Pharmacokinetics, pharmacodynamics, and tolerability of an aqueous formulation of rusfertide (PTG-300), a hepcidin mimetic, in healthy volunteers: A double-blind first-in-human study.

OBJECTIVES: Rusfertide is a potent peptide mimetic of hepcidin being investigated for the treatment of polycythemia vera. This randomized, placebo-controlled, double-blind study evaluated the safety, pharmacokinetics, and pharmacodynamics of single and repeated subcutaneous doses of an aqueous formulation of rusfertide in healthy adult males. METHODS: Subjects received single doses of 1, 3, 10, 20, 40, or 80 mg rusfertide or placebo. A separate cohort of subjects received two doses of 40 mg rusfertide or placebo 1 week apart. Blood samples for pharmacokinetics and pharmacodynamics were collected, and adverse events, clinical laboratory tests, 12-lead electrocardiograms, and vital signs were monitored. RESULTS: Rusfertide was well tolerated. There were no serious or severe treatment-emergent adverse events, and no patterns of clinically important adverse events, or laboratory, vital sign, or electrocardiogram abnormalities. Mean maximum rusfertide plasma concentration (Cmax) and area under the concentration-time curve increased with dose, but less than dose proportionally. Median time to Cmax was 2-4.5 h for 40 and 80 mg rusfertide and 8-24 h for lower doses. Apparent clearance and half-life increased with dose. Single doses of rusfertide 1-80 mg were associated with dose-dependent decreases in serum iron and transferrin-iron saturation. CONCLUSIONS: Rusfertide was well tolerated and showed dose-dependent pharmacokinetics and pharmacodynamics.

Rusfertide, a Hepcidin Mimetic, for Control of Erythrocytosis in Polycythemia Vera.

BACKGROUND: Polycythemia vera is a chronic myeloproliferative neoplasm characterized by erythrocytosis. Rusfertide, an injectable peptide mimetic of the master iron regulatory hormone hepcidin, restricts the availability of iron for erythropoiesis. The safety and efficacy of rusfertide in patients with phlebotomy-dependent polycythemia vera are unknown. METHODS: In part 1 of the international, phase 2 REVIVE trial, we enrolled patients in a 28-week dose-finding assessment of rusfertide. Part 2 was a double-blind, randomized withdrawal period in which we assigned patients, in a 1:1 ratio, to receive rusfertide or placebo for 12 weeks. The primary efficacy end point was a response, defined by hematocrit control, absence of phlebotomy, and completion of the trial regimen during part 2. Patient-reported outcomes were assessed by means of the modified Myeloproliferative Neoplasm Symptom Assessment Form (MPN-SAF) patient diary (scores range from 0 to 10, with higher scores indicating greater severity of symptoms). RESULTS: Seventy patients were enrolled in part 1 of the trial, and 59 were assigned to receive rusfertide (30 patients) or placebo (29 patients) in part 2. The estimated mean (±SD) number of phlebotomies per year was 8.7±2.9 during the 28 weeks before the first dose of rusfertide and 0.6±1.0 during part 1 (estimated difference, 8.1 phlebotomies per year). The mean maximum hematocrit was 44.5±2.2% during part 1 as compared with 50.0±5.8% during the 28 weeks before the first dose of rusfertide. During part 2, a response was observed in 60% of the patients who received rusfertide as compared with 17% of those who received placebo (P = 0.002). Between baseline and the end of part 1, rusfertide treatment was associated with a decrease in individual symptom scores on the MPN-SAF in patients with moderate or severe symptoms at baseline. During parts 1 and 2, grade 3 adverse events occurred in 13% of the patients, and none of the patients had a grade 4 or 5 event. Injection-site reactions of grade 1 or 2 in severity were common. CONCLUSIONS: In patients with polycythemia vera, rusfertide treatment was associated with a mean hematocrit of less than 45% during the 28-week dose-finding period, and the percentage of patients with a response during the 12-week randomized withdrawal period was greater with rusfertide than with placebo. (Funded by Protagonist Therapeutics; REVIVE ClinicalTrials.gov number, NCT04057040.).

Tissue-targeted and localized AAV5-DCN and AAV5-PEDF combination gene therapy abrogates corneal fibrosis and concurrent neovascularization in rabbit eyes in vivo.

PURPOSE: Corneal fibrosis and neovascularization (CNV) after ocular trauma impairs vision. This study tested therapeutic potential of tissue-targeted adeno-associated virus5 (AAV5) mediated decorin (DCN) and pigment epithelium-derived factor (PEDF) combination genes in vivo. METHODS: Corneal fibrosis and CNV were induced in New Zealand White rabbits via chemical trauma. Gene therapy in stroma was delivered 30-min after chemical-trauma via topical AAV5-DCN and AAV5-PEDF application using a cloning cylinder. Clinical eye examinations and multimodal imaging in live rabbits were performed periodically and corneal tissues were collected 9-day and 15-day post euthanasia. Histological, cellular, and molecular and apoptosis assays were used for efficacy, tolerability, and mechanistic studies. RESULTS: The AAV5-DCN and AAV5-PEDF combination gene therapy significantly reduced corneal fibrosis (p < 0.01 or p < 0.001) and CNV (p < 0.001) in therapy-given (chemical-trauma and AAV5-DCN + AAV5-PEDF) rabbit eyes compared to the no-therapy given eyes (chemical-trauma and AAV5-naked vector). Histopathological analyses demonstrated significantly reduced fibrotic α-smooth muscle actin and endothelial lectin expression in therapy-given corneas compared to no-therapy corneas on day-9 (p < 0.001) and day-15 (p < 0.001). Further, therapy-given corneas showed significantly increased Fas-ligand mRNA levels (p < 0.001) and apoptotic cell death in neovessels (p < 0.001) compared to no-therapy corneas. AAV5 delivered 2.69 × 107 copies of DCN and 2.31 × 107 copies of PEDF genes per µg of DNA. AAV5 vector and delivered DCN and PEDF genes found tolerable to the rabbit eyes and caused no significant toxicity to the cornea. CONCLUSION: The combination AAV5-DCN and AAV5-PEDF topical gene therapy effectively reduces corneal fibrosis and CNV with high tolerability in vivo in rabbits. Additional studies are warranted.

Rusfertide for the treatment of iron overload in HFE-related haemochromatosis: an open-label, multicentre, proof-of-concept phase 2 trial.

BACKGROUND: Hereditary haemochromatosis protein (HFE)-related haemochromatosis, an inherited iron overload disorder caused by insufficient hepcidin production, results in excessive iron absorption and tissue and organ injury, and is treated with first-line therapeutic phlebotomy. We aimed to investigate the efficacy and safety of rusfertide, a peptidic mimetic of hepcidin, in patients with HFE-related haemochromatosis. METHODS: This open-label, multicentre, proof-of-concept phase 2 trial was done across nine academic and community centres in the USA and Canada. Adults (aged ≥18 years) with HFE-related haemochromatosis on a stable therapeutic phlebotomy regimen (maintenance phase) for at least 6 months before screening and who had a phlebotomy frequency of at least 0·25 per month (eg, at least three phlebotomies in 12 months or at least four phlebotomies in 15 months) and less than one phlebotomy per month, with serum ferritin of less than 300 ng/mL and haemoglobin of more than 11·5 g/dL, were eligible. Patients initiated 24 weeks of subcutaneous rusfertide treatment within 7 days of a scheduled phlebotomy at 10 mg once weekly. Rusfertide doses and dosing schedules could be adjusted to maintain serum transferrin iron saturation (TSAT) at less than 40%. During rusfertide treatment, investigators were to consider the need for phlebotomy when the serum ferritin and TSAT values exceeded the patient's individual pre-phlebotomy serum ferritin and TSAT values. No primary endpoint or testing hierarchy was prespecified. Prespecified efficacy endpoints included the change in the frequency of phlebotomies; the proportion of patients achieving phlebotomy independence; change in serum iron, TSAT, serum transferrin, serum ferritin, and liver iron concentration (LIC) as measured by MRI; and treatment-emergent adverse events (TEAEs). The key efficacy analyses for phlebotomy rate and LIC were conducted by use of paired t tests in the intention-to-treat population, defined as all patients who received any study drug and who had pretreatment and at least one post-dose measurement. We included all participants who received at least one dose of rusfertide in the safety analyses. This trial is closed and completed and is registered with ClinicalTrials.gov, NCT04202965. FINDINGS: Between March 11, 2020, and April 23, 2021, 28 patients were screened and 16 (ten [63%] men and six [38%] women) were enrolled. 16 were included in analyses of phlebotomy endpoints and 14 for the LIC endpoint. 12 (75%) patients completed 24 weeks of treatment. The mean number of phlebotomies was significantly reduced during the 24-week rusfertide treatment (0·06 phlebotomies [95% CI -0·07 to 0·20]) compared with 24 weeks pre-study (2·31 phlebotomies [95% CI 1·77 to 2·85]; p<0·0001). 15 (94%) of 16 patients were phlebotomy-free during the treatment period. Mean LIC in the 14 patients in the intention-to-treat population was 1·4 mg iron per g dry liver weight (95% CI 1·0 to 1·8) at screening and 1·1 mg iron per g dry liver weight (95% CI 0·9 to 1·3) at the end of treatment (p=0·068). Mean TSAT was 45·3% (95% CI 33·2 to 57·3) at screening, 36·7% (24·2 to 49·2) after the pretreatment phlebotomy, 21·8% (15·8 to 27·9) 24 h after the first dose of rusfertide, 40·4% (27·1 to 53·8) at the end of treatment, and 32·6% (25·0 to 40·1) over the treatment duration. Mean serum iron was 24·6 µmol/L (95% CI 18·6 to 30·6), 20·1 µmol/L (14·8 to 25·3), 11·9 µmol/L (9·2 to 14·7), 22·5 µmol/L (15·9 to 29·1), and 19·0 µmol/L (15·3 to 22·6) at these same timepoints, respectively. Mean serum ferritin was 83·3 µg/L (52·2 to 114.4), 65·5 µg/L (32·1 to 98·9), 62·8 µg/L (33·8 to 91·9), 150·0 µg/L (86·6 to 213.3), and 94·3 µg/L (54·9 to 133.6) at these same timepoints, respectively. There were only minor changes in serum transferrin concentration. 12 (75%) patients had at least one TEAE, the most common of which was injection site pain (five [31%] patients). All TEAEs were mild or moderate in severity, except for a serious adverse event of pancreatic adenocarcinoma, which was considered severe and unrelated to treatment and was pre-existing and diagnosed 21 days after starting rusfertide treatment. INTERPRETATION: Rusfertide prevents iron re-accumulation in the absence of phlebotomies and could be a viable therapeutic option for selected patients with haemochromatosis. FUNDING: Protagonist Therapeutics.

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.

Toxicological effects of ocular acrolein exposure to eyelids in rabbits in vivo.

Acrolein is a highly reactive volatile toxic chemical that injures the eyes and many organs. It has been used in wars and terrorism for wounding masses on multiple occasions and is readily accessible commercially. Our earlier studies revealed acrolein's toxicity to the cornea and witnessed damage to other ocular tissues. Eyelids play a vital role in keeping eyes mobile, moist, lubricated, and functional utilizing a range of diverse lipids produced by the Meibomian glands located in the upper and lower eyelids. This study sought to investigate acrolein's toxicity to eyelid tissues by studying the expression of inflammatory and lipid markers in rabbit eyes in vivo utilizing our reported vapor-cap model. The study was approved by the institutional animal care and use committees and followed ARVO guidelines. Twelve New Zealand White Rabbits were divided into 3 groups: Naïve (group 1), 1-min acrolein exposure (group 2), or 3-min acrolein exposure (group 3). The toxicological effects of acrolein on ocular health in live animals were monitored with regular clinical eye exams and intraocular pressure measurements and eyelid tissues post-euthanasia were subjected to H&E and Masson's trichrome histology and qRT-PCR analysis. Clinical eye examinations witnessed severely swollen eyelids, abnormal ocular discharge, chemosis, and elevated intraocular pressure (p < 0.001) in acrolein-exposed eyes. Histological studies supported clinical findings and exhibited noticeable changes in eyelid tissue morphology. Gene expression studies exhibited significantly increased expression of inflammatory and lipid mediators (LOX, PAF, Cox-2, and LTB4; p < 0.001) in acrolein-exposed eyelid tissues compared to naïve eyelid tissues. The results suggest that acrolein exposure to the eyes causes acute damage to eyelids by altering inflammatory and lipid mediators in vivo.

Corneal injury is associated with stromal and vascular alterations within cranial dura mater.

The cornea and cranial dura mater share sensory innervation. This link raises the possibility that pathological impulses mediated by corneal injury may be transmitted to the cranial dura, trigger dural perivascular/connective tissue nociceptor responses, and induce vascular and stromal alterations affecting dura mater blood and lymphatic vessel functionality. In this study, using a mouse model, we demonstrate for the first time that two weeks after the initial insult, alkaline injury to the cornea leads to remote pathological changes within the coronal suture area of the dura mater. Specifically, we detected significant pro-fibrotic changes in the dural stroma, as well as vascular remodeling characterized by alterations in vascular smooth muscle cell (VSMC) morphology, reduced blood vessel VSMC coverage, endothelial cell expression of the fibroblast specific protein 1, and significant increase in the number of podoplanin-positive lymphatic sprouts. Intriguingly, the deficiency of a major extracellular matrix component, small leucine-rich proteoglycan decorin, modifies both the direction and the extent of these changes. As the dura mater is the most important route for the brain metabolic clearance, these results are of clinical relevance and provide a much-needed link explaining the association between ophthalmic conditions and the development of neurodegenerative diseases.

Real-world treatments and thrombotic events in polycythemia vera patients in the USA.

Polycythemia vera (PV) is a myeloproliferative neoplasm associated with increased risk of thrombotic events (TE) and death. Therapeutic interventions, phlebotomy and cytoreductive medications, are targeted to maintain hematocrit levels < 45% to prevent adverse outcomes. This retrospective observational study examined medical and pharmacy claims of 28,306 PV patients initiating treatment for PV in a data period inclusive of 2011 to 2019. Study inclusion required ≥ 2 PV diagnosis codes in the full data period, at least 1 year of PV treatment history, and ≥ 1 prescription claim and medical claim in both 2018 and 2019. Patients having ≥ 2 hematocrit (HCT) test results in linked outpatient laboratory data (2018-2019) were designated as the HCT subgroup (N = 4246). Patients were characterized as high- or low-risk at treatment initiation based on age and prior thrombotic history. The majority of patients in both risk groups (60% of high-risk and 83% of low-risk) initiated treatment with phlebotomy monotherapy, and during a median follow-up period of 808 days, the vast majority (81% low-risk, 74% high-risk) maintained their original therapy during the follow-up period. Hematocrit control was suboptimal in both risk groups; 54% of high-risk patients initiating with phlebotomy monotherapy sometimes/always had HCT levels > 50%; among low-risk patients, 64% sometimes/always had HCT levels above 50%. Overall, 16% of individuals experienced at least 1 TE subsequent to treatment initiation, 20% (n = 3920) among high-risk and 8% (n = 629) among low-risk patients. This real-world study suggests that currently available PV treatments may not be used to full advantage.

Role of aquaporins in corneal healing post chemical injury.

Aquaporins (AQPs) are transmembrane water channel proteins that regulate the movement of water through the plasma membrane in various tissues including cornea. The cornea is avascular and has specialized microcirculatory mechanisms for homeostasis. AQPs regulate corneal hydration and transparency for normal vision. Currently, there are 13 known isoforms of AQPs that can be subclassified as orthodox AQPs, aquaglyceroporins (AQGPs), or supraquaporins (SAQPs)/unorthodox AQPs. AQPs are implicated in keratocyte function, inflammation, edema, angiogenesis, microvessel proliferation, and the wound-healing process in the cornea. AQPs play an important role in wound healing by facilitating the movement of corneal stromal keratocytes by squeezing through tight stromal matrix and narrow extracellular spaces to the wound site. Deficiency of AQPs can cause reduced concentration of hepatocyte growth factor (HGF) leading to reduced epithelial proliferation, reduced/impaired keratocyte migration, reduced number of keratocytes in the injury site, delayed and abnormal wound healing process. Dysregulated AQPs cause dysfunction in osmolar homeostasis as well as wound healing mechanisms. The cornea is a transparent avascular tissue that constitutes the anterior aspect of the outer covering of the eye and aids in two-thirds of visual light refraction. Being the outermost layer of the eye, the cornea is prone to injury. Of the 13 AQP isoforms, AQP1 is expressed in the stromal keratocytes and endothelial cells, and AQP3 and AQP5 are expressed in epithelial cells in the human cornea. AQPs can facilitate wound healing through aid in cellular migration, proliferation, migration, extracellular matrix (ECM) remodeling and autophagy mechanism. Corneal wound healing post-chemical injury requires an integrative and coordinated activity of the epithelium, stromal keratocytes, endothelium, ECM, and a battery of cytokines and growth factors to restore corneal transparency. If the chemical injury is mild, the cornea will heal with normal clarity, but severe injuries can lead to partial and/or permanent loss of corneal functions. Currently, the role of AQPs in corneal wound healing is poorly understood in the context of chemical injury. This review discusses the current literature and the role of AQPs in corneal homeostasis, wound repair, and potential therapeutic target for acute and chronic corneal injuries.

Characterization of C-X-C chemokine receptor type 5 in the cornea and role in the inflammatory response after corneal injury.

C-X-C chemokine receptor type 5 (CXCR5) regulates inflammatory responses in ocular and non-ocular tissues. However, its expression and role in the cornea are still unknown. Here, we report the expression of CXCR5 in human cornea in vitro and mouse corneas in vivo, and its functional role in corneal inflammation using C57BL/6J wild-type (CXCR5+/+) and CXCR5-deficient (CXCR5-/-) mice, topical alkali injury, clinical eye imaging, histology, immunofluorescence, PCR, qRT-PCR, and western blotting. Human corneal epithelial cells, stromal fibroblasts, and endothelial cells demonstrated CXCR5 mRNA and protein expression in PCR, and Western blot analyses, respectively. To study the functional role of CXCR5 in vivo, mice were divided into four groups: Group-1 (CXCR5+/+ alkali injured cornea; n = 30), Group-2 (CXCR5-/- alkali injured cornea; n = 30), Group-3 (CXCR5+/+ naïve cornea; n = 30), and Group-4 (CXCR5-/- naïve cornea; n = 30). Only one eye was wounded with alkali. Clinical corneal evaluation and imaging were performed before and after injury. Mice were euthanized 4 h, 3 days, or 7 days after injury, eyes were excised and used for histology, immunofluorescence, and qRT-PCR. In clinical eye examinations, CXCR5-/- mouse corneas showed ocular health akin to the naïve corneas. Alkali injured CXCR5+/+ mouse corneas showed significantly increased mRNA (p < 0.001) and protein (p < 0.01 or p < 0.0001) levels of the CXCR5 compared to the naïve corneas. Likewise, alkali injured CXCR5-/- mouse corneas showed remarkably amplified inflammation in clinical eye exams in live animals. The histological and molecular analyses of these corneas post euthanasia exhibited markedly augmented inflammatory cells in H&E staining and significant CD11b + cells in immunofluorescence (p < 0.01 or < 0.05); and tumor necrosis factor-alpha (TNFα; p < 0.05), cyclooxygenase 2 (COX-2; p < 0.0001), interleukin (IL)-1ß (p < 0.0001), and IL-6 (p < 0.0001 or < 0.01) mRNA expression compared to the CXCR5+/+ mouse corneas. Interestingly, CXCR5-/- alkali injured corneas also showed altered mRNA expression of fibrotic alpha smooth muscle actin (α-SMA; p > 0.05) and angiogenic vascular endothelial growth factor (VEGF; p < 0.01) compared to the CXCR5+/+ alkali injured corneas. In summary, the CXCR5 gene is expressed in all three major layers of the cornea and appears to influence corneal inflammatory and repair events post-injury in vivo. More studies are warranted to tease the mechanistic role of CXCR5 in corneal inflammation and wound healing.

Carbofuran pesticide toxicity to the eye.

Pesticide exposure to eyes is a major source of ocular morbidities in adults and children all over the world. Carbofuran (CF), N-methyl carbamate, pesticide is most widely used as an insecticide, nematicide, and acaricide in agriculture, forestry, and gardening. Contact or ingestion of carbofuran causes high morbidity and mortality in humans and pets. Pesticides are absorbed in the eye faster than other organs of the body and damage ocular tissues very quickly. Carbofuran exposure to eye causes blurred vision, pain, loss of coordination, anti-cholinesterase activities, weakness, sweating, nausea and vomiting, abdominal pain, endocrine, reproductive, and cytotoxic effects in humans depending on amount and duration of exposure. Pesticide exposure to eye injures cornea, conjunctiva, lens, retina, and optic nerve and leads to abnormal ocular movement and vision impairment. Additionally, anticholinesterase pesticides like carbofuran are known to cause salivation, lacrimation, urination, and defecation (SLUD). Carbofuran and its two major metabolites (3-hydroxycarbofuran and 3-ketocarbofuran) are reversible inhibitors of acetylcholinesterase (AChE) which regulates acetylcholine (ACh), a neurohumoral chemical that plays an important role in corneal wound healing. The corneal epithelium contains high levels of ACh whose accumulation by AChE inhibition after CF exposure overstimulates muscarinic ACh receptors (mAChRs) and nicotinic ACh receptors (nAChRs). Hyper stimulation of mAChRs in the eye causes miosis (excessive constriction of the pupil), dacryorrhea (excessive flow of tears), or chromodacryorrhea (red tears). Recent studies reported alteration of autophagy mechanism in human cornea in vitro and ex vivo post carbofuran exposure. This review describes carbofuran toxicity to the eye with special emphasis on corneal morbidities and blindness.

Corneal fibrosis abrogation by a localized AAV-mediated inhibitor of differentiation 3 (Id3) gene therapy in rabbit eyes in vivo.

Previously we found that inhibitor of differentiation 3 (Id3) gene, a transcriptional repressor, efficiently inhibits corneal keratocyte differentiation to myofibroblasts in vitro. This study evaluated the potential of adeno-associated virus 5 (AAV5)-mediated Id3 gene therapy to treat corneal scarring using an established rabbit in vivo disease model. Corneal scarring/fibrosis in rabbit eyes was induced by alkali trauma, and 24 h thereafter corneas were administered with either balanced salt solution AAV5-naked vector, or AAV5-Id3 vector (n = 6/group) via an optimized reported method. Therapeutic effects of AAV5-Id3 gene therapy on corneal pathology and ocular health were evaluated with clinical, histological, and molecular techniques. Localized AAV5-Id3 gene therapy significantly inhibited corneal fibrosis/haze clinically from 2.7 to 0.7 on the Fantes scale in live animals (AAV5-naked versus AAV5-Id3; p < 0.001). Furthermore, AAV5-Id3 treatment significantly reduced profibrotic gene mRNA levels: α-smooth muscle actin (α-SMA) (2.8-fold; p < 0.001), fibronectin (3.2-fold; p < 0.001), collagen I (0.8-fold; p < 0.001), and collagen III (1.4-fold; p < 0.001), as well as protein levels of α-SMA (23.8%; p < 0.001) and collagens (1.8-fold; p < 0.001). The anti-fibrotic activity of AAV5-Id3 is attributed to reduced myofibroblast formation by disrupting the binding of E-box proteins to the promoter of α-SMA, a transforming growth factor-ß signaling downstream target gene. In conclusion, these results indicate that localized AAV5-Id3 delivery in stroma caused no clinically relevant ocular symptoms or corneal cellular toxicity in the rabbit eyes.

Effects of Regular/Dilute Proparacaine Anesthetic Eye Drops in Combination with Ophthalmic Antibiotics on Corneal Wound Healing.

Purpose: Topical, local anesthetic eye drops in conjunction with antibiotics are commonly used to reduce ocular pain and treat patients in emergency clinics; however, their effects on corneal healing are poorly understood. This study examined whether regular or diluted proparacaine eye drops given in combination with common ophthalmic antibiotics affect corneal wound healing parameters using in vitro and in vivo models. Methods: Primary human corneal fibroblasts generated from donor corneas and New Zealand white rabbits were used. Regular (0.5%) and diluted (0.05%) proparacaine eye drops, twice daily for 3 days, were applied to cultures and rabbit eyes, with or without ophthalmic antibiotics (polymyxin B sulfate and trimethoprim). Trypan blue, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and scratch wound assays measured cellular viability, proliferation, and migration, respectively, in vitro. Slit lamp biomicroscopy, tonometry, fluorescein eye test, hematoxylin and eosin (H&E) staining, and 4',6-diamidino-2-phenylindole (DAPI) immunofluorescence were used for in vivo studies. Results: Both regular and diluted proparacaine affected wound healing response in the cornea in vitro and in vivo in a time-dependent manner. Adjunct antibiotic treatments had additive effects characterized by reduced corneal fibroblast viability, proliferation, and migration in vitro and corneal epithelial recovery in vivo. Regular proparacaine with antibiotics showed most pronounced effects on corneal wound healing parameters, and diluted proparacaine without antibiotics had minimal negative effects in vitro and in vivo. Conclusion: Both methods of regular (0.5%) and diluted (0.05%) proparacaine topical application to the cornea are safe, but impede corneal wound healing in vitro and in vivo. Adjunct antibiotic treatments had additive negative effects on corneal wound repair.

Decorin regulates collagen fibrillogenesis during corneal wound healing in mouse in vivo.

A characteristic rigid spatial arrangement of collagen fibrils in the stroma is critical for corneal transparency. This unique organization of collagen fibrils in corneal stroma can be impacted by the presence and interactions of proteoglycans and extracellular matrix (ECM) proteins in a corneal microenvironment. Earlier studies revealed that decorin, a leucine-rich proteoglycan in stroma, regulates keratocyte-collagen matrix assembly and wound healing in the cornea. This study investigated the role of decorin in the regulation of stromal fibrillogenesis and corneal transparency in vivo employing a loss-of-function genetic approach using decorin null (dcn-/-) and wild type (dcn+/+) mice and a standard alkali-injury model. A time-dependent ocular examinations with Slit lamp microscope in live animals assessed corneal clarity, haze, and neovascularization levels in normal and injured eyes. Morphometric changes in normal and injured dcn+/+ and dcn-/- corneas, post-euthanasia, were analyzed with Masson's Trichrome and Periodic Acid-Schiff (PAS) histology evaluations. The ultrastructure changes in all corneas were investigated with transmission electron microscopy (TEM). Injury to eye produced clinically relevant corneal haze and neovascularization in dcn-/- and dcn+/+ mice while corneas of uninjured eyes remained clear and avascular. A clinically significant haze and neovascularization appeared in injured dcn-/- corneas compared to the dcn+/+ corneas at day 21 post-injury and not at early tested times. Histological examinations revealed noticeably abnormal morphology and compromised collagen levels in injured dcn-/- corneas compared to the injured/normal dcn+/+ and uninjured dcn-/- corneas. TEM analysis exhibited remarkably uneven collagen fibrils size and distribution in the stroma with asymmetrical organization and loose packing in injured dcn-/- corneas than injured/normal dcn+/+ and uninjured dcn-/- corneas. The minimum and maximum inter-fibril distances were markedly irregular in injured dcn-/- corneas compared to all other corneas. Together, results of clinical, histological, and ultrastructural investigations in a genetic knockout model suggested that decorin influenced stromal fibrillogenesis and transparency in healing cornea.

Reputational impact of Covid-19 pandemic management on World Health Organization among Indian public health professionals.

Organizational reputation is critical for successful stakeholder engagement. A crisis can affect the organizational reputation and alter stakeholder perception about organizations. The current study investigates the impact of the Covid-19 pandemic and its management on the World Health Organization's (WHO's) reputation among Indian public health professionals (PHPs). The study applies the situational crisis communication theory (SCCT) model to investigate the reputational impact of the pandemic on WHO among the study subjects. The study results indicate that most Indian PHPs attribute the current Pandemic to WHO. Their current reputation has dropped compared to their earlier reputation among Indian PHPs. The same is reflected in their behavioral intent, with the PHP's willingness to follow WHO guidelines on public health issues significantly reduced. The study also finds empirical support for the SCCT Model.

PTG-100, an Oral α4ß7 Antagonist Peptide: Preclinical Development and Phase 1 and 2a Studies in Ulcerative Colitis.

BACKGROUND & AIMS: Oral therapies targeting the integrin α4ß7 may offer unique advantages for the treatment of inflammatory bowel disease. We characterized the oral α4ß7 antagonist peptide PTG-100 in preclinical models and established safety, pharmacokinetic/pharmacodynamic relationships, and efficacy in a phase 2a trial in patients with ulcerative colitis (UC). METHODS: In vitro studies measured binding properties of PTG-100. Mouse studies measured biomarkers and drug concentrations in blood and tissues. The phase 1 study involved healthy volunteers. In phase 2a, patients with moderate to severe active UC were randomized to receive PTG-100 (150, 300, or 900 mg) or placebo once daily for 12-weeks. RESULTS: PTG-100 potently and selectively blocks α4ß7. Oral dosing of PTG-100 in mice showed high levels of target engagement and exposure in gut-associated lymphoid tissues. In healthy volunteers, PTG-100 showed dose-dependent increases in plasma exposure and blood target engagement. Although this phase 2a study initially did not meet the primary endpoint, a blinded reread of the endoscopy videos by a third party indicated clinical efficacy in conjunction with histologic remission at doses correlating with less than 100% receptor occupancy in peripheral blood. CONCLUSIONS: PTG-100 showed local gastrointestinal tissue target engagement and inhibition of memory T-cell trafficking in mice. It was safe and well tolerated in phase 1 and 2 studies. Phase 2a data are consistent with biological and clinical response and showed a dose response reflecting similar activities in preclinical models and healthy individuals. These data suggest that local gut activity of an oral α4ß7 integrin antagonist, distinct from full target engagement in blood, are important for efficacy and the treatment of UC. (ClinicalTrials.gov, Number NCT02895100; EudraCT, Number 2016-003452-75).

Long-Term Safety and Tolerability of BMP7 and HGF Gene Overexpression in Rabbit Cornea.

Purpose: Tissue-targeted localized BMP7+HGF genes delivered into the stroma via nanoparticle effectively treats corneal fibrosis and rehabilitates transparency in vivo without acute toxicity. This study evaluated the long-term safety and tolerability of BMP7+HGF nanomedicine for the eye in vivo. Methods: One eye each of 36 rabbits received balanced salt solution (group 1, naïve; n = 12), naked vector with polyethylenimine-conjugated gold nanoparticles (PEI2-GNP; group 2, naked-vector; n = 12), or BMP7+HGF genes with PEI2-GNP (group 3, BMP7+HGF; n = 12) via a topical delivery technique. Safety and tolerability measurements were performed by clinical biomicroscopy in live rabbits at predetermined time intervals up to 7 months. Corneal tissues were collected at 2 months and 7 months after treatment and subjected to histology, immunofluorescence, and quantitative real-time PCR analyses. Results: Clinical ophthalmic examinations and modified MacDonald-Shadduck scores showed no significant changes in corneal thickness (P = 0.3389), tear flow (P = 0.2121), intraocular pressure (P = 0.9958), epithelial abrasion, or ocular abnormality. Slit-lamp, stereo, confocal, and specular biomicroscopy showed no signs of blepharospasm chemosis, erythema, epiphora, abnormal ocular discharge, or changes in epithelium, stroma, and endothelium after BMP7+HGF therapy for up to 7 months, as compared with control groups. Throughout the 7-month period, no significant changes were recorded in endothelial density (P = 0.9581). Histological and molecular data were well corroborated with the subjective clinical analyses and showed no differences in the naïve, naked-vector, and BMP7+HGF groups. Conclusions: Localized BMP7+HGF therapy is a safe, tolerable, and innovative modality for the treatment of corneal fibrosis. Translational Relevance: Nanoparticle-mediated BMP7+HGF combination gene therapy has the potential to treat corneal fibrosis in vivo without short- or long-term toxicity.

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.

Case reports of chronic myeloid leukemia and tuberculosis: Is imatinib the link between the two?

Current standard of care for treatment of CML is based on tyrosine kinase inhibitors (TKI's). Imatinib is most frequently used first line tyrosine kinase inhibitor. Various side effects of TKI's are known, but some may still be unknown. We are reporting three cases of CML who developed tuberculosis while on treatment with imatinib or dasatinib. Two cases developed CNS tuberculosis and other one was tubercular pleural effusion. These cases indicate that imatinib and other TKI's probably interfere with immunological functions and predispose patients for tuberculosis.