Impact of concomitant dexamethasone dosing schedule on bortezomib-induced peripheral neuropathy in multiple myeloma.

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.

Evidence for a functional adrenomedullin signaling pathway in the mouse retina.

PURPOSE: Adrenomedullin (ADM) is a small, secreted peptide often associated with vasodilation. However, ADM can also function as a neurotransmitter/neuromodulator, and studies suggest ADM is upregulated in the eye in several ocular diseases. However, no studies to date have described an ADM signaling pathway in the retina. METHODS: PCR, immunocytochemistry, nitric oxide imaging, western blots, and a nitrite assay were used to determine the localization of the components of the ADM signaling pathway in the mouse retina. RESULTS: We used reverse-transcriptase polymerase chain reaction to show that ADM and its primary receptor, calcitonin-receptor-like receptor, along with its associated receptor activity modifying proteins 2 and 3 are expressed in the retina. Using immunocytochemistry, we detected ADM staining throughout the retina in the photoreceptor outer segments, the outer nuclear layer, Müller and amacrine cell somata in the inner nuclear layer, and some somata in the ganglion cell layer. We found that calcitonin-receptor-like receptor and receptor activity modifying protein 2 had localization patterns similar to ADM, especially in somata in the inner nuclear and ganglion cell layers. Finally, we showed that the ADM receptor was functional in the retina. Stimulation of isolated retinas with ADM increased cyclic adenosine monophosphate- and cyclic guanosine monophosphate-like immunoreactivity, as well as nitric oxide production. CONCLUSIONS: These results are the first to show that ADM and functional ADM receptors are present in the retina. Since ADM is increased in eyes with ocular pathologies such as diabetic retinopathy, glaucoma, retinitis pigmentosa, and uveitis, the ADM signaling pathway may provide a new target for ameliorating these retinal pathologies.

Inhibition of the adrenomedullin/nitric oxide signaling pathway in early diabetic retinopathy.

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 ß 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.

Transduction of the inner mouse retina using AAVrh8 and AAVrh10 via intravitreal injection.

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.

Increased neuronal nitric oxide synthase activity in retinal neurons in early diabetic retinopathy.

PURPOSE: There are increased levels of nitric oxide (NO) in diabetic retinas. The purpose of this study was to determine the extent that neuronal nitric oxide synthase (nNOS) contributes to the increased levels of retinal NO in early diabetic retinopathy by examining the expression and activity of nNOS in retinal neurons after 5 weeks of diabetes. METHODS: Changes in NO levels were measured using NO imaging of retinal neurons in mice with streptozotocin-induced diabetes for five weeks. NO imaging was compared to nNOS localization using immunocytochemistry, and nNOS message and protein levels were measured using quantitative real-time PCR and western blots. RESULTS: There was a close anatomic correlation between the localization of the increased NO production and the nNOS immunoreactivity in the retinal plexiform layers of diabetic retinas. There was no change in nNOS message, but nNOS protein was decreased and its subcellular localization was altered. Treatment with insulin or aminoguanidine partially ameliorated the increase in NO in diabetic retinas. CONCLUSIONS: These results suggest that increased nNOS activity is responsible for the majority of increased NO in retinal neurons in early diabetic retinopathy. This supports a role for increased nNOS activity in the early neuronal dysfunction in the diabetic retina.

Identification of alternate transcripts of neuronal nitric oxide synthase in the mouse retina.

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.