Differential gene expression and protein-protein interaction network profiling of sulfur mustard-exposed rabbit corneas employing RNA-seq data and bioinformatics tools.

Sulfur mustard (SM) ocular exposure severely damages the cornea and causes vision impairment. At present, no specific therapy exists to mitigate SM-induced corneal injury and vision loss. This study performed transcriptome profiling of naïve, SM-damaged, and SM-undamaged rabbit corneas using RNA-seq analysis and bioinformatic tools to gain a better mechanistic understanding and develop SM-specific medical countermeasures. The mRNA profiles of rabbit corneas 4 weeks post SM vapor exposure were generated using Illumina-NextSeq deep sequencing (Gene Expression Omnibus accession # GSE127708). The RNA sequences of naïve (n = 4), SM-damaged (n = 5), and SM-undamaged (n = 5) corneas were subjected to differential expression (DE) analysis after quality control profiling with FastQC. DE analysis was performed using HISAT2, StringTie, and DESeq2. The log2(FC)±2 and adjusted p˂0.05 were chosen to identify the most relevant genes. A total of 5930 differentially expressed genes (DEGs) (upregulated: 3196, downregulated: 2734) were found in SM-damaged corneas compared to naïve corneas, whereas SM-undamaged corneas showed 1884 DEGs (upregulated: 1029, downregulated: 855) compared to naïve corneas. DE profiling of SM-damaged corneas to SM-undamaged corneas revealed 985 genes (upregulated: 308, downregulated: 677). The DE profiles were subsequently subjected to signaling pathway enrichment, and protein‒protein interactions (PPIs) were analyzed. Pathway enrichment was performed for the genes associated with cellular apoptosis, death, adhesion, migration, differentiation, proliferation, extracellular matrix, and tumor necrosis factor production. To identify novel targets, we narrowed the pathway analysis to upregulated and downregulated genes associated with cell proliferation and differentiation, and PPI networks were developed. Furthermore, protein targets associated with cell differentiation and proliferation that may play vital roles in corneal fibrosis and wound healing post SM injury were identified.

Mustard Gas Exposure Actuates SMAD2/3 Signaling to Promote Myofibroblast Generation in the Cornea.

Sulfur mustard gas (SM) is a vesicating and alkylating agent used as a chemical weapon in many mass-casualty incidents since World War I. Ocular injuries were reported in >90% of exposed victims. The mechanisms underlying SM-induced blindness remain elusive. This study tested the hypothesis that SM-induced corneal fibrosis occurs due to the generation of myofibroblasts from resident fibroblasts via the SMAD2/3 signaling pathway in rabbit eyes in vivo and primary human corneal fibroblasts (hCSFs) isolated from donor corneas in vitro. Fifty-four New Zealand White Rabbits were divided into three groups (Naïve, Vehicle, SM-Vapor treated). The SM-Vapor group was exposed to SM at 200 mg-min/m3 for 8 min at the MRI Global facility. Rabbit corneas were collected on day 3, day 7, and day 14 for immunohistochemistry, RNA, and protein lysates. SM caused a significant increase in SMAD2/3, pSMAD, and ɑSMA expression on day 3, day 7, and day 14 in rabbit corneas. For mechanistic studies, hCSFs were treated with nitrogen mustard (NM) or NM + SIS3 (SMAD3-specific inhibitor) and collected at 30 m, 8 h, 24 h, 48 h, and 72 h. NM significantly increased TGFß, pSMAD3, and SMAD2/3 levels. On the contrary, inhibition of SMAD2/3 signaling by SIS3 treatment significantly reduced SMAD2/3, pSMAD3, and ɑSMA expression in hCSFs. We conclude that SMAD2/3 signaling appears to play a vital role in myofibroblast formation in the cornea following mustard gas exposure.

Time-dependent in situ structural and cellular aberrations in rabbit cornea in vivo after mustard gas exposure.

An array of corneal pathologies collectively called mustard gas keratopathy (MGK) resulting from ocular exposure to sulfur mustard (SM) gas are the most prevalent chemical warfare injury. MGK involves chronic ocular discomfort that results in vision impairment. The etiology of MGK remains unclear and poorly understood primarily due to a lack of scientific data regarding structural and cellular changes in different layers of the cornea altered by mustard vapor exposure in vivo. The goals of this study were to (a) characterize time-dependent changes in different layers of corneal epithelium, stroma, and endothelium in live animals in situ by employing state-of-the-art multimodal clinical ophthalmic imaging techniques and (b) determine if SM-induced acute changes in corneal cells could be rescued by a topical eye drop (TED) treatment using in an established rabbit in vivo model. Forty-five New Zealand White Rabbit eyes were divided into four groups (Naïve, TED, SM, and SM + TED). Only one eye was exposed to SM (200 mg-min/m3 for 8 min), and each group had three time points with six eyes each (Table-1). TED was topically applied twice a day for seven days. Clinical eye examinations and imaging were performed in live rabbits with stereo, Slit-lamp, HRT-RCM3, and Spectralis microscopy system. Fantes grading, fluorescein staining, Schirmer's tests, and applanation tonometry were conducted to measure corneal haze, ocular surface aberrations, tears, and intraocular pressure respectively. H&E and PSR staining were used for histopathological cellular changes in the cornea. In vivo confocal and OCT imaging revealed significant changes in structural and morphological appearance of corneal epithelium, stroma, and endothelium in vivo in SM-exposed rabbit corneas in a time-dependent manner compared to naïve cornea. Also, SM-exposed eyes showed loss of corneal transparency characterized by increased stromal thickness and light-scattering myofibroblasts or activated keratocytes, representing haze formation in the cornea. Neither naive nor TED-alone treated eyes showed any structural, cellular, and functional abnormalities. Topical TED treatment significantly reduced SM-induced abnormalities in primary corneal layers. We conclude that structural and cellular changes in primary corneal layers are early pathological events contributing to MGK in vivo, and efficient targeting of them with suitable agents has the potential to mitigate SM ocular injury.

Evaluation of CRISPR/Cas9 mediated TGIF gene editing to inhibit corneal fibrosis in vitro.

Corneal wound healing is influenced by many factors including transcriptional co-repressors and co-activators. Interactions of co-activators and co-repressors with Smads influence mechanistic loop facilitating transcription of alpha-smooth muscle actin (α-SMA), a key profibrotic gene, in corneal repair. The role of a transcriptional repressor, 5'TG3'-interacting factor (TGIF), in the regulation of α-SMA and myofibroblast formation in the cornea was shown previously by our group. This study tested a hypothesis if TGIF1 gene editing via CRISPR/Cas9 can ease myofibroblast formation in the cornea using an in vitro model. Primary human corneal stromal fibroblasts (hCSFs) generated from donor corneas received gene-editing plasmid facilitating loss (CRISPR/Cas9 knockout) or gain (CRISPR activation) of TGIF function by UltraCruz transfection reagent. Phase-contrast microscopy, immunoblotting, immunocytochemistry and quantitative polymerase chain reaction (qPCR) were used to measure levels of myofibroblast profibrotic genes (α-SMA, fibronectin, Collagen-I, and Collagen-IV) in hCSFs lacking or overexpressing TGIF1 after growing them in± transforming growth factor beta1 (TGF-ß1) under serum-free conditions. The CRISPR-assisted TGIF1 activation (gain of function) in hCSFs demonstrated significantly decreased myofibroblast formation and messenger ribonucleic acid (mRNA) and protein levels of profibrotic genes. Conversely, CRISPR/Cas9-assisted TGIF knockdown (loss of function) in hCSFs demonstrated no significant change in the levels of myofibroblast formation or profibrotic genes under similar conditions. These results suggest that TGIF gene-editing approach can be employed to modulate the transcriptional activity of α-SMA in controlling pathological and promoting physiological wound healing in an injured cornea.

Six-Month In Vivo Safety Profiling of Topical Ocular AAV5-Decorin Gene Transfer.

Purpose: A significant remission of corneal fibrosis and neovascularization in rabbit eye in vivo was observed from a tissue-selective localized adeno-associated virus (AAV)5-Decorin (Dcn) gene therapy. This study sought to investigate 6-month toxicity profiling of this gene therapy for the eye in vivo using a rabbit model. Methods: A small epithelial scrape followed by corneal drying was performed unilaterally in 12 rabbit eyes and either AAV5-Dcn (n = 6) or naked vector (n = 6) was delivered topically using a cloning cylinder technique. Contralateral eyes served as naïve control (n = 6). Safety and tolerability measurements in live rabbits were performed periodically until month 6 using multimodel clinical ophthalmic imaging tools-a slit lamp, stereomicroscope, and HRT3-RCM in vivo confocal microscope. Thereafter, corneas were excised and subjected to hematoxylin and eosin staining, Mason trichome staining, propidium iodide nuclear staining, and quantitative real-time polymerase chain reaction analyses. Results: Clinical eye examinations based on the modified Hackett-McDonald ocular scoring system, and in vivo confocal imaging of the cornea showed no signs of ocular toxicity in rabbit eyes given AAV5-Dcn gene transfer vs control eyes (P > 0.05) through 6 months after treatment. The histologic and molecular analyses showed no significant differences in AAV5-Dcn vs AAV naked or naïve control groups (P > 0.05) and were in accordance with the masked clinical ophthalmic observations showing no abnormalities. Conclusions: Topical tissue-targeted localized AAV5-Dcn gene therapy seems to be safe and nontoxic to the rabbit eye in vivo. Translational Relevance: AAV5-Dcn gene therapy has the potential to treat corneal fibrosis and neovascularization in vivo safely without significant ocular toxicity.

A Novel Topical Ophthalmic Formulation to Mitigate Acute Mustard Gas Keratopathy In Vivo: A Pilot Study.

Purpose: This pilot study investigated the in vivo therapeutic potential and tolerability of a multimodal ophthalmic formulation, topical eye drops (TED), for acute mustard gas keratopathy (MGK) using a rabbit model. Methods: Twenty New Zealand White rabbits were used. Only right eyes of 18 rabbits (oculus dexter [OD]) received single sulfur mustard gas (SM) vapor injury, whereas contralateral eyes were left untreated or received TED for tolerabilty evaluation. Two rabbit eyes received no treatment and served as age-matched naive control. The four groups were: Naive (oculus sinister [OS] untreated eyes; n = 9); TED (OS treated only with TED BID for 3 days; n = 9); SM (OD exposed to SM vapor; n = 9); and SM+TED (OD exposed to SM+TED BID for 3 days; n = 9). Ocular examination in live rabbits were performed utilizing slit-lamp biomicroscopy, Fantes grading system, fluorescein staining, Schirmer's tests, pachymetry, and applanation tonometry. Cellular and molecular changes in rabbit corneas were assessed after humane euthanasia on day-3 and day-7 with histopathological and real-time polymerase chain reaction PCR techniques. Results: TED to rabbit eyes was found tolerable in vivo. SM-exposed eyes showed significant increase in Fantes scores, central corneal thickness (CCT), Schirmer's test, epithelium-stroma separation, and corneal edema. TED mitigated clinical symptoms by reducing corneal edema, Fantes scores, CCT, and Schirmer's test. Further, TED decreased SM-induced corneal haze, inflammatory and profibrotic markers, transforming growth factor-TGF-ß1 and cyclooxygenase-2COX-2, and damage to corneal structure, including epithelial-stromal integrity. Conclusions: The developed multimodal eyedrop formulation, TED, has potential to mitigate acute MGK effectively in vivo. Translational Relevance: TED is effective against MGK.

Is sex a biological variable in corneal wound healing?

Wound healing differs significantly between men and women in a tissue-dependent manner. Dermal wounds heal faster in women whereas mucosal wounds heal faster in men. However, the effect of sex as a variable in corneal wound healing is largely unknown. The primary objective of this study was to test whether sex is a biological variable in corneal wound healing activated by the trauma or injury using an established in vivo rabbit model with male and female New Zealand White rabbits. Corneal wounds in rabbits were produced by a single topical alkali (0.5N Sodium hydroxide) application. Serial slit-lamp, stereo biomicroscopy, and applanation tonometry evaluated corneal opacity, anterior segment ocular health, and intraocular pressure (IOP), respectively, at various times during the study. Fourteen days after alkali-wound, corneal tissues were collected after humane euthanasia to examine cellular and molecular wound healing parameters. Quantitative PCR (qPCR) and immunofluorescence were used to quantify changes in the extracellular modeling protein levels of alpha-smooth muscle actin (α-SMA), Fibronectin (FN), Collagen-I (Col-I), and Transforming growth factor beta 1 (TGFß1) involved in corneal healing. Hematoxylin and Eosin (H&E) staining was used to study histopathological changes in morphology and TUNEL assay to evaluate levels of apoptotic cell death. Male and female rabbits showed no significant differences in corneal opacity (Fantes score) or intraocular pressure (IOP) values (9.5 ±â€¯0.5 mm Hg) in live animals. Likewise, no statistically significant sex-based differences in the mRNA levels of α-SMA (male = 5.95 ±â€¯0.21 fold vs. female = 5.32 ±â€¯0.043), FN (male = 3.02 ±â€¯0.24 fold vs. female = 3.23 ±â€¯0.27), Col-I (male = 3.12 ±â€¯0.37 fold vs. female = 3.31 ±â€¯0.24), TGFß1 (male = 1.65 ±â€¯0.06 fold vs. female = 1.59 ±â€¯0.053); and protein levels of α-SMA (male = 74.16 ±â€¯4.6 vs. female = 71.58 ±â€¯7.1), FN (male = 60.11 ±â€¯4.6 vs. female = 57.41 ±â€¯8.3), Col-I (male = 84.11 ±â€¯2.8 vs. female = 84.55 ±â€¯3.6), TGFß1 (male = 11.61 ±â€¯2.8 vs. female = 9.5 ±â€¯3.04) were observed. Furthermore, H&E and TUNEL analyses found no statistically significant differences in cellular structures and apoptosis, respectively, in male vs. female corneas. Consistent with earlier reports, wounded corneas showed significantly increased levels of these parameters compared to the unwounded corneas. Our data suggest that sex is not a major biological variable during active early stages of corneal wound healing in rabbits in vivo.

Linear Polyethylenimine-DNA Nanoconstruct for Corneal Gene Delivery.

PURPOSE: This study investigated the efficiency and potential toxicity of a linear 22-kDa polyethylenimine (PEI)-DNA nanoconstruct for delivering genes to corneal cells and the effects of PEI nitrogen-to-DNA phosphate (N:P) ratio on gene transfer efficiency in vitro and in vivo. METHODS: A gel retardation assay, zeta potential measurement, bright-field microscopy, transfection with green fluorescent protein (GFP), immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) were used to characterize the physicochemical and biological properties and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), and reactive oxygen species (ROS) assay for cytotoxicity of the linear PEI-DNA nanoconstruct using in vitro cultured primary human corneal fibroblast and in vivo mouse models. RESULTS: Of the several evaluated N:P ratios, the highest gene transfection efficiency achieved without any notable cytotoxicity was observed at an N:P ratio of 30:1 (N:P 30). In vivo gene transfer studies revealed substantial GFP gene delivery into the corneas of mice 3 days after a single 5-min topical application without any significant adverse ocular effects. Slit-lamp biomicroscope ophthalmic examination of the mouse exposed to the linear PEI-DNA nanoconstruct showed no evidence of hyperemia (redness), corneal edema, ocular inflammation, or epiphora (excessive tearing). CONCLUSIONS: The 22-kDa linear PEI-DNA nanoconstruct is an efficient and well-tolerated vector for corneal gene therapy in vitro and in vivo and could be used as a platform for developing novel gene-based nanomedicine approaches for corneal diseases.

Autophagy in corneal health and disease: A concise review.

Autophagy is a well-conserved self-eating mechanism of cell survival during periods of nutrient deprivation, stress and injury. Autophagy is implicated in many pathophysiological conditions across all organ systems. The cornea is an avascular transparent tissue that is prone to damage by trauma, injury and infection. Following insult, the cornea undergoes a complex wound healing process, which is regulated by multiple factors including autophagy. The involvement of autophagy in keratoconus and HSV-1 infection has been demonstrated, underlining the importance of this mechanism in corneal disorders. However, the role of autophagy in corneal wound repair, fibrosis and angiogenesis is still unclear. Recently, we characterized the expression of autophagy-related genes in cornea and are studying their role in the modulation of corneal conditions including fibrosis and dystrophies. Preliminary results presented within this review article support further investigation of the dynamic modulation of autophagy-related genes in corneal health and disease. This article provides an overview of how autophagy modulates corneal function.

Decorin antagonizes corneal fibroblast migration via caveolae-mediated endocytosis of epidermal growth factor receptor.

Decorin (Dcn), a small leucine-rich proteoglycan, is involved in the regulation of corneal wound healing. Epidermal growth factor receptor (EGFR) plays a critical role in corneal fibroblasts proliferation, migration and extracellular matrix (ECM) modulation upon injury or infection. The present study aimed to investigate the mechanistic role of Dcn in EGFR internalization to the regulation of corneal stromal fibroblasts (CSFs) migration, a key step in the corneal wound healing. Human corneal stromal fibroblasts (hCSF) cultures were generated from donor corneas. At 70% confluence, cells were switched to serum-free conditions for 48 h and then treated with decorin (250 nM) in the presence or absence of EGF (100 ng/ml) for various time points (10-60 min). Cell lysates were subjected to proteome array analysis screening for 42 different phosphorylated human receptor tyrosine kinases (RTKs), immunocytochemistry, and western blots to analyze EGFR phosphorylation. The scratch-wound assay was performed to evaluate the effects of decorin on EGF-mediated hCSF migration. Dcn caused a rapid EGFR phosphorylation within 10 min of exposure in RTK blot defining its role as a biological ligand for EGFR in hCSFs. Prolonged exposure to Dcn caused complete disappearance of EGFR and inhibition of the hCSF migration in the scratch wound assay suggesting Dcn binding to EGFR causes EGFR down-regulation. Immunostaining studies indicated that Dcn-treatment to hCSFs internalizes Dcn-EGFR complex, which does not require tyrosine kinase activity when treated with the AG1478 inhibitor and co-localizes the complex to the perinuclear region. Next, we found that Dcn-EGFR complex does not follow canonical early endosome internalization as revealed by the EEA1 antibody instead binds to the CD63 antibody directed for degradation by the late endosome. We also found that Dcn regulates the EGFR recycling by preventing its binding to Rab11, a specific antibody for recycling endosome. Further, hCSFs-pretreated with pharmacological inhibitors, methyl-ß-cyclodextrin and chlorpromazine and supplemented with Dcn suggested EGFR trafficking via the caveolae-mediated pathway. These results suggest that Dcn acts as a biological ligand for EGFR and modulates hCSF migration via EGFR down-regulation, thus playing a vital role in corneal wound healing.

Age-dependent alterations in expression and co-localization of Pax6 and Ras-GAP in brain of aging mice.

As the brain ages, the survival and plasticity of neurons and glia are compromised. The data-mining and in silico studies suggest interactions of Pax6 with Ras and binding sites in Ras-GAP promoter. The Pax6 also shows age-dependent alterations. Therefore, it is presumed that Pax6 may be associated with the Ras-GAP, a synaptic protein, either directly or indirectly in brain. The expression, co-localization and interaction of Pax6 and Ras-GAP in different regions of brain of mice during aging were investigated through immunofluorescence assay, co-immunoprecipitation and western blotting, respectively. The co-localization of Pax6 and Ras-GAP were observed in dentate gyrus (DG) and sub-granular zone (SGZ) of hippocampus, in glomerular (GlLa) and mitral cells (MiCe) of olfactory lobe, granular cells (GrCe), Purkinje cell (PuCe) and molecular cell layer (MoLa) of cerebellum, internal plexiform layer (InPl), molecular layer (MoLa) of cerebral cortex and in intercalated cells of amygdala (ITC), caudate nucleus regions in brain of aging mice. The expression of Pax6 and Ras-GAP was altered in hippocampus, amygdala, caudate nucleus, olfactory lobe, cerebral cortex and cerebellum from young to old mice. The Pax6 interacts with Ras-GAP in brain of mice. Results indicate impact of Pax6 on Ras-GAP-mediated activities of synapses, learning and memory, emotions and fear as well as motor functions. Alterations in expression and co-localization of Pax6 and Ras-GAP during aging may be responsible for age-associated compromised survival and plasticity of neurons and glia.

Novel Combination BMP7 and HGF Gene Therapy Instigates Selective Myofibroblast Apoptosis and Reduces Corneal Haze In Vivo.

Purpose: We tested the potential of bone morphogenic protein 7 (BMP7) and hepatocyte growth factor (HGF) combination gene therapy to treat preformed corneal fibrosis using established rabbit in vivo and human in vitro models. Methods: Eighteen New Zealand White rabbits were used. Corneal fibrosis was produced by alkali injury. Twenty-four hours after scar formation, cornea received topically either balanced salt solution (BSS; n = 6), polyethylenimine-conjugated gold nanoparticle (PEI2-GNP)-naked plasmid (n = 6) or PEI2-GNP plasmids expressing BMP7 and HGF genes (n = 6). Donor human corneas were used to obtain primary human corneal fibroblasts and myofibroblasts for mechanistic studies. Gene therapy effects on corneal fibrosis and ocular safety were evaluated by slit-lamp microscope, stereo microscopes, quantitative real-time PCR, immunofluorescence, TUNEL, modified MacDonald-Shadduck scoring system, and Draize tests. Results: PEI2-GNP-mediated BMP7+HGF gene therapy significantly decreased corneal fibrosis in live rabbits in vivo (Fantes scale was 0.6 in BMP7+HGF-treated eyes compared to 3.3 in -therapy group; P < 0.001). Corneas that received BMP7+HGF demonstrated significantly reduced mRNA levels of profibrotic genes: α-SMA (3.2-fold; P < 0.01), fibronectin (2.3-fold, P < 0.01), collagen I (2.1-fold, P < 0.01), collagen III (1.6-fold, P < 0.01), and collagen IV (1.9-fold, P < 0.01) compared to the -therapy corneas. Furthermore, BMP7+HGF-treated corneas showed significantly fewer myofibroblasts compared to the -therapy controls (83%; P < 0.001). The PEI2-GNP introduced >104 gene copies per microgram DNA of BMP7 and HGF genes. The recombinant HGF rendered apoptosis in corneal myofibroblasts but not in fibroblasts. Localized topical BMP7+HGF therapy showed no ocular toxicity. Conclusions: Localized topical BMP7+HGF gene therapy treats corneal fibrosis and restores transparency in vivo mitigating excessive healing and rendering selective apoptosis in myofibroblasts.

Altering equine corneal fibroblast differentiation through Smad gene transfer.

OBJECTIVE: To explore the impact of equine corneal fibroblast (ECF) to myofibroblast (ECM) differentiation by altering the expression of the Smad genes either individually or in combination. Specifically, we sought to examine the ECF differentiation after (a) silencing of Smad2, 3, and 4 profibrotic genes individually and (b) overexpression of antifibrotic Smad7 gene and in a combination with pro- and antifibrotic Smad genes. METHODS: Equine corneal fibroblast primary cultures were generated as previously described. ECFs were transfected with individual plasmids which silenced gene expression of either Smad2, 3, or 4 or in combination with a plasmid overexpressing Smad7 using Lipofectamine 2000™ or Lipofectamine BLOCK-iT™. Smad-transfected clones were then exposed to TGF-ß1 to induce differentiation to myofibroblasts. Immunofluorescence and qRT-PCR techniques quantified levels of ECF differentiation to ECM by measuring alpha smooth muscle actin, a known marker of ECM transdifferentiation. RESULTS: Silencing of individual Smad2, 3, or 4 genes or overexpression of Smad7 showed significant inhibition of ECF transdifferentiation (73-83% reduction). Silencing of Smad2 showed the greatest inhibition of ECF transdifferentiation in (a) and was therefore utilized for the combination gene transfer testing. The combination gene transfer consisting of Smad7 overexpression and Smad2 silencing attenuated ECF differentiation significantly; however, the level was not significant compared to the overexpression of Smad7 individually. CONCLUSIONS: Using gene transfer technology involving profibrotic Smad silencing, antifibrotic Smad overexpression or its combination is a novel strategy to control TGF-ß1-mediated fibrosis in equine fibroblasts. Combination gene therapy was not better than single gene therapy in this study.

Blockade of KCa3.1: A novel target to treat TGF-ß1 induced conjunctival fibrosis.

Postoperative conjunctival fibrosis is common in patients after glaucoma filtration surgery. The calcium activated potassium (KCa3.1) channel has been shown to inhibit fibrosis in many non-ocular tissues. However, its potential in treating ocular fibrosis remains unknown. We tested the anti-fibrotic potential of TRAM34, a selective blocker of KCa3.1 channel, in treating conjunctival fibrosis. Primary human conjunctival fibroblast (HCF) cultures derived from donor tissues. Myofibroblasts causing conjunctival fibrosis were generated by growing HCFs in the presence of TGFß1 for 72 h. KCa3.1 mRNA and protein expression in HCF was examined with PCR and western blot. The anti-fibrotic potential of TRAM34 was examined by measuring fibrotic gene expression with quantitative PCR (qPCR), immunofluorescence, and western blotting in HCFs in ±â€¯TGFß1 (5 ng/ml) and TRAM34 (0-25 µM). The cytotoxicity of Tram34 was analyzed with trypan blue assay and its role in Smad signaling was studied with immunofluorescence. Expression of KCa3.1 mRNA and protein was detected in HCFs and TGFß1 treatment to HCFs significantly increased expression of KCa3.1. TRAM34 treatment attenuated transcription of fibrotic markers, αSMA (p < .001), fibronectin (p < .05), collagen I (p < .001) and collagen IV (p < .001) in TGFß1-induced HCFs. Further, TRAM34 significantly inhibited TGFß1-stimulated αSMA protein expression (p < .01) and nuclear translocation of fibrotic Smad2/3 in HCFs and showed no significant cytotoxicity (p < .05). The KCa3.1 potassium channel plays a significant role in the prevention of conjunctival fibrosis and TRAM34 has potential to control post surgical bleb fibrosis in patients. In vivo studies are warranted.

Level of PAX5 in differential diagnosis of non-Hodgkin's lymphoma.

BACKGROUND & OBJECTIVES: The PAX5, a paired box transcription factor and B-cell activator protein (BSAP), activates B-cell commitment genes and represses non-B-cell lineage genes. About 14 transcript variants of PAX5 have been observed in human. Any alteration in its expression pattern leads to lymphogenesis or associated diseases and carcinogenesis in non-lymphoid tissues. Its mechanisms of function in pathophysiology of non-Hodgkin's lymphoma (NHL) are unclear. This study was intended to explore influence of PAX5 in cascade of NHL pathogenesis and diagnosis. METHODS: Samples of 65 patients were evaluated by immunohistochemical staining for cellular localization of PAX5, CD19, CD3, cABL, p53, Ras and Raf and by TUNEL assay, RNA-isolation and reverse transcriptase (RT)-PCR, w0 estern blot analysis, and lactate dehydrogenase (LDH) specific staining. RESULTS: B-cell type NHL patients were positive for PAX5, p53, Ras, CD19, Raf and CD3. All of them showed TUNEL-positive cells. The differential expression pattern of PAX5, CD19, p53, CD3, Zap700 , HIF 1α, Ras, Raf and MAPK (mitogen-activated protein kinase) at the levels of transcripts and proteins was observed. The LDH assay showed modulation of LDH4 and LDH5 isoforms in the lymph nodes of NHL patients. INTERPRETATION & CONCLUSIONS: The histological observations suggested that the patients represent diverse cases of NHL like mature B-cell type, mature T-cell type and high grade diffuse B-cell type NHL. The findings indicate that patients with NHL may also be analyzed for status of PAX5, CD19 and ZAP70, and their transcriptional and post-translational variants for the differential diagnosis of NHL and therapy.

Chromosomal structural analysis in carcinoma of the gallbladder.

BACKGROUND: The etiopathogenesis of gallbladder cancer is still unknown. Both environmental and patient factors have been incriminated in its cause. That it is found in pockets of epidemiological distribution raises an issue of genetic changes associated with it. The aim of this study was to find out the chromosomal changes in gallbladder cancer. METHODS: Lymphocyte cell culture was carried out on blood of gallbladder cancer patients to determine chromosomal banding abnormalities. Native PAGE was also evaluated to analyze lactate dehydrogenase (LDH), superoxide dismutase (SOD) and catalase enzyme activity from the same blood of gallbladder cancer patients. RESULTS: Out of 30 gallbladder cancer patients, 4 male showed breakage on the long arm of chromosome 1 while only one male patient showed the translocation from the long arm of chromosome 4 to the long arm of chromosome 6 in a male patient. CONCLUSION: The aberrations found in our study may suggest underlying genetic predisposition for the development of gallbladder cancer. They can act as a marker for gallbladder cancer, which needs further study.

Aging-associated modulation in the expression of Pax6 in mouse brain.

Symptoms like mental retardation, depression, and anxiety have been observed during aging. Almost similar phenotypes have been evident in patients having haploinsufficiency or mutations in Pax6, a transcriptional regulator. Since Pax6 regulates axon guidance, differentiation of neurons from glia, and neuronal migration, it has been considered as a marker of newly generated neurons. The immunohistochemical analysis of Pax6 positive cells and expression pattern of Pax6 in olfactory lobe, hippocampus, and cerebellum of aging mouse brain have been investigated. The number of Pax6 positive cells and level of Pax6 were reduced progressively in olfactory lobe, cerebellum, and hippocampus from postnatal day-zero (P0) to old age mice. Pax6 positive cells were significantly lower in dentate gyrus, CA1, CA2, and CA3 regions of hippocampus, in mitral cell (MiCe), and internal plexiform (InPl) layers of olfactory lobe, and in granular cell (GrLa), and Purkinje's cell (PuCe) layers of cerebellum from P0 to old age. Thus, modulation in the expression of Pax6 and reduction in Pax6 positive cells show direct association of Pax6 with aging-related neuronal dystrophy.

Interaction of Pax6 with SPARC and p53 in brain of mice indicates Smad3 dependent auto-regulation.

Pax6 regulates formation of cerebral cortex, axon guidance, differentiation of neurons from glia and neuronal migration in the cerebellum. Although Pax6 is known to be a nuclear protein, it is presumed that it may interact with matricelluar proteins like SPARC during transport and processing of Pax6. The proteins involved in cell survival and cell proliferation can also not be ignored. The present study demonstrates co-localization of Pax6, secreted protein acidic rich in cysteine (SPARC), and p53 in astrocytes of primary culture, cerebellum and cortex region of mouse brain. The physical interaction of Pax6 with SPARC and p53 in brain of mice is also evident. The Pax6 probably participates with p53 to regulate neuronal survival. It indicates that the interaction of Pax6, SPARC and p53 may influence Smad3-dependent auto-regulation of Pax6.