Polymer coated polymeric microneedles for intravitreal delivery of dexamethasone.

The polymer coated polymeric (PCP) microneedles (MNs) is a novel approach for controlled delivery of drugs (without allowing release of the excipients) to the target site. PCP MNs was explored as an approach to deliver the drug intravitreally to minimize the risks associated with conventional intravitreal injections. The core MNs was fabricated with polyvinyl pyrrolidone K30 (PVP K30) and coating was with Eudragit E100. Preformulation studies revealed that the films prepared using Eudragit E 100 exhibited excellent integrity in the physiological medium after prolonged exposure. FTIR studies were performed to investigate the possible interaction between the API and the polymer. The PCP MNs fabricated with different drug loads (dexamethasone sodium phosphate) were subjected to in vitro drug release studies. The drug release from uncoated MNs was instantaneous and complete. On the other hand, a controlled release profile was observed in case of PCP MNs. Likewise, even in the ex vivo porcine eye model, the drug release was gradual into the vitreous humor in case of PCP MNs. The uncoated microneedles released all the drug instantaneously where the PCP MNs retarded the release up to 3 h.

Faunipollenites Bharadwaj 1962 and Protohaploxypinus Samoilovich 1953 emend. Morbey 1975: morphologic comparison of oxidized and non- oxidized specimens from India and Brazil, and its taxonomic importance.

Faunipollenites Bharadwaj is considered a junior synonym of Protohaploxypinus Samoilovich emend. Morbey. However, Indian workers claim it is a valid genus due to a poorly defined corpus and absence of folds in distal attachment. In India, a standard method is applied including the oxidization with HNO3 more than 48 hours (+10' of KOH). We analyze the effects of that treatment on the morphology of pollen grains of both genera in samples from the Permian of India and Brazil. The same samples are also processed with HCl, HF, two hours of HNO3 and 2' of KOH and slides are mounted after each step. Our analysis reveals that distinct or indistinct central body and presence/absence of folds in distal attachment do not change in contrast to the indistinct central body and mostly absence of folds from samples that underwent a longer period of oxidization (24-48 hours and KOH 10'). The synonymization of Faunipollenites to Protohaploxypinus is confirmed. Species of Faunipollenites are reassigned to the revised species of Protohaploxypinus. The usage of the latter genus and its species in Permian biostratigraphic studies of India will improve Gondwanan correlations and paleobiogeographic reconstructions in future studies.

Ocular Ciliopathies: Genetic and Mechanistic Insights into Developing Therapies.

Developing suitable medicines for genetic diseases requires a detailed understanding of not only the pathways that cause the disease, but also the identification of the genetic components involved in disease manifestation. This article focuses on the complexities associated with ocular ciliopathies - a class of debilitating disorders of the eye caused by ciliary dysfunction. Ciliated cell types have been identified in both the anterior and posterior segments of the eye. Photoreceptors (rods and cones) are the most studied ciliated neurons in the retina, which is located in the posterior eye. The photoreceptors contain a specialized lightsensing outer segment, or cilium. Any defects in the development or maintenance of the outer segment can result in severe retinal ciliopathies, such as retinitis pigmentosa and Leber congenital amaurosis. A role of cilia in the cell types involved in regulating aqueous fluid outflow in the anterior segment of the eye has also been recognized. Defects in these cell types are frequently associated with some forms of glaucoma. Here, we will discuss the significance of understanding the genetic heterogeneity and the pathogenesis of ocular ciliopathies to develop suitable treatment strategies for these blinding disorders.

Histone deacetylase expression patterns in developing murine optic nerve.

BACKGROUND: Histone deacetylases (HDACs) play important roles in glial cell development and in disease states within multiple regions of the central nervous system. However, little is known about HDAC expression or function within the optic nerve. As a first step in understanding the role of HDACs in optic nerve, this study examines the spatio-temporal expression patterns of methylated histone 3 (K9), acetylated histone 3 (K18), and HDACs 1-6 and 8-11 in the developing murine optic nerve head. RESULTS: Using RT-qPCR, western blot and immunofluorescence, three stages were analyzed: embryonic day 16 (E16), when astrocyte precursors are found in the optic stalk, postnatal day 5 (P5), when immature astrocytes and oligodendrocytes are found throughout the optic nerve, and P30, when optic nerve astrocytes and oligodendrocytes are mature. Acetylated and methylated histone H3 immunoreactivity was co-localized in the nuclei of most SOX2 positive glia within the optic nerve head and adjacent optic nerve at all developmental stages. HDACs 1-11 were expressed in the optic nerve glial cells at all three stages of optic nerve development in the mouse, but showed temporal differences in overall levels and subcellular localization. HDACs 1 and 2 were predominantly nuclear throughout optic nerve development and glial cell maturation. HDACs 3, 5, 6, 8, and 11 were predominantly cytoplasmic, but showed nuclear localization in at least one stage of optic nerve development. HDACs 4, 9 and10 were predominantly cytoplasmic, with little to no nuclear expression at any time during the developmental stages examined. CONCLUSIONS: Our results showing that HDACs 1, 2, 3, 5, 6, 8, and 11 were each localized to the nuclei of SOX2 positive glia at some stages of optic nerve development and maturation and extend previous reports of HDAC expression in the aging optic nerve. These HDACs are candidates for further research to understand how chromatin remodeling through acetylation, deacetylation and methylation contributes to glial development as well as their injury response.

Role of MMP-9 in the breakdown of barrier integrity of the corneal endothelium in response to TNF-α.

TNF-α induces loss of barrier integrity of the corneal endothelium through mechanisms involving the activation of p38 MAP kinase. This study has investigated the role of matrix metalloproteinase-9 (MMP-9), known to be activated by mechanisms downstream of p38 MAP kinase, on the breakdown of the barrier integrity. Experiments were performed with primary cultures of bovine corneal endothelium. Changes in the trans-endothelial electrical resistance (TER), a measure of barrier integrity, were measured by electric cell-substrate impedance sensing. The integrity of the apical junctional assembly was imaged by immunolocalization of ZO-1. MMP-9 activity in the conditioned medium of cells treated with TNF-α was visualized by gelatin zymography. Transcriptional activation of MMP-9 was assessed by real-time RT-PCR. Exposure to TNF-α led to significant disruption of ZO-1 and also caused a continuous decline in TER for more than 20 h. These effects were opposed by cycloheximide (protein synthesis inhibitor), GM-6001 (broad spectrum inhibitor of MMPs), minocycline (MMP-2 and MMP-9 inhibitor), and MMP-9 inhibitor I (selective MMP-9 inhibitor). Cycloheximide, GM-6001, and MMP-9 inhibitor I also attenuated the increase in permeability to FITC-dextran (10 kDa). In addition, TNF-α led to an increased MMP-9 activity in the conditioned medium as well as a nearly 20-fold increase in mRNA for MMP-9 but not for MMP-2. The functional activity and increase in mRNA levels of MMP-9 were blocked by SB-203580 (selective p38 MAP kinase inhibitor) and cycloheximide. In conclusion, transcriptional and translational activation of MMP-9, downstream of p38 MAP kinase signaling, is involved in the (TNF-α)-induced loss of corneal endothelial barrier integrity.

Megakaryocytes regulate expression of Pyk2 isoforms and caspase-mediated cleavage of actin in osteoblasts.

The proliferation and differentiation of osteoblast (OB) precursors are essential for elaborating the bone-forming activity of mature OBs. However, the mechanisms regulating OB proliferation and function are largely unknown. We reported that OB proliferation is enhanced by megakaryocytes (MKs) via a process that is regulated in part by integrin signaling. The tyrosine kinase Pyk2 has been shown to regulate cell proliferation and survival in a variety of cells. Pyk2 is also activated by integrin signaling and regulates actin remodeling in bone-resorbing osteoclasts. In this study, we examined the role of Pyk2 and actin in the MK-mediated increase in OB proliferation. Calvarial OBs were cultured in the presence of MKs for various times, and Pyk2 signaling cascades in OBs were examined by Western blotting, subcellular fractionation, and microscopy. We found that MKs regulate the temporal expression of Pyk2 and its subcellular localization. We also found that MKs regulate the expression of two alternatively spliced isoforms of Pyk2 in OBs, which may regulate OB differentiation and proliferation. MKs also induced cytoskeletal reorganization in OBs, which was associated with the caspase-mediated cleavage of actin, an increase in focal adhesions, and the formation of apical membrane ruffles. Moreover, BrdU incorporation in MK-stimulated OBs was blocked by the actin-polymerizing agent, jasplakinolide. Collectively, our studies reveal that Pyk2 and actin play an important role in MK-regulated signaling cascades that control OB proliferation and may be important for therapeutic interventions aimed at increasing bone formation in metabolic diseases of the skeleton.

Dynamin and PTP-PEST cooperatively regulate Pyk2 dephosphorylation in osteoclasts.

Bone loss is caused by the dysregulated activity of osteoclasts which degrade the extracellular bone matrix. The tyrosine kinase Pyk2 is highly expressed in osteoclasts, and mice lacking Pyk2 exhibit an increase in bone mass, in part due to impairment of osteoclast function. Pyk2 is activated by phosphorylation at Y402 following integrin activation, but the mechanisms leading to Pyk2 dephosphorylation are poorly understood. In the current study, we examined the mechanism of action of the dynamin GTPase on Pyk2 dephosphorylation. Our studies reveal a novel mechanism for the interaction of Pyk2 with dynamin, which involves the binding of Pyk2's FERM domain with dynamin's plextrin homology domain. In addition, we demonstrate that the dephosphorylation of Pyk2 requires dynamin's GTPase activity and is mediated by the tyrosine phosphatase PTP-PEST. The dephosphorylation of Pyk2 by dynamin and PTP-PEST may be critical for terminating outside-in integrin signaling, and for stabilizing cytoskeletal reorganization during osteoclast bone resorption.

Elevated cAMP opposes (TNF-alpha)-induced loss in the barrier integrity of corneal endothelium.

PURPOSE: Elevated cyclic adenosine monophosphate (cAMP) enhances the barrier integrity of the corneal endothelium and thereby facilitates stromal hydration control, which is necessary for corneal transparency. This study investigates whether elevated cAMP is effective against the tumor necrosis factor-alpha (TNF-alpha)-induced loss of barrier integrity in monolayers of bovine corneal endothelial cells (BCEC). METHODS: BCEC in primary culture were used for the study. Trans-endothelial electrical resistance (TER), a measure of barrier integrity, was determined by electrical cell-substrate impedance sensing. The changes were also ascertained by measuring paracellular permeability to fluorescein isothiocyanate (FITC)-dextran (10 kDa) across cells grown on porous culture inserts, and by immunofluorescence imaging of the apical junctional complex (AJC). The activation of p38 MAP kinase was assessed using western blotting. RESULTS: Co-treatment with forskolin, which activates adenylate cyclase, and rolipram, which inhibits cAMP-dependent phosphodiesterase PDE4, reduced the TNF-alpha-induced increase in the flux of FITC-dextran. Similar co-treatment also prevented the TNF-alpha-induced disorganization of zona occludens-1 (ZO-1) and cadherins at the AJC. Co-treatment, as well pre-treatment, with forskolin plus rolipram prevented the TNF-alpha-induced decrease in TER. The influence of the agents was significant after 12 h of exposure to the cytokine. This effect was also mimicked by A2B agonists, adenosine and 5'-N-ethylcarboxamidoadenosine (NECA), which are known to mobilize cAMP in BCEC. Elevated cAMP also inhibited the cytokine-induced activation of p38 MAP kinase, and further blocked the disassembly of microtubules as well as the disruption of the PAMR (peri-junctional actomyosin ring) at the AJC. CONCLUSIONS: These results suggest that elevated cAMP opposes the TNF-alpha-induced loss in barrier integrity of the corneal endothelium. This effect follows inhibition of the cytokine-induced activation of p38 MAP kinase and its downstream signaling involved in the disruption of AJC and PAMR, as well as the disassembly of microtubules.

Lovastatin inhibits the thrombin-induced loss of barrier integrity in bovine corneal endothelium.

PURPOSE: Increased actomyosin contraction of the dense band of actin cytoskeleton at the apical junctional complex (perijunctional actomyosin ring, PAMR) breaks down the barrier integrity of corneal endothelium. This study has investigated the efficacy of statins, which inhibit activation of RhoA, in opposing the thrombin-induced loss of barrier integrity of monolayers of cultured bovine corneal endothelium. METHODS: Myosin light chain (MLC) phosphorylation, a biochemical measure of actomyosin contraction, was assayed by urea-glycerol gel electrophoresis, followed by western blot analysis. The locus of MLC phosphorylation and changes in the organization of the PAMR were visualized by immunostaining. Phosphorylation of MYPT1, a regulatory subunit of myosin light-chain phosphatase (MLCP), was assessed by Western blot analysis to determine down-regulation of RhoA. The barrier integrity was assessed in terms of trans-endothelial electrical resistance (TER), and further confirmed by determining permeability to FITC dextran (10 kDa) and distribution of ZO-1, a marker of tight junctional assembly. RESULTS: Lovastatin, a prototype of lipophilic statins, induced MLC dephosphorylation under basal conditions. It opposed increase in phosphorylation of MLC and MYPT1 in response to thrombin and nocodazole, agents known to activate RhoA in the endothelium. Pretreatment with the statin opposed the thrombin- and nocodazole-induced disruption of the PAMR and the thrombin-induced decline in TER. Lovastatin also opposed the thrombin- and nocodazole-induced increase in permeability to FITC dextran and redistribution of ZO-1. However, upon supplementation with GGPP (geranylgeranyl pyrophosphate), lovastatin failed to oppose the effects of thrombin and nocodazole on the PAMR, ppMLC, and ZO-1 distribution. CONCLUSIONS: Lovastatin attenuates RhoA activation in the corneal endothelium presumably by reducing its isoprenylation. This underlies the suppression of the thrombin-induced loss in barrier integrity of the corneal endothelium.

Barrier dysfunction of the corneal endothelium in response to TNF-alpha: role of p38 MAP kinase.

PURPOSE: TNF-alpha is elevated in the cornea and aqueous humor during allograft rejection and anterior uveitis. The authors investigated the involvement of p38 MAP kinase in the TNF-alpha-induced loss of barrier integrity in monolayers of cultured bovine corneal endothelial cells. METHODS: Transendothelial electrical resistance (TER), a measure of barrier integrity, was determined by electrical cell-substrate impedance sensing. Barrier integrity was further assessed in terms of permeability to FITC dextran. Reorganization of the apical junctional complex (AJC) in response to TNF-alpha was visualized by immunofluorescence. The expression of TNF-alpha receptors was confirmed by RT-PCR. Activation of p38 MAP kinase in response to TNF-alpha was determined by Western blot analysis. RESULTS: Exposure to TNF-alpha induced a continuous decline in TER that persisted for more than 20 hours. It also led to a significant increase in permeability to FITC dextran. At the AJC, the cytokine caused disassembly of microtubules, disruption of perijunctional actomyosin ring (PAMR), and dislocation of ZO-1 and cadherins. Western blot analysis showed that TNF-alpha also led to the activation of p38 MAP kinase. All these responses to the cytokine were opposed by treatment with SB-203580, a selective p38 MAP kinase inhibitor. TNFR1, but not TNFR2, was expressed in untreated cells with no change in the expression pattern on treatment with the cytokine. CONCLUSIONS: TNF-alpha breaks down the barrier integrity of corneal endothelium, concomitant with the disruption of PAMR, remodeling of AJC, and disassembly of microtubules. These effects are mediated by transient activation of p38 MAP kinase. Thus, the TNF-alpha-induced barrier dysfunction in the corneal endothelium can be suppressed by inhibitors of p38 MAP kinase and agents downstream of the kinase that affect the cytoskeleton.

Microtubule stabilization opposes the (TNF-alpha)-induced loss in the barrier integrity of corneal endothelium.

Microtubule disassembly breaks down the barrier integrity in a number of epithelial and endothelial monolayers. This study has investigated effects of TNF-alpha, which is implicated in corneal allograft rejection, on microtubules and barrier integrity in cultured bovine corneal endothelial cells. Exposure to TNF-alpha led to disassembly of the microtubules, and also caused disruption of the perijunctional actomyosin ring (PAMR). As a measure of barrier integrity, trans-endothelial electrical resistance (TER) was determined based on electrical cell-substrate impedance sensing in realtime. Exposure to TNF-alpha caused a slow decline in TER for > 20 h, and a similar exposure to cells grown on porous culture inserts led to a significant increase in permeability to FITC dextran. These changes, indicating a loss of barrier integrity, were also reflected by dislocation of ZO-1 at the cell border and disassembly of cadherins. These effects of TNF-alpha were inhibited upon stabilization of microtubules by pre-treatment with paclitaxel or epothilone B. Microtubule stabilization may be a useful strategy to overcome (TNF-alpha)-induced loss of the barrier integrity of corneal endothelium during inflammation associated with transplant rejection and uveitis.

Microtubule disassembly breaks down the barrier integrity of corneal endothelium.

Increased contractility of the peri-junctional actomyosin ring (PAMR) breaks down the barrier integrity of corneal endothelium. This study has examined the effects of microtubule disassembly on Myosin Light Chain (MLC) phosphorylation, a biochemical marker of actomyosin contraction, and barrier integrity in monolayers of cultured bovine corneal endothelial cells (BCEC). Exposure to nocodazole, which readily induced microtubule disassembly, led to disruption of the characteristically dense assembly of cortical actin cytoskeleton at the apical junctional complex (i.e., PAMR) and dispersion of ZO-1 from its normal locus. Nocodazole also led to an increase in phosphorylation of MLC. Concomitant with these changes, nocodazole caused an increase in permeability to HRP and FITC dextran (10 kDa) and a decrease in trans-endothelial electrical resistance (TER). Y-27632 (a Rho kinase inhibitor) and forskolin (known to inhibit activation of RhoA through direct elevation of cAMP) opposed the nocodazole-induced MLC phosphorylation, decrease in TER, and dispersion of ZO-1. Thrombin, which breaks down the barrier integrity of BCEC monolayers, also induced microtubule disassembly and MLC phosphorylation. Pre-treatment with paclitaxel to stabilize microtubules opposed the thrombin effects. These results suggest that microtubule disassembly breaks down the barrier integrity of BCEC through activation of RhoA and subsequent disruption of the PAMR. The thrombin effect also highlights that signaling downstream of GPCRs can also influence the organization of microtubules.