Nitric oxide (NO) is a major signaling molecule in the retina and CNS, with physiological roles in every cell type in the retina. Previous work shows that neuronal nitric oxide synthase (nNOS) is an important source of NO in the vertebrate retina. There are distinct, active alternative transcripts of nNOS observed in many tissues, including testes and brain, that may differ in both localization and enzyme kinetics. The present study characterized nNOS and the NO production from nNOS in the mouse retina in terms of its alternate transcripts, namely, nNOS alpha, nNOS beta, and nNOS gamma. We examined both basal and light-stimulated NO production as imaged using the NO-sensitive dye 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate-FM (DAF-FM), and we compared the NO production with the immunocytochemical localization of nNOS using antisera that recognize nNOS alpha/beta or nNOS alpha/beta/gamma. Western blots suggested the presence of NOS alpha/gamma protein in retina, but not nNOS beta, and we confirmed this at the message level by using a combination of RT-PCR and quantitative real-time PCR. Our findings indicated that the primary source of NO in the mammalian retina is nNOS alpha and that nNOS gamma may contribute to NO production as well.

Adeno-associated virus (AAV) is a proven, safe and effective vector for gene delivery in the retina. There are over 100 serotypes of AAV, and AAV2 through AAV9 have been evaluated in the retina. Each AAV serotype has different cell tropism and transduction efficiency. Intravitreal injections of AAV into the eye tend to transduce cells in the ganglion cell layer (GCL), while subretinal injections tend to transduce retinal pigment epithelium and photoreceptors. Efficient transduction of the inner retina beyond the GCL is not well established with the current methodologies and serotypes used to date. In this study, we compared the cellular tropism of AAVrh8 and AAVrh10 vectors encoding enhanced green fluorescent protein (EGFP) using intravitreal injections. We found that AAVrh8 largely transduced cells in the GCL and also amacrine cells in the inner nuclear layer (INL), as well as Müller and horizontal cells. Inner retinal transduction with AAVrh10 was similar to AAVrh8, but AAVrh10 appeared to also transduce bipolar cells. The transduction efficiency as measured by the intensity of EGFP signal was 3.5 fold higher in horizontal cells transduced with AAVrh10 than AAVrh8. Glial fibrillary accessory protein (GFAP) levels were increased in Müller cells in transduced areas for both serotypes. The results of this study suggest that AAVrh8 and AAVrh10 may be excellent vector candidates to deliver genetic material to the INL, particularly for amacrine and horizontal cells, however they may also cause cellular stress as shown by increased glial GFAP expression.

Peripheral neuropathy (PN) is the most troublesome adverse event associated with the proteasome inhibitor bortezomib. Studies suggest an inflammatory aetiology for bortezomib-induced PN (BiPN) and it has been hypothesized that reducing inflammation with concomitant dexamethasone may reduce BiPN incidence and/or severity. We retrospectively analysed PN rates from 32 studies (2697 patients with previously untreated multiple myeloma) incorporating bortezomib and differing dexamethasone schedules: partnered dosing (days of and after bortezomib), weekly dosing, and other dosing schedules (e.g. days 1-4, 8-11). Pooled overall PN rates were 45·5%, 63·9%, and 47·5%, respectively, with 5·3%, 11·0%, and 9·6% grade ≥3. Adjusting for potential confounders (age, gender, presence of thalidomide, bortezomib treatment duration), PN rates in patients on partnered dosing schedules appeared lower than in patients on weekly or other dosing schedules. Analyses conducted using patient-level data suggest that cumulative dexamethasone dose, a potential confounding factor, is unlikely to have influenced the analyses. Findings were similar in a separate pooled analysis excluding data from regimens incorporating thalidomide, when pooled overall PN rates were 50·1%, 63·9%, and 48·3%, respectively, with 4·2%, 11·0%, and 8·6% grade ≥3. These findings suggest that partnered dexamethasone dosing may result in less severe BiPN compared with alternative dexamethasone dosing schedules.

The nitric oxide (NO) signaling pathway is integrally involved in visual processing and changes in the NO pathway are measurable in eyes of diabetic patients. The small peptide adrenomedullin (ADM) can activate a signaling pathway to increase the enzyme activity of neuronal nitric oxide synthase (nNOS). ADM levels are elevated in eyes of diabetic patients and therefore, ADM may play a role in the pathology of diabetic retinopathy. The goal of this research was to test the effects of inhibiting the ADM/NO signaling pathway in early diabetic retinopathy. Inhibition of this pathway decreased NO production in high-glucose retinal cultures. Treating diabetic mice with the PKC β inhibitor ruboxistaurin for 5 weeks lowered ADM mRNA levels and ADM-like immunoreactivity and preserved retinal function as assessed by electroretinography. The results of this study indicate that inhibiting the ADM/NO signaling pathway prevents neuronal pathology and functional losses in early diabetic retinopathy.

296 Background: Immune checkpoint (PD-1, PD-L1) inhibitors are important in the treatment (Tx) of aNSCLC, as reflected in European Society for Medical Oncology (ESMO) guidelines. Pembrolizumab (pembro) combined with pemetrexed and platinum-based chemotherapy is a standard induction option in 1L Tx of NSQ aNSCLC. Decisions about 1L maintenance consider response and toxicity after induction, performance status, and patient preference. This study documents recent real-world 1L MT patient characteristics and Tx in EU4 (France, Germany, Italy, Spain) and UK. Methods: This retrospective analysis used patient chart review completed by treating physicians in EU4 and UK between 1 Jan 2018 and 31 Dec 2020 in the IQVIA Oncology Dynamics (OD) database. Adults (≥21 years) with confirmed NSQ aNSCLC (stage IIIB/C or IV) without actionable oncogenic driver mutations and with Eastern Cooperative Oncology Group (ECOG) status of 0 or 1 were included. 1L MT and Tx characteristics and trends over time were evaluated overall and by country. Results: In total, 1,814 NSQ aNSCLC patients receiving 1L MT Tx met the inclusion criteria; 29% of patients entered the analysis in 2018, 40% in 2019, and 31% in 2020. Most patients were male (61%), in the 46–65 age group (51%), current/ex-smokers (87%), and treated in the UK (29%) or Germany (24%); 94% were metastatic (stage IV), 10% presented with CNS/brain metastases, and 77% had ECOG score of 1. Overall, 91% were tested for PD-L1; of these, 47% had tumor proportion score (TPS) of ≥1% to 49%, 22% had TPS of ≥50%, and 21% tested negative. Baseline demographic and clinical characteristics were generally similar among countries. The top two Tx in 1L MT were pemetrexed (40%; range: 12% in Germany to 66% in Italy) and pembro (28%; range: 14% in Spain to 49% in Germany) monotherapies. Overall use of pemetrexed-based Tx in 1L MT decreased from 68% in 2018 Q1 to 56% in 2020 Q4, while pembro-based Tx increased from 18% in 2018 Q1 to 73% in 2020 Q4 in 1L MT. Conclusions: Despite ESMO guidelines, this real-world data suggests notable differences between European countries in 1L MT Tx for NSQ aNSCLC, with trends over time likely reflecting introduction and adoption of pembro regardless of PD-L1 status. Awareness of country and time-variability in clinical practice are critical for implementing clinical trials and updating clinical practice guidelines to maximize Tx benefits with improved tolerability.[Table: see text]