Silibinin alleviates silica-induced pulmonary fibrosis: Potential role in modulating inflammation and epithelial-mesenchymal transition.

Pulmonary fibrosis (PF) is a devastating interstitial lung disease resulting from indefinite causes with very few limited, those too ineffective therapeutic options. Earlier evidence reported inflammation and epithelial-mesenchymal transition (EMT) are the major threats in PF. The present study was aimed to examine the anti-fibrotic activity of silibinin (SB) in PF. PF was induced by administering oropharyngeal 1.5 mg/mice silica on day 1, followed by treatment with and without oral SB for 14 days. Lung injury was assessed by x-ray analysis on day 14 and all the animals were sacrificed on day 15. The results showed that silica remarkably altered the histoarchitecture and induced the expression of inflammatory components in BALF and pulmonary tissue. Immunoblotting investigation quantified the expression of TGF-ß, p-smad2/3, collagen-I, fibronectin, and α-SMA in the pulmonary tissue. To this end, treatment with SB alleviated inflammatory components, including IL-1ß, IL-6, and TNF-α in the fibrotic tissue. Moreover, SB harnessed the tissue architecture, improved diffusive scattering of x-ray signals, and modulated epithelial-mesenchymal phenotypic alterations, including TGF-ß, p-smad2/3, and collagen-I. Altogether, the significant reduction of inflammatory signaling, collagen deposition, and epithelial-mesenchymal transdifferentiation by SB suggested that it could be used as a potential therapeutic candidate to treat pulmonary inflammation and fibrosis.

Ferulic acid ameliorates the progression of pulmonary fibrosis via inhibition of TGF-ß/smad signalling.

Natural products are one of the best sources for the discovery of novel drugs and compounds for multiple diseases. Pulmonary fibrosis (PF) is a chronic, progressive, irreversible, and fatal fibrotic disorder of lungs with unknown etiology and finite therapeutic choices. The use of naturally occurring phytomedicines has emerged to counteract many fibrotic disorders involving oxidative stress and inflammation. In the present study, we evaluated the protective effects of ferulic acid (FA), in an animal model of silica-induced PF. Pulmonary function of mice was evaluated by performing radiological analysis, bronchoalveolar lavage fluid (BALF), inflammatory cytokines, histology and protein expression studies. Our findings revealed that mice challenged with silica displayed characteristic features of pulmonary injury and fibrosis. However, treatment with FA significantly restored the accumulation of inflammatory cells in BALF. FA led to a partial reversal of silica-induced fibrotic changes in the pulmonary tissue. Subsequently, FA halts the progression of PF in a dose-dependent manner by ameliorating the expression of fibrotic proteins including collagen-I, TGF-ß, p-smad2/3 and prevented epithelial-mesenchymal transition (EMT). Collectively, the present study suggests that the inhibition of oxidative stress, inflammatory and TGF-ß/smad signalling might be involved in the observed anti-fibrotic benefits of FA against silica-induced PF in mice.

Synthesis and in Vitro Cytotoxicity Evaluation of Phenanthrene Linked 2,4- Thiazolidinediones as Potential Anticancer Agents.

OBJECTIVE: To synthesize a series of phenanthrene-thiazolidinedione hybrids and explore their cytotoxic potential against human cancer cell lines of A-549 (lung cancer), HCT-116 and HT-29 (colon cancer), MDA MB-231 (triple-negative breast cancer), BT-474 (breast cancer) and (mouse melanoma) B16F10 cells. METHODS: A new series of phenanthrene-thiazolidinedione hybrids was synthesized via Knoevenagel condensation of phenanthrene-9-carbaldehyde and N-alkylated thiazolidinediones. The cytotoxicity (IC50) of the synthesized compounds was determined by MTT assay. Apoptotic assays like (AO/EB) and DAPI staining, cell cycle analysis, JC-1 staining and Annexin V binding assay studies were performed for the most active compound (Z)- 3-(4-bromobenzyl)-5-((2,3,6,7-tetramethoxyphenanthren-9-yl)methylene)thiazolidine-2,4-dione (17b). Molecular docking, dynamics and evaluation of pharmacokinetic (ADME/T) properties were also carried out by using Schrödinger. RESULTS AND DISCUSSION: From the series of tested compounds, 17b unveiled promising cytotoxic action with an IC50 value of 0.985±0.02µM on HCT-116 human colon cancer cells. The treatment of HCT-116 cells with 17b demonstrated distinctive apoptotic morphology like shrinkage of cells, horseshoe-shaped nuclei formation and chromatin condensation. The flow-cytometry analysis revealed the G0/G1 phase cell cycle arrest in a dosedependent fashion. The AO/EB, DAPI, DCFDA, Annexin-V and JC-1 staining studies were performed in order to determine the effect of the compound on cell viability. Computational studies were performed by using Schrödinger to determine the stability of the ligand with the DNA. CONCLUSION: The current study provides an insight into developing a series of phenanthrene thiazolidinedione derivatives as potential DNA interactive agents which might aid in colon cancer therapy.

Novel anticancer Hsp90 inhibitor disubstituted pyrazolyl 2-aminopyrimidine compound 7t induces cell cycle arrest and apoptosis via mitochondrial pathway in MCF-7 cells.

Compound 7t, 4-(4-bromophenyl)-6-(1-(4-chlorophenyl)-3-(4-nitrophenyl)-1H-pyrazol-4-yl) pyrimidin-2-amine, is a proven potent anticancer agent exhibiting Hsp90 inhibition in our previous studies. Herein, we explored the apoptotic potential of compound 7t by Annexin V assay. The mechanism underlining the apoptosis process is elucidated. As a potent Hsp90 inhibitor, compound 7t would induce the mitochondrial stress leading to increased permeability of its membrane, that would subsequently initiate the apoptosis in MCF-7 cells. This was proven by increased J-monomer formation using JC-1 stain. Moreover, due to the impaired mitochondrial function, compound 7t also exaggerated the apoptosis process by ROS generation as proved by DCFDA staining. The morphological and nuclear changes in MCF-7 cells following apoptosis were identified by AO/EB and DAPI staining techniques. It also induced subG1 phase cell cycle arrest. Thus, compound 7t could serve as potential drug in the treatment regimen of breast cancer.

Targeted Krüppel-Like Factor 4 Gene Knock-Out in Retinal Ganglion Cells Improves Visual Function in Multiple Sclerosis Mouse Model.

Axonal demyelination injury and neuronal degeneration are the primary causes of visual disability in multiple sclerosis (MS)-linked optic neuritis patients. Immunomodulatory therapies targeting inflammation have failed to avert the disease progression and no therapies exist to prevent the neuronal deficits seen in MS to date. Neuroprotective strategies targeting oligodendrocytes and astroglia have shown limited success due to a lack of axonal regeneration from injured neurons. In this study, we used the chronic experimental autoimmune encephalomyelitis (EAE) mouse model of MS to investigate the axonal regenerative approach to improve the neuronal function. Our approach focused on targeted knock-out (KO) of the developmentally regulated axon growth inhibitory Krüppel-like factor 4 (Klf4) gene in retinal ganglion cells (RGCs) of Klf4fl/fl mice by intravitreal delivery of AAV2-Cre-ires-EGFP recombinant virus (1) at the time of EAE sensitization and (2) after the onset of optic neuritis-mediated visual defects in the mice. Klf4 gene KO performed simultaneous with EAE sensitization prevented the visual loss as assessed by pattern electroretinograms (PERGs) in the mice and protected the RGCs from EAE-mediated death. More importantly, however, Klf4 gene KO after the onset of optic neuritis also resulted in RGC neuroprotection with additional restoration of their function, thereby improving the visual function outcomes in the EAE model. This study establishes the efficacy of Klf4 targeted knock-down in EAE even after the onset of disease symptoms, and thus should be further explored as a potential treatment strategy for MS/optic neuritis patients.

Gene Therapy with Single-Subunit Yeast NADH-Ubiquinone Oxidoreductase (NDI1) Improves the Visual Function in Experimental Autoimmune Encephalomyelitis (EAE) Mice Model of Multiple Sclerosis (MS).

Mitochondrial dysfunction mediated loss of respiration, oxidative stress, and loss of cellular homeostasis contributes to the neuronal and axonal degenerations permanent loss of function in experimental autoimmune encephalomyelitis model (EAE) of multiple sclerosis (MS). To address the mitochondrial dysfunction mediated visual loss in EAE mice, self-complementary adeno-associated virus (scAAV) containing the NADH-dehydrogenase type-2 (NDI1) complex I gene was intravitreally injected into the mice after the onset of visual defects. Visual function assessed by pattern electroretinogram (PERGs) showed progressive loss of function in EAE mice were improved significantly in NDI1 gene therapy-treated mice. Serial optical coherence tomography (OCT) revealed that progressive thinning of inner retinal layers in EAE mice was prevented upon NDI1 expression. The 45% optic nerve axonal and 33% retinal ganglion cell (RGC) loss contributed to the permanent loss of visual function in EAE mice were ameliorated by NDI1-mediated prevention of mitochondrial cristae dissolution and improved mitochondrial homeostasis. In conclusion, targeting the dysfunctional complex I using NDI1 gene can be an approach to address axonal and neuronal loss responsible for permanent disability in MS that is unaltered by current disease modifying drugs.

Mechanistic investigations on substituted benzene sulphonamides as apoptosis inducing anticancer agents.

In an approach to develop potent cytotoxic compounds with targeted action, a systematic methodology was employed to design and initially synthesize parent compounds A1, A8, A13 and A14 followed by synthesis of further analogs of A1 (A2-A7) and A8 (A9-A12) with characterization by IR, NMR, mass and elemental techniques. These compounds were evaluated for their in vitro anti-proliferative activities against DU-145, MCF-7, HCT-15, HT-29 cell lines and apoptosis inducing potential via various mechanistic studies. Compounds A2, A9, A10 exhibited significant cytotoxic activities compared to their parent compounds and standard drug 5-fluorouracil. Compound A2 displayed superior cytotoxicity with IC50 values less than 1 µM in most of the tested cell lines. Further, compound A2 also induced apoptosis in DU-145 cells as exemplified from DAPI staining, Annexin V-FITC assay, ROS generation and mitochondrial membrane alteration studies. The above studies depict the synthesized compound A2 as potent anticancer agent with the ability to induce apoptosis in prostate cancerous cells.

Design and synthesis of substituted dihydropyrimidinone derivatives as cytotoxic and tubulin polymerization inhibitors.

An operationally simple Biginelli protocol was employed for the synthesis of new C6-carbon based aryl α-haloacrylamide-linked dihydropyrimidinone derivatives. The synthesized compounds were appraised for their in vitro antiproliferative potential against a selected panel of human cancer cell lines especially MCF-7 (human breast cancer), MDA-MB-231 (human breast cancer), HCT-116 (human colon cancer), HCT-15 (human colorectal adenocarcinoma), HT-29 (human colon adenocarcinoma) and DU145 (human prostate cancer) along with normal lung fibroblasts (HFL-1). Preferably, compounds containing α-haloacrylamide (10a-g) functionality were found to exhibit most significant cytotoxicity (IC50 value 0.54 ±â€¯0.12 to 8.35 ±â€¯0.82 µM) against the listed cancer cell lines, particularly towards breast cancer cell lines MCF-7 and MDA-MB-231 (IC50 value 0.54 ±â€¯0.12 to 3.70 ±â€¯0.24 µM). In the seam of synthesized compounds, compound 10f exhibited potent antiproliferative activity against breast cancer cell lines namely MCF-7 (IC50 value 0.54 ±â€¯0.12 µM) and MDA-MB-231 (IC50 value 1.18 ±â€¯0.32 µM). Further to understand the underlying apoptosis mechanisms, different staining techniques such as AO/EB, DCFDA, and DAPI staining were performed. To know the extent of apoptosis and loss of mitochondrial membrane potential in MCF-7 cell lines, annexin V-FITC/PI and JC-1 were performed. Cell cycle analysis revealed that compound 10f arrested the cells at G2/M phase in a dose-dependent manner. The compound 10f also found to exhibit significant inhibition of tubulin polymerization (IC50 of 6.91 ±â€¯0.43 µM) with microtubule destabilizing properties. Molecular docking studies also revealed that compound 10f efficiently interacted with critical catalytically active residues Ser178, Val238, and Val318 of the α/ß-tubulin by a hydrogen bond.

Design, synthesis, and molecular docking studies of novel pyrazolyl 2-aminopyrimidine derivatives as HSP90 inhibitors.

A series of novel pyrazolyl 2-aminopyrimidine derivatives (7a-t) were designed based on scaffold hopping techniques, synthesized and biologically evaluated for their HSP90 inhibition and anticancer activity. Several compounds exhibited potent HSP90 inhibition with IC50 values less than that of the reference standard 17-AAG (1.25 µM). The most potent compound 7t displayed excellent HSP90 inhibition with an IC50 of 20 nM and in vitro antiproliferative potential against three cancer cell lines (IC50 < 5 µM). 7t also induced dose dependent degradation of client proteins (pHER2 and pERK1/2) in Western blot analysis. Several structural features of 7p-t oriented the molecules to retain all the essential binding interactions with HSP90, as observed by rationalized docking studies. Therefore, the para-nitrophenyl ring on the central pyrazole ring along with the 2-amino group on the pyrimidine ring are the crucial features in the development of novel HSP90 inhibitors based on this scaffold for targeted anticancer therapy.

Synthesis of 1-benzyl-1H-benzimidazoles as galectin-1 mediated anticancer agents.

In our pursuit to develop novel non-carbohydrate small molecule Galectin-1 Inhibitors, we have designed a series of 1-benzyl-1H-benzimidazole derivatives and demonstrated their anticancer activity. The compound 6g, 4-(1-benzyl-5-chloro-1H-benzo[d]imidazol-2-yl)-N-(4-hydroxyphenyl) benzamide was found to be most potent with an IC50 of 7.01 ±â€¯0.20 µM and arresting MCF-7 cell growth at G2/M phase and S phase. Induction of apoptosis was confirmed by morphological changes like cell shrinkage, blebbing and cell wall deformation, dose dependent increase in the mitochondrial membrane potential (ΔΨm) and ROS levels. Further, dose dependent decrease in Gal-1 protein levels proves Gal-1 mediated apoptosis by 6g. Molecular docking studies were performed to understand the Gal-1 interaction with compound 6g. In addition, RP-HPLC studies showed 85.44% of 6g binding to Gal-1. Binding affinity studies by fluorescence spectroscopy and Surface Plasmon Resonance (SPR) showed that 6g binds to Gal-1 with binding constant (Ka) of 1.2 × 104 M-1 and equilibrium constant KD value of 5.76 × 10-4 M respectively.

Synthesis and Biological Evaluation of Novel Heterocyclic Imines Linked Coumarin- Thiazole Hybrids as Anticancer Agents.

BACKGROUND: Human Galectin-1, a protein of lectin family showing affinity towards ß-galactosides has emerged as a critical regulator of tumor progression and metastasis, by modulating diverse biological events including homotypic cell aggregation, migration, apoptosis, angiogenesis and immune escape. Therefore, galectin-1 inhibitors might represent novel therapeutic agents for cancer. METHODS: A new series of heterocyclic imines linked coumarin-thiazole hybrids (6a-6r) was synthesized and evaluated for its cytotoxic potential against a panel of six human cancer cell lines namely, lung (A549), prostate (DU-145), breast (MCF-7 & MDA-MB-231), colon (HCT-15 & HT-29) using MTT assay. Characteristic apoptotic assays like DAPI staining, cell cycle, annexin V and Mitochondrial membrane potential studies were performed for the most active compound. Furthermore, Gal-1 inhibition was confirmed by ELISA and fluorescence spectroscopy. RESULTS: Among all, compound 6g {3-(2-(2-(pyridin-2-ylmethylene) hydrazineyl) thiazol-4-yl)-2H-chromen-2- one} exhibited promising growth inhibition against HCT-15 colorectal cancer cells with an IC50 value of 1.28 ± 0.14 µM. The characteristic apoptotic morphological features like chromatin condensation, membrane blebbing and apoptotic body formation were clearly observed with compound 6g on HCT-15 cells using DAPI staining studies. Further, annexin V-FITC/PI assay confirmed effective early apoptosis induction by treatment with compound 6g. Loss of mitochondrial membrane potential and enhanced ROS generation were confirmed with JC-1 and DCFDA staining method, respectively by treatment with compound 6g, suggesting a possible mechanism for inducing apoptosis. Moreover, flow cytometric analysis revealed that compound 6g blocked G0/G1 phase of the cell cycle in a dose-dependent manner. Compound 6g effectively reduced the levels of Gal-1 protein in a dose-dependent manner. The binding constant (Ka) of 6g with Gal-1 was calculated from the intercept value which was observed as 1.9 x 107 M-1 by Fluorescence spectroscopy. Molecular docking studies showed strong interactions of compound 6g with Gal-1 protein. CONCLUSION: Our studies demonstrate the anticancer potential and Gal-1 inhibition of heterocyclic imines linked coumarin-thiazole hybrids.

Andrographolide ameliorates silica induced pulmonary fibrosis.

The purpose of this study was to investigate the protective effect of andrographolide in silica-induced pulmonary fibrosis (PF) in mice and its underlying mechanisms. Male Swiss albino mice were divided into five groups: Normal control group, disease control group (1.5 mg silica/60 µL/mice) via oropharyngeal route, low dose (LD) group received silica + andrographolide (3 mg/kg), high dose (HD) group received silica + andrographolide (10 mg/kg), andrographolide per se group received 10 mg/kg andrographolide. Various bronchoalveolar lavage fluid (BALF) and biochemical parameters, inflammatory cytokines, histology and protein expression studies were carried out. Andrographolide significantly reduced total protein concentration, albumin, accumulation of inflammatory cells and lactate dehydrogenase (LDH) level in BALF. We found that andrographolide intervention led to decreased levels of the inflammatory cells including neutrophils, macrophages and lymphocytes in the BALF of the treated animals. In addition, andrographolide significantly reduced nitrite (p < 0.01 at HD), malondialdehyde (p < 0.01 at HD) and upregulated glutathione (p < 0.01 at HD) in silica challenged animals. Andrographolide showed anti-fibrotic activity by reducing collagen deposition and inflammation in lung. Histopathology revealed that andrographolide decreased irregular cellular nodules, inflammatory infiltration and fibrosis. Andrographolide intervention significantly reduced the expression of N-cadherin, α-SMA and vimentin (mesenchymal markers) and upregulated the expression of E-cadherin (an epithelial marker). Hence, andrographolide elicits its anti-pulmonary fibrotic effect by halting the progression of epithelial-to-mesenchymal transition (EMT) via affecting fibroblasts. We, to the best of our knowledge prove for the first time that andrographolide possesses potent antifibrotic activity by targeting inflammatory cells and EMT associated fibroblasts.

Oropharyngeal administration of silica in Swiss mice: A robust and reproducible model of occupational pulmonary fibrosis.

Pulmonary fibrosis (PF) is a lethal end stage of interstitial lung disease with increasing prevalence. The disease burden of PF has seen a sharp surge in the past two decades owing to entry of heavy amount of particulate matter due to industrialization and urbanization. In this work, we developed an oropharyngeal aspiration model of silica (1.5 mg/mice) induced pulmonary fibrosis as a homogeneous, reproducible, simple and alternative strategy in Swiss albino mice. Various BALF (protein, albumin, cell count), biochemical parameters (MDA, GSH, hydroxyproline), cytokines (IL-1ß, IL-6, TNF-α and TGF-ß1), histological (H&E and PSR staining) and protein expression (N-cadherin, vimentin, α-SMA, CTGF, collagen-1) studies were conducted to validate the model. Oropharyngeal administration of silica in Swiss mice produced significantly changes in lung morphology with statistically higher lung weights compared to normal control animals. The silica treated mice showed profoundly elevated BALF soluble and cytological parameters and enhanced oxidative and nitrosative stress in lungs. The levels of hydroxyproline were increased by 2.6 fold in the silica treated mice. The expression of pro-inflammatory cytokines were profoundly increased in silica treated mice. The histology and PSR staining indicated increased inflammatory infiltration and staggering fibrosis in silica treated group. In addition, the expression of EMT markers (N-cadherin, vimentin, α-SMA and CTGF) were significantly increased indicating their role in silica induced pulmonary fibrosis. Our work clearly demonstrates the superiority of stress free oropharyngeal instillation of silica with dose reduction over the conventional invasive and non-homogeneous intratracheal route.

Lacritin and other autophagy associated proteins in ocular surface health.

Advantage may be taken of macroautophagy ('autophagy') to promote ocular health. Autophagy continually captures aged or damaged cellular material for lysosomal degradation and recyling. When autophagic flux is chronically elevated, or alternatively deficient, health suffers. Chronic elevation of flux and stress are the consequence of inflammatory cytokines or of dry eye tears but not normal tears invitro. Exogenous tear protein lacritin transiently accelerates flux to restore homeostasis invitro and corneal health invivo, and yet the monomeric active form of lacritin appears to be selectively deficient in dry eye. Tissue transglutaminase-dependent cross-linking of monomer decreases monomer quantity and monomer affinity for coreceptor syndecan-1 thereby abrogating activity. Tissue transglutaminase is elevated in dry eye. Mutation of arylsulfatase A, arylsulfatase B, ceroid-lipofuscinosis neuronal 3, mucolipin, or Niemann-Pick disease type C1 respectively underlie several diseases of apparently insufficient autophagic flux that affect the eye, including: metachromatic leukodystrophy, mucopolysaccharidosis type VI, juvenile-onset Batten disease, mucolipidosis IV, and Niemann-Pick type C associated with myelin sheath destruction of corneal sensory and ciliary nerves and of the optic nerve; corneal clouding, ocular hypertension, glaucoma and optic nerve atrophy; accumulation of 'ceroid-lipofuscin' in surface conjunctival cells, and in ganglion and neuronal cells; decreased visual acuity and retinal dystrophy; and neurodegeneration. For some, enzyme or gene replacement, or substrate reduction, therapy is proving to be successful. Here we discuss examples of restoring ocular surface homeostasis through alteration of autophagy, with particular attention to lacritin.

Complex I subunit gene therapy with NDUFA6 ameliorates neurodegeneration in EAE.

PURPOSE: To address the permanent disability induced by mitochondrial dysfunction in experimental autoimmune encephalomyelitis (EAE). METHODS: Mice sensitized for EAE were rescued by intravitreal injection of adeno-associated viral vector serotype 2 with the complex I subunit gene scAAV-NDUFA6Flag. Controls were injected with a mitochondrially targeted red fluorescent protein (scAAV-COX8-cherry). Another group received scAAV-COX8-cherry, but was not sensitized for EAE. Serial pattern electroretinograms (PERGs) and optical coherent tomography (OCT) evaluated visual function and structure of the retina at 1, 3, and 6 months post injection (MPI). Treated mice were killed 6 MPI for histopathology. Immunodetection of cleaved caspase 3 gauged apoptosis. Complex I activity was assessed spectrophotometrically. Expression of NDUFA6Flag in the retina and optic nerve were evaluated between 1 week to 1 month post injection by RT-PCR, immunofluorescence and immunoblotting. RESULTS: Reverse transcription-PCR and immunoblotting confirmed NDUFA6Flag overexpression with immunoprecipitation and blue native PAGE showing integration into murine complex I. Overexpression of NDUFA6Flag in the visual system of EAE mice rescued retinal complex I activity completely, axonal loss by 73%, and retinal ganglion cell (RGC) loss by 88%, RGC apoptosis by 66%, and restored the 33% loss of complex I activity in EAE to normal levels; thereby, preventing loss of vision indicated by the 43% reduction in the PERG amplitudes of EAE mice. CONCLUSIONS: NDUFA6 gene therapy provided long-term suppression of neurodegeneration in the EAE animal model suggesting that it may also ameliorate the mitochondrial dysfunction associated with permanent disability in optic neuritis and MS patients.

LHON gene therapy vector prevents visual loss and optic neuropathy induced by G11778A mutant mitochondrial DNA: biodistribution and toxicology profile.

PURPOSE: To demonstrate safety and efficacy of allotopic human ND4 for treatment of a Leber's hereditary optic neuropathy (LHON) mouse model harboring the G11778A mitochondrial mutation. METHODS: We induced LHON in mice by intravitreal injection of mutant (G11778A) human ND4 DNA, responsible for most cases of LHON, that was directed to mitochondria using an AAV2 vector to which we appended a mitochondrial targeting sequence to the VP2 capsid. We then attempted rescue of visual loss using our test article (ScAAV2-P1ND4v2) containing a synthetic nuclear encoded G11778G ND4 gene that was allotopically expressed. Control mice either were uninjected or received AAV2-GFP or AAV2-mCherry. We performed RT-PCR and confocal microscopy at 2 weeks post injection. Pattern electroretinograms (PERGs), spectral-domain optical coherence tomography (SD-OCT), histology, and transmission electron microscopy (TEM) were performed. For toxicology and biodistribution studies, the test article was administered intravitreally to rats and rhesus macaques at different doses. RESULTS: Mutant and wild-type ND4 were efficiently expressed in the mitochondria of retinal ganglion cells (RGCs). Visual function assessed by serial PERGs and retinal structure by serial SD-OCT showed a significant rescue by the test article. Histology and ultrastructural analysis confirmed that loss of RGCs and demise of axons was prevented by ScAAV2-P1ND4v2. Rat and nonhuman primate biodistribution studies showed that vector spread outside the injected eye into spleen and lymph nodes was minimal. Histopathology of tissues and organs including the eyes was comparable to that of uninfected and saline-injected eyes. CONCLUSIONS: Allotopically expressed wild-type ND4 prevents the phenotype induced by G11778A mitochondrial DNA with a toxicology profile acceptable for testing in a phase I clinical trial.

Gene therapy with mitochondrial heat shock protein 70 suppresses visual loss and optic atrophy in experimental autoimmune encephalomyelitis.

PURPOSE: To rescue visual loss and optic neuropathy in experimental autoimmune encephalomyelitis (EAE). METHODS: Encephalomyelitis was induced in mice that received intravitreal injections of AAV2-mtHSP70Flag or AAV2-Cox8-mCherry. Additional mice were injected with AAV2-Cox8-mCherry, but not sensitized for EAE. Visual function was assessed by pattern electroretinograms (PERG) at 1, 3, and 6 months post injection (MPI). Optical coherence tomography (OCT) evaluated the thickness of the inner plexiform layer + nerve fiber layers at 1, 3, and 6 MPI. Retinas and optic nerves (ONs) of mice euthanized 6 MPI were processed for light and electron microscopy. Expression of mtHSP70Flag in the retina and ONs was evaluated by RT-PCR, immunofluorescence, and Western blotting. The activities of respiratory complexes I and III, as well as mitochondrial protein import were quantitated. RESULTS: Expression: immunofluorescence revealed punctate and perinuclear expression of mtHSP70Flag that colocalized with mitochondrial porin in thy1.2 labeled retinal ganglion cells (RGCs). Immunoblotting and RT-PCR confirmed mtHSP70Flag expression in the retina and ON. Rescue: treatment with mtHSP70Flag resulted in a 44% increase in PERG amplitude and less delays in latency relative to the EAE-mCherry group that also showed progressive inner retinal thinning. At 6 MPI, the almost 50% loss of RGCs and optic nerve axons in EAE mice was suppressed by mtHSP70Flag. In addition, retinas of EAE-mtHSP70Flag mice showed nearly complete rescue of complex I and III activities that was reduced by one-third in the EAE-mCherry retinas. Lastly, reductions in import of COX8-mCherry into mitochondria of mice sensitized for EAE improved by 30% with mtHSP70Flag gene therapy. CONCLUSIONS: Mitochondrial HSP70 ameliorates mitochondrial dysfunction that culminates in irreversible visual loss and atrophy of the optic nerve in EAE suggesting that it may be useful to prevent irreversible disability in patients with optic neuritis and multiple sclerosis (MS).

Structural integrity of the Greek key motif in ßγ-crystallins is vital for central eye lens transparency.

BACKGROUND: We highlight an unrecognized physiological role for the Greek key motif, an evolutionarily conserved super-secondary structural topology of the ßγ-crystallins. These proteins constitute the bulk of the human eye lens, packed at very high concentrations in a compact, globular, short-range order, generating transparency. Congenital cataract (affecting 400,000 newborns yearly worldwide), associated with 54 mutations in ßγ-crystallins, occurs in two major phenotypes nuclear cataract, which blocks the central visual axis, hampering the development of the growing eye and demanding earliest intervention, and the milder peripheral progressive cataract where surgery can wait. In order to understand this phenotypic dichotomy at the molecular level, we have studied the structural and aggregation features of representative mutations. METHODS: Wild type and several representative mutant proteins were cloned, expressed and purified and their secondary and tertiary structural details, as well as structural stability, were compared in solution, using spectroscopy. Their tendencies to aggregate in vitro and in cellulo were also compared. In addition, we analyzed their structural differences by molecular modeling in silico. RESULTS: Based on their properties, mutants are seen to fall into two classes. Mutants A36P, L45PL54P, R140X, and G165fs display lowered solubility and structural stability, expose several buried residues to the surface, aggregate in vitro and in cellulo, and disturb/distort the Greek key motif. And they are associated with nuclear cataract. In contrast, mutants P24T and R77S, associated with peripheral cataract, behave quite similar to the wild type molecule, and do not affect the Greek key topology. CONCLUSION: When a mutation distorts even one of the four Greek key motifs, the protein readily self-aggregates and precipitates, consistent with the phenotype of nuclear cataract, while mutations not affecting the motif display 'native state aggregation', leading to peripheral cataract, thus offering a protein structural rationale for the cataract phenotypic dichotomy "distort motif, lose central vision".

Anti-inflammatory treatments for chronic diseases: a review.

Inflammation is viewed as one of the major causes for the development of different diseases like cancer, cardiovascular disease, diabetes, obesity, osteoporosis, rheumatoid arthritis, inflammatory bowel disease, asthma, and CNS related diseases such as depression and parkinson's disease; and this fervent phenomenon provides space for understanding different inflammatory markers. Increasing evidences have elucidated the outcome of inflammatory pathways dysregulation resulting in many symptoms of chronic diseases. The detection of transcription factors such as nuclear factor kappa-B (NF-κB), STAT and their gene products such as COX-2, cytokines, chemokines and chemokine receptors has laid molecular foundation for the important role of inflammation in chronic diseases in which the NF-κB is reported as a major mediator which makes a possible way for the development of new therapeutic approaches using synthetic and natural compounds that might eventually decrease the prevalence of these diseases. Even if many inflammatory markers like TNF-α, IL-1, IL-6, IL-8 and C-reactive protein (CRP) are reported to be the major key factors with proved role in several inflammatory diseases, IL-1 and TNF-α are the important cytokines that can induce the expression of NF-κB which is the potential target in these inflammatory diseases. This review aims to explore and summarize that how some drugs and natural compounds show their modulatory activity in inflammatory pathways and chronic inflammatory markers in these inflammatory diseases.

Noninvasive assessments of optic nerve neurodegeneration in transgenic mice with isolated optic neuritis.

PURPOSE: To determine if phosphorylated neurofilament heavy chain (pNF-H) released into the bloodstream and the pattern ERG are noninvasive indicators of neurodegeneration in experimental optic neuritis. METHODS: Serum from Myelin oligodendrocyte glycoprotein (MOG)-specific T cell receptor-positive (TCR+) transgenic mice that develop isolated optic neuritis usually without any other characteristic lesions of inflammation or demyelination in the spinal cord and littermates negative for the transgene were assayed for the presence of serum phosphorylated neurofilament H (pNF-H). In vivo measurements of optic nerve and retinal ganglion cell injury were assessed by magnetic resonance imaging (MRI), optical coherence tomography (OCT), and pattern electroretinogram (PERG). Automated two dimensional fluorescence differential in-gel electrophoresis (2D-DIGE) of pooled optic nerve samples, light, and transmission electron micrographs were used to evaluate optic atrophy postmortem. RESULTS: We found an almost 3-fold elevation in serum pNF-H levels in MOG+ mice relative to MOG-littermates (P = 0.02). 2D-DIGE revealed a 3-fold reduction in optic nerve neurofilaments. Visual function assessed by the PERG was reduced by one-quarter (P = 0.033) and latencies increased by 38% (P = 0.036). MOG+ mice with the lowest PERG amplitudes had optic nerve atrophy visualized by MRI. Optic nerve diameters were reduced by one-third (P = 0.0001) and axon counts reduced by more than two-thirds. Histopathology of the spinal cords was normal. CONCLUSIONS: Elevated serum pNF-H levels and the PERG are useful markers of neurodegeneration of the optic nerve in isolated experimental optic neuritis. Our findings suggest that elevations of this axonal protein in patients with optic neuritis who had a poor visual outcome are likely also due to demise of optic nerve axons.