortical Arp2/3-nucleated actin networks remain unclear. Here using asymmetrically dividing Drosophila neural stem cells as a model system, we identify a pool of membrane protrusions that form at the apical cortex of neuroblasts as they enter mitosis. Strikingly, these apically localized protrusions are enriched in SCAR, and depend on SCAR and Arp2/3 complexes for their formation. Because compromising SCAR or the Arp2/3 complex delays the apical clearance of Myosin II at the onset of anaphase and induces cortical instability at cytokinesis, these data point to a role for an apical branched actin filament network in fine-tuning the actomyosin cortex toenable the precise control of cell shape changes during an asymmetric cell division.

Rigid Gas-Permeable Semi-Scleral Contact Lenses after Radial Keratotomy: Apical Space, Lens Diameter, Limbal Clearance, Peripheral O-Rings, and Tear Exchange as Contact-Lens-Fitting Success Factors

The purpose of this case report is to verify if the adaptation of a rigid gas-permeable contact lens can improve VA and comfort in a patient with complications derived from radial keratotomy (RK) surgery. A semi-scleral contact lens was fitted in a 46-year-old female patient who had undergone bilateral RK before 30 years. The uncorrected distance visual acuity in the right eye (RE) and left eye (LE) was 0.5 and 0.6 logMAR (minimum angle resolution), respectively. The RE and LE manifest refractions were +2.00 − 4.25 × 155 diopters (D) and +2.00 − 3.00 × 15 D, respectively. A semi-scleral rigid gas-permeable Rose K2 XL™ lens was fitted in both eyes. The central clearance was 400–450 µm in the RE and 300–350 µm in the LE. The semi-scleral corrected distance visual acuity in the RE and LE was 0.1 and 0.16 logMAR, respectively. Semi-scleral contact lenses are comfortable and a beneficial choice for patients after RK when associated with visual problems and intolerance to other therapeutic options. Semi-scleral lenses permit prolonged duration of use without discomfort, adverse alterations, and improve the feeling of glare in scotopic vision. In the present case, there were multiple factors that influenced the resolution, such as the amount of apical space, lens diameter, limbal clearance, peripheral O-rings, and tear exchange.

The impact of hybrid contact lenses on keratoconus progression after accelerated transepithelial corneal cross-linking.

To investigate the effect of hybrid contact lenses (HCLs) on keratoconus (KCN) progression after accelerated transepithelial cross-linking (A-TE CXL). Thirty-five eyes of 26 patients who preferred Ultrahealth HCLs for an optical correction after A-TE CXL formed the study group, and 45 eyes of 34 patients who preferred spectacle correction were age- and sex-matched to form the control group. Corrected distance visual acuity (CDVA), maximum keratometry, mean keratometry, apical posterior keratometry, cylindrical power, minimum corneal thickness, keratoconus vertex indices and curvature asymmetry indices obtained by Scheimpflug corneal topography were compared before, 6 and 12months after the procedure. Anterior segment optic coherence tomography (AS-OCT) was performed to measure the apical corneal clearance of HCL-wearing patients. The median pre-CXL CDVA value of the patients in the HCL group was logMAR 0.30 (0.20-1.0), and it was logMAR 0.30 (0.10-1.0) in the spectacle-corrected group. There was a significant increase in CDVA 6 and 12months after CXL procedure in both groups (p < 0.001, 0.003, respectively). The median front curve asymmetry index (FCAsym) significantly improved after A-TE CXL in the HCL group. The pre-CXL and 12th-month topographic comparisons of the spectacle-corrected group revealed no significant difference. In addition, no significant difference was observed between topographic alterations of two groups (p > 0.05). The CDVA significantly improved, and KCN progression was halted in patients wearing HCL 12months after A-TE CXL. Besides, FCAsym indices can be considered for follow-up of the HCL-wearing patients as an assistive parameter to AS-OCT measurements.

Short-term changes in topometric indices after discontinuation of rigid gas permeable lens wear in keratoconic eyes.

Purpose:To estimate the time taken for topometric indices to stabilize in keratoconic corneas following cessation of habitual RGP contact lens wear.Methods:A total of 29 eyes of 20 patients, diagnosed with mild to severe keratoconus were included in this prospective observational study. All patients were experienced RGP contact lens wearers (either conventional RGP or Rose K2 lens) with each patient having used these lenses for at least a year. Corneal topography was performed immediately following discontinuation of habitual contact lens wear at baseline and each of four consecutive visits, 1-week apart.Results:An overall reduction in the keratometry and thickness values were noted on tomography immediately following cessation of contact lens wear and these indices increased significantly in the 1st week (P < 0.001). Consecutive visits following the first visit did not show any significant change in the topometric parameters (P > 0.05). Subgroup analysis revealed a similar trend in eyes with “severe” keratoconus and in eyes fitted with the “three-point touch” philosophy. However, eyes with “mild-moderate” keratoconus and those fitted with “apical clearance” fitting philosophy showed marginal differences even within the 1-week period of lens cessation.Conclusion:Maximum changes in keratometry and pachymetry values following discontinuation of RGP lens wear stabilize within the 1st week of cessation of rigid lens wear in a keratoconic cornea. These changes were more pronounced in patients with severe keratoconus and those with an apical bearing fit. This information would be useful for practitioners to assess the progression of keratoconus in RGP lens users before collagen cross-linking.

Scleral lens centration: The influence of centre thickness, scleral topography, and apical clearance

PurposeTo investigate the effect of lens centre thickness (and mass) upon short-term horizontal and vertical scleral lens decentration, and the association between both scleral topography and apical clearance, with lens decentration. MethodsLens decentration was measured using over-topography data from 9 healthy young participants (25 ± 4 years) with normal corneae fitted with ICD 16.5 scleral lenses (hexafocon B material) with centre thicknesses of 150, 250, and 350 μm, while controlling for other lens parameters. Scleral toricity and elevation were determined from sagittal height data over a 15 mm chord obtained from a corneo-scleral topographer and central apical clearance was quantified using anterior segment optical coherence tomography. ResultsThe mean lens decentration was 0.55 ± 0.19 mm temporally and 0.84 ± 0.35 mm inferiorly, which did not vary significantly with centre thickness (p > 0.05). The mean nasal-temporal asymmetry in scleral elevation data was substantially greater (619 ± 67 μm) compared to the vertical meridian (369 ± 57 μm) (p < 0.01), and this variation in scleral topography along the horizontal meridian was associated with the magnitude of horizontal lens decentration (r = 0.68, p = 0.04). Greater initial central apical clearance was associated with more inferior lens decentration (r = −0.78, p = 0.01). ConclusionLens centre thickness and mass did not significantly influence centration. Horizontal lens decentration was associated with the nasal-temporal asymmetry in scleral elevation, while vertical lens decentration correlated with initial central apical clearance. Factors affecting scleral lens centration may vary between the horizontal and vertical meridians.

Anterior segment optical coherence tomography angiography to evaluate the peripheral fitting of scleral contact lenses.

PurposeThe aim of this study was to show the potential applicability of optical coherence tomography angiography (OCTA) for the evaluation of the peripheral fitting of fully scleral contact lenses.MethodsA pilot study was proposed fitting three different scleral contact lenses (Irregular Corneal Design [ICD]) with different sagittal heights (4200, 4800, and 5600 mm) in a healthy volunteer of 27 years old. We evaluated by means of optical coherence tomography (OCT, DRI Triton) the apical clearance achieved with each of the three lenses fitted. The impact over scleral flow was assessed with the OCTA module of the same device.ResultsThe apical clearance was 310, 901, and 1680 µm with the scleral lenses of sagittal heights 4200, 4800, and 5600 µm, respectively. With OCTA, we evaluated the impact of the lens bearing on the conjunctival vascular flow, observing an area of vascular interruption of 0, 25, and 75% with the lenses of 4200, 4800, and 5600 µm of sagittal heights, respectively. The vascular interruption was induced in the perilimbar area, suggesting the need of readjusting the limbal clearance zone of the lens.ConclusionFully scleral contact lens fitting may be optimized with the use of OCTA, allowing the practitioner to perform the fitting with better control of the peripheral bearing of the lens on the conjunctival tissue, assessing the impact on vascular structures. This potential use of OCTA must be investigated further in future studies including large samples of eyes.

Influence of Apical Clearance on Mini-Scleral Lens Settling, Clinical Performance, and Corneal Thickness Changes.

The purpose of this work was to define the timing and magnitude of scleral contact lens settling, the factors influencing settling, and to examine whether the amount of apical clearance has an impact on clinical performance or is associated with hypoxia-induced corneal changes. Eleven patients (22 eyes) with keratoconus were fitted with a mini-scleral lens (Esclera, Brazil). Three different lenses with successively greater sagittal depths were applied to achieve 3 levels of initial apical clearance: 100 to 200 μm (low), 200 to 300 μm (medium), and greater than 300 μm (high). Corneal apical clearance was measured at 15 min, 1, 2, 4, 6, and 8 hr with optical coherence tomography (OCT). Central corneal thickness was measured with OCT and Pentacam, in the morning and immediately after removal of the contact lens. Mean settling was 26.8±18.8 μm (42.7%) at 1 hr, 39.5±26.5 μm (62.9%) at 2 hr, 50.7±31.6 μm (80.8%) at 4 hr, 57.4±34.6 μm (91.4%) at 6 hr, and 62.8±38.4 μm (100%) at 8 hr. Settling rate was significantly lower in low apical clearance group (P=0.01). The smaller diameter lenses settled more (P=0.03). There was a slight statistically significant increase of 1.3% in central corneal thickness measured with OCT (P=0.03). Central corneal thickness measured with Pentacam at three locations increased slightly (P=0.001). Settling showed significant intrasubject and intersubject variations. The amount of corneal swelling and comfort scores did not differ significantly according to apical clearance. The average amount of settling was 62.8 μm after 8 hr, 80% of which occurred during the first 4 hr. Slight corneal swelling (1.3%) occurred after 8 hr of wear.

Rasip1-Mediated Rho GTPase Signaling Regulates Blood Vessel Tubulogenesis via Nonmuscle Myosin II.

Vascular tubulogenesis is essential to cardiovascular development. Within initial vascular cords of endothelial cells, apical membranes are established and become cleared of cell-cell junctions, thereby allowing continuous central lumens to open. Rasip1 (Ras-interacting protein 1) is required for apical junction clearance, as well as for regulation of Rho GTPase (enzyme that hydrolyzes GTP) activity. However, it remains unknown how activities of different Rho GTPases are coordinated by Rasip1 to direct tubulogenesis. The aim of this study is to determine the mechanisms downstream of Rasip1 that drive vascular tubulogenesis. Using conditional mouse mutant models and pharmacological approaches, we dissect GTPase pathways downstream of Rasip1. We show that clearance of endothelial cell apical junctions during vascular tubulogenesis depends on Rasip1, as well as the GTPase Cdc42 (cell division control protein 42 homolog) and the kinase Pak4 (serine/threonine-protein kinase 4). Genetic deletion of Rasip1 or Cdc42, or inhibition of Pak4, all blocks endothelial cell tubulogenesis. By contrast, inactivation of RhoA (Ras homologue gene family member A) signaling leads to vessel overexpansion, implicating actomyosin contractility in control of lumen diameter. Interestingly, blocking activity of NMII (nonmuscle myosin II) either before, or after, lumen morphogenesis results in dramatically different tubulogenesis phenotypes, suggesting time-dependent roles. Rasip1 controls different pools of GTPases, which in turn regulate different pools of NMII to coordinate junction clearance (remodeling) and actomyosin contractility during vascular tubulogenesis. Rasip1 promotes activity of Cdc42 to activate Pak4, which in turn activates NMII, clearing apical junctions. Once lumens open, Rasip1 suppresses actomyosin contractility via inhibition of RhoA by Arhgap29, allowing controlled expansion of vessel lumens during embryonic growth. These findings elucidate the stepwise processes regulated by Rasip1 through downstream Rho GTPases and NMII.

Fitting of a New Design of Full Scleral Contact Lens in Advanced Keratoconus with Previous Implantation of Intracorneal Ring Segments

ABSTRACT This case report reports the visual rehabilitation obtained with the fitting of a new design of full scleral contact lens (ICD 16.5 contact lens, Paragon Vision Sciences, distributed by Lenticon, Madrid, Spain) in a cornea with advanced keratoconus and previous implantation of intracorneal ring segment with a very limited effect. This eye had a refraction of −3.00 × 55° cylinder, providing a visual acuity of 0.5 LogMAR. The topographic pattern was very irregular with the presence of a significant central protrusion and a significant central corneal thinning. Some previous unsuccessful fittings have been performed with corneal and corneal-scleral lenses. A comfortable wearing was achieved with a fully scleral contact lens of 4600 µm of sagittal height, optical power of −11.25 D, and providing an apical clearance of 196 µm. A visual acuity of 0.0 LogMAR combined with a relevant aberrometric improvement was achieved with this contact lens. The patient was completely satisfied with the fitting. The result was maintained during 1 year after the fitting. Full scleral lenses are then able to provide comfortable wear and a significant increase in visual acuity combined with a significant improvement in the visual quality in eyes with advanced keratoconus. How to cite this article Llorens DPP. Fitting of a New Design of Full Scleral Contact Lens in Advanced Keratoconus with Previous Implantation of Intracorneal Ring Segments. Int J Kerat Ect Cor Dis 2015;4(2):56-59.

Short-term corneal changes with gas-permeable contact lens wear in keratoconus subjects: A comparison of two fitting approaches

PurposeTo evaluate changes in anterior corneal topography and higher-order aberrations (HOA) after 14-days of rigid gas-permeable (RGP) contact lens (CL) wear in keratoconus subjects comparing two different fitting approaches. MethodsThirty-one keratoconus subjects (50 eyes) without previous history of CL wear were recruited for the study. Subjects were randomly fitted to either an apical-touch or three-point-touch fitting approach. The lens’ back optic zone radius (BOZR) was 0.4mm and 0.1mm flatter than the first definite apical clearance lens, respectively. Differences between the baseline and post-CL wear for steepest, flattest and average corneal power (ACP) readings, central corneal astigmatism (CCA), maximum tangential curvature (KTag), anterior corneal surface asphericity, anterior corneal surface HOA and thinnest corneal thickness measured with Pentacam were compared. ResultsA statistically significant flattening was found over time on the flattest and steepest simulated keratometry and ACP in apical-touch group (all p<0.01). A statistically significant reduction in KTag was found in both groups after contact lens wear (all p<0.05). Significant reduction was found over time in CCA (p=0.001) and anterior corneal asphericity in both groups (p<0.001). Thickness at the thinnest corneal point increased significantly after CL wear (p<0.0001). Coma-like and total HOA root mean square (RMS) error were significantly reduced following CL wearing in both fitting approaches (all p<0.05). ConclusionShort-term rigid gas-permeable CL wear flattens the anterior cornea, increases the thinnest corneal thickness and reduces anterior surface HOA in keratoconus subjects. Apical-touch was associated with greater corneal flattening in comparison to three-point-touch lens wear.

Spectral optical coherence tomography vs. fluorescein pattern for rigid gas-permeable lens fit.

BackgroundThis study aimed to evaluate anterior segment spectral optical coherence tomography (AS SOCT) for assessing the lens-to-cornea fit of rigid gas-permeable (RGP) lenses. The results were verified with the fluorescein pattern method, considered the criterion standard for RGP lens alignment evaluations.Material/MethodsTwenty-six eyes of 14 patients were enrolled in the study. Initial base curve radius (BCR) of each RGP lens was determined on the basis of keratometry readings. The fluorescein pattern and AS SOCT tomograms were evaluated, starting with an alignment fit, and subsequently, with BCR reductions in increments of 0.1 mm, up to 3 consecutive changes. AS SOCT examination was performed with the use of RTVue (Optovue, California, USA).ResultsThe average BCR for alignment fits, defined according to the fluorescein pattern, was 7.8 mm (SD=0.26). Repeatability of the measurements was 18.2%. BCR reductions of 0.1, 0.2, and 0.3 mm resulted in average apical clearances detected with AS SOCT of 12.38 (SD=9.91, p<0.05), 28.79 (SD=15.39, p<0.05), and 33.25 (SD=10.60, p>0.05), respectively.ConclusionsBCR steepening of 0.1 mm or more led to measurable changes in lens-to-cornea fits. Although AS SOCT represents a new method of assessing lens-to-cornea fit, apical clearance detection with current commercial technology showed lower sensitivity than the fluorescein pattern assessment.

Innovative in Vitro Method To Predict Rate and Extent of Drug Delivery to the Brain across the Blood–Brain Barrier

The relevant parameters for predicting rate and extent of access across the blood-brain barrier (BBB) are fu,plasma (unbound fraction in plasma), Vu,brain (distribution volume in brain) and Kp,uu,brain (ratio of free concentrations in plasma and brain). Their estimation still requires animal studies and in vitro low throughput experiments which make difficult the screening of new CNS candidates. The aim of the present work was to develop a new whole in vitro high throughput method to predict drug rate and extent of access across the BBB. The system permits estimation of fu,plasma, Vu,brain and Kp,uu,brain in a single experimental system, using in vitro cell monolayers in different conditions. From the ratios of the apparent permeability values (Papp) with the adequate mathematical analysis the relevant parameters can be estimated. Papp of ten model compounds has been obtained in MDCKII and MDCK-Mdr1cell monolayers in the absence and presence of albumin and brain homogenate. The ratio of Papp in the absence and presence of albumin allows estimation of in vitro fu,plasma. Papp in the presence of brain homogenate is used to estimate fu,brain and Vu,brain. Kp,uu,brain is estimated from the apical to basal versus basal to apical clearances. The BBB parameters obtained with the new method were predictive of the in vivo behavior of candidates. In vitro fu,plasma, Kp,uu,brain and Vu,brain (calculated with Papp from MDCKII cell line) presented a good correlation with in vivo fu,plasma, Kp,uu,CSF and Vu,brain published values (r=0.92; r=0.85; and r=0.99 respectively). Despite its simplicity the predictive performance is fairly good considering the reduced number of tested compounds with different physicochemical and transport properties. Further experimental modifications could be checked to optimize the method, but the present data support its feasibility. As other in vitro cell culture models, the system is suitable for miniaturization and robotization to allow high throughput screening of CNS candidates.

An Assessment of the Optimal Lens Fit Rate in Keratoconus Subjects Using Three-Point-Touch and Apical Touch Fitting Approaches With the Rose K2 Lens

To compare the number of ordered lenses required to achieve an optimal lens fit between three-point-touch and apical touch fittings in keratoconus subjects with nipple and oval cones using the first definite apical clearance lens (FDACL) as a starting point. First trial lens was selected following manufacturer's guidelines and the back optic zone radius (BOZR) was flattened or steepened in 0.10 mm steps until a FDACL was found. Subsequently, subjects were randomly allocated to three-point-touch (BOZR 0.10 mm flatter than FDACL) and apical touch fittings (BOZR 0.40 mm flatter than FDACL). Changes were made solely in total lens diameter, edge lift, and/or back vertex power until an optimal lens fit was achieved. Differences between fitting approaches were compared in terms of optimal lens fit rates defined as the percentage of subjects successfully fitted with the first lens ordered from the manufacturer. Sixty-one subjects (109 eyes) were randomly allocated to three-point-touch and apical touch fitting approaches. An average of 2.3 trial lenses were necessary to achieve the FDACL. An optimal lens fit rate was achieved with the first lens in 84 of 109 eyes (77%). No statistically significant differences in optimal lens fit rates were found between three-point-touch and apical touch fitting approaches (83% and 71%, respectively; P = 0.12) and between nipple and oval cones (81% and 68%, respectively; P = 0.12). The use of the FDACL provides a systematic, reliable, and repeatable starting point for Rose K2 contact lens fitting in keratoconus. Eye care practitioners should anticipate higher optimal lens fit rates when using three-point-touch (83%) in comparison with apical touch contact lens fittings (71%).

Apical Pressure and Extent of Irrigant Flow beyond the Needle Tip during Positive-pressure Irrigation in an In Vitro Root Canal Model

IntroductionThis study aimed to measure the pressure generated during positive-pressure irrigation at the periapex of an in vitro tooth model using a novel method of measurement, investigating the effect of flow rate and needle design. Apical pressure was correlated with the extent of dye clearance from the end of a needle tip in a plastic root canal model with similar dimensions. MethodsThe mesiobuccal canal of a mandibular molar was instrumented to #35/06 and placed into a chamber coupled to a pressure transducer. Irrigation was performed using a digital peristaltic pump using flow rates from 1–15 mL/min with irrigation needles of different sizes and designs. A plastic root canal model instrumented to the same size filled with dye was used to measure the extent of dye clearance beyond the needle tip using the same irrigation conditions. ResultsPositive-pressure irrigation revealed a flow rate–dependent increase in apical pressure (P < .05). The apical pressure at high irrigation flow rates was several times higher than at low flow rates. Needle designs with safety features yielded statistically significant lower apical pressures than needles without safety features (P < .05). There was no further increase in dye clearance from the end of the needle tip in a plastic root canal model at flow rates higher than 4 mL/min. ConclusionsIf apical clearance of dye beyond the needle tip is a measure of irrigation effectiveness, then maximum effectiveness with safe apical pressures can be gained at specific flow rates using specific needle tip designs. The use of an irrigation flow rate of 4 mL/min was able to achieve maximum effectiveness in this study.

Neurulation: coordinating cell polarisation and lumen formation

EMBO J (2013) 32 1, 30–44 doi:10.1038/emboj.2012.305; published online November302012 Cell polarisation in development is a common and fundamental process underlying embryo patterning and morphogenesis, and has been extensively studied over the past years. Our current knowledge of cell polarisation in development is predominantly based on studies that have analysed polarisation of single cells, such as eggs, or cellular aggregates with a stable polarising interface, such as cultured epithelial cells (St Johnston and Ahringer, 2010). However, in embryonic development, particularly of vertebrates, cell polarisation processes often encompass large numbers of cells that are placed within moving and proliferating tissues, and undergo mesenchymal-to-epithelial transitions with a highly complex spatiotemporal choreography. How such intricate cell polarisation processes in embryonic development are achieved has only started to be analysed. By using live imaging of neurulation in the transparent zebrafish embryo, Buckley et al (2012) now describe a novel polarisation strategy by which cells assemble an apical domain in the part of their cell body that intersects with the midline of the forming neural rod. This mechanism, along with the previously described mirror-symmetric divisions (Tawk et al, 2007), is thought to trigger formation of both neural rod midline and lumen.

Use of Physiologically Based Pharmacokinetic Models to Evaluate the Impact of Intestinal Glucuronide Hydrolysis on the Pharmacokinetics of Aglycone

Drug elimination via glucuronidation pathway is a complex process involving glucuronide excretion. Glucuronide excreted into the gut lumen either directly from the enterocytes or from the hepatobiliary route can be recovered back to the precursor (aglycone) through bacteria-mediated hydrolysis. As a result, the pharmacokinetics [e.g., plasma terminal half-life (T(1/2))] of aglycone might be altered. Here, impact of intestinal glucuronide hydrolysis on the pharmacokinetics of aglycone is evaluated using physiologically based pharmacokinetic (PBPK) models with liver and/or intestine as eliminating organs. It is found that compared with its absence, the presence of intestinal glucuronide hydrolysis leads to increases in the oral systemic bioavailability (F(sys)) of aglycone. The magnitude of fold increase is positively correlated with the level of metabolism, as metabolic clearance mainly contributes to recycled amount of glucuronide. Although F(sys) is independent of the glucuronide efflux in a traditional model and a segregated-flow model of the intestine, dependence of F(sys) on the glucuronide efflux can be observed in a segmental segregated-flow model of the intestine and whole-body PBPK models. Interestingly, when the ratio of apical versus basolateral efflux intrinsic clearances (of glucuronide) is fixed, their effects on the intestinal bioavailability and F(sys) cease to exist. In addition, glucuronide hydrolysis can lead to a significantly delayed elimination of the aglycone as evidenced by a prolonged (e.g., a 2.1-fold increase) T(1/2). Surprisingly, when a pharmacokinetic profile for aglycone is simulated with a flat terminal portion (a reflection of the experimental observations), changes in the aglycone bioavailabilities are limited (i.e., ≤ 1.3-fold). In conclusion, this study explores the possible role of intestinal glucuronide hydrolysis in the disposition of aglycone via simulations utilizing various PBPK models. The mechanistic observations should be helpful to better understand the complex glucuronidation in vivo.

Models to Predict Unbound Intracellular Drug Concentrations in the Presence of Transporters

Knowledge of free drug intracellular concentration is necessary to predict the impacts of drugs on intracellular targets. The goal of this study was to develop models to predict free intracellular drug concentrations in the presence of apical efflux transporters. The apical efflux transporter P-glycoprotein (P-gp), encoded by human gene multidrug resistance 1 (MDR1), was studied. Apparent permeabilities for 10 compounds in Madin-Darby canine kidney (MDCK) and MDR1-MDCK cell monolayers were obtained experimentally. Six of these compounds were evaluated additionally in the presence of the P-gp inhibitor cyclosporine A. A three-compartment model was developed, and passive and apical efflux clearances (CL(d) and CL(ae), respectively) were estimated. Endogenous canine transporters also were delineated. The three-compartment model was unable to simulate experimentally observed lag times and exhibited systematic bias across the simulations. Next, a five-compartment model with explicit membrane compartments was developed. This model resulted in lower systematic errors and simulated the lag time observed experimentally. Apical efflux was modeled out of the cell or out of the membrane. The five-compartment model with apical efflux out of the membrane predicted marked differences in unbound intracellular concentrations between the apical-to-basolateral and the basolateral-to-apical directions. Upon apical drug addition, large decreases in intracellular concentrations were observed with the efflux transporter. No such difference was predicted upon basolateral drug addition. This is consistent with experimental differences in the impact of P-gp on hepatic and brain distribution and supports the hypothesis that apical efflux occurs out of the apical membrane.

Fluorescein Pattern Interpretation in Keratoconus

The purpose of this study is to assess the effect of disease severity on how accurately contact lens fluorescein patterns can be interpreted in keratoconus by clinician assessment. Two clinicians evaluated fluorescein patterns on 111 eyes of 60 patients with mild (<45 D, 14 eyes), moderate (45 D to 52 D, 61 eyes,) and severe (>52 D, 36 eyes) keratoconus. The masked clinicians were given six contact lenses in random order, the lens that just cleared the corneal apex (the first definite apical clearance lens), three lenses flatter (in 0.1 mm increments), and two lenses steeper (in 0.1 mm increments) than the first definite apical clearance lens. They ranked the lenses from flattest to steepest, based on the fluorescein patterns. The percentage of lenses correctly ranked was determined using (1) exact match with actual; (2) within 0.1 mm of actual; and (3) within 0.2 mm of actual. Accuracy was assessed as the sum of the squared differences between the actual base curve value and each clinician's ranking. Comparison of the mean percentage correctly ranked and accuracy for each keratoconus severity groups was performed using a mixed linear model. Neither percentage correctly ranked (using any of the three protocols) nor accuracy was found to be related to severity of keratoconus (p > 0.15 for all comparisons). Accuracy of ranking contact lenses in order of base curve radius based on fluorescein pattern assessment by clinicians does not seem to be related to severity of keratoconus. Many factors influence interpretation of fluorescein patterns including all components of the system, fluorescein, tears, cornea, contact lens, external forces, and technique.

Directional Transcellular Transport of Bisoprolol in P-glycoprotein-expressed LLC-GA5-COL150 Cells but not in Renal Epithelial LLC-PK1 Cells

To evaluate the mechanism responsible for the tubular secretion of bisoprolol, we compared transcellular transport of bisoprolol with that of tetraethylammonium (TEA), cimetidine, and quinidine across LLC-PK1 cell monolayers grown on porous membrane filters. TEA and cimetidine were actively transported in the basolateral-to-apical direction by the specific transport system. Pharmacokinetic analysis indicated that basolateral influx and apical efflux were cooperatively responsible for the directional transport of TEA and cimetidine. Lipophilic cationic drugs, quinidine, S-nicotine, and bisoprolol, significantly diminished basolateral influx and apical efflux clearance of cimetidine. However, transcellular transport of quinidine in the basolateral-to-apical direction was similar to that in the opposite direction in LLC-PK1 cells. In contrast, quinidine was transported actively in the basolateral-to-apical direction in P-glycoprotein-expressed LLC-GA5-COL150 cells. Pharmacokinetic analysis indicated that P-glycoprotein increased the apical efflux of quinidine and also decreased the apical influx of the drug. Basolateral-to-apical transport of bisoprolol was also similar to apical-to-basolateral transport in LLC-PK1 cells, whereas the drug was directionally transported from the basolateral to the apical side in LLC-GA5-COL150 cells. These results suggested that bisoprolol was not significantly transported via transport systems involved in the directional transport of TEA and cimetidine, but that P-glycoprotein was responsible for the directional transport of bisoprolol as well as quinidine in renal epithelial cells.

Resolvin E1 promotes mucosal surface clearance of neutrophils: a new paradigm for inflammatory resolution

Migration of neutrophils (PMN) across epithelia is a pathological hallmark of numerous mucosal diseases. Whereas lesions at mucosal surfaces are generally self-limiting, endogenous mechanisms of resolution are incompletely understood. Previous studies revealed that resolvins directly act on PMN to attenuate transendothelial migration, less is known about the influence of resolvins on PMN-epithelial interactions and whether they act on epithelia. We studied the dynamics of resolvin E1 (RvE1) actions on leukocyte transepithelial migration. PMN exposure to RvE1 or chemerin (peptide agonist of ChemR23) reduced transepithelial migration in a concentration-dependent manner. Conversely, activation of epithelial ChemR23 promoted apical clearance of PMN. A nonbiased screen of known PMN ligands expressed on epithelial cells in response to RvE1 revealed selective induction of CD55, an apically expressed antiadhesive molecule. CD55 promoter analysis demonstrated that both RvE1 and chemerin activate the CD55 promoter. Inhibition of CD55 by neutralizing antibody prevented RvE1-dependent augmentation of apical PMN clearance. Taken together these findings implicate a "two-hit" model of inflammatory resolution, whereby activation of the PMN RvE1 receptor attenuates transepithelial migration and subsequent actions on the epithelium promote CD55-dependent clearance of PMN across the epithelial cell surface promoting active inflammatory resolution.