Inhibition of experimental autoimmune uveitis by intravitreal AAV-Equine-IL10 gene therapy.

Equine recurrent uveitis (ERU) is a spontaneous, painful, and vision threatening disease affecting up to 25% of equine populations worldwide. Current treatments of ERU are non-specific and have many side effects which limits them to short-term use. In order to develop an effective therapy for ERU, we investigated the use of adeno-associated virus (AAV) gene therapy, exploiting a natural immune tolerance mechanism induced by equine interleukin-10 (Equine-IL10). The purpose of this study was to evaluate the therapeutic efficacy of a single intravitreal (IVT) dose of AAV8-Equine-IL10 gene therapy for inhibition of experimental autoimmune uveitis (EAU) in rats. Each rat was dosed intravitreally (IVT) in both eyes with either balanced salt solution (BSS) (control; n = 4), AAV8-Equine-IL10 at a low dose (2.4x109 vg; n = 5) or high dose (2.4x1010 vg; n = 5). EAU was induced in all groups of rats 7 days after IVT injections and euthanized 21 days post-injection. Ophthalmic examination and aqueous humor (AH) cell counts were recorded with the observer blinded to the treatment groups. Histopathology and qPCR were performed on selected ocular tissues. Data presented herein demonstrate that AAV8-Equine-IL10 treated rats exhibited a significant decrease in clinical inflammatory scores and AH cell counts compared to BSS-treated EAU eyes on days 10, 12 and 14 post EAU induction at both administered vector doses. Mean cellular histologic infiltrative scores were also significantly less in AAV8-Equine-IL10 dosed rats compared to the BSS group. Intravitreal injection of AAV8-Equine-IL10 resulted in Equine-IL10 cDNA expression in the ciliary body, retina, cornea, and optic nerve in a dose-dependent manner. A single IVT injection of AAV8-Equine-IL10 appeared to be well-tolerated and inhibited EAU even at the lowest administered dose. These results demonstrate safety and efficacy of AAV8-Equine-IL10 to prevent EAU and support continued exploration of AAV gene therapy for the treatment of equine and perhaps human recurrent uveitis.

Conjunctival staining with lissamine green as a predictor of tear film deficiency in dogs.

OBJECTIVE: To evaluate a grading scheme for conjunctival staining patterns with lissamine green ocular dye in the diagnosis of tear film deficiencies in dogs. PROCEDURES: Client-owned and research colony dogs were enrolled in a prospective study between February and October 2018 in which slit-lamp biomicroscopy, Schirmer tear test (STT), tear film breakup time (TFBUT), conjunctival lissamine green staining (LGS), and intraocular pressure (IOP) measurement were performed in both eyes of all dogs. Lissamine green staining of the temporal bulbar conjunctiva was graded from 0-3, with a higher grade corresponding to an increased stain intensity. RESULTS: Fifty-four dogs (107 eyes), comprising 31 males and 23 females with a mean age of 5.0 ± 3.9 years (range 0.5-14.3), were enrolled in the study. STT was <15 mm/min in 21 eyes and ≥15 mm/min in 86 eyes. Lissamine green staining grade for eyes with a STT of <15 mm/min (2.0 ± 0.9) was significantly higher than for eyes with a STT ≥15 mm/min (0.2 ± 0.7) (P < .001). TFBUT for eyes with a STT <15 mm/min (6.5 ± 4.4 seconds) was significantly shorter than for eyes with a STT ≥ 15 mm/min (16.1 ± 3.6 seconds) (P < .001). As LGS grade increased, both STT (P < .001) and TFBUT (P < .001) significantly decreased. CONCLUSIONS: A higher LGS grade was significantly associated with a lower STT and more rapid TFBUT in dogs. Lissamine green ocular dye can be considered as an adjunctive diagnostic test when evaluating tear film deficiency in dogs.

Treatment of spontaneous chronic corneal epithelial defects (SCCEDs) with diamond burr debridement vs combination diamond burr debridement and superficial grid keratotomy.

OBJECTIVE: To evaluate the efficacy of diamond burr debridement (DBD) vs a combination of diamond burr debridement with superficial grid keratotomy (DBD+SGK) for the treatment of spontaneous chronic corneal epithelial defects (SCCEDs) in dogs. PROCEDURE: Medical records of dogs diagnosed with SCCEDs from three different institutions that received a DBD or DBD+SGK between 2003 and 2015 were reviewed. Age, breed, sex, history of a previous SCCED, procedures performed, time to healing, and complications were statistically analyzed. RESULTS: One hundred and ninety-four dogs met the inclusion criteria. Eighty-two of 106 eyes (77.4%) received a DBD and healed following the first treatment (13.3 ± 4.9 days to recheck, range 2-27). Sixty-eight of 88 eyes (77.3%) received a DBD+SGK and healed following the first treatment (15.4 ± 5.0 days to recheck, range 5-45). No significant difference in healing outcome was found between the two treatments (P = 1). For SCCEDs that healed after a single treatment (n = 150), complications occurred in 13.3% (n = 20) of eyes with no difference in complications between the DBD and DBD+SGK groups (P = .86). Thirty-five of 44 eyes (80.0%) healed after the second treatment (16 ± 8.2 days from second treatment to third visit, range 5-47); nine of 44 eyes (20.0%) were not healed (12 ± 6.2 days from second treatment to third visit, range 5-25). The second treatment method did not influence healing rates (P = .64). CONCLUSIONS: DBD and DBD+SGK are equally effective treatment methods for canine SCCEDs. No differences in complication rates after one treatment were observed between DBD and DBD+SGK.

Optic neuritis in dogs: 96 cases (1983-2016).

OBJECTIVE: To characterize ocular and neurologic findings, causes, and treatment outcomes of dogs with optic neuritis. PROCEDURE: Medical records from dogs with a diagnosis of optic neuritis at North Carolina State University, College of Veterinary Medicine, Veterinary Hospital between 1983 and 2016 were reviewed. RESULTS: Ninety-six cases (20 unilateral, 76 bilateral), comprised of 38 males and 58 females with a mean age of 6.1 ± 3.0 years (range 0.5-13), were identified. Seventy-four cases were presented for vision loss, and 42 had other concurrent neurologic abnormalities. Funduscopic findings included optic nerve head elevation (n = 92), peripapillary retinal edema or separation (n = 37), retinal hemorrhage or dilation of retinal vasculature (n = 23), and multiple inflammatory foci in the peripapillary region (n = 13). Retrobulbar optic neuritis was diagnosed in four cases. The final diagnoses included the following: multifocal meningoencephalitis of unknown etiology (MUE, n = 35), isolated optic neuritis (I-ON, n = 42), neoplasia (n = 10), microbial infection (n = 6), orbital inflammation (n = 2), and suspected ivermectin toxicosis (n = 1). Dogs with I-ON were more commonly male, and medium-to-large breed, when compared to dogs with MUE. Follow-up was available in 72 cases, 50 of which remained blind, 10 had partial visual improvement, and 12 were assessed as having normal vision in the affected eye(s). CONCLUSION: Optic neuritis was most commonly associated with multifocal MUE or was isolated as the sole neurologic finding, with a similar incidence between the two groups. Findings suggest that a clinical syndrome of isolated optic neuritis, distinct from multifocal MUE, occurs in dogs.

AAV vector-meditated expression of HLA-G reduces injury-induced corneal vascularization, immune cell infiltration, and fibrosis.

Over 1.5 million individuals suffer from cornea vascularization due to genetic and/or environmental factors, compromising visual acuity and often resulting in blindness. Current treatments of corneal vascularization are limited in efficacy and elicit undesirable effects including, ironically, vision loss. To develop a safe and effective therapy for corneal vascularization, adeno-associated virus (AAV) gene therapy, exploiting a natural immune tolerance mechanism induced by human leukocyte antigen G (HLA-G), was investigated. Self-complementary AAV cassettes containing codon optimized HLA-G1 (transmembrane) or HLA-G5 (soluble) isoforms were validated in vitro. Then, following a corneal intrastromal injection, AAV vector transduction kinetics, using a chimeric AAV capsid, were determined in rabbits. One week following corneal trauma, a single intrastromal injection of scAAV8G9-optHLA-G1 + G5 prevented corneal vascularization, inhibited trauma-induced T-lymphocyte infiltration (some of which were CD8+), and dramatically reduced myofibroblast formation compared to control treated eyes. Biodistribution analyses suggested AAV vectors persisted only in the trauma-induced corneas; however, a neutralizing antibody response to the vector capsid was observed inconsistently. The collective data demonstrate the clinical potential of scAAV8G9-optHLA-G to safely and effectively treat corneal vascularization and inhibit fibrosis while alluding to broader roles in ocular surface immunity and allogenic organ transplantation.

Evaluation of Intracameral Pentablock Copolymer Thermosensitive Gel for Sustained Drug Delivery to the Anterior Chamber of the Eye.

PURPOSE: To investigate PTSgels (Pentablock copolymers) as an injectable formulation technology for sustained ocular drug delivery. Drug release profile, tolerability, and polymer degradation for one of the thermosensitive, biodegradable, and biocompatible compositions were investigated through intracameral (IC) injection in rabbits. METHODS: New Zealand White rabbit eyes were injected IC (50 µL) with 100 µg near-infrared-immunoglobulin G (NIR-IgG) in balanced salt solution (BSS) or 20% PTSgel; or with PTSgel or BSS alone. Ocular irritation scoring, intraocular pressure (IOP), and corneal thickness (CT) measurement, as well as color and infrared photography, were performed for up to 28 days postinjection. Upon euthanasia at 7, 14, or 28 days, eyes underwent ex vivo imaging (Xenogen IVIS) followed by tissue fixation and histopathology. RESULTS: IC injection of PTSgel (liquid at room temperature) was performed without difficulty using a 31G needle. The polymer quickly gelled in the IC space resulting in an inferior anterior chamber deposit. The tested PTSgel was well tolerated, with no significant changes in IOP or CT. Eyes injected with NIR-IgG in PTSgel had visible NIR-IgG through 9 days postinjection, and ex vivo imaging detected a strong NIR-IgG signal in the anterior chamber through day 28. The gel deposit steadily decreased in size over time and was nearly eliminated by 28 days. CONCLUSIONS: The PTSgel released IgG for 28 days and was well tolerated. The polymer degraded in parallel with drug release. These results demonstrate the potential of intracameral PTSgel formulations for sustained delivery of biologic therapies to the ocular anterior segment.

Adjusting Cyclophosphamide Dose in Obese Patients with Lymphoma Is Safe and Yields Favorable Outcomes after Autologous Hematopoietic Cell Transplantation.

No clear dosing guidelines exist for cyclophosphamide (Cy) dose adjustments in obese patients treated with high-dose chemoradiotherapy followed by autologous hematopoietic cell transplantation (HCT). We prospectively compared the outcomes of high-dose Cy/total body irradiation (TBI) conditioning in 147 non-Hodgkin lymphoma (NHL) patients in 3 weight groups: nonobese (<120% ideal body weight [IBW]; n = 72), overweight (120% to 149% IBW; n = 46), and obese (≥150% IBW; n = 29). Nonobese and overweight patients received Cy (120 mg/kg of total body weight, intravenously) and TBI (1320 cGy), whereas obese patients (median body mass index, 36) received an adjusted Cy dose based on IBW plus 50% of the difference between total body weight and IBW (AdjBW50). The median patient age was 57 years (range, 19 to 73). The most common diagnoses were diffuse large B cell lymphoma (n = 57) and mantle cell lymphoma (n = 51). Three-year overall survival was 61% (95% confidence interval [CI], 48% to 72%) for nonobese patients, 68% (95% CI, 52% to 82%) for overweight patients, and 80% (95% CI, 62% to 93%) for obese patients. Cumulative incidence of relapse (48%, 43%, and 38%, respectively) and nonrelapse mortality (∼4%) were similar in all groups. Hemorrhagic cystitis and cardiac toxicity were rare events. Our data show that the AdjBW50 formula can be safely and effectively used for Cy dose adjustments in obese patients treated for NHL with high-dose Cy/TBI conditioning followed by autologous HCT.

Removal of fast flowing nitrogen from marshes restored in sandy soils.

Groundwater flow rates and nitrate removal capacity from an introduced solution were examined for five marsh restoration designs and unvegetated plots shortly after planting and 1 year post-planting. The restoration site was a sandy beach with a wave-dampening fence 10 m offshore. Simulated groundwater flow into the marsh was introduced at a rate to mimic intense rainfall events. Restoration designs varied in initial planting density and corresponded to 25%, 50%, 75% and 100% of the plot area planted. In general, groundwater flow was slower with increasing planting density and decreased from year 0 to year 1 across all treatments. Nevertheless, removal of nitrate from the introduced solution was similar and low for all restoration designs (3-7%) and similar to the unvegetated plots. We suggest that the low NO3(-) removal was due to sandy sediments allowing rapid flow of groundwater through the marsh rhizosphere, thereby decreasing the contact time of the NO3(-) with the marsh biota. Our findings demonstrate that knowledge of the groundwater flow regime for restoration projects is essential when nutrient filtration is a target goal of the project.

Chimeric porcine circoviruses (PCV) containing amino acid epitope tags in the C terminus of the capsid gene are infectious and elicit both anti-epitope tag antibodies and anti-PCV type 2 neutralizing antibodies in pigs.

A chimeric porcine circovirus (PCV1-2) with the capsid gene of pathogenic PCV2 cloned into the genomic backbone of nonpathogenic PCV1 is attenuated in pigs but elicits protective immunity against PCV2. In this study, short epitope tags were inserted into the C terminus of the capsid protein of the chimeric PCV1-2 vaccine virus, resulting in a tractable marker virus that is infectious both in vitro and in vivo. Pigs experimentally infected with the epitope-tagged PCV1-2 vaccine viruses produced tag-specific antibodies, as well as anti-PCV2 neutralizing antibodies, indicating that the epitope-tagged viruses could potentially serve as a positive-marker modified live-attenuated vaccine.