Selective pressure mediated by influenza virus M158-66 epitope-specific CD8+T cells promotes accumulation of extra-epitopic amino acid substitutions associated with viral resistance to these T cells.

Influenza viruses are notorious for their capacity to evade host immunity. Not only can they evade recognition by virus-neutralizing antibodies, there is also evidence that they accumulate mutations in epitopes recognized by virus-specific CD8+T cells. In addition, we have shown previously that human influenza A viruses were less well recognized than avian influenza viruses by CD8+T cells directed to the highly conserved, HLA-A*02:01 restricted M158-66 epitope located in the Matrix 1 (M1) protein. Amino acid differences at residues outside the epitope were responsible for the differential recognition, and it was hypothesized that this reflected immune adaptation of human influenza viruses to selective pressure exerted by M158-66-specific CD8+T cells in the human population. In the present study, we tested this hypothesis and investigated if selective pressure exerted by M158-66 epitope-specific CD8+T cells could drive mutations at the extra-epitopic residues in vitro. To this end, isogenic influenza A viruses with the M1 gene of a human or an avian influenza virus were serially passaged in human lung epithelial A549 cells that transgenically express the HLA-A*02:01 molecule or not, in the presence or absence of M158-66 epitope-specific CD8+T cells. Especially in the virus with the M1 gene of an avian influenza virus, variants emerged with mutations at the extra-epitopic residues associated with reduced recognition by M158-66-specific T cells as detected by Next Generation Sequencing. Although the emergence of these variants was observed in the absence of selective pressure exerted by M158-66 epitope-specific CD8+T cells, their proportion was much larger in the presence of this selective pressure.

A Comparative Preclinical Evaluation of a Novel Difluprednate 0.04% BID Ophthalmic Solution and Marketed 0.05% QID Ophthalmic Emulsion.

Purpose: The purpose of this study was to compare the efficacy, and ocular pharmacokinetics of a new 0.04% w/v bis in die means twice a day (BID) ophthalmic solution and marketed 0.05% w/v quater in die means four times a day (QID) ophthalmic emulsion of difluprednate in New Zealand white (NZW) rabbits. Methods: The preclinical proof of concept was established in paracentesis-induced acute inflammation, endotoxin-induced acute uveitis, and bovine serum albumin-induced chronic uveitis in NZW rabbit animal models. A comparison of clinical score, total cell count, and total protein was performed to determine efficacy. An ocular pharmacokinetic study was conducted to study the influence of the vehicle on the ocular absorption of the drug. Results: In both uveitis models, the new solution formulation and marketed emulsion formulation inhibited total clinical score, total cell count, PGE2, and total protein significantly more than the placebo and lipopolysaccharide (disease control) groups and were comparable. In an ocular pharmacokinetic study, the Cmax and AUC0-t of difluoroprednisolone 17-butyrate in humor were ∼2-fold higher after 14 days' instillation of new solution formulation (0.04% w/v, BID) compared with 14 days' instillation of marketed emulsion (0.05% w/v, QID). Conclusions: The study demonstrated that the efficacy of the solution formulation at a lower dose and reduced dosing regimen were comparable to that of the emulsion formulation. The reduction in strength and regimen may result in improved patient adherence and outcomes.

HPLC-UV Method Development and Validation to Monitor Difluprednate Synthesis.

During the synthesis of active pharmaceutical ingredients (APIs) there is a need for the development and validation of a simple and rapid high performance liquid chromatography (HPLC) method for the determination and quantification of the synthesized product and related by-products. An HPLC method gives a better understanding of how a synthesis is proceeding. A rapid and easy to use HPLC-UV (ultraviolet) method for the determination of difluprednate and monitoring of impurities generated during synthesis was developed and validated. A Shimadzu VP Series HPLC equipped with a LabSolutions software and UV detector set at 240 nm was used for analysis. The mobile phase consisted of phosphate buffer (pH 6) and acetonitrile 50:50 (v/v) and was eluted at a flow rate of 1.2 mL/min. Separation took place on a reversed-phase Kinetex C18 column (150 × 4.60 mm; 5 µm i.d.). Column temperature was set at 40°C. The developed method was found to have good linearity and acceptable accuracy and precision. The developed method may be effectively applied to determine products and by-products formed during synthetic reactions of steroids and to calculate the yield of the products obtained during each step of the synthesis.

[Preparation and characterization of a recombinant poly-epitopic vaccine EgG1Y162-2 (4) against cystic echinococcosis based on the linker GSGGSG].

OBJECTIVE: To perform prokaryotic expression and preliminary characterization of the recombinant poly-epitope vaccine EgG1Y162-2 (4) against cystic echinococcosis. METHODS: The recombinant poly-epitope vaccine EgG1Y162-2 (4) against Echinococcus granulosus based on the linker GSGGSG was subjected to structural three-dimensional (3D) modeling using immunoinformatics to analyze the structural changes and evaluate the antigenicity of the vaccine. The pET30a-EgG1Y162-2 (4) recombinant plasmid was generated using double digestion with EcoR I and Sal I, and then transformed into competent cells. Following protein induction with isopropyl-ß-D-thiogalactoside (IPTG), the prokaryotic expression proteins were characterized using Western blotting, and the antigenicity of the recombinant protein was analyzed using sera from cystic echinococcosis patients and health volunteers. RESULTS: The four EgG1Y162-2 proteins coupled by the 3D structure of the recombinant vaccine EgG1Y162-2 (4) presented independent and effective expression and good antigenicity. The highest protein expression was detected in the supernatant following induction of the recombinant plasmid pET30a-EgG1Y162-2 (4) by 0.2 mmol/L IPTG at 37 °C for 4 h, and a pure protein component was seen following elution with 60 mmol/L imidazole. Western blotting analysis of the recombinant multiepitope protein HIS-EgG1Y162-2 (4) showed a band at approximately 39 kDa, and this band was recognized by sera from cystic echinococcosis patients. CONCLUSIONS: A recombinant poly-epitope vaccine EgG1Y162-2 (4) against cystic echinococcosis has been successfully constructed, which provides a preliminary basis for researches on recombinant multi-epitope vaccine against cystic echinococcosis.

EpiTopics: A dynamic machine learning model to predict and inform non-pharmacological public health interventions from global news reports.

Non-pharmacological interventions (NPIs) are important for controlling infectious diseases such as COVID-19, but their implementation is currently monitored in an ad hoc manner. To address this issue, we present a three-stage machine learning framework called EpiTopics to facilitate the surveillance of NPI. In this protocol, we outline the use of transfer-learning to address the limited number of NPI-labeled documents and topic modeling to support interpretation of the results. For complete details on the use and execution of this protocol, please refer to Wen et al. (2022).

Risk of Elevated Intraocular Pressure With Difluprednate in Patients With Non-Infectious Uveitis.

PURPOSE: To evaluate the risk factors associated with clinically important intraocular pressure (IOP) elevation with topical difluprednate treatment in patients with non-infectious uveitis. DESIGN: Retrospective cohort study. METHODS: Fifty-four eyes of 54 patients with non-infectious uveitis treated with topical difluprednate at the current institution were included. Demographics and clinical characteristics of uveitis patients were collected. The main outcome measure was development of clinically important IOP elevation defined as IOP ≥21 mmHg and an increase of ≥10 mmHg from baseline. RESULTS: A clinically important IOP elevation was observed in 17 patients (31.5%). The mean time to clinically important IOP elevation was 7.4±4.8 weeks (range 3-19). Statistically significant risk factors for incident clinically important IOP elevation were being a child (adjusted hazard ratio [aHR] 7.85 [95% CI 1.48-41.56], P = .02) and concurrent use of systemic steroids (aHR 5.31 [95% CI 1.18-24.00], P = .03). Patients with concurrent systemic corticosteroids developed clinically important IOP elevation earlier than those without systemic corticosteroid (mean 5.7±3.4 [range 3-14] vs 10.4±5.7 [range 4-19] weeks, P = .05). Incident IOP ≥30 mmHg occurred in 7 patients (13.0%). All patients responded well to the cessation of difluprednate and/or use of topical antiglaucomatous agents and no eyes required glaucoma surgery. CONCLUSIONS: This study demonstrated that clinically important IOP elevation is common in uveitis patients with topical difluprednate treatment. Children and patients with concurrent systemic corticosteroids are at substantial risk of developing clinically important IOP elevation.

Surgical Outcomes of Ahmed Glaucoma Valve Implantation with Postoperative Use of Prednisolone Acetate versus Difluprednate.

PURPOSE: To evaluate the effect of postoperative topical prednisolone acetate and difluprednate on surgical outcomes of Ahmed glaucoma valve (AGV) implantation. DESIGN: Retrospective, comparative case series. PARTICIPANTS: The study population consisted of 102 eyes of 90 patients, including 52 eyes that received 1% prednisolone acetate (Pred Forte [PF]; Allergan Inc) and 50 eyes that received 0.05% difluprednate (Durezol [DZ]; Novartis Inc). METHODS: The medical records of consecutive patients who underwent AGV implantation at the University of California, San Francisco, were retrospectively reviewed. Patients in the PF group received 1% prednisolone acetate 6 to 8 times per day tapered over 5 to 6 months postoperatively, and patients in the DZ group received 0.05% difluprednate 4 times daily tapered over 4 months postoperatively. MAIN OUTCOME MEASURES: Intraocular pressure (IOP), number of glaucoma medications, visual acuity (VA), postoperative complications, and the rate of treatment success. RESULTS: At 1 year, the IOPs (mean ± standard deviation) were 12.4 ± 3.7 mmHg in the DZ group and 13.0 ± 4.0 mmHg in the PF group (P = 0.49). The numbers of glaucoma medications were 0.72 ± 0.71 in the DZ group and 1.09 ± 0.91 in the PF group (P = 0.04), with reductions from baseline of 2.5 ± 1.0 glaucoma medications in the DZ group and 1.8 ± 1.6 glaucoma medications in the PF group (P = 0.01). The logarithm of the minimum angle of resolution VAs (mean ± standard deviation) were 0.55 ± 0.80 in the DZ group and 0.59 ± 0.65 in the PF group after 1 year of follow-up (P = 0.81). The cumulative probabilities of success were 95.8% in the DZ group and 93.5% in the PF group at 1 year (P = 0.61). Postoperative complications occurred in 4 eyes (7.7%) in the DZ group and 6 eyes (12%) in the PF group (P = 0.52). CONCLUSIONS: After 1 year, postoperative treatment with 0.05% difluprednate after AGV implantation resulted in a similar IOP, with the use of fewer glaucoma medications, compared with postoperative treatment with 1% prednisolone acetate. The rates of treatment success and surgical complications were comparable between the 2 groups during the first year of follow-up.

Sustained Release Biodegradable Microneedles of Difluprednate for Delivery to Posterior Eye.

Purpose: Difluprednate ophthalmic emulsion (Durezol®) is currently used for the treatment of anterior uveitis; however, recent studies have shown that difluprednate can treat posterior eye conditions. Topical formulations limit the amount of drug capable of permeating to the posterior segment due to permeation barriers, lacrimation, and lymphatic clearance. Methods: Resomer®-based microneedle patches were fabricated for difluprednate using poly(acrylic acid) (PAA) for the rapidly dissolvable backing. The patches were analyzed for microneedle uniformity and sharpness using scanning electron microscopy, and the penetration depth was analyzed by confocal microscopy. Failure force necessary to break the microneedles and force needed to penetrate the sclera were analyzed by the texture analyzer. Difluprednate release and trans-scleral permeation studies on microneedles were performed using Franz diffusion cells. Results: The microneedles were uniform, sharp, and penetrated to 500 µm depth on sclera. The microneedles have a failure force proportional to the molecular weight (MW) of the polymer used. There was no correlation between failure force and the penetration force of the microneedles. The PAA backing dissolved within 30-40 min, while release studies showed a matrix diffusion-controlled release over the 7-day study. The amount of drug permeation and retention in the sclera were decreased with an increase in the MW of the Resomer and failure force of each array. Conclusions: Resomer-based microneedles have a potential application for the sustained release of difluprednate for posterior segment conditions.

TN strain proteome mediated therapeutic target mapping and multi-epitopic peptide-based vaccine development for Mycobacterium leprae.

Leprosy is a significant universal health problem that is remarkably still a concern in developing countries due to infection frequency. New therapeutic molecules and next-generation vaccines are urgently needed to accelerate the leprosy-free world. In this direction, the present study was performed using two routes: proteome-mediated therapeutic target identification and mapping as well as multi-epitopic peptide-based novel vaccine development using state of the art of computational biology for the TN strain of M. leprae. The TN strain was selected from 65 Mycobacterium strains, and TN strain proteome mediated 83 therapeutic protein targets were mapped and characterized according to subcellular localization. Also, drug molecules were mapped with respect to protein targets localization. The Druggability potential of proteins was also evaluated. For multi-epitope peptide-based vaccine development, the four common types of B and T cell epitopes were identified (SLFQSHNRK, VVGIGQHAA, MMHRSPRTR, LGVDQTQPV) and combined with the suitable peptide linker. The vaccine component had an acceptable protective antigenic score (0.9751). The molecular docking of vaccine components with TLR4/MD2 complex exhibited a low ACE value (-244.12) which signifies the proper binding between the two molecules. The estimated free Gibbs binding energy ensured accurate protein-protein interactions (-112.46 kcal/mol). The vaccine was evaluated through adaptive immunity stimulation as well as immune interactions. The molecular dynamic simulation was carried out by using CHARMM topology-based parameters to minimize the docked complex. Subsequently, the Normal Mode Analysis in the internal coordinates showed a low eigen-value (1.3982892e-05), which also signifies the stability of molecular docking. Finally, the vaccine components were adopted for reverse transcription and codon optimization in E. coli strain K12 for the pGEX-4T1 vector, which supports in silico cloning of the vaccine components against the pathogen. The study directs the experimental study for therapeutics molecules discovery and vaccine candidate development with higher reliability.

Difluprednate 0.05% twice a day vs prednisolone acetate 1% 4 times a day for cataract postsurgical inflammation treatment: noninferiority trial.

PURPOSE: To establish whether difluprednate 0.05% nanoemulsion (DIFL) twice a day is as effective as prednisolone acetate 1% + phenylephrine hydrochloride 0.12% suspension (PRED) 4 times a day for postsurgical inflammation treatment. SETTING: 4 private Argentine ophthalmological centers. DESIGN: Noninferiority, prospective, multicenter, double-blind, randomized, parallel-group, comparative trial. METHODS: A total of 259 patients who underwent phacoemulsification randomly received DIFL or PRED, starting the day before surgery and continuing for 28 days. The primary endpoint was central corneal thickness. Noninferior anti-inflammatory efficacy was considered if the difference of corneal thickness between baseline and day 4 did not differ beyond 17 µm between treatments. Secondary endpoints were cell and flare, corrected distance visual acuity (CDVA), endothelial cell count, optical coherence tomography (OCT) central macular thickness, and intraocular pressure. All outcomes were evaluated at baseline and day 1, 4, and 28 postoperatively. RESULTS: 225 patients finished the study. The difference in corneal thickness at baseline and day 4 did not differ beyond 17 µm between treatments (95% CI -2.78 µm to 14.84 µm), with no statistically significant difference ( P = .523). No statistically significant differences were found between groups in total anterior chamber clearance at any study timepoint ( P > .05). Moreover, no statistically significant differences were reported between treatments in CDVA ( P = .455), endothelial cell count ( P = .811), OCT central macular thickness ( P = .869), and intraocular pressure outcome ( P = .316). CONCLUSIONS: Difluprednate administered twice a day was at least as effective as prednisolone acetate administered 4 times a day for inflammatory treatment after cataract surgery.

Difluprednate-Hydroxypropyl-ß-Cyclodextrin-Based Ophthalmic Solution for Improved Delivery in a Porcine Eye Model.

Purpose: Difluprednate (DFP) is an approved corticosteroid, available as an ophthalmic emulsion (Durezol®), used to treat pain and inflammation of the eye following ocular surgeries. This study utilized hydroxypropyl-ß-cyclodextrin (HPBCD)-based DFP ophthalmic solution for improved ocular delivery. Methods: The DFP-HPBCD complex formation was studied in the liquid and solid states. Phase solubility, molecular docking studies, differential scanning calorimetry, and Fourier transform infrared spectroscopy suggested inclusion complexation of DFP and HPBCD. Results: DFP-HPBCD-based eye drops (solution) provided 16 and 26 times higher transcorneal permeation when compared to the suspension (no HPBCD, control) and Durezol, respectively (P < 0.001). In addition, ocular drug distribution studies conducted in continuously perfused whole porcine eyes showed DFP permeated into all of the ocular tissues in significantly higher amounts than Durezol. Conclusions: The solution-based eye drops in this study is iso-osmotic, safe, and more permeable in porcine eyes compared to Durezol.

HLA-EPI: A new EPIsode in exploring donor/recipient epitopic compatibilities.

The HLA system plays a pivotal role both in transplantation and immunology. While classical HLA genotypes matching is made at the allelic level, recent progresses were developed to explore antibody-antigen recognition by studying epitopes. Donor to recipient matching at the epitopic level is becoming a trending topic in the transplantation research field because anti-HLA antibodies are epitope-specific rather than allele-specific. Indeed, different HLA alleles often share common epitopes. We present the HLA-Epi tool (hla.univ-nantes.fr) to study an HLA genotype at the epitope level. Using the international HLA epitope registry (Epregistry.com.br) as a reference, we developed HLA-Epi to easily determine epitopic and allelic compatibility levels between several HLA genotypes. The epitope database covers the most common HLA alleles (N = 2976 HLA alleles), representing more than 99% of the total observed frequency of HLA alleles. The freely accessible web tool HLA-Epi calculates an epitopic mismatch load between different sets of potential recipient-donor pairs at different resolution levels. We have characterized the epitopic mismatches distribution in a cohort of more than 10,000 kidney transplanted pairs from European ancestry, which showed low number of epitopic mismatches: 56.9 incompatibilities on average. HLA-Epi allows the exploration of epitope pairing matching to better understand epitopes contribution to immune responses regulation, particularly during transplantation. This free and ready-to-use bioinformatics tool not only addresses limitations of other related tools, but also offers a cost-efficient and reproducible strategy to analyze HLA epitopes as an alternative to HLA allele compatibility. In the future, this could improve sensitization prevention for allograft allocation decisions and reduce the risk of alloreactivity.

Core amino acid substitutions in HCV-3a isolates from Pakistan and opportunities for multi-epitopic vaccines.

Hepatitis C virus (HCV), which infected 71 million worldwide and about 5%-6% are from Pakistan, is an ssRNA virus, responsible for end-stage liver disease. To date, no effective therapy is available to cure this disease. Hence, it is important to study the most prevalent genotypes infecting human population and design novel vaccine or small molecule inhibitors to control the infections associated with HCV. Therefore, in this study clinical samples (n = 35; HCV-3a) from HCV patients were subjected to Sanger sequencing method. The sequencing of the core gene, which is generally considered as conserved, involved in the detection, quantitation and genotyping of HCV was performed. Multiple mutations, that is, R46C, R70Q, L91C, G60E, N/S105A, P108A, N110I, S116V, G90S, A77G and G145R that could be linked with response to antiviral therapies were detected. Phylogenetic analysis suggests emerging viral isolates are circulating in Pakistan. Using ab initio modelling technique, we predicted the 3D structure of core protein and subjected to molecular dynamics simulation to extract the most stable conformation of the structure for further analysis. Immunoinformatic approaches were used to propose a multi-epitopes vaccine against HCV by using core protein. The vaccine constructs consist of nine CTL and three HTL epitopes joined by different linkers were docked against the two reported Toll-like receptors (TLR-3 and TLR-8). Docking of vaccine construct with TLR-3 and TLR-8 shows proper binding and in silico expression of the vaccine resulted in a CAI value of 0.93. These analyses suggest that specific immune responses may be produced by the proposed vaccine.Communicated by Ramaswamy H. Sarma.

Effectiveness of Difluprednate for the Treatment of Anterior Scleritis.

PURPOSE: To describe the effectiveness and side effect profile of difluprednate therapy in a series of patients with anterior scleritis. DESIGN: Retrospective, interventional case series. METHODS: Data collected from all patients with anterior scleritis who used difluprednate as a single treatment agent from January 1, 2018, to January 1, 2020, including demographics, scleritis type, presence of nodules or necrosis, changes in scleritis activity, intraocular pressure (IOP), number of difluprednate drops used, best-corrected visual acuity (BCVA), and lens status. The primary outcome was clinical resolution of scleritis. Secondary outcomes included BCVA loss ≥2 lines, change in lens status or cataract surgery, and IOP ≥24 mm Hg. RESULTS: Twenty-five patients (35 eyes) were analyzed. The median age was 60 years (range 13-78); 60% were female; 64% were White. Forty percent had bilateral disease, and 44% of patients had an associated systemic disease. The majority of eyes (66%) had diffuse anterior scleritis. Eighty-three percent of eyes achieved resolution of scleritis, with a median time of resolution of 6 weeks. Eyes treated with an initial dose of ≥4 times daily were more likely to achieve disease resolution (hazard ratio [HR] = 3.43, 95% confidence interval [CI] 1.19, 9.88, P = .02). Nine eyes had IOP elevation. Four eyes lost ≥2 lines of BCVA, and 1 due to cataract progression. One eye underwent cataract surgery. CONCLUSIONS: Difluprednate alone may effectively treat non-infectious anterior scleritis with a tolerable side effect profile.

Localized alopecia and suppression of hypothalamic-pituitary-adrenal (HPA) axis in dogs following treatment with difluprednate 0.05% ophthalmic emulsion (Durezol®).

BACKGROUND: Despite the common use of topical ophthalmic corticosteroids in dogs, detailed reports on systemic and dermatologic adverse effects are limited. RESULTS: Nine purpose-bred research Beagles were treated with difluprednate 0.05% ophthalmic emulsion in one or both eyes 2-3 times daily. Some difluprednate treated dogs developed mild to severe alopecia of the periocular region, face, and distal pinna (5/9). The median duration of treatment prior to onset of dermatologic signs for difluprednate treated dogs was 550 days (453-1160 days). Diagnostic testing included complete blood count (CBC) and serum biochemistry, adrenocorticotropic hormone (ACTH) stimulation testing combined with endogenous ACTH measurement, and skin biopsy. The CBC and chemistry were within normal limits for all dogs. There were varying degrees of suppression of the hypothalamic-pituitary-adrenocortical (HPA) axis with difluprednate treatment. Dogs with the most profound alopecic changes had less pronounced HPA axis suppression compared to dogs with no integumentary changes. Skin biopsies demonstrated follicular atrophy and follicular keratosis. When topical difluprednate was reduced to unilateral therapy, the hair regrew on the untreated side of the face. In addition to the affected research dogs, a 7-year old female spayed Chihuahua that was being treated as a clinical patient with long-term difluprednate 0.05% ophthalmic emulsion developed generalized hypotrichosis on the head and body and a potbellied appearance. ACTH stimulation testing revealed suppression of the HPA axis with a mild increase in serum alkaline phosphatase (ALP) activity and a urine specific gravity of 1.016. The combination of clinical signs and laboratory abnormalities was supportive of iatrogenic hyperadrenocorticism. CONCLUSIONS: In dogs long-term use of difluprednate ophthalmic emulsion results in HPA axis suppression and in some cases iatrogenic hyperadrenocorticism. A novel pattern of localized alopecia is suspected to be related to dermal absorption and local action due to superior potency and penetration compared to other commonly utilized ophthalmic corticosteroids.

Structural Phylogeny of Different Allergens May Reveal Common Epitopic Footprint.

BACKGROUND: The incidence of allergy has been increasing at an alarming rate over the last few decades. OBJECTIVE: Our present study aims to find out the structurally homologous motifs present in different proteinaceous allergens. METHODS: Significant number of protein sequences and their corresponding structures of various pollen, fungal, bacterial, and food allergens were retrieved and the sequence and structural identity were analyzed. RESULTS: Intra- and inter-sequence along with their structural analysis of the proteinaceous allergens revealed that no significant relationships exist among them. A few, but not the negligible number of high structural similarities, were observed within different groups of allergens from fungus, angiosperms, and animals (Aves and Mammalia). CONCLUSION: Our in silico study on thirty-six different allergens showed a significant level of structural similarities among themselves, regardless of their sequences.

Are Epitopic Sites of 3AB and 3D Nonstructural Proteins Sufficient for Detection of Foot and Mouth Disease?

An efficient method for detection of foot and mouth disease (FMD) and, particularly, differentiation of vaccinated from infected animals is the use of nonstructural (NS) proteins as antigens in Enzyme-Linked Immunosorbent Assay (ELISA) Kits. In this study, only epitopic regions of 3AB and 3D NS proteins were used for recombinant protein production, as a cost-effective method instead of peptide synthesis, for application in in-house ELISA diagnostic kits. Specific primers were designed according to the antigenic regions of 3AB (C-terminus of 3A and the whole 3B) and 3D (N-terminus) proteins, and the polymerase chain reaction (PCR) amplification was performed. Purified amplicons were cloned into pET21a (+) vectors and then transformed into Escherichia coli (BL21). Thereafter, bacteria were induced with 1 mM isopropyl ß-d-1-thiogalactopyranoside (IPTG) for expression of antigenic proteins. Antigenic 3AB protein was expressed in soluble form, but 3D protein was extracted from the bacterial lysate. Protein expression was confirmed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses. An indirect ELISA was developed for each protein, and the diagnostic sensitivity and specificity were determined. The 3AB-ELISA showed higher sensitivity and specificity than 3D-ELISA (95.24% and 100%, compared with 90.48% and 88.71%, respectively). The epitopic 3AB-ELISA developed here can be used for detection and differentiation of FMD infected from vaccinated animals, but the epitopic 3D-ELISA showed lower efficiency in screening for FMD status.

A multi-technique analytical approach for impurity profiling during synthesis: The case of difluprednate.

A methodology for the qualitative analysis of a mixture of compounds obtained during the synthesis of difluprednate is described herein for the first time. For this scope a multi-technique analytical approach was developed, combining Liquid Chromatography/Mass Spectrometry (LC/MS), Nuclear Magnetic Resonance (NMR) and computational chemistry. Separation of isomers is frequently required for the identification of impurities in active pharmaceutical ingredients (APIs) to assess the impact they may exhibit on public health. During the final step of the difluprednate synthesis apart from the desired product, various by-products may be obtained. Structural analysis of the products using LC/MS and NMR indicated that the steroid difluprednate was obtained along with its acetyl/butyryl regional isomers, whereas the results were further supported by semi-empirical calculations of the MS-derived data. Following the proposed approach, we managed to elucidate the structures of the challenging 11-acetate, 17-butyrate from the 17-acetate, 21-butyrate, 6α,9α-difluoro prednisolone isomers. The approach utilized may be of general applicability for the analysis of impurities in active pharmaceutical ingredients obtained during chemical synthesis.

Treatment of Secondary Full-Thickness Macular Holes with Topical Therapy.

PURPOSE: To report the outcomes of secondary full-thickness macular holes (FTMHs) treated with topical therapy. DESIGN: Retrospective case series. PARTICIPANTS: Patients with secondary FTHMs initially treated with topical therapy in a referral-based retina practice. METHODS: Patients evaluated between April 14, 2016, and February 22, 2019, for secondary FTMHs who underwent topical therapy were included. Patient demographics, ocular history, type of drops used, duration of therapy, and duration of follow-up as well as anatomic features on OCT, including hole diameter and presence of vitreomacular traction, epiretinal membrane (ERM), and cystoid macular edema (CME), were analyzed. MAIN OUTCOME MEASURES: Closure rate of FTMHs and change in visual acuity. RESULTS: A total of 123 FTMHs were seen during the study, of which 12 were secondary macular holes. Topical therapy was attempted in 9 eyes (8 patients). Six of these 9 FTMHs were associated with prior retinal detachment (RD). Previous pars plana vitrectomy (PPV) had been performed in 3 eyes (1 for RD, 2 for ERM). One eye had vitreomacular traction and a remote history of blunt trauma. Average initial hole diameter was 79.6 µm (range, 44-132 µm). Average follow-up was 53 weeks (range, 5-153 weeks). All FTMHs had some element of ERM and CME. All patients received difluprednate with the addition of a topical carbonic anhydrase inhibitor in 6 eyes and nonsteroidal anti-inflammatory drug (NSAID) drops in 2 eyes. Eight eyes (89%) achieved successful hole closure and resolution of CME with concurrent improvement in visual acuity after an average of 6 weeks of therapy (range, 2-19 weeks). Average vision among all 9 eyes improved from 0.69 to 0.37 logarithm of the minimum angle of resolution (Snellen equivalent from approximately 20/100 to approximately 20/50). No episodes of corneal melts or ulcers occurred. One patient showed mild keratopathy and elevation of intraocular pressure with topical NSAID and steroid therapy that resolved when the topical NSAID was stopped and difluprednate was tapered down to once weekly. CONCLUSIONS: Topical therapy achieved high closure rates in secondary FTMH and can be considered as an initial treatment option especially in those with small holes and CME.

Comparison of topically administered 0.05% difluprednate and 1% prednisolone acetate for inhibition of aqueocentesis-induced breakdown of the blood-aqueous barrier in healthy dogs.

OBJECTIVE: To compare the efficacy of 0.05% difluprednate ophthalmic emulsion and 1% prednisolone acetate ophthalmic suspension for controlling aqueocentesis-induced breakdown of the blood-aqueous barrier in healthy dogs. ANIMALS: 34 healthy dogs. PROCEDURES: Dogs were allocated to 5 groups (6 to 8 dogs/group) to receive 0.05% difluprednate, 1% prednisolone acetate, or saline (0.9% NaCl) solution (control treatment) in both eyes 2 or 4 times daily. Eye drops were administered topically for 5 consecutive days. Anterior chamber paracentesis (aqueocentesis) was performed in 1 eye on the third day. Automated fluorophotometry was performed immediately before and 20 minutes and 24 and 48 hours after aqueocentesis. Relative fluorescence (RF), defined as fluorescence of the eye that had undergone aqueocentesis divided by fluorescence of the contralateral eye, was calculated to help control for variation among dogs. RESULTS: Mean RF was significantly lower at 24 hours after aqueocentesis in dogs treated twice daily with 0.05% difluprednate or 4 times daily with 1% prednisolone acetate than in dogs receiving the control treatment. At 48 hours after aqueocentesis, mean RF was significantly lower in dogs treated 4 times daily with 1% prednisolone acetate than in control dogs. Mean RF differed over time in dogs treated 4 times daily with 0.05% difluprednate but did not differ over time for any of the other treatments. CONCLUSIONS AND CLINICAL RELEVANCE: All 4 treatments were effective for reducing aqueocentesis-induced anterior uveitis in healthy dogs regardless of the drug or frequency of administration. Topical ophthalmic administration of 0.05% difluprednate may be a viable treatment option for dogs with anterior uveitis and warrants further study.