Passive lipoidal diffusion and carrier-mediated cell uptake are both important mechanisms of membrane permeation in drug disposition.

Recently, it has been proposed that drug permeation is essentially carrier-mediated only and that passive lipoidal diffusion is negligible. This opposes the prevailing hypothesis of drug permeation through biological membranes, which integrates the contribution of multiple permeation mechanisms, including both carrier-mediated and passive lipoidal diffusion, depending on the compound's properties, membrane properties, and solution properties. The prevailing hypothesis of drug permeation continues to be successful for application and prediction in drug development. Proponents of the carrier-mediated only concept argue against passive lipoidal diffusion. However, the arguments are not supported by broad pharmaceutics literature. The carrier-mediated only concept lacks substantial supporting evidence and successful applications in drug development.

Sorting mechanisms regulating membrane protein traffic in the apical transcytotic pathway of polarized MDCK cells.

The transcytotic pathway followed by the polymeric IgA receptor (pIgR) carrying its bound ligand (dIgA) from the basolateral to the apical surface of polarized MDCK cells has been mapped using morphological tracers. At 20 degreesC dIgA-pIgR internalize to interconnected groups of vacuoles and tubules that comprise the endosomal compartment and in which they codistribute with internalized transferrin receptors (TR) and epidermal growth factor receptors (EGFR). Upon transfer to 37 degreesC the endosome vacuoles develop long tubules that give rise to a distinctive population of 100-nm-diam cup-shaped vesicles containing pIgR. At the same time, the endosome gives rise to multivesicular endosomes (MVB) enriched in EGFR and to 60-nm-diam basolateral vesicles. The cup-shaped vesicles carry the dIgA/pIgR complexes to the apical surface where they exocytose. Using video microscopy and correlative electron microscopy to study cells grown thin and flat we show that endosome vacuoles tubulate in response to dIgA/pIgR but that the tubules contain TR as well as pIgR. However, we show that TR are removed from these dIgA-induced tubules via clathrin-coated buds and, as a result, the cup-shaped vesicles to which the tubules give rise become enriched in dIgA/pIgR. Taken together with the published information available on pIgR trafficking signals, our observations suggest that the steady-state concentrations of TR and unoccupied pIgR on the basolateral surface of polarized MDCK cells are maintained by a signal-dependent, clathrin-based sorting mechanism that operates along the length of the transcytotic pathway. We propose that the differential sorting of occupied receptors within the MDCK endosome is achieved by this clathrin-based mechanism continuously retrieving receptors like TR from the pathways that deliver pIgR to the apical surface and EGFR to the lysosome.

Characterization of Adult Stem/Progenitor Cell Populations From Bone Marrow in a Three-Dimensional Collagen Gel Culture System.

Stem cell transplantation therapy using mesenchymal stem cells (MSCs) is considered a useful strategy. Although MSCs are commonly isolated by exploiting their plastic adherence, several studies have suggested that there are other populations of stem and/or osteoprogenitor cells which are removed from primary culture during media replacement. Therefore, we developed a three-dimensional (3D) culture system in which adherent and non-adherent stem cells are selected and expanded. Here, we described the characterization of 3D culture-derived cell populations in vitro and the capacity of these cells to differentiate into bone and/or cartilage tissue when placed inside of demineralized bone matrix (DBM) cylinders, implanted subcutaneously into the backs of rat for 2, 4 and 8 weeks. Our results demonstrates that 3D culture cells were a heterogeneous population of uncommitted cells that express pluripotent, hematopoietic, mesenchymal and endothelial specific markers in vitro and can undergo osteogenic differentiation in vivo.

Ocular toxoplasmosis: evaluation of lacrimal - specific secretory IgA levels in both patients with active and inactive phases of the disease

Ocular toxoplasmosis can result in recurrent uveitis. Studies have shown that a correlation between active ocular toxoplasmosis and the presence of anti-Toxoplasma gondii secretory IgA (SIgA) in tears. This study compares anti-T. gondii SIgA levels in patients' tears during the acute and inactive phases of toxoplasmic uveitis. Twenty-nine positive tear specific SIgA for T. gondii patients with acute toxoplasmic uveitis were selected and were followed-up for at least two years, when the anti-T. gondii SIgA tears levels were determined. Specific SIgA for T. gondii was negative in 22 patients (75.86%) and positive in seven patients (24.13%) of whom six (85.7%) were followed over three years. Average SIgA levels during the acute phase are 1.54 and decrease significantly to 0.72 (p = 0.0001) during the inactive phase of disease. Because anti-T. gondii SIgA in the tear is negative in 75.86% of patients after the acute phase of infection, T. gondii SIgA levels may be used as a complementary diagnostic marker for active ocular toxoplasmosis.

Use of active personal dosemeters in interventional radiology and cardiology: Tests in hospitals - ORAMED project

Although active personal dosemeters (APDs) are not used quite often in hospital environments, the possibility to assess the dose and/or dose rate in real time is particularly interesting in interventional radiology and cardiology (IR/IC) since operators can receive relatively high doses while standing close to the primary radiation field.A study concerning the optimization of the use of APDs in IR/IC was performed in the framework of the ORAMED project, a Collaborative Project (2008-2011) supported by the European Commission within its 7th Framework Program. This paper reports on tests performed with APDs on phantoms using an X-ray facility in a hospital environment and APDs worn by interventionalists during routine practice in different European hospitals.The behaviour of the APDs is more satisfactory in hospitals than in laboratories with respect to the influence of the tube peak high voltage and pulse width, because the APDs are tested in scattered fields with dose equivalent rates generally lower than 1 Sv.h(-1).

Kaposi sarcoma incidence in the Swiss HIV Cohort Study before and after highly active antiretroviral therapy.

Between 1984 and 2006, 12 959 people with HIV/AIDS (PWHA) in the Swiss HIV Cohort Study contributed a total of 73 412 person-years (py) of follow-up, 35 551 of which derived from PWHA treated with highly active antiretroviral therapy (HAART). Five hundred and ninety-seven incident Kaposi sarcoma (KS) cases were identified of whom 52 were among HAART users. Cox regression was used to estimate hazard ratios (HR) and corresponding 95% confidence intervals (CI). Kaposi sarcoma incidence fell abruptly in 1996-1998 to reach a plateau at 1.4 per 1000 py afterwards. Men having sex with men and birth in Africa or the Middle East were associated with KS in both non-users and users of HAART but the risk pattern by CD4 cell count differed. Only very low CD4 cell count (<50 cells microl(-1)) at enrollment or at HAART initiation were significantly associated with KS among HAART users. The HR for KS declined steeply in the first months after HAART initiation and continued to be low 7-10 years afterwards (HR, 0.06; 95% CI, 0.02-0.17). Thirty-three out of 52 (63.5%) KS cases among HAART users arose among PWHA who had stopped treatment or used HAART for less than 6 months.

NS2 Proteins of GB Virus B and Hepatitis C Virus Share Common Protease Activities and Membrane Topologies.

GB virus B (GBV-B), which is hepatotropic in experimentally infected small New World primates, is a member of the Hepacivirus genus but phylogenetically relatively distant from hepatitis C virus (HCV). To gain insights into the role and specificity of hepaciviral nonstructural protein 2 (NS2), which is required for HCV polyprotein processing and particle morphogenesis, we investigated whether NS2 structural and functional features are conserved between HCV and GBV-B. We found that GBV-B NS2, like HCV NS2, has cysteine protease activity responsible for cleavage at the NS2/NS3 junction, and we experimentally confirmed the location of this junction within the viral polyprotein. A model for GBV-B NS2 membrane topology was experimentally established by determining the membrane association properties of NS2 segments fused to green fluorescent protein (GFP) and their nuclear magnetic resonance structures using synthetic peptides as well as by applying an N-glycosylation scanning approach. Similar glycosylation studies confirmed the HCV NS2 organization. Together, our data show that despite limited amino acid sequence similarity, GBV-B and HCV NS2 proteins share a membrane topology with 3 N-terminal transmembrane segments, which is also predicted to apply to other recently discovered hepaciviruses. Based on these data and using trans-complementation systems, we found that intragenotypic hybrid NS2 proteins with heterologous N-terminal membrane segments were able to efficiently trans-complement an assembly-deficient HCV mutant with a point mutation in the NS2 C-terminal domain, while GBV-B/HCV or intergenotypic NS2 chimeras were not. These studies indicate that virus- and genotype-specific intramolecular interactions between N- and C-terminal domains of NS2 are critically involved in HCV morphogenesis. IMPORTANCE: Nonstructural protein 2 (NS2) of hepatitis C virus (HCV) is a multifunctional protein critically involved in polyprotein processing and virion morphogenesis. To gain insights into NS2 mechanisms of action, we investigated whether NS2 structural and functional features are conserved between HCV and GB virus B (GBV-B), a phylogenetically relatively distant primate hepacivirus. We showed that GBV-B NS2, like HCV NS2, carries cysteine protease activity. We experimentally established a model for GBV-B NS2 membrane topology and demonstrated that despite limited sequence similarity, GBV-B and HCV NS2 share an organization with three N-terminal transmembrane segments. We found that the role of HCV NS2 in particle assembly is genotype specific and relies on critical interactions between its N- and C-terminal domains. This first comparative analysis of NS2 proteins from two hepaciviruses and our structural predictions of NS2 from other newly identified mammal hepaciviruses highlight conserved key features of the hepaciviral life cycle.

Targeting aquaporin function: potent inhibition of aquaglyceroporin-3 by a gold-based compound.

Aquaporins (AQPs) are membrane channels that conduct water and small solutes such as glycerol and are involved in many physiological functions. Aquaporin-based modulator drugs are predicted to be of broad potential utility in the treatment of several diseases. Until today few AQP inhibitors have been described as suitable candidates for clinical development. Here we report on the potent inhibition of AQP3 channels by gold(III) complexes screened on human red blood cells (hRBC) and AQP3-transfected PC12 cells by a stopped-flow method. Among the various metal compounds tested, Auphen is the most active on AQP3 (IC(50) = 0.8±0.08 µM in hRBC). Interestingly, the compound poorly affects the water permeability of AQP1. The mechanism of gold inhibition is related to the ability of Au(III) to interact with sulphydryls groups of proteins such as the thiolates of cysteine residues. Additional DFT and modeling studies on possible gold compound/AQP adducts provide a tentative description of the system at a molecular level. The mapping of the periplasmic surface of an homology model of human AQP3 evidenced the thiol group of Cys40 as a likely candidate for binding to gold(III) complexes. Moreover, the investigation of non-covalent binding of Au complexes by docking approaches revealed their preferential binding to AQP3 with respect to AQP1. The high selectivity and low concentration dependent inhibitory effect of Auphen (in the nanomolar range) together with its high water solubility makes the compound a suitable drug lead for future in vivo studies. These results may present novel metal-based scaffolds for AQP drug development.

Kinetoplastid membrane protein-11 exacerbates infection with Leishmania amazonensis in murine macrophages

In Leishmania amazonensis, kinetoplastid membrane protein-11 (KMP-11) expression increases during metacyclogenesis and is higher in amastigotes than in promastigotes, suggesting a role for this protein in the infection of the mammalian host. We show that the addition of KMP-11 exacerbates L. amazonensis infection in peritoneal macrophages from BALB/c mice by increasing interleukin (IL)-10 secretion and arginase activity while reducing nitric oxide (NO) production. The doses of KMP-11, the IL-10 levels and the intracellular amastigote loads were strongly, positively and significantly correlated. The increase in parasite load induced by KMP-11 was inhibited by anti-KMP-11 or anti-IL-10 neutralising antibodies, but not by isotype controls. The neutralising antibodies, but not the isotype controls, were also able to significantly decrease the parasite load in macrophages cultured without the addition of KMP-11, demonstrating that KMP-11-induced exacerbation of the infection is not dependent on the addition of exogenous KMP-11 and that the protein naturally expressed by the parasite is able to promote it. In this study, the exacerbating effect of KMP-11 on macrophage infection with Leishmania is for the first time demonstrated, implicating it as a virulence factor in L. amazonensis. The stimulation of IL-10 production and arginase activity and the inhibition of NO synthesis are likely involved in this effect.

A morphologically distinct Phlebotomus argentipes population from active cutaneous leishmaniasis foci in central Sri Lanka

Although the reported aetiological agent of cutaneous leishmaniasis (CL) in Sri Lanka is Leishmania donovani, the sandfly vector remains unknown. Ninety-five sandflies, 60 females and 35 males, collected in six localities in the district of Matale, central Sri Lanka, close to current active transmission foci of CL were examined for taxonomically relevant characteristics. Eleven diagnostic morphological characters for female sandflies were compared with measurements described for Indian and Sri Lankan sandflies, including the now recognised Phlebotomus argentipes sensu lato species complex. The mean morphometric measurements of collected female sandflies differed significantly from published values for P. argentipes morphospecies B, now re-identified as Phlebotomus annandalei from Delft Island and northern Sri Lanka, from recently re-identified P. argentipes s.s. sibling species and from Phlebotomus glaucus. Furthermore, analysis of underlying variation in the morphometric data through principal component analysis also illustrated differences between the population described herein and previously recognised members of the P. argentipes species complex. Collectively, these results suggest that a morphologically distinct population, perhaps most closely related to P. glaucus of the P. argentipess. I. species complex, exists in areas of active CL transmission. Thus, research is required to determine the ability of this population of flies to transmit cutaneous leishmaniasis.

The impact of highly active antiretroviral therapy on the survival of vertically HIV-infected children and adolescents in Belo Horizonte, Brazil

The use of highly active antiretroviral therapy (HAART) for human immunodeficiency virus (HIV)-infected patients has reduced the number of acquired immune deficiency syndrome-related deaths worldwide. This study assessed the impact of HAART on the survival and death rates of vertically HIV-infected children and adolescents in Belo Horizonte, Brazil. Data were obtained from a historic cohort of vertically HIV-infected children and adolescents aged zero-19 years old who were admitted from March 1989-December 2004 and were followed until June 2006. Patients who used HAART were included if they were treated for at least 12 weeks. Of 359 patients, 320 patients met the inclusion criteria. The overall mortality rate was 9.7% [31/320; 95% confidence interval (CI): 6.0-13%]. The median survival for the non-HAART and HAART groups was 31.5 and 55.9 months, respectively (log rank = 22.11, p < 0.0001). In the multivariate analysis, the statistically significant variables were HAART and the weight-for-age Z score < -2, with HAART constituting a protective factor [relative risk (RR): 0.13; CI 95%: 0.05-0.33] and malnutrition constituting a risk factor (RR: 3.44; CI 95%: 1.60-7.40) for death. The incidence of death was 5.1/100 person-years in the non-HAART group and 0.8/100 person-years in the HAART group (p < 0.0001).

Anatomic and functional outcome after 23-gauge vitrectomy, peeling, and intravitreal triamcinolone for idiopathic macular epiretinal membrane.

PURPOSE: To report both the functional and anatomic outcome and safety profile of 23-gauge pars plana vitrectomy combined with membrane peeling and intravitreal injection of triamcinolone acetonide in eyes with idiopathic macular epiretinal membranes. METHODS: Retrospective study of 39 consecutive patients who underwent 23-gauge transconjunctival sutureless vitrectomy, membrane peeling, and intravitreal triamcinolone acetonide injection for an idiopathic macular epiretinal membrane between February 2007 and February 2008. Minimum follow-up was 6 months. RESULTS: Thirty-nine eyes of 39 patients were included in the study. The mean follow-up was 7 +/- 2.2 months (range, 6-15 months). Twenty-two eyes (56%) were pseudophakic and 17 (44%) were phakic at the time of surgery. Five of the phakic eyes (29.4%) had worsening of cataracts during the follow-up period. Mean preoperative intraocular pressure was 14 +/- 3.5 mmHg. At the final follow-up, mean intraocular pressure was 14.5 +/- 2.7 mmHg, which did not differ significantly from the intraocular pressure at baseline (P = 0.14, two-tailed t-test). Five (13%) patients needed topical antiglaucoma treatment. Mean preoperative best-corrected visual acuity (BCVA) was 0.28 decimal equivalent (20/71 Snellen equivalent; logarithm of the minimum angle of resolution 0.54 +/- 0.2, range: 1.0-0.2) and improved significantly (P < 0.0001, two-tailed t-test) to a mean of 0.6 decimal equivalent (20/33 Snellen equivalent; logarithm of the minimum angle of resolution 0.22 +/- 0.16, range: 0.6-0) at the final follow-up. The BCVA improved by a mean of 3.2 +/- 2.1 lines (range: 0-8). Twenty-nine patients (74%) demonstrated a gain of > or =3 lines. Mean central macular thickness was 456 +/- 77 microm (mean +/- SD) at baseline, which was significantly reduced at the final follow-up to 327 +/- 79 microm (mean +/- SD; P < 0.0001, two-tailed t-test). Average central macular thickness reduction was 131 +/- 77 microm (mean +/- SD; range: 36-380 microm). A subgroup analysis of 15 selected cases, which had central macular thickness and BCVA measurements after the first postoperative week, demonstrated that 84% of the total final reduction in central macular thickness and 84% of the total final improvement in BCVA occurred already during the first postoperative week. CONCLUSION: Twenty-three-gauge sutureless transconjunctival vitrectomy is a safe and effective technique for the treatment of idiopathic macular epiretinal membranes. The concomitant administration of intravitreal triamcinolone acetonide after pars plana vitrectomy may speed up and improve the anatomic and functional outcome.

Light-induced phosphorylation of a membrane protein plays an early role in signal transduction for phototropism in Arabidopsis thaliana.

Blue light is known to cause rapid phosphorylation of a membrane protein in etiolated seedlings of several plant species, a protein that, at least in etiolated pea seedlings and maize coleoptiles, has been shown to be associated with the plasma membrane. The light-driven phosphorylation has been proposed on the basis of correlative evidence to be an early step in the signal transduction chain for phototropism. In the Arabidopsis thaliana mutant JK224, the sensitivity to blue light for induction of first positive phototropism is known to be 20- to 30-fold lower than in wild type, whereas second positive curvature appears to be normal. While light-induced phosphorylation can be demonstrated in crude membrane preparations from shoots of the mutant, the level of phosphorylation is dramatically lower than in wild type, as is the sensitivity to blue light. Another A. thaliana mutant, JK218, that completely lacks any phototropic responses to up to 2 h of irradiation, shows a normal level of light-induced phosphorylation at saturation. Since its gravitropic sensitivity is normal, it is presumably blocked in some step between photoreception and the confluence of the signal transduction pathways for phototropism and gravitropism. We conclude from mutant JK224 that light-induced phosphorylation plays an early role in the signal transduction chain for phototropism in higher plants.