Preliminary brain-targeting studies on intranasal mucoadhesive microemulsions of sumatriptan

The aim of this investigation was to prepare microemulsions containing sumatriptan (ST) and sumatriptan succinate (SS) to accomplish rapid delivery of drug to the brain in acute attacks of migraine and perform comparative in vivo evaluation in rats. Sumatriptan microemulsions (SME)/sumatriptan succinate microemulsions (SSME) were prepared using titration method and characterized for drug content, globule size and size distribution, and zeta potential. Biodistribution of SME, SSME, sumatriptan solution (SSS), and marketed product (SMP) in the brain and blood of Swiss albino rats following intranasal and intravenous (IV) administrations were examined using optimized technetium-labeled (^99mTc-labeled) ST formulations. The pharmacokinetic parameters, drug targeting efficiency (DTE), and direct drug transport (DTP) were derived. Gamma scintigraphy imaging of rat brain following IV and intranasal administrations were performed to ascertain the localization of drug. SME and SSME were transparent and stable with mean globule size 38±20 nm and zeta potential between −35 to −55 mV. Brain/blood uptake ratios at 0.5 hour following IV administration of SME and intranasal administrations of SME, SMME, and SSS were found to be 0.20, 0.50, 0.60, and 0.26, respectively, suggesting effective transport of drug following intranasal administration of microemulsions. Higher DTE and DTP for mucoadhesive microemulsions indicated more effective targeting following intranasal administration and best brain targeting of ST from mucoadhesive microemulsions. Rat brain scintigraphy endorsed higher uptake of ST into the brain. Studies conclusively demonstrated rapid and larger extent of transport of microemulsion of ST compared with microemulsion of SS, SMP, and SSS into the rat brain. Hence, intranasal delivery of ST microemulsion developed in this investigation can play a promising role in the treatment of acute attacks of migraine. Published: January 20, 2006     

Effects of the HIV Protease Inhibitor Ritonavir on GLUT4 Knock-out Mice

HIV protease inhibitors acutely block glucose transporters (GLUTs) in vitro, and this may contribute to altered glucose homeostasis in vivo. However, several GLUT-independent mechanisms have been postulated. To determine the contribution of GLUT blockade to protease inhibitor-mediated glucose dysregulation, the effects of ritonavir were investigated in mice lacking the insulin-sensitive glucose transporter GLUT4 (G4KO). G4KO and control C57BL/6J mice were administered ritonavir or vehicle at the start of an intraperitoneal glucose tolerance test and during hyperinsulinemic-euglycemic clamps. G4KO mice exhibited elevated fasting blood glucose compared with C57BL/6J mice. Ritonavir impaired glucose tolerance in control mice but did not exacerbate glucose intolerance in G4KO mice. Similarly, ritonavir reduced peripheral insulin sensitivity in control mice but not in G4KO mice. Serum insulin levels were reduced in vivo in ritonavir-treated mice. Ritonavir reduced serum leptin levels in C57BL/6J mice but had no effect on serum adiponectin. No change in these adipokines was observed following ritonavir treatment of G4KO mice. These data confirm that a primary effect of ritonavir on peripheral glucose disposal is mediated through direct inhibition of GLUT4 activity in vivo. The ability of GLUT4 blockade to contribute to derangements in the other molecular pathways that influence insulin sensitivity remains to be determined.     

Quantifying and imaging NY-ESO-1/LAGE-1-derived epitopes on tumor cells using high affinity T cell receptors

Presentation of intracellular tumor-associated Ags (TAAs) in the context of HLA class I molecules offers unique cancer-specific cell surface markers for the identification and targeting of tumor cells. For most peptide Ags, the levels of and variations in cell surface presentation remain unknown, yet these parameters are of crucial importance when considering specific TAAs as targets for anticancer therapy. Here we use a soluble TCR with picomolar affinity for the HLA-A2-restricted 157-165 epitope of the NY-ESO-1 and LAGE-1 TAAs to investigate presentation of this immunodominant epitope on the surface of a variety of cancer cells. By single molecule fluorescence microscopy, we directly visualize HLA-peptide presentation for the first time, demonstrating that NY-ESO-1/LAGE-1-positive tumor cells present 10-50 NY-ESO-1/LAGE-1(157-165) epitopes per cell.     

Development of a fluorescence assay for the detection of L-ficolin-MASP in serum or purified samples

A fluorescence assay for the detection of L-ficolin-MASP in human serum or purified sample was developed by measuring the cleavage of fluorescent amide substrate by L-ficolin associated MASPs bound to the lipoteichoic acid (LTA). LTA (Staphylococcus aureus DSM 20233) was coated on NuncMaxisorp microtiter plates and serum or purified sample incubated overnight at 4 degrees C to allow the L-ficolin-MASP to bind LTA. Assay conditions for binding and complete cleavage of fluorescent amide substrate were standardized. The optimum temperature, incubation time and molarity of NaCl for LTA-ficolin binding were found to be 4 degrees C for 6 h at 1 M NaCl concentration. The optimum incubation time and pH for complete cleavage of fluorescent amide substrate by LTA bound L-ficolin associated MASP were found to be 2 h at pH 8.5. LTA-ficolin binding was found to be highly specific and was inhibited completely by LTA but not with mannose. A calibration curve was prepared by using the purified ficolin-MASP complex (1 to 12 mug/ml) and could be used to find concentration of ficolin-MASP complex in normal human serum.     

Effect of controlled carbon dioxide on in vitro shoot multiplication in Feronia limonia (L.) Swingle

The effect of controlled carbon dioxide environment on in vitro shoot growth and multiplication in Feronia limonia (a tropical fruit plant, Family- Rutaceae) was studied. Carbon dioxide available in the ambient air of the growth room was insufficient for in vitro growth of the shoots alone. Also, the presence of sucrose only as the C-source in the medium (without CO₂), was found to be inadequate for sustainable growth and multiplication of shoots. The carbon dioxide enrichment promoted shoot multiplication and overall growth. The promotory effect of CO₂ was independent of the presence of sucrose in the medium. In the presence of both CO₂ and sucrose, an additive effect was observed producing maximum shoot growth. In the absence of sucrose a higher concentration of CO₂ (10.0)g m⁻³ was required to achieve photoautotrophic shoot multiplication comparable to ambient air controls. Highest leaf area per shoot cluster promoting shoot growth and multiplication was recorded under this treatment. Shoots growing on sucrose containing medium under controlled CO₂ environment of 0.6 g m⁻³ concentration evoked better response than ambient air controls (shoots growing on sucrose containing medium) in growth room. This treatment produced the overall best response. The present study highlighted the possibility of photoautotrophic multiplication which might prove useful for successful hardening and acclimatization in tissue culture plants.     

The monomer/dimer transition of enzyme I of the Escherichia coli phosphotransferase system

Enzyme I (EI) is the first protein in the phosphotransfer sequence of the bacterial phosphoenolpyruvate:glycose phosphotransferase system. This system catalyzes sugar phosphorylation/transport and is stringently regulated. Since EI homodimer accepts the phosphoryl group from phosphoenolpyruvate (PEP), whereas the monomer does not, EI may be a major factor in controlling sugar uptake. Previous work from this and other laboratories (e.g. Dimitrova, M. N., Szczepanowski, R. H., Ruvinov, S. B., Peterkofsky, A., and Ginsburg A. (2002) Biochem. 41, 906-913), indicate that K(a) is sensitive to several parameters. We report here a systematic study of K(a) determined by sedimentation equilibrium, which showed that it varied by 1000-fold, responding to virtually every parameter tested, including temperature, phosphorylation, pH (6.5 versus 7.5), ionic strength, and especially the ligands Mg(2+) and PEP. This variability may be required for a regulatory protein. Further insight was gained by analyzing EI by sedimentation velocity, by near UV CD spectroscopy, and with a nonphosphorylatable active site mutant, EI-H189Q, which behaved virtually identically to EI. The singular properties of EI are explained by a model consistent with the results reported here and in the accompanying paper (Patel, H. V., Vyas, K. A., Mattoo, R. L., Southworth, M., Perler, F. B., Comb, D., and Roseman, S. (2006) J. Biol. Chem. 281, 17579-17587). We suggest that EI and EI-H189Q each comprise a multiplicity of conformers and progressively fewer conformers as they dimerize and bind Mg(2+) and finally PEP. Mg(2+) alone induces small or no detectable changes in structure, but large conformational changes ensue with Mg(2+)/PEP. This effect is explained by a "swiveling mechanism" (similar to that suggested for pyruvate phosphate dikinase (Herzberg, O., Chen, C. C., Kapadia, G., McGuire, M., Carroll, L. J., Noh, S. J., and Dunaway-Mariano, D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2652-2657)), which brings the C-terminal domain with the two bound ligands close to the active site His(189).     

Development, characterization and in vivo assessment of effective lipidic nanoparticles for dermal delivery of fluconazole against cutaneous candidiasis

The nanoparticulate carrier systems as solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) have gained interest for the topical treatment of skin associated fungal infection as they facilitate the skin penetration of loaded drugs. Therefore in this study, SLNs and NLCs loaded fluconazole (FLZ) were prepared by solvent diffusion method in an aqueous system and characterized for different parameters. In addition, antifungal activity was carried out on experimentally induced cutaneous candidiasis in immunosuppressed albino rats. The results showed that SLNs and NLCs represent the respective mean particle sizes of approx. 178 and 134 nm with encapsulation efficiency of 75.7±4.94% and 81.4±3.89%, respectively. The skin-retention studies of FLZ from in vitro and in vivo experiments revealed significantly higher accumulation of drug in the case of NLCs formulation. The in vivo cumulative amount of FLZ retention from NLCs was more than 5-fold that of the plain solution, while it was 3.3-fold more in the case of an equivalent-dose application in the form of SLNs at 12h after administration. The antifungal study also confirmed the maximum therapeutic efficacy of NLCs, as the lowest number of cfu/ml was recorded. It can be concluded from this study that NLCs provide a good skin targeting effect and may be a promising carrier for topical delivery of FLZ offering the sustained release and maintain the localized effect, resulting in an effective treatment of a life-threatening cutaneous fungal infection.     

The Candida glabrata adhesin Epa1p causes adhesion, phagocytosis, and cytokine secretion by innate immune cells

While Candida albicans is the most significant fungal pathogen for humans, Candida glabrata accounts for an increasing number of infections. Little is known about how C.?glabrata interacts with the innate immune system, the first line of defense against such organisms. The C.?glabrata adhesin Epa1p was previously shown to bind mammalian epithelial cells. We hypothesized that Epa1p mediates unique, nonopsonic binding to macrophages, leading to induction of immune responses. We found that Epa1p mediated adhesion by both C.?glabrata (Cg) and transformed Saccharomyces cerevisiae (Sc(EPA1) ) to human macrophage-like cells, including Thp1 and U937 lines, and donor PBMCs. Adhesion was distinct from described mechanisms such as Dectin-1. Epa1p expression was necessary and sufficient for S.?cerevisiae binding and phagocytosis, the latter of which was actin-mediated. Sc(EPA1) induced inflammatory cytokine production (IL-8 and TNF-α) by human PBMC-derived macrophages. Despite expressing Epa1p and binding to macrophages, Cg avoided phagocytosis and cytokine induction. In contrast to human results, in murine cell models (RAW264.7, J774A.1, and C57BL/6-derived cells), Epa1p-mediated binding was only revealed after blocking the Dectin-1 system. Recognition of Epa1p represents a novel mechanism by which human innate immune cells bind fungi, and for Sc(EPA1) results in phagocytosis and subsequent cytokine production.     

Possible transmission of human immunodeficiency virus-1 infection from an elite controller to a patient who progressed to acquired immunodeficiency syndrome: a case report

ABSTRACT: INTRODUCTION: Most individuals infected with human immunodeficiency virus-1, in the absence of antiretroviral therapy, exhibit persistent virus replication and declining CD4+ cell numbers, and progress to acquired immunodeficiency syndrome within 10 years of infection. Elite controllers are rare individuals with human immunodeficiency virus-1 infection who can maintain undetectable plasma virus levels and remain asymptomatic without antiretroviral therapy. It has been proposed that elite controllers benefit from being infected with attenuated human immunodeficiency virus-1 variants. CASE PRESENTATION: A 31-year-old African woman presented with human immunodeficiency virus-1 infection during pregnancy and was diagnosed with acquired immunodeficiency syndrome. Subsequently, her husband, a 31-year-old African man, was tested and found to be seropositive for human immunodeficiency virus-1. His plasma human immunodeficiency virus-1 ribonucleic acid level was found to be below the limit of detection of the clinical assay. CONCLUSION: This report provides evidence for the first described case of human immunodeficiency virus-1 infection possibly transmitted from an elite controller to a patient who progressed to acquired immunodeficiency syndrome. This observation strengthens the case against avirulence as a mechanism that protects elite controllers.     

Nanoscale organization of hedgehog is essential for long-range signaling

Hedgehog (Hh) plays crucial roles in tissue-patterning and activates signaling in Patched (Ptc)-expressing cells. Paracrine signaling requires release and transport over many cell diameters away by a process that requires interaction with heparan sulfate proteoglycans (HSPGs). Here, we examine the organization of functional, fluorescently tagged variants in living cells by using optical imaging, FRET microscopy, and mutational studies guided by bioinformatics prediction. We find that cell-surface Hh forms suboptical oligomers, further concentrated in visible clusters colocalized with HSPGs. Mutation of a conserved Lys in a predicted Hh-protomer interaction interface results in an autocrine signaling-competent Hh isoform--incapable of forming dense nanoscale oligomers, interacting with HSPGs, or paracrine signaling. Thus, Hh exhibits a hierarchical organization from the nanoscale to visible clusters with distinct functions.