Nanomicellar Topical Aqueous Drop Formulation of Rapamycin for Back-of-the-Eye Delivery

The objective of this study was to develop a clear, aqueous rapamycin-loaded mixed nanomicellar formulations (MNFs) for the back-of-the-eye delivery. MNF of rapamycin (0.2%) was prepared with vitamin E tocopherol polyethylene glycol succinate (TPGS) (Vit E TPGS) and octoxynol-40 (Oc-40) as polymeric matrix. MNF was characterized by various parameters such as size, charge, shape, and viscosity. Proton nuclear magnetic resonance (¹H NMR) was used to identify unentrapped rapamycin in MNF. Cytotoxicity was evaluated in human retinal pigment epithelial (D407) and rabbit primary corneal epithelial cells (rPCECs). In vivo posterior ocular rapamycin distribution studies were conducted in male New Zealand white rabbits. The optimized MNF has excellent rapamycin entrapment and loading efficiency. The average size of MNF was 10.98 ± 0.089 and 10.84 ± 0.11 nm for blank and rapamycin-loaded MNF, respectively. TEM analysis revealed that nanomicelles are spherical in shape. Absence of free rapamycin in the MNF was confirmed by ¹H NMR studies. Neither placebo nor rapamycin-loaded MNF produced cytotoxicity on D407 and rPCECs indicating formulations are tolerable. In vivo studies demonstrated a very high rapamycin concentration in retina-choroid (362.35 ± 56.17 ng/g tissue). No drug was identified in the vitreous humor indicating the sequestration of rapamycin in lipoidal retinal tissues. In summary, a clear, aqueous MNF comprising of Vit E TPGS and Oc-40 loaded with rapamycin was successfully developed. Back-of-the-eye tissue distribution studies demonstrated a very high rapamycin levels in retina-choroid (place of drug action) with a negligible drug partitioning into vitreous humor.KEY WORDS: back-of-the-eye, drug delivery, formulation, mixed nanomicelles, posterior, rabbits, rapamycin/sirolimus, retina/choroid, sclera, topical eye drops     

Bacterial Ghosts of Escherichia coli Drive Efficient Maturation of Bovine Monocyte-Derived Dendritic Cells

Bacterial ghosts (BGs) are empty cell envelopes derived from Gram-negative bacteria. They not only represent a potential platform for development of novel vaccines but also provide a tool for efficient adjuvant and antigen delivery system. In the present study, we investigated the interaction between BGs of Escherichia coli (E. coli) and bovine monocyte-derived dendritic cells (MoDCs). MoDCs are highly potent antigen-presenting cells and have the potential to act as a powerful tool for manipulating the immune system. We generated bovine MoDCs in vitro from blood monocytes using E. coli expressed bovine GM-CSF and IL-4 cytokines. These MoDCs displayed typical morphology and functions similar to DCs. We further investigated the E. coli BGs to induce maturation of bovine MoDCs in comparison to E. coli lipopolysaccharide (LPS). We observed the maturation marker molecules such as MHC-II, CD80 and CD86 were induced early and at higher levels in BG stimulated MoDCs as compared to the LPS stimulated MoDCs. BG mediated stimulation induced significantly higher levels of cytokine expression in bovine MoDCs than LPS. Both pro-inflammatory (IL-12 and TNF-α) and anti-inflammatory (IL-10) cytokines were induced in MoDCs after BGs stimulation. We further analysed the effects of BGs on the bovine MoDCs in an allogenic mixed lymphocyte reaction (MLR). We found the BG-treated bovine MoDCs had significantly (p<0.05) higher capacity to stimulate allogenic T cell proliferation in MLR as compared to the LPS. Taken together, these findings demonstrate the E. coli BGs induce a strong activation and maturation of bovine MoDCs.     

Evaluation of Hs-CRP Levels and Interleukin 18 (-137G/C) Promoter Polymorphism in Risk Prediction of Coronary Artery Disease in First Degree Relatives

Background

Coronary Artery Disease (CAD) is clearly a multifactorial disease that develops from childhood and ultimately leads to death. Several reports revealed having a First Degree Relatives (FDRS) with premature CAD is a significant autonomous risk factor for CAD development. C - reactive protein (CRP) is a member of the pentraxin family and is the most widely studied proinflammatory biomarker. IL-18 is a pleiotrophic and proinflammatory cytokine which is produced mainly by macrophages and plays an important role in the inflammatory cascade.

Methods and Results

Hs-CRP levels were estimated by ELISA and Genotyping of IL-18 gene variant located on promoter -137 (G/C) by Allele specific PCR in blood samples of 300 CAD patients and 300 controls and 100 FDRS. Promoter Binding sites and Protein interacting partners were identified by Alibaba 2.1 and Genemania online tools respectively. Hs-CRP levels were significantly high in CAD patients followed by FDRS when compared to controls. In IL-18 -137 (G/C) polymorphism homozygous GG is significantly associated with occurrence of CAD and Hs-CRP levels were significantly higher in GG genotype subjects when compared to GC and CC. IL-18 was found to be interacting with 100 protein interactants.

Conclusion

Our results indicate that Hs-CRP levels and IL-18-137(G/C) polymorphism may help to identify risk of future events of CAD in asymptomatic healthy FDRS.     

Lipid Phosphate Phosphatase 3 Stabilization of β-Catenin Induces Endothelial Cell Migration and Formation of Branching Point Structures

Endothelial cell (EC) migration, cell-cell adhesion, and the formation of branching point structures are considered hallmarks of angiogenesis; however, the underlying mechanisms of these processes are not well understood. Lipid phosphate phosphatase 3 (LPP3) is a recently described p120-catenin-associated integrin ligand localized in adherens junctions (AJs) of ECs. Here, we tested the hypothesis that LPP3 stimulates β-catenin/lymphoid enhancer binding factor 1 (β-catenin/LEF-1) to induce EC migration and formation of branching point structures. In subconfluent ECs, LPP3 induced expression of fibronectin via β-catenin/LEF-1 signaling in a phosphatase and tensin homologue (PTEN)-dependent manner. In confluent ECs, depletion of p120-catenin restored LPP3-mediated β-catenin/LEF-1 signaling. Depletion of LPP3 resulted in destabilization of β-catenin, which in turn reduced fibronectin synthesis and deposition, which resulted in inhibition of EC migration. Accordingly, reexpression of β-catenin but not p120-catenin in LPP3-depleted ECs restored de novo synthesis of fibronectin, which mediated EC migration and formation of branching point structures. In confluent ECs, however, a fraction of p120-catenin associated and colocalized with LPP3 at the plasma membrane, via the C-terminal cytoplasmic domain, thereby limiting the ability of LPP3 to stimulate β-catenin/LEF-1 signaling. Thus, our study identified a key role for LPP3 in orchestrating PTEN-mediated β-catenin/LEF-1 signaling in EC migration, cell-cell adhesion, and formation of branching point structures.Angiogenesis, the formation of new blood vessels, involves several well-coordinated cellular processes, including endothelial cell (EC) migration, synthesis and deposition of extracellular matrix proteins, such as fibronectin, cell-cell adhesion, and formation of branching point structures (1-3, 19, 33); however, less is known about the underlying mechanisms of these processes (6, 8, 12, 14, 16, 17). For example, adherens junctions (AJs), which mediate cell-cell adhesion between ECs, may be involved in limiting the extent of cell migration (2, 14, 38, 40). VE-cadherin, a protein found in AJs, is a single-pass transmembrane polypeptide responsible for calcium-dependent homophilic interactions through its extracellular domains (2, 38, 40). The VE-cadherin cytoplasmic domain interacts with the Armadillo domain-containing proteins, β-catenin, γ-catenin (plakoglobin), and p120-catenin (p120ctn) (2, 15, 38, 40, 43). Genetic and biochemical evidence documents a crucial role of β-catenin in regulating cell adhesion as well as proliferation secondary to the central position of β-catenin in the Wnt signaling pathway (13, 16, 25, 31, 44). In addition, the juxtamembrane protein p120ctn regulates AJ stability via binding to VE-cadherin (2, 7, 9, 15, 21, 28, 32, 43). The absence of regulation or inappropriate regulation of β-catenin and VE-cadherin functions is linked to cardiovascular disease and tumor progression (2, 6).We previously identified lipid phosphate phosphatase 3 (LPP3), also known as phosphatidic acid phosphatase 2b (PAP2b), in a functional assay of angiogenesis (18, 19, 41, 42). LPP3 not only exhibits lipid phosphatase activity but also functions as a cell-associated integrin ligand (18, 19, 35, 41, 42). The known LPPs (LPP1, LPP2, and LPP3) (20-23) are six transmembrane domain-containing plasma membrane-bound enzymes that dephosphorylate sphingosine-1-phosphate (S1P) and its structural homologues, and thus, these phosphatases generate lipid mediators (4, 5, 23, 35, 39). All LPPs, which contain a single N-glycosylation site and a putative lipid phosphatase motif, are situated such that their N and C termini are within the cell (4, 5, 22, 23, 35, 39). Only the LPP3 isoform contains an Arg-Gly-Asp (RGD) sequence in the second extracellular loop, and this RGD sequence enables LPP3 to bind integrins (18, 19, 22). Transfection experiments with green fluorescent protein (GFP)-tagged LPP1 and LPP3 showed that LPP1 is apically sorted, whereas LPP3 colocalized with E-cadherin at cell-cell contact sites with other Madin-Darby canine kidney (MDCK) cells (22). Mutagenesis and domain swapping experiments established that LPP1 contains an apical targeting signal sequence (FDKTRL) in its N-terminal segment. In contrast, LPP3 contains a dityrosine (109Y/110Y) basolateral sorting motif (22). Interestingly, conventional deletion of Lpp3 is embryonic lethal, since the Lpp3 gene plays a critical role in extraembryonic vasculogenesis independent of its lipid phosphatase activity (11). In addition, an LPP3-neutralizing antibody was shown to prevent cell-cell interactions (19, 42) and angiogenesis (42). Here, we addressed the hypothesis that LPP3 plays a key role in EC migration, cell-cell adhesion, and formation of branching point structures by stimulating β-catenin/lymphoid enhancer binding factor 1 (β-catenin/LEF-1) signaling.     

Genetic diversity of <Emphasis Type="Italic">Macrophomina phaseolina</Emphasis> isolates from certain agro-climatic regions of India by using RAPD markers

Genetic diversity analysis of Macrophomina phaseolina isolates obtained from different host range and diverse geographical locations in India was carried out using RAPD fingerprinting. Of the thirteen 10-mer random primers used, primer OPB-08 gave the maximum polymorphism and the UPGMA clustering could separate 50 isolates in to ten groups at more than 65% similarity level. The ten clusters correlated well with the geographical locations with exceptions for isolates obtained from Eastern and Western Ghats. There was a segregation of isolates from these two geographical locations in to two clusters thus, distributing 10 genotypes in to eight geographical locations. All the isolates M. phaseolina irrespective of their host and geographical origin, exhibited two representative monomorphic bands at 250 bp and 1 kb, presence of these bands suggests that isolates might have evolved from a common ancestor but due to geographical isolation fallowed by natural selection and genetic drift might have segregated in to subpopulations. Genetic similarity in the pathogenic population reflects the dispersal of single lineage in all locations in India.     

Effects of GsMTx4 on bacterial mechanosensitive channels in inside-out patches from giant spheroplasts

GsMTx4 is a 34-residue peptide isolated from the tarantula Grammostola spatulata folded into an inhibitory cysteine knot and it selectively affects gating of some mechanosensitive channels. Here we report the effects of cytoplasmic GsMTx4 on the two bacterial channels, MscS and MscL, in giant Escherichia coli spheroplasts. In excised inside-out patches, GsMTx4 sensitized both channels to tension by increasing the opening rate and decreasing the closing rate. With ascending and descending pressure ramps, GsMTx4 increased the gating hysteresis for MscS, a consequence of slower gating kinetics. Quantitative kinetic analysis of the primary C↔O transition showed that the hysteresis is a result of the decreased closing rate. The gating barrier location relative to the open state energy well was unaffected by GsMTx4. A reconstructed energy profile suggests that the peptide prestresses the resting state of MscS, lowering the net barrier to opening and stabilizes the open conformation by ∼8 kT. In excised patches, both MscL and MscS exhibit reversible adaptation, a process separable from inactivation for MscS. GsMTx4 decreased the rate of reversible adaptation for both channels and the MscS recovery rate from the inactivation. These measurements support a mechanism where GsMTx4 binds to the lipid interface of the channel, increasing the local stress that is sensed by the channels and stabilizing the expanded conformations.     

Cardioprotective potential of ocimum sanctum in isoproterenol induced myocardial infarction in rats.

Myocardial infarction (MI) was produced in rats with 85, 200 and 300 mg/kg of isoproterenol (ISO) administered subcutaneously (sc) twice at an interval of 24 h. Shift in antioxidant parameters, lactate dehydrogenase (LDH) together with morphological and histopathological changes were investigated. Two hundred mg/kg ISO dose was selected for the present study as this dose offered significant alteration in biochemical parameters along with moderate necrosis in heart. Effect of pre- and co-treatment of hydroalcoholic extract of Ocimum sanctum (Os) at different doses (25, 50, 75, 100, 200 and 400 mg/kg) was investigated against ISO (200 mg/kg) induced myocardial infarction in rats. Modulation of various biochemical parameters and membrane integrity was studied. Os at the dose of 25, 50, 75 and 100 mg/kg reduced significantly glutathione (GSH), superoxide dismutase (SOD) and LDH levels. It also inhibited the lipid peroxidation as observed by the reduced thiobarbituric acid reactive substances (TBARS) levels. In the present study Os at the dose of 50 mg/kg was found to demonstrate maximum cardioprotective effect. Above results were further confirmed by histopathological findings. Thus from the present study it is concluded that Os may be of therapeutic and prophylactic value in the treatment of MI.     

Preparation of a monolithic capillary column with immobilized alpha-mannose for affinity chromatography of lectins

A simple method for the preparation of an affinity monolithic (also called continuous bed) capillary column for alpha-mannose-specific lectins is described. 2-Hydroxyethyl methacrylate in combination with (+)-N,N -diallyltartardiamide (DATD) and piperazine diacrylamide (PDA, 1,4-bisacryloyl-piperazine) as crosslinkers, were used as monomers for the monolith. After oxidation of DATD with periodate, alpha-mannose with spacer was bound to the aldehyde groups of the polymeric skeleton via reductive amination to form an affinity column for the separation, enrichment or binding studies of mannose-specific lectins. The permeability of the column was excellent. The porosity of the monolith was investigated by scanning electron microscope (SEM) and inverse size exclusion chromatography (ISEC). The affinity of the monolith was evaluated by frontal analysis (FA) and fluorescence microscopy (FM) using fluorescently labeled concanavalin (Con A). Frontal affinity chromatography showed a specific interaction of two different lectins with the alpha-mannose-modified monolith. According to FM the affinity sites were evenly distributed over the monolithic bed.     

Increased intrahepatic triglyceride is associated with peripheral insulin resistance: in vivo MR imaging and spectroscopy studies

Recent studies have indicated that the mass/content of intramyocellular lipid (IMCL), intrahepatic triglyceride (IHTG), visceral fat (VF), and even deep abdominal subcutaneous fat (SF) may all be correlated with insulin resistance. Since simultaneous measurements of these parameters have not been reported, the relative strength of their associations with insulin action is not known. Therefore, the goals of this study were 1) to simultaneously measure IMCL, IHTG, VF, and abdominal SF in the same nondiabetic individuals using noninvasive (1)H-magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) and 2) to examine how these fat stores are correlated with systemic insulin sensitivity as measured by whole body glucose disposal (R(d)) during euglycemic-hyperinsulinemic clamp studies. Positive correlations were observed among IMCL, IHTG, and VF. There were significant inverse correlations between whole body R(d) and both IMCL and VF. Notably, there was a particularly tight inverse correlation between IHTG and whole body R(d) (r = -0.86, P < 0.001), consistent with an association between liver fat and peripheral insulin sensitivity. This novel finding suggests that hepatic triglyceride accumulation has important systemic consequences that may adversely affect insulin sensitivity in other tissues.