Netarsudil: a novel intra-ocular pressure lowering agent

Optic disc health is an important indicator of visual functions. The first line of management to prevent/halt the damage to optic disc is to control responsible pathological condition, if identified. In absence of identifiable cause, the most validated approach is lowering of intra-ocular pressure (IOP). Individually, as well as combinations of currently available drugs are not fully effective in all patients of glaucoma in achieving desired IOP control. Hence, there is a need of newer alternatives which address this unmet need. Recently, a newer IOP lowering agent with a novel mechanism of action, netarsudil, has been approved by United States Food and Drug Administration (US-FDA) in December 2017. Netarsudil acts by inhibiting Rho-associated protein kinase resulting in lowering of overall tone of the contractible cells in trabecular meshwork thereby improving drainage of aqueous humor outflow and lowering of IOP. Though in its early days, this drug gives an armamentarium to ophthalmologists and physicians to control IOP in patients of open-angle glaucoma and ocular hypertension

Studies in Sulphonamides- Part IX

Four differently substituted benzoylacetones, viz., 1-(4'-methylpheny1)-, 1-(4'-ethylpheny1)-, 1-(4'-methoxyphenyI)-, and 1-(4'-ethoxypheny1)-3-methy1 propane-1,3-diones have been synthesised and coupled with fourteen different diazotised sulphonamide bases. All the resulting sulphonamidobenzeneazo derivatives when subjected to their screening in vitro against S. aureus and E. coli were found to passes considerable activity

Studies in Sulphonamides- Part VIII

1-Ary1-3-methypropane-1,3-diones, viz.,1-(4/Sup1 chloropheny1),1-4'-bromopheny1)-1-(4'-chloro-3'-methy1-pheny1)-and -bipheny1-3-methy1Propane-1,3-dions have been synthesised and coupled with different dinzotises sulphonamied, bases in presence of sodium acetate to furnish the respective 1-ary1-3- methy1-2(subsituted sulphonamidobe-nzeneazo propane-1,3-diones.All these azo compounds have been tested in vitro for their antibacterial properties against S. aureus and E. coli and majority of these are found active against S. aureus

Studies in Sulphonamides- Part IX

Four differently substituted benzoylacetones, viz., 1-(4'-methylpheny1)-, 1-(4'-ethylpheny1)-, 1-(4'-methoxyphenyI)-, and 1-(4'-ethoxypheny1)-3-methy1 propane-1,3-diones have been synthesised and coupled with fourteen different diazotised sulphonamide bases. All the resulting sulphonamidobenzeneazo derivatives when subjected to their screening in vitro against S. aureus and E. coli were found to passes considerable activity

Role of 5\u27TG3\u27-Interacting Factors (TGIFs) in Vorinostat (HDAC Inhibitor)-Mediated Corneal Fibrosis Inhibition

Purpose: We have previously reported that vorinostat, an FDA-approved, clinically used histone deacetylase (HDAC) inhibitor, attenuates corneal fibrosis in vivo in rabbits by blocking transforming growth factor β (TGFβ). The 5′TG3′-interacting factors (TGIFs) are transcriptional repressors of TGFβ1 signaling via the Smad pathway. The present study was designed to explore the expression of TGIFs in human corneal fibroblasts and to investigate their role in mediating the antifibrotic effect of vorinostat. Methods: Human corneal fibroblast cultures were generated from donor corneas. RNA isolation, cDNA preparation, and PCR were performed to detect the presence of TGIF1 and TGIF2 transcripts. The cultures were exposed to vorinostat (2.5 μM) to test its effect on TGIF mRNA and protein levels using qPCR and immunoblotting. Myofibroblast formation was induced with TGFβ1 (5 ng/ml) treatment under serum-free conditions. The changes in fibrosis parameters were quantified by measuring fibrosis marker α-smooth muscle actin (αSMA) mRNA and protein levels with qPCR, immunostaining, and immunoblotting. Smad2/3/4 and TGIF knockdowns were performed using pre-validated RNAi/siRNAs and a commercially available transfection reagent. Results: Human corneal fibroblasts showed the expression of TGIF1 and TGIF2. Vorinostat (2.5 μM) caused a 2.8–3.3-fold increase in TGIF1 and TGIF2 mRNA levels and a 1.4–1.8-fold increase in TGIF1 and TGIF2 protein levels. Vorinostat treatment also caused a significant increase in acetylhistone H3 and acetylhistone H4. Vorinostat-induced increases in TGIF1 and TGIF2 were accompanied by a concurrent decrease in corneal fibrosis, as indicated by a decrease in αSMA mRNA by 83±7.7% and protein levels by 97±5%. The RNAi-mediated knockdown of Smad2, Smad3, and Smad4 markedly attenuated TGFβ1-evoked transdifferentiation of fibroblasts to myofibroblasts. The siRNA-mediated knockdown of TGIF1 and TGIF2 neutralized vorinostat-evoked decreases in αSMA mRNA by 31%–45% and protein levels by 12%–23%. Conclusions: Human corneal fibroblasts demonstrate the expression of TGIF1 and TGIF2 transcription factors. These transcriptional repressors are critical, at least partially, in mediating the antifibrotic effect of vorinostat in the cornea

Assessment of knowledge, attitude and practice about dengue in factory workers of Jammu region, India

Background: Dengue, a mosquito borne, arboviral disease has become a major cause of health concern in the recent times throughout the world. In India, we have been witnessing annual outbreaks for the past few years and lack of knowledge about prevention and treatment of dengue among majority of the population leads to increased mortality. In spite of this fact, very few studies have been done to know about the knowledge of people regarding dengue fever and whether proper preventive measures are being practiced by the community to limit its spread. The objective of the study is to assess the knowledge, attitude and practices (KAP) regarding dengue in factory workers in Jammu.Methods: An observational study was conducted in a factory of Jammu to assess knowledge, attitude and practices of factory workers about dengue.Results: Majority of workers had knowledge about dengue (92.56%), source (81.81%), nature of disease, symptoms, but complications were not known. Majority of the workers had no idea that laboratory test for dengue is not available in every laboratory.63.63% of the workers knew that papaya is useful in dengue. 74.38% of the workers knew that low platelet count is found in dengue. There were 82.64% of the workers told that they think dengue is curable and preventable. There were 54.54% of the workers told that dengue can be prevented by avoiding stagnation of water. 57.85% of the workers told that they are aware about the sprays used by govt. to kill mosquito.Conclusions: There is a need to bring awareness about dengue, prevention and treatment as it is a prevalent disease now

Vector Delivery Technique Affects Gene Transfer in the Cornea \u3cem\u3ein vivo\u3c/em\u3e

Purpose: This study tested whether controlled drying of the cornea increases vector absorption in mouse and rabbit corneas in vivo and human cornea ex vivo, and studied the effects of corneal drying on gene transfer, structure and inflammatory reaction in the mouse cornea in vivo. Methods: Female C57 black mice and New Zealand White rabbits were used for in vivo studies. Donor human corneas were used for ex vivo experiments. A hair dryer was used for drying the corneas after removing corneal epithelium by gentle scraping. The corneas received no, once, twice, thrice, or five times warm air for 10 s with a 5 s interval after each 10 s hair dryer application. Thereafter, balanced salt solution (BSS) was topically applied immediately on the cornea for 2 min using a custom-cloning cylinder. The absorbed BSS was quantified using Hamilton microsyringes. The adenoassociated virus 8 (AAV8) vector (1.1×108 genomic copies/μl) expressing marker gene was used to study the effect of corneal drying on gene transfer. Animals were sacrificed on day 14 and gene expression was analyzed using commercial staining kit. Morphological changes and infiltration of inflammatory cells were examined with H & E staining and immunocytochemistry. Results: Mice, rabbit or human corneas subjected to no or 10 s drying showed 6%–8% BSS absorption whereas 20, 30, or 50 s corneal drying showed significantly high 14%–19% (pin vivowith mild-to-moderate changes in corneal morphology. The 30 s of drying also showed significantly (pin vivowithout jeopardizing corneal morphology whereas 10 or 20 s drying showed moderate degree of gene transfer with no altered corneal morphology. Corneas that underwent 50 s drying showed high CD11b-positive cells (p Conclusions: Controlled corneal drying with hair dryer increases vector absorption significantly. The dispensing of efficacious AAV serotype into cornea with optimized minimally invasive topical application technique could provide high and targeted expression of therapeutic genes in the stroma in vivo without causing significant side effects

Molecular Mechanisms of Suberoylanilide Hydroxamic Acid in the Inhibition of TGF-β1-Mediated Canine Corneal Fibrosis

Objective—To investigate molecular mechanisms mediating anti-fibrotic effect of SAHA in the canine cornea using an in vitro model. We hypothesized that SAHA attenuates corneal fibrosis by modulating Smad-dependent and, to a lesser extent, Smad-independent signaling pathways activated by TGF-β1, as well as matrix metalloproteinase (MMP) activity. Methods—Cultured canine corneal fibroblasts (CCF) were incubated in the presence/absence of TGF-β1 (5ng/ml) and SAHA (2.5μM) for 24hrs. Western blot analysis was used to quantify non-phosphorylated and phosphorylated isoforms of Smad2/3, p38 MAP kinase (MAPK), ERK1/2 and JNK1. Real-time PCR and zymography were utilized to quantify MMP1, MMP2, MMP8 and MMP9 mRNA expression and MMP2 and MMP9 protein activity, respectively. Results—TGF-β1 treatment caused a significant increase in phospho-Smad2/3 and phospho-p38 MAPK. SAHA treatment reduced TGF-β1-induced phosphorylation of Smad2/3 but not of p38 MAPK. TGF-β1 did not modulate the phosphorylation of ERK1/2 or JNK1. SAHA caused a significant reduction in phospho-ERK1/2 expression regardless of concurrent TGF-β1 treatment. Neither SAHA alone nor in combination with TGF-β1 altered phospho-JNK1 expression. TGF-β1 significantly increased MMP1 and MMP9 mRNA expression but did not alter MMP2 mRNA. SAHA treatment attenuated TGF-β1-induced MMP9 mRNA expression while significantly enhancing TGF-β1-induced MMP1 mRNA expression. Zymography detected reduced expression of MMP2 and MMP9 proteins in untreated control CCF. TGF-β1 treatment did not alter their expression but SAHA treatment +/−TGF-β1 significantly increased MMP2 and MMP9 protein expression. Conclusions—The corneal anti-fibrotic effects of SAHA involve multiple mechanisms including modulation of canonical and non-canonical components of TGF-β1 intracellular signaling and MMP activity

AAV Serotype Influences Gene Transfer in Corneal Stroma \u3cem\u3ein vivo\u3c/em\u3e

This study evaluated the cellular tropism and relative transduction efficiency of three AAV serotypes, AAV6, AAV8 and AAV9, for corneal gene delivery using mouse cornea in vivo and donor human cornea ex vivo. The AAV6, AAV8 and AAV9 serotypes having AAV2 plasmid encoding for alkaline phosphatase (AP) gene were generated by transfecting HEK293 cell line with pHelper, pARAP4 and pRep/Cap plasmids. Viral vectors (109 vg/μl) were topically applied onto mouse cornea in vivo and human cornea ex vivo after removing the epithelium. Human corneas were processed for transgene delivery at day 5 after viral vector application. Mouse corneas were harvested at 4, 14 and 30 days after vector application for AP staining. Transduction efficiency was calculated by quantifying pixels of AP-stained area using Image J software and also confirmed by functional AP enzyme activity in the corneal lysates. Cellular toxicity of the three AAV serotypes was tested with TUNEL assay. Inflammatory response was detected by immunostaining for CD11b and F4/80. All three AAV serotypes successfully transduced mouse and human corneas. The order of transduction efficiency was AAV9\u3eAAV8\u3eAAV6. The transduction efficiency of AAV9 was 1.1–1.4 fold higher (p\u3e0.05) as compared to AAV8 and 3.5–5.5 fold higher (p\u3c0.01) as compared to AAV6. The level of transgene expression for all the three serotypes was greater at 14 days compared to 4 days and this high level of transgene expression was maintained up to the tested time point of 30 days. Corneas exposed to any of the three AAV serotypes did not show significant TUNEL positive cells or any inflammatory response as tested by CD11b or F4/80 staining suggesting that tested AAV serotypes do not induce cell death or inflammation and are safe for corneal gene therapy

PDGF-driven proliferation, migration, and IL8 chemokine secretion in human corneal fibroblasts involve JAK2-STAT3 signaling pathway

Purpose: Platelet-derived growth factor (PDGF) is associated with corneal fibroblast migration and proliferation and plays an important role in corneal wound healing. However, the intracellular mechanisms of PDGF-mediated functions in corneal fibroblasts are poorly understood. We tested the hypothesis that PDGF functional activities in the cornea involve the Janus kinase-2/signal transducers and activators of transcription-3 (JAK2-STAT3) signaling pathway and whether PDGF induces the expression of suppressors of cytokine signaling 3 (SOCS3), belonging to the novel family of feedback regulators of cytokine and growth factor activities. Methods: Human corneal fibroblast (HSF) cultures were used as an in vitro model for functional analysis. Real-time polymerase chain reactions were performed to quantify gene expression. Immunoprecipitation and immunoblotting techniques were used to measure protein expression. Cell growth, migration, and ELISA assays were used for functional validation. Results: Low endogenous levels of STAT3 and SOCS3 mRNA and protein expression were noted in HSFs. PDGF treatment of HSF significantly induced SOCS3 mRNA (3.0–4.5 fold) and protein (1.5–2.5 fold) expression in a timedependent manner. Similarly, PDGF treatment of HSF significantly increased STAT3 protein expression at two tested time points (2.5–2.96 fold). Cultures exposed to vehicle (control) did not show any change in SOCS3 and STAT3 mRNA or protein expression. An addition of AG-490, a selective inhibitor of the JAK2-STAT3 pathway, significantly inhibited PDGF-mediated STAT3 induction and cell growth and migration in HSF. We also observed that PDGF induced interleukin-8 (IL8) chemokine secretion (2 fold) and AG-490 inhibited IL8 secretion. Conclusions: Our data showed that PDGF induced STAT3, SOCS3, and IL8 chemokine secretion in human corneal fibroblasts. Further, PDGF-induced cell growth, migration, and IL8 secretion in corneal fibroblast involve the JAK2- STAT3 signaling pathway