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Darby D Miller,1 Syed A Hasan,1 Nathaniel L Simmons,2 Michael W Stewart1 1Department of Ophthalmology, Mayo Clinic, Jacksonville, FL 32224, USA; 2Department of Ophthalmology, University of Rochester, Rochester, NY 14642, USA Purpose: To comprehensively review the literature regarding recurrent corneal erosion (RCE) and to present treatment options and recommendations for management.Overview: RCE usually presents with sharp, unilateral pain upon awakening, in an eye with an underlying basement membrane dystrophy, prior ocular trauma, stromal dystrophy or degeneration, or prior surgery for refractive errors, cataracts, or corneal transplantation. Making the correct diagnosis requires a careful slit-lamp examination of both eyes coupled with a high degree of suspicion. Several treatments are commonly used for RCE but new therapies have been introduced recently. Conservative treatment consists of antibiotic and preservative-free lubricating drops, with topical cycloplegics and oral analgesics to control pain. Patients who are unresponsive to these therapies may benefit from therapeutic bandage contact lenses (BCL). Newer therapies include oral matrix metalloproteinase (MMP) inhibitors, blood-derived eye drops, amniotic membrane graft application, and judicious application of topical corticosteroids. Once the epithelium is healed, a course of hypertonic saline solution and/or ointment can be used. Surgical procedures may be performed in patients who fail conservative therapy. Punctal occlusion with plugs increases the tear film volume. Epithelial debridement with diamond burr polishing (DBP), anterior stromal puncture (ASP), or alcohol delamination should be considered in selected patients. DBP can be used for patients with basement membrane dystrophies and is the preferred treatment overall due to a low recurrence rate. ASP can be used for erosions outside the central visual axis. Excimer laser phototherapeutic keratectomy is an attractive option in eyes with central RCE since it precisely removes tissue while preserving corneal transparency. In patients with RCE who are also candidates for refractive surgery, photorefractive keratectomy can be considered.Summary: Newly introduced therapies for RCE enable therapy to be individualized and lower the recurrence rate. Keywords: recurrent corneal erosion, anterior basement membrane dystrophy, map-dot-fingerprint dystrophy, epithelial basement membrane dystrophy, corneal abrasion

Purpose: To highlight the novel application of anterior segment optical coherence tomography (AS-OCT) to detect corneal silver deposition in a case of ocular argyrosis., Methods: This is a case report and review of the literature., Results: A 67-year-old man with a 30-year history of chronic occupational exposure to silver-halides secondary to photographic film manufacturing presented with significant ocular argyrosis. His ophthalmic examination was notable for bilateral, widespread pinpoint gray deposits throughout his conjunctiva and cornea at the level of Descemet membrane. Further evaluation with AS-OCT showed deposition in 2 distinct, diffusely hyperreflective bands, corresponding to Bowman layer and Descemet membrane., Conclusions: AS-OCT provides a useful imaging modality in evaluating corneal deposition diseases. In this case of ocular argyrosis, AS-OCT led to the detection of a hyperreflective Bowman layer and Descemet membrane, representing silver deposition in both these anatomic locations.

Efficient nanomaterials for artificial photosynthesis require fast and robust unidirectional electron transfer (ET) from photosensitizers through charge-separation and accumulation units to redox-active catalytic sites. We explored the ultrafast time-scale limits of photo-induced charge transfer between a Ru(II)tris(bipyridine) derivative photosensitizer and PpcA, a 3-heme c-type cytochrome serving as a nanoscale biological wire. Four covalent attachment sites (K28C, K29C, K52C, and G53C) were engineered in PpcA enabling site-specific covalent labeling with expected donor-acceptor (DA) distances of 4-8 Å. X-ray scattering results demonstrated that mutations and chemical labeling did not disrupt the structure of the proteins. Time-resolved spectroscopy revealed three orders of magnitude difference in charge transfer rates for the systems with otherwise similar DA distances and the same number of covalent bonds separating donors and acceptors. All-atom molecular dynamics simulations provided additional insight into the structure-function requirements for ultrafast charge transfer and the requirement of van der Waals contact between aromatic atoms of photosensitizers and hemes in order to observe sub-nanosecond ET. This work demonstrates opportunities to utilize multi-heme c-cytochromes as frameworks for designing ultrafast light-driven ET into charge-accumulating biohybrid model systems, and ultimately for mimicking the photosynthetic paradigm of efficiently coupling ultrafast, light-driven electron transfer chemistry to multi-step catalysis within small, experimentally versatile photosynthetic biohybrid assemblies.

We analyzed clinical and histopathologic data of 97 pediatric patients who underwent excision of dermoid cysts. On review, 16.5% of the sample population demonstrated localized chronic inflammatory changes, including the presence of giant cells and epithelial disruption. These features were considered indicative of prior cyst rupture. Age at time of initial presentation was significantly older and cyst size was significantly larger in patients with histopathologic signs of previous rupture. Longer time to presentation and time to excision were associated with increased odds of spontaneous rupture., (Copyright © 2019 American Association for Pediatric Ophthalmology and Strabismus. Published by Elsevier Inc. All rights reserved.)

Retrobulbar injection of anesthesia is one of the most common procedures performed for ophthalmic surgery. Complications are rare but can be potentially serious, including retrobulbar hemorrhage, brainstem anesthesia, and inadvertent globe perforation. This is the preliminary report describing branch retinal vein occlusion (BRVO) secondary to accidental retrobulbar needle laceration with subsequent preretinal neovascularization., (Copyright 2016, SLACK Incorporated.)

A series of novel marinopyrroles with sulfide and sulphone spacers were designed and synthesized. Their activity to disrupt the binding of the pro-apoptotic protein, Bim, to the pro-survival proteins, Mcl-1 and Bcl-xL, was evaluated using ELISA assays. Fluorescence-quenching (FQ) assays confirmed the direct binding of marinopyrroles to Mcl-1. Benzyl- and benzyl methoxy-containing sulfide derivatives 4 and 5 were highly potent dual Mcl-1/Bim and Bcl-xL/Bim disruptors (IC50 values of 600 and 700 nM), whereas carboxylate-containing sulfide derivative 9 exhibited 16.4-fold more selectivity for disrupting Mcl-1/Bim over Bcl-xL/Bim binding. In addition, a nonsymmetrical marinopyrrole 12 is as equally potent as the parent marinopyrrole A (1) for disrupting both Mcl-1/Bim and Bcl-xL/Bim binding. Some of the derivatives were also active in intact human breast cancer cells where they reduced the levels of Mcl-1, induced programd cell death (apoptosis) and inhibited cell proliferation.

Nephronophthisis (NPHP) is the major cause of pediatric renal failure, yet the disease remains poorly understood, partly due to the lack of appropriate animal models. Joubert syndrome (JBTS) is an inherited ciliopathy giving rise to NPHP with cerebellar vermis aplasia and retinal degeneration. Among patients with JBTS and a cerebello-oculo-renal phenotype, mutations in CEP290 (NPHP6) are the most common genetic lesion. We present a Cep290 gene trap mouse model of JBTS that displays the kidney, eye, and brain abnormalities that define the syndrome. Mutant mice present with cystic kidney disease as neonates. Newborn kidneys contain normal amounts of lymphoid enhancer-binding factor 1 (Lef1) and transcription factor 1 (Tcf1) protein, indicating normal function of the Wnt signaling pathway; however, an increase in the protein Gli3 repressor reveals abnormal Hedgehog (Hh) signaling evident in newborn kidneys. Collecting duct cells from mutant mice have abnormal primary cilia and are unable to form spheroid structures in vitro. Treatment of mutant cells with the Hh agonist purmorphamine restored normal spheroid formation. Renal epithelial cells from a JBTS patient with CEP290 mutations showed similar impairments to spheroid formation that could also be partially rescued by exogenous stimulation of Hh signaling. These data implicate abnormal Hh signaling as the cause of NPHP and suggest that Hh agonists may be exploited therapeutically.

Rumen fermentation of plant-based forage in bovines is the major site for generation and absorption of short-chain fatty acids. Consequentially, the rumen is also the site for initial exposure to toxins released from diet. Accordingly, we have investigated the expression of bovine ABC transporters in the rumen associated with cytoprotection against xenobiotic exposure, namely MDR1 (ABCB1), MRP2 (ABCC2) and BCRP (ABCG2). Bovine rumen samples from the ventral sac were obtained post-mortem from a commercial slaughterhouse after humane killing. Rumen papilla samples were then prepared for total RNA isolation for RT-PCR, SDS-PAGE/Western blotting and immunohistochemistry. PCR products of the predicted size were observed for both MDR1 and BCRP, but not for MRP2 using bovine-specific primers. β-actin was used as a control transcript. Western blot analysis using C219 primary monoclonal antibody revealed MDR1 protein expression in bovine rumen (Mapp, of ~170-180 kD). Immunolocalisation of MDR1 using UIC2 monoclonal antibody within cryosections of bovine rumen showed extensive membrane staining in the cells of the stratum granulosum, stratum spinosum and stratum basale. MDR1 expression was absent from outer stratum corneum. Protein expression and immunolocalisation were also confirmed for BCRP, with prevalent staining in the stratum basale, becoming weaker in the stratum spinosum and stratum granulosum.

A series of novel cyclic marinopyrroles were designed and synthesized. Their activity to disrupt the binding of the pro-apoptotic protein, Bim, to the pro-survival proteins, Mcl-1 and Bcl-x(L), was evaluated using ELISA assays. Both atropisomers of marinopyrrole A (1) show similar potency. A tetrabromo congener 9 is two-fold more potent than 1. Two novel cyclic marinopyrroles (3 and 4) are two- to seven-fold more potent than 1.

The failure of the adenovirus serotype 5 (Ad5) vector-based human immunodeficiency virus type 1 (HIV-1) vaccine in the STEP study has led to the development of adenovirus vectors derived from alternative serotypes, such as Ad26, Ad35, and Ad48. We have recently demonstrated that vaccines using alternative-serotype Ad vectors confer partial protection against stringent simian immunodeficiency virus (SIV) challenges in rhesus monkeys. However, phenotypic differences between the T cell responses elicited by Ad5 and those of alternative-serotype Ad vectors remain unexplored. Here, we report the magnitude, phenotype, functionality, and recall capacity of memory T cell responses elicited in mice by Ad5, Ad26, Ad35, and Ad48 vectors expressing lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP). Our data demonstrate that memory T cells elicited by Ad5 vectors were high in magnitude but exhibited functional exhaustion and decreased anamnestic potential following secondary antigen challenge compared to Ad26, Ad35, and Ad48 vectors. These data suggest that vaccination with alternative-serotype Ad vectors offers substantial immunological advantages over Ad5 vectors, in addition to circumventing high baseline Ad5-specific neutralizing antibody titers.

A global HIV-1 vaccine will likely need to induce immune responses against conserved HIV-1 regions to contend with the profound genetic diversity of HIV-1. Here we evaluated the capacity of immunogens consisting of only highly conserved HIV-1 sequences that are aimed at focusing cellular immune responses on these potentially critical regions. We assessed in rhesus monkeys the breadth and magnitude of T lymphocyte responses elicited by adenovirus vectors expressing either full-length HIV-1 Gag/Pol/Env immunogens or concatenated immunogens consisting of only highly conserved HIV-1 sequences. Surprisingly, we found that the full-length immunogens induced comparable breadth (P = 1.0) and greater magnitude (P = 0.01) of CD8(+) T lymphocyte responses against conserved HIV-1 regions compared with the conserved-region-only immunogens. Moreover, the full-length immunogens induced a 5-fold increased total breadth of HIV-1-specific T lymphocyte responses compared with the conserved-region-only immunogens (P = 0.007). These results suggest that full-length HIV-1 immunogens elicit a substantially increased magnitude and breadth of cellular immune responses compared with conserved-region-only HIV-1 immunogens, including greater magnitude and comparable breadth of responses against conserved sequences.

In order to determine the capacity and regulation of the breast cancer resistance protein (BCRP)-mediated transport in intact human intestinal epithelial monolayers (Caco-2) in which multiple ABC transporters are expressed, nitrofurantoin has been used as a selective transported substrate. Nitrofurantoin transepithelial secretion was confirmed in both human BCRP and mouse bcrp-transfected MDCKII epithelia, whereas no net transepithelial secretion was observed in native or human MDR1-MDCKII epithelia. Furthermore, nitrofurantoin transepithelial secretion by BCRP-MDCKII monolayers was inhibited by Ko143 (10 μM), but not verapamil (100 μM). In Caco-2 cells grown upon permeable supports, nitrofurantoin displayed a dose-dependent transepithelial secretion with an apparent Km=69.41 ± 22.3 μM and Vmax=14.03 ± 2.27 nmol/(cm(2).h). Net nitrofurantoin transepithelial secretion by Caco-2 epithelia was inhibited 92% by 10 μM Ko143. Regulation of expression and function of BCRP in Caco-2 epithelial monolayers was determined after 72-h pre-exposure of the monolayers to a number of potential inducing agents. Quantitative real-time PCR and Western blotting were used to correlate induction of BCRP transcript and protein levels with transport activity. 72-h pre-treatment with β-napthoflavone and rosiglitazone up-regulates BCRP mRNA and protein expression and transport of nitrofurantoin. Ko143-sensitive transepithelial secretion of the bi-substrate (MDR1/BCRP) prazosin was also increased in the presence of rosiglitazone. We conclude that nitrofurantoin may be used to unambiguously measure BCRP-mediated fluxes in Caco-2 epithelial layers. Since dynamic regulation of BCRP expression and function is retained, the Caco-2 cell-line is useful as a screen for drug-drug and drug-diet interactions mediated by BCRP., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Intestinal secretory movement of the fluoroquinolone antibiotic, ciprofloxacin, may limit its oral bioavailability. Active ATP-binding cassette (ABC) transporters such as breast cancer resistance protein (BCRP) have been implicated in ciprofloxacin transport. The aim of this study was to test the hypothesis that BCRP alone mediates intestinal ciprofloxacin secretion. The involvement of ABC transport proteins in ciprofloxacin secretory flux was investigated with the combined use of transfected cell lines [bcrp1/BCRP-Madin-Darby canine kidney II (MDCKII) and multidrug resistance-related protein 4 (MRP4)-human embryonic kidney (HEK) 293] and human intestinal Caco-2 cells, combined with pharmacological inhibition using 3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6, 7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)-propionic acid tert-butyl ester (Ko143), cyclosporine, 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK571), and verapamil as ABC-selective inhibitors. In addition, the regional variation in secretory capacity was investigated using male Han Wistar rat intestine mounted in Ussing chambers, and the first indicative measurements of ciprofloxacin transport by ex vivo human jejunum were made. Active, Ko143-sensitive ciprofloxacin secretion was observed in bcrp1-MDCKII cell layers, but in low-passage (BCRP-expressing) Caco-2 cell layers only a 54% fraction was Ko143-sensitive. Ciprofloxacin accumulation was lower in MRP4-HEK293 cells than in the parent line, indicating that ciprofloxacin is also a substrate for this transporter. Ciprofloxacin secretion by Caco-2 cell layers was not inhibited by MK571. Secretory flux showed marked regional variability in the rat intestine, increasing from the duodenum to peak in the ileum. Ciprofloxacin secretion was present in human jejunum and was reduced by Ko143 but showed marked interindividual variability. Ciprofloxacin is a substrate for human and rodent BCRP. An additional pathway for ciprofloxacin secretion exists in Caco-2 cells, which is unlikely to be MRP(4)-mediated. BCRP is likely to be the dominant transport mechanism for ciprofloxacin efflux in both rat and human jejunum.

Vitamin A deficiency is highly prevalent in much of the developing world, where vaccination programs are of paramount importance to public health. However, the impact of vitamin A deficiency on the immunogenicity and protective efficacy of vaccines has not been defined previously. In this article, we show that the vitamin A metabolite retinoic acid is critical for trafficking of vaccine-elicited T lymphocytes to the gastrointestinal mucosa and for vaccine protective efficacy in mice. Moderate vitamin A deficiency abrogated Ag-specific T lymphocyte trafficking to the gastrointestinal tract, gastrointestinal cellular immune responses, and protection against a mucosal challenge following immunization with a recombinant adenovirus vaccine vector. Oral vitamin A supplementation as well as retinoic acid administration fully restored the mucosal immune responses and vaccine protective efficacy. These data suggest that oral vitamin A supplementation may be important for optimizing the success of vaccines against HIV-1 and other mucosal pathogens in the developing world, highlighting a critical relationship between host nutritional status and vaccine efficacy.

The development of an enantioselective allylic alcohol dichlorination catalyzed by dimeric cinchona alkaloid derivatives and employing aryl iododichlorides as chlorine sources is reported. Reaction optimization, exploration of the substrate scope, and a model for stereoinduction are presented., (© 2011 American Chemical Society)

A five-step total synthesis of the antibiotic marinopyrrole A (1) is described. The developed synthetic technology enabled the synthesis of several marinopyrrole A analogues whose antibacterial properties against methicillin-resistant Staphylococcus aureus TCH1516 were evaluated.

The innate immune pathways that contribute to the potent immunogenicity of recombinant adenovirus (rAd) vaccine vectors remain largely undefined. Previous studies assessing innate immunity triggered by vaccine vectors have largely focused on in vitro studies involving antigen-presenting cells and on early in vivo inflammatory responses. Here, we systematically explore the Toll-like receptor (TLR) signaling requirements for the generation of cellular immune responses by intramuscular immunization with common and alternative serotype rAd vectors in mice. Antigen-specific CD8(+) T-lymphocyte responses elicited by these rAd vectors were significantly diminished in MyD88(-/-) mice but not in TRIF(-/-) or TLR3(-/-) mice, suggesting the importance of MyD88-dependent TLR signaling. However, the absence of each individual TLR resulted in minimal to no effect on vaccine-elicited cellular immune responses. Moreover, responses were not diminished in IL-1R(-/-) or IL-18R(-/-) mice. These data suggest that rAd vectors engage multiple MyD88-dependent signaling pathways, none of which are individually critical; rather, they are integrated to contribute to the potent immunogenicity of rAd vectors. Stimulation of multiple innate immune mechanisms may prove a generalizable property of potent vaccines, and this strategy could be harnessed in the development of next-generation vaccine vectors and adjuvants.

Toll-like receptor (TLR) ligands are critical activators of innate immunity and are being developed as vaccine adjuvants. However, their utility in conjunction with viral vector-based vaccines remains unclear. In this study, we evaluated the impact of a variety of TLR ligands on antigen-specific CD8(+) T lymphocyte responses elicited by a recombinant adenovirus serotype 26 (rAd26) vector expressing simian immunodeficiency virus Gag in mice. The TLR3 ligand poly(I:C) suppressed Gag-specific cellular immune responses, whereas the TLR4 ligands lipopolysaccharide and monophosphoryl lipid A substantially augmented the magnitude and functionality of these responses by a MyD88- and TRIF-dependent mechanism. These data demonstrate that TLR ligands can modulate the immunogenicity of viral vaccine vectors both positively and negatively. Moreover, these findings suggest the potential utility of TLR4 ligands as adjuvants for rAd vector-based vaccines.

Topical zinc applications promote wound healing and epithelialization. "Leaky" MDCKII epithelia exposed to apical ZnCl₂ (10 mM) showed a time-dependent increase (t (0.5) 22.2 ± 2.7 min) of transepithelial resistance (R (t)) from 82.3 ± 2.4 Ω cm² to 1,551 ± 225.6 Ω cm²; the increase was dose-dependent, being observed at 3 mM but not at 1 mM. Basal Zn²+ applications also increased epithelial resistance (at 10 mM to 323 ± 225.6 Ω cm²). The linear current-voltage relationship in control epithelia changed after apical 10 mM ZnCl₂ to show rectification. Voltage deflections resulting from inward currents showed time-dependent relaxation (basal potential difference (p.d.)-positive), with outward currents being time-independent. Cation selectivity was tested after apical ZnCl₂ elevated resistance; both the NaCl:mannitol (basal replacement) dilution p.d. and the choline:Na bi-ionic p.d. decreased (P(Na)/P(Cl) from 4.9 to 2.3 and P(Na)/P(choline) from 3.8 to 2.1, respectively). Transepithelial paracellular basal to apical ⁴⁵Ca fluxes increased approximately twofold when driven by a basal positive Na:NMDG bi-ionic p.d., but with basal 10 mM ZnCl₂, ⁴⁵Ca fluxes decreased approximately twofold. Neither ZO-1 nor occludin distribution was altered after ~2-h exposure to apical 10 mM ZnCl₂. However, claudin-2, though present at the tight junction, increased within the cell. Increased epithelial barrier resistance by Zn²+ is due to modification of the paracellular pathway, most probably by multiple mechanisms.

Candidate HIV-1 vaccine regimens utilizing intramuscularly (i.m.) administered recombinant adenovirus (rAd)-based vectors can induce potent mucosal cellular immunity. However, the degree to which mucosal rAd vaccine routing might alter the quality and anatomic distribution of vaccine-elicited CD8(+) T lymphocytes remains unclear. We show that the route of vaccination critically impacts not only the magnitude but also the phenotype and trafficking of antigen-specific CD8(+) T lymphocytes in mice. I.m. rAd immunization induced robust local transgene expression and elicited high-frequency, polyfunctional CD8(+) T lymphocytes that trafficked broadly to both systemic and mucosal compartments. In contrast, intranasal (i.n.) rAd immunization led to similarly robust local transgene expression but generated low-frequency, monofunctional CD8(+) T lymphocytes with restricted anatomic trafficking patterns. Respiratory rAd immunization elicited systemic and mucosal CD8(+) T lymphocytes with phenotypes and trafficking properties distinct from those elicited by i.m. or i.n. rAd immunization. Our findings indicate that the anatomic microenvironment of antigen expression critically impacts the phenotype and trafficking of antigen-specific CD8(+) T lymphocytes.

Although mucosal CD8(+) T-cell responses are important in combating mucosal infections, the generation of such immune responses by vaccination remains problematic. In the present study, we evaluated the ability of plasmid DNA to induce local and systemic antigen-specific CD8(+) T-cell responses after pulmonary administration. We show that the pulmonary delivery of plasmid DNA formulated with polyethyleneimine (PEI-DNA) induced robust systemic CD8(+) T-cell responses that were comparable in magnitude to those generated by intramuscular (i.m.) immunization. Most importantly, we observed that the pulmonary delivery of PEI-DNA elicited a 10-fold-greater antigen-specific CD8(+) T-cell response in lungs and draining lymph nodes of mice than that of i.m. immunization. The functional evaluation of these pulmonary CD8(+) T cells revealed that they produced type I cytokines, and pulmonary immunization with PEI-DNA induced lung-associated antigen-specific CD4(+) T cells that produced higher levels of interleukin-2 than those induced by i.m. immunization. Pulmonary PEI-DNA immunization also induced CD8(+) T-cell responses in the gut and vaginal mucosa. Finally, pulmonary, but not i.m., plasmid DNA vaccination protected mice from a lethal recombinant vaccinia virus challenge. These findings suggest that pulmonary PEI-DNA immunization might be a useful approach for immunizing against pulmonary pathogens and might also protect against infections initiated at other mucosal sites.

The native envelope (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) is trimeric, and thus trimeric Env vaccine immunogens are currently being explored in preclinical immunogenicity studies. Key challenges have included the production and purification of biochemically homogeneous and stable trimers and the evaluation of these immunogens utilizing standardized virus panels for neutralization assays. Here we report the binding and neutralizing antibody (NAb) responses elicited by clade A (92UG037.8) and clade C (CZA97.012) Env gp140 trimer immunogens in guinea pigs. These trimers have been selected and engineered for optimal biochemical stability and have defined antigenic properties. Purified gp140 trimers with Ribi adjuvant elicited potent, cross-clade NAb responses against tier 1 viruses as well as detectable but low-titer NAb responses against select tier 2 viruses from clades A, B, and C. In particular, the clade C trimer elicited NAbs that neutralized 27%, 20%, and 47% of tier 2 viruses from clades A, B, and C, respectively. Heterologous DNA prime, protein boost as well as DNA prime, recombinant adenovirus boost regimens expressing these antigens, however, did not result in an increased magnitude or breadth of NAb responses in this system. These data demonstrate the immunogenicity of stable, homogeneous clade A and clade C gp140 trimers and exemplify the utility of standardized tier 1 and tier 2 virus panels for assessing the NAb responses of candidate HIV-1 Env immunogens.

The effect of Pgp induction in rats by pregnenolone 16α-carbonitrile (PCN) (3 days, 35 mg/kg/d, p.o.) on digoxin pharmacokinetics and intestinal transport has been assessed. After intravenous or oral digoxin dosing the arterial and hepatic portal vein (oral) AUC (0-24h) were significantly reduced by PCN pre-treatment. Biliary digoxin clearance increased 2-fold following PCN treatment. PCN significantly increased net digoxin secretion (2.05- and 4.5-fold respectively) in ileum and colon but not in duodenum or jejunum. This increased secretion correlated with increased Pgp protein expression in ileum and colon. Both intestinal and biliary excretion therefore contribute to altered digoxin disposition following PCN.

The worldwide diversity of HIV-1 presents an unprecedented challenge for vaccine development. Antigens derived from natural HIV-1 sequences have elicited only a limited breadth of cellular immune responses in nonhuman primate studies and clinical trials to date. Polyvalent 'mosaic' antigens, in contrast, are designed to optimize cellular immunologic coverage of global HIV-1 sequence diversity. Here we show that mosaic HIV-1 Gag, Pol and Env antigens expressed by recombinant, replication-incompetent adenovirus serotype 26 vectors markedly augmented both the breadth and depth without compromising the magnitude of antigen-specific T lymphocyte responses as compared with consensus or natural sequence HIV-1 antigens in rhesus monkeys. Polyvalent mosaic antigens therefore represent a promising strategy to expand cellular immunologic vaccine coverage for genetically diverse pathogens such as HIV-1.

Facilitative UT-B urea transporters have been located in the gastrointestinal tract of numerous mammalian species. We have previously identified UT-B urea transporters within the epithelial layers of the bovine (b) rumen. The aim of this study was to test the hypothesis that ruminal bUT-B urea transporters are regulated by dietary intake. Six Limousine-cross steers (initial BW = 690 +/- 51 kg) were separated into 2 groups fed a basic silage-based diet (RS) or a concentrate-based diet (RC) for 37 d and compared for ruminal morphology, content, and bUT-B expression. Analysis by reverse transcription-PCR showed that ruminal bUT-B2 mRNA expression was greater in RC-fed than RS-fed animals. Utilizing an anti-bUT-B antibody, we also detected a significant increase in bUT-B2 protein expression in RC-fed rumen (P < 0.05, n = 3). In agreement with these findings, immunolocalization studies of RC-fed ruminal tissue showed strong bUT-B signals throughout all epithelial layers, in contrast to weaker staining in RS-fed rumen that was more localized to the stratum basale. This study therefore confirmed that ruminal bUT-B urea transporter expression and localization were indeed altered by changes in dietary intake. We conclude that UT-B transporters play a significant role in the dietary regulation of bovine nitrogen balance.

Rare serotype and chimeric recombinant adenovirus (rAd) vectors that evade anti-Ad5 immunity are currently being evaluated as potential vaccine vectors for human immunodeficiency virus type 1 and other pathogens. We have recently reported that a heterologous rAd prime-boost regimen expressing simian immunodeficiency virus (SIV) Gag afforded durable partial immune control of an SIV challenge in rhesus monkeys. However, single-shot immunization may ultimately be preferable for global vaccine delivery. We therefore evaluated the immunogenicity and protective efficacy of a single immunization of chimeric rAd5 hexon hypervariable region 48 (rAd5HVR48) vectors expressing SIV Gag, Pol, Nef, and Env against a homologous SIV challenge in rhesus monkeys. Inclusion of Env resulted in improved control of peak and set point SIV RNA levels following challenge. In contrast, DNA vaccine priming did not further improve the protective efficacy of rAd5HVR48 vectors in this system.

We have previously shown that the membrane conductance of mIMCD-3 cells at a holding potential of 0 mV is dominated by a Ca2+-dependent Cl(-) current (I(CLCA)). Here we report that I(CLCA) activity is also voltage dependent and that this dependence on voltage is linked to the opening of a novel Al3+-sensitive, voltage-dependent, Ca2+ influx pathway. Using whole-cell patch-clamp recordings at a physiological holding potential (-60 mV), ICLCA was found to be inactive and resting currents were predominantly K+ selective. However, membrane depolarization to 0 mV resulted in a slow, sigmoidal, activation of ICLCA (T(0.5) approximately 500 s), while repolarization in turn resulted in a monoexponential decay in I(CLCA) (T (0.5) approximately 100 s). The activation of I(CLCA) by depolarization was reduced by lowering extracellular Ca2+ and completely inhibited by buffering cytosolic Ca2+ with EGTA, suggesting a role for Ca2+ influx in the activation of I(CLCA). However, raising bulk cytosolic Ca2+ at -60 mV did not produce sustained I(CLCA) activity. Therefore I(CLCA) is dependent on both an increase in intracellular Ca2+ and depolarization to be active. We further show that membrane depolarization is coupled to opening of a Ca2+ influx pathway that displays equal permeability to Ca2+ and Ba2+ ions and that is blocked by extracellular Al3+ and La3+. Furthermore, Al3+ completely and reversibly inhibited depolarization-induced activation of ICLCA, thereby directly linking Ca2+ influx to activation of I(CLCA). We speculate that during sustained membrane depolarization, calcium influx activates ICLCA which functions to modulate NaCl transport across the apical membrane of IMCD cells.

A recombinant adenovirus serotype 5 (rAd5) vector-based vaccine for HIV-1 has recently failed in a phase 2b efficacy study in humans. Consistent with these results, preclinical studies have demonstrated that rAd5 vectors expressing simian immunodeficiency virus (SIV) Gag failed to reduce peak or setpoint viral loads after SIV challenge of rhesus monkeys (Macaca mulatta) that lacked the protective MHC class I allele Mamu-A*01 (ref. 3). Here we show that an improved T-cell-based vaccine regimen using two serologically distinct adenovirus vectors afforded substantially improved protective efficacy in this challenge model. In particular, a heterologous rAd26 prime/rAd5 boost vaccine regimen expressing SIV Gag elicited cellular immune responses with augmented magnitude, breadth and polyfunctionality as compared with the homologous rAd5 regimen. After SIV(MAC251) challenge, monkeys vaccinated with the rAd26/rAd5 regimen showed a 1.4 log reduction of peak and a 2.4 log reduction of setpoint viral loads as well as decreased AIDS-related mortality as compared with control animals. These data demonstrate that durable partial immune control of a pathogenic SIV challenge for more than 500 days can be achieved by a T-cell-based vaccine in Mamu-A*01-negative rhesus monkeys in the absence of a homologous Env antigen. These findings have important implications for the development of next-generation T-cell-based vaccine candidates for HIV-1.

Background/aims: ANKH encodes a putative pyrophosphate transporter named ANKH, which regulates tissue calcification. ANKH is a transmembrane protein with at least 8 predicted transmembrane domains. Sequence analysis reveals a possible cilial localisation motif immediately after the last transmembrane segment. Here we aim to determine the subcellular localisation of ANKH in ciliated epithelial cells and murine tissue and identify colocalisation using ciliary/basal body markers., Methods: Using murine kidney, renal epithelial cells and osteoblast cells we investigated the expression and localisation of ANKH using RT-PCR, Western blotting and immunocytochemistry., Results: Here we confirm endogenous expression of ANKH mRNA and protein in whole mouse kidney as well as mouse renal epithelial cell lines M1 and mpkCCDcl4 and the osteoblast cell line MC3T3-E1. Using antibodies directed towards ANKH, we confirm cilial and basal body localisation in renal tissues and renal epithelial cells, in addition to a centrosomal localisation in dividing mpkCCDcl4 cells. We also establish that the osteoblast cell line MC3T3-E1 forms an epithelioid cell layer, with junctional complex formation and primary cilia expression. ANKH is also seen within cilial and basal body structures of MC3T3-E1 cells. An ANKH-3XFLAG construct expressed in mpkCCDcl4 cells also localises to the primary cilium/basal body complex confirming this localisation., Conclusion: We conclude that the transmembrane protein ANKH is expressed in cilia and basal body structures, and postulate a sensory role at this location., (2009 S. Karger AG, Basel.)

Intestinal induction of Pgp is known to limit the oral availability of certain drug compounds and give rise to detrimental drug-drug interactions. We have investigated the induction of P-glycoprotein (Pgp; MDR1) activity in a human intestinal epithelial cell line (T84) following pre-exposure to a panel of drug compounds, reported to be Pgp substrates, inhibitors or inducers. Human MDR1-transfected MDCKII epithelial monolayers were used to assess Pgp substrate interactions and inhibition of digoxin secretion by the selected drug compounds. The T84 cell line was used to assess induction of Pgp-mediated digoxin secretion following pre-exposure to the same compounds. Changes in gene expression (MDR1, MRP2, PXR and CAR) were determined by quantitative RT-PCR. Net transepithelial digoxin secretion was increased (1.3 fold, n=6, P<0.05) following pre-exposure to the PXR activator hyperforin (100nM, 72h), as was MDR1 mRNA expression (3.0 fold, n=4, P<0.05). A number of Pgp substrates (quinidine, amprenavir, irinotecan, topotecan, atorvastatin and erythromycin) induced net digoxin secretion, as did the non-Pgp substrate artemisinin. Various non-Pgp substrates demonstrated inhibition of digoxin secretion (verapamil, mifepristone, clotrimazole, mevastatin, diltiazem and isradipine) but did not induce Pgp-mediated digoxin secretion. Of the compounds that increased Pgp secretion, quinidine, topotecan, atorvastatin and amprenavir pre-exposure also elevated MDR1 mRNA levels, whereas erythromycin, irinotecan and artemisinin displayed no change in transcript levels. This indicates possible post-translational regulation of digoxin secretion. Finally, a strong correlation between drug modulation of MRP2 and PXR mRNA expression levels was evident.

Background and Purpose: Oral drug bioavailability is limited by intestinal expression of P-glycoprotein (MDR1, Pgp, ABCB1) whose capacity is regulated via nuclear receptors e.g. the pregnane X receptor (PXR, SXR, NR1I2). In order to study dynamic regulation of MDR1 transport capacity we have identified the T84 epithelial cell-line as a model for human intestine co-expressing MDR1 with PXR. The ability of rifampin, a known PXR agonist and digoxin, a model MDR1 substrate, to regulate MDR1 expression and transport activity has been tested, in these T84 cells., Experimental Approach: Transport was assayed by bi-directional [(3)H]-digoxin transepithelial fluxes across epithelial layers of T84 cells seeded onto permeable filter supports following pre-exposure to rifampin and digoxin. Quantitative real-time PCR, Western blotting and immunocytochemistry were used to correlate induction of MDR1 transcript and protein levels with transport activity., Key Results: Rifampin exposure (10 microM, 72 hours) increased MDR1 transcript levels (3.4 fold), MDR1 total protein levels (4.4 fold), apical MDR1 protein (2.7 fold) and functional activity of MDR1 (1.2 fold). Pre-incubation with digoxin (1 microM, 72 hours) potently induced MDR1 transcript levels (92 fold), total protein (7 fold), apical MDR1 protein (4.7 fold) and functional activity (1.75 fold). Whereas PXR expression was increased by rifampin incubation (2 fold), digoxin reduced PXR expression (0.3 fold)., Conclusions and Implications: Chronic digoxin pre-treatment markedly upregulates MDR1 expression and secretory capacity of T84 epithelia. Digoxin-induced changes in MDR1 levels are distinct from PXR-mediated changes resulting from rifampin exposure.

Extracellular pyrophosphate (PPi) plays a central role in the control of normal bone mineralization since it antagonizes inorganic phosphate in the promotion of hydroxyapatite deposition. Studies using knock-out mice have established the functional importance of PPi generation via nucleotide pyrophosphatase phosphodiesterases (NPP) and of PPi transmembrane transport by the progressive ankylosis (ANK) protein. Tissue non-specific alkaline phosphatase activity counteracts this by hydrolysis of PPi to inorganic phosphate. The molecular nature and transport function of ANK are reviewed. A close parallel is drawn between the controlled mineralization of bone and the prevention of abnormal calcium crystal deposition within the kidney, especially when concentrated urine is produced. Pyrophosphate is present in urine, and ANK is expressed in the cortical collecting duct where PPi transport to both the tubular lumen and the renal interstitium may occur. Pyrophosphate may also be generated here by nucleoside triphosphate diphosphohydrolases (NTPD2 and 3) together with NPP1. Alkaline phosphatase activity is restricted to the proximal nephron, remote from these sites of PPi generation, transport and function. The physiological importance of PPi generation and transport in preventing idiopathic calcium renal stone disease and nephrocalcinosis now needs to be established.

Monocarboxylate-H+ cotransporters, such as monocarboxylate transporter (MCT) SLC16A, have been suggested to mediate transruminal fluxes of short-chain fatty acids, ketone bodies, and lactate. Using an RT-PCR approach, we demonstrate expression of MCT1 (SLC16A1) and MCT2 (SLC16A7) mRNA in isolated bovine rumen epithelium. cDNA sequence from these PCR products combined with overlapping expressed sequence tag data allowed compilation of the complete open reading frames for MCT1 and MCT2. Immunohistochemical localization of MCT1 shows plasma membrane staining in cells of the stratum basale, with intense staining of the basal aspects of the cells. Immunostaining decreased in the cell layers toward the rumen lumen, with weak staining in the stratum spinsoum. Immunostaining in the stratum granulosum and stratum corneum was essentially negative. Since monocarboxylate transport will load the cytosol with acid, expression and location of Na+/H+ exchanger (NHE) family members within the rumen epithelium were determined. RT-PCR demonstrates expression of multiple NHE family members, including NHE1, NHE2, NHE3, and NHE8. In contrast to MCT1, immunostaining showed that NHE1 was predominantly localized to the stratum granulosum, with a progressive decrease toward the stratum basale. NHE2 immunostaining was observed mainly at an intracellular location in the stratum basale, stratum spinosum, and stratum granulosum. Given the anatomic localization of MCT1, NHE1, and NHE2, the mechanism of transruminal short-chain fatty acid, ketone body, and lactate transfer is discussed in relation to a functional model of the rumen epithelium comprising an apical permeability barrier at the stratum granulosum, with a cell syncitium linking the stratum granulosum to the blood-facing stratum basale.

We have used the perforated patch clamp and fura-2 fluorescence techniques to study the effect of extracellular Zn(2+) on whole-cell Ca(2+)-activated Cl(-) currents (I (CLCA)) in mouse inner medullary collecting duct cells (mIMCD-3). I (CLCA) was spontaneously active in 74% of cells under basal conditions and displayed time and voltage-independent kinetics and an outwardly rectifying current/voltage relationship (I/V). Addition of zinc chloride (10-400 microM) to the bathing solution resulted in a dose-dependent increase in I (CLCA) with little change in Cl(-) selectivity or biophysical characteristics, whereas gadolinium chloride (30 microM) and lanthanum chloride (100 microM) had no significant effect on the whole-cell current. Using fura-2-loaded mIMCD-3 cells, extracellular Zn(2+) (400 microM) stimulated an increase in intracellular Ca(2+) to an elevated plateau. The Zn(2+)-stimulated [Ca(2+)](i) increase was inhibited by thapsigargin (200 nM), the IP(3) receptor antagonist 2-aminoethoxydiphenyl borate (10 microM) and removal of bath Ca(2+). Pre-exposure to Zn(2+) (400 microM) markedly attenuated the ATP (100 microM)-stimulated [Ca(2+)](i) increase. These data are consistent with the hypothesis that extracellular Zn(2+) stimulates an increase in [Ca(2+)](i) by a release of calcium from thapsigargin/IP(3) sensitive stores. A possible physiological role for a divalent metal ion receptor, distinct from the extracellular Ca(2+)-sensing receptor, in IMCD cells is discussed.

Background/aims: Mutation of the pyrophosphate transporter, ANK, results in progressive arthritis in mice. ANK is expressed in non-skeletal tissues including kidney. The aim was therefore to investigate ANK location at the cellular and subcellular level in renal cells., Methods: RT-PCR identified a murine cell-line, mIMCD3, expressing ANK. The intra-renal distribution of ANK was determined by immunohistochemistry and the subcellular distribution in mIMCD3 cells by transfection of an ANK-NT-GFP fusion protein. Furthermore, an inactivating mutation of murine ank, Glu440X, and a gain of function mutation, Met48Thr, were tested to determine whether membrane traffic contributed to a transport defect., Results: ANK is expressed in cells of the cortical collecting duct, as assessed by colocalisation with aquaporin 2 and at the lateral and apical plasma membranes of mIMCD-3 epithelial cells, as assessed by colocalisation with wheat germ agglutinin lectin (WGA). ANK-NT-GFP was also present in endoplasmic reticulum, Golgi, acidic endosomes and mitochondria. mIMCD3 expression of Glu440X ANK-NT-GFP shows evidence of Golgi retention whereas Met48Thr ANK-NT-GFP is unaltered at the plasma membrane compared to wild type., Conclusion: The intra-renal and subcellular localisation of ANK is consistent with pyrophosphate export from collecting duct cells and supports a role for ANK in limiting intra-renal calcium-crystal formation., (Copyright (c) 2007 S. Karger AG, Basel.)

Purpose: The aim of this study was to investigate transepithelial ionic permeation via the paracellular pathway of human Caco-2 epithelial monolayers and its contribution to absorption of the base guanidine., Methods: Confluent monolayers of Caco-2 epithelial cells were mounted in Ussing chambers and the transepithelial conductance and electrical potential difference (p.d.) determined after NaCl dilution or medium Na substitution (bi-ionic conditions). Guanidine absorption (Ja-b) was measured +/- transepithelial potential gradients using bi-ionic p.d.'s., Results: Basal NaCl replacement with mannitol gives a transepithelial dilution p.d. of 28.0 +/- 3.1 mV basal solution electropositive (PCl/PNa = 0.34). Bi-ionic p.d.'s (basal replacements) indicate a cation selectivity of NH4+ > K+ approximately Cs+ > Na+ > Li+ > tetraethylammonium+ > N-methyl-D: -glucamine+ approximately choline+. Transepithelial conductances show good correspondence with bi-ionic potential data. Guanidine Ja-b was markedly sensitive to imposed transepithelial potential difference. The ratio of guanidine to mannitol permeability (measured simultaneously) increased from 3.6 in the absence of an imposed p.d. to 13.8 (basolateral negative p.d.)., Conclusions: Hydrated monovalent ions preferentially permeate the paracellular pathway (Eisenman sequence 2 or 3). Guanidine may access the paracellular pathway because absorptive flux is sensitive to the transepithelial potential difference. An alternative method to assess paracellular-mediated flux of charged organic molecules is suggested.

Mutations in CLCN5, which encodes the voltage-dependent Cl(-)/H(+)antiporter, CLC-5, cause Dent's disease. This disorder is characterized by low molecular-weight proteinuria, hypercalciuria, nephrocalcinosis and nephrolithiasis. Using a collecting duct cell model (mIMCD-3) in which endogenous clc-5 is disrupted by antisense clc-5 or overexpression of truncated clc-5, we demonstrate altered expression of the crystal adhesion molecule, annexin A2. Endogenously expressed annexin A2 is intracellular with limited plasma membrane localization. Following clc-5 disruption, there is both a marked increase in plasma membrane annexin A2 and an increase in cell surface crystal retention and agglomeration, which may be attenuated using pretreatment with anti-annexin A2 antibodies or wheat germ agglutinin lectin but not by concanavalin A. We hypothesize that in Dent's disease, endocytic failure leads to an accumulation at the plasma membrane of crystal-binding molecules that include annexin A2 leading to retention of calcium crystals and ultimately nephrocalcinosis and nephrolithiasis.

The UT-A (SLC14a2) and UT-B (SLC14a1) genes encode a family of specialized urea transporter proteins that regulate urea movement across plasma membranes. In this report, we describe the structure of the bovine UT-B (bUT-B) gene and characterize UT-B expression in bovine rumen. Northern analysis using a full-length bUT-B probe detected a 3.7-kb UT-B signal in rumen. RT-PCR of bovine mRNA revealed the presence of two UT-B splice variants, bUT-B1 and bUT-B2, with bUT-B2 the predominant variant in rumen. Immunoblotting studies of bovine rumen tissue, using an antibody targeted to the NH2-terminus of mouse UT-B, confirmed the presence of 43- to 54-kDa UT-B proteins. Immunolocalization studies showed that UT-B was mainly located on cell plasma membranes in epithelial layers of the bovine rumen. Ussing chamber measurements of ruminal transepithelial transport of (14)C-labeled urea indicated that urea flux was characteristically inhibited by phloretin. We conclude that bUT-B is expressed in the bovine rumen and may function to transport urea into the rumen as part of the ruminant urea nitrogen salvaging process.

The functional organization of the bovine rumen epithelium has been examined by electron and light microscopy combined with immunocytochemistry to define a transport model for this epithelium. Expression of connexin 43, an integral component of gap junctions, the tight-junction molecules claudin-1 and zonula occludens 1 (ZO-1), and the catalytic alpha-subunit of Na(+)-K(+)-ATPase was demonstrated by SDS-PAGE and Western blotting. From the lumen surface, four cell layers can be distinguished: the stratum corneum, the stratum granulosum, the stratum spinosum, and the stratum basale. Both claudin-1 and ZO-1 immunostaining showed plasma membrane staining, which was present at the stratum granulosum with decreasing intensity through the stratum spinosum to the stratum basale. The stratum corneum was negative for claudin-1 immunostaining. Transmission electron microscopy confirmed that occluding tight junctions were present at the stratum granulosum. Plasma membrane connexin 43 immunostaining was most intense at the stratum granulosum and decreased in intensity through stratum spinosum and stratum basale. There was intense immunostaining of the stratum basale for Na(+)-K(+)-ATPase, with weak staining of the stratum spinosum. Both the stratum granulosum and the stratum corneum were essentially negative. Stratum basale cells also displayed a high mitochondrial density relative to more apical cell layers. We conclude that epithelial barrier function may be attributed to the stratum granulosum and that cell-cell gap junctions allow diffusion to interconnect the barrier cell layer with the stratum basale where Na(+)-K(+)-ATPase is concentrated.