Estimating ranibizumab injection numbers and visual acuity at 12 months based on 2-month data on branch retinal vein occlusion treatment.

Anti-vascular endothelial growth factor treatment for macular edema secondary to branch retinal vein occlusion generally provides good visual acuity (VA) improvement but may require repeated injections for years. To reduce the number of patients who suffer from avoidable VA loss caused by treatment drop-out, providing prospects of the correlation between expected vision improvement and required number of injections at the early stages of treatment may be helpful. In this post hoc analysis of the phase IV, randomized, open-label ZIPANGU study, we investigated the correlation between the data from Month 2 and Month 12 in terms of VA and required ranibizumab injection numbers. Fifty-nine patients were evaluated (ranibizumab monotherapy, 29; combination therapy, 30). In the monotherapy group, patients who received 1 and 3 injections by Month 2 received a mean total of 2.8 and 8.3 injections during the year, respectively. Data from the combination group were similar. The correlation coefficients for VA scores at Months 2 and 12 were 0.60 and 0.51 for the monotherapy and combination groups, respectively (both p < 0.01). Based on VA and injection numbers at Month 2 of treatment, physicians could provide rough prospects on patients' expected final VA and required number of injections.

Impact on visual acuity and psychological outcomes of ranibizumab and subsequent treatment for diabetic macular oedema in Japan (MERCURY).

PURPOSE: The MERCURY study aimed to evaluate the effects on visual acuity and psychological symptoms, and safety, of ranibizumab and subsequent treatment in patients with diabetic macular oedema (DME) and impaired visual acuity (VA). We report data from the prespecified 12-month interim analysis. METHODS: This was a 24-month, phase 4, open-label, single-arm, prospective, observational study conducted at 20 specialised retinal centres in Japan. Participants were 209 patients with DME and impaired VA, not previously treated with either intravitreal or systemic anti-vascular endothelial growth factor (anti-VEGF) agents, who initiated ranibizumab 0.5 mg per investigator discretion. Following ranibizumab administration, patients were treated per routine clinical practice. Other treatments were allowed. The main outcome measure was the mean change in best-corrected VA (BCVA) in logarithmic minimum angle of resolution (logMAR) from baseline to month 12. An exploratory objective was to assess patients' psychological status using the Hospital Anxiety and Depression Scale (HADS). RESULTS: The mean ± standard deviation BCVA at baseline was 0.43 ± 0.39 logMAR. The mean number of injections of ranibizumab and anti-VEGF agents from baseline to month 11 was 3.2 ± 2.0 and 3.6 ± 2.4, respectively. The BCVA change from baseline to 12 months was - 0.08 ± 0.34 logMAR (p = 0.011), showing a significant improvement; the HADS-anxiety score also decreased significantly (p = 0.001) and the depression score decreased numerically (p = 0.080). CONCLUSION: MERCURY study data confirm the effectiveness of real-world treatment initiated with ranibizumab in Japanese patients with DME. In addition, treatment was able to positively influence anxiety via VA improvement.

The randomized ZIPANGU trial of ranibizumab and adjunct laser for macular edema following branch retinal vein occlusion in treatment-naïve patients.

The ZIPANGU study assessed the efficacy and safety of ranibizumab as a one loading dose + pro re nata (one + PRN) regimen with/without focal/grid laser among treatment-naïve patients suffering from macular edema (ME) following branch retinal vein occlusion (BRVO). ZIPANGU was a phase IV, prospective, randomized, open-label, active-controlled, 12-month, two-arm, multicenter study. Treatment-naïve patients with visual impairment (19-73 letters) caused by ME, defined as central subfield thickness (CSFT) > 300 µm, due to BRVO were randomly assigned to ranibizumab monotherapy (n = 29) or combination therapy (ranibizumab + focal/grid short-pulse laser, n = 30). The primary endpoint was the number of ranibizumab injections. Secondary endpoints were mean changes in best-corrected visual acuity (BCVA) and CSFT, and safety. There were no statistically significant differences in the mean number of ranibizumab injections between monotherapy (4.3 injections) vs. combination (4.1 injections) therapy, or in CSFT. BCVA improvement in the monotherapy arm (22.0 letters) was better than the combination therapy arm (15.0 letters) (p = 0.035). Overall, both regimens appeared to be safe and well tolerated. One + PRN ranibizumab is safe and efficacious in treatment-naïve patients with ME secondary to BRVO. A conjunctive laser treatment did not lead to better functional outcomes or fewer ranibizumab injections.

In-depth Analysis of the Lid Subunits Assembly Mechanism in Mammals.

The 26S proteasome is a key player in the degradation of ubiquitinated proteins, comprising a 20S core particle (CP) and a 19S regulatory particle (RP). The RP is further divided into base and lid subcomplexes, which are assembled independently from each other. We have previously demonstrated the assembly pathway of the CP and the base by observing assembly intermediates resulting from knockdowns of each proteasome subunit and the assembly chaperones. In this study, we examine the assembly pathway of the mammalian lid, which remains to be elucidated. We show that the lid assembly pathway is conserved between humans and yeast. The final step is the incorporation of Rpn12 into the assembly intermediate consisting of two modular complexes, Rpn3-7-15 and Rpn5-6-8-9-11, in both humans and yeast. Furthermore, we dissect the assembly pathways of the two modular complexes by the knockdown of each lid subunit.

Assembly mechanisms of specialized core particles of the proteasome.

The 26S proteasome has a highly complicated structure comprising the 20S core particle (CP) and the 19S regulatory particle (RP). Along with the standard CP in all eukaryotes, vertebrates have two more subtypes of CP called the immunoproteasome and the thymoproteasome. The immunoproteasome has catalytic subunits ß1i, ß2i, and ß5i replacing ß1, ß2, and ß5 and enhances production of major histocompatibility complex I ligands. The thymoproteasome contains thymus-specific subunit ß5t in place of ß5 or ß5i and plays a pivotal role in positive selection of CD8+ T cells. Here we investigate the assembly pathways of the specialized CPs and show that ß1i and ß2i are incorporated ahead of all the other ß-subunits and that both ß5i and ß5t can be incorporated immediately after the assembly of ß3 in the absence of ß4, distinct from the assembly of the standard CP in which ß-subunits are incorporated in the order of ß2, ß3, ß4, ß5, ß6, ß1, and ß7. The propeptide of ß5t is a key factor for this earlier incorporation, whereas the body sequence seems to be important for the earlier incorporation of ß5i. This unique feature of ß5t and ß5i may account for preferential assembly of the immunoproteasome and the thymoproteasome over the standard type even when both the standard and specialized subunits are co-expressed.

Using siRNA techniques to dissect proteasome assembly pathways in mammalian cells.

The 26S proteasome is an ATP-dependent protease known to collaborate with ubiquitin, the polymerization of which acts as a marker for protein degradation in eukaryotic cells, and is involved in a diverse array of biological processes, such as the cell-cycle progression, DNA repair, apoptosis, immune response, signal transduction, transcription, metabolism, protein quality control, and developmental program. The 26S proteasome is a huge protease complex and consists of one catalytic core called the 20S proteasome (or 20S core particle) and one or two 19S regulatory particles (19S RP), which include 14 and 19 different subunits, respectively. Recent studies have revealed that the proteasome formation requires multiple assembly factors and that the assembly pathways are highly conserved between yeast and mammalian cells. This chapter is focused on experimental approaches to reveal the assembly pathways of the proteasome using small interfering RNA techniques in mammalian cells. Knockdown of a proteasome subunit causes arrest of the assembly process before incorporation of the targeted subunit and accumulation of a specific intermediate.

Proteasome assembly defect due to a proteasome subunit beta type 8 (PSMB8) mutation causes the autoinflammatory disorder, Nakajo-Nishimura syndrome.

Nakajo-Nishimura syndrome (NNS) is a disorder that segregates in an autosomal recessive fashion. Symptoms include periodic fever, skin rash, partial lipomuscular atrophy, and joint contracture. Here, we report a mutation in the human proteasome subunit beta type 8 gene (PSMB8) that encodes the immunoproteasome subunit ß5i in patients with NNS. This G201V mutation disrupts the ß-sheet structure, protrudes from the loop that interfaces with the ß4 subunit, and is in close proximity to the catalytic threonine residue. The ß5i mutant is not efficiently incorporated during immunoproteasome biogenesis, resulting in reduced proteasome activity and accumulation of ubiquitinated and oxidized proteins within cells expressing immunoproteasomes. As a result, the level of interleukin (IL)-6 and IFN-γ inducible protein (IP)-10 in patient sera is markedly increased. Nuclear phosphorylated p38 and the secretion of IL-6 are increased in patient cells both in vitro and in vivo, which may account for the inflammatory response and periodic fever observed in these patients. These results show that a mutation within a proteasome subunit is the direct cause of a human disease and suggest that decreased proteasome activity can cause inflammation.

Assembly pathway of the Mammalian proteasome base subcomplex is mediated by multiple specific chaperones.

The 26S proteasome is an enzymatic complex that degrades ubiquitinated proteins in eukaryotic cells. It is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). The latter is further divided into the lid and base subcomplexes. While the mechanism involved in the assembly of the CP is well investigated, that of the RP is poorly understood. Here, we show that the formation of the mammalian base subcomplex involves three distinct modules, where specific pairs of ATPase subunits are associated with the distinct chaperones p28, S5b, or p27. The process of base formation starts from association of the p28-Rpt3-Rpt6-Rpn14 complex with the S5b-Rpt1-Rpt2-Rpn1 complex, followed by incorporation of the p27-Rpt5-Rpt4 complex and Rpn2, where p28, S5b, and p27 regulate the associations between the modules. These chaperones dissociate before completion of 26S proteasome formation. Our results demonstrate that base assembly is facilitated by multiple proteasome-dedicated chaperones, like CP assembly.

Dissecting beta-ring assembly pathway of the mammalian 20S proteasome.

The 20S proteasome is the catalytic core of the 26S proteasome. It comprises four stacked rings of seven subunits each, alpha(1-7)beta(1-7)beta(1-7)alpha(1-7). Recent studies indicated that proteasome-specific chaperones and beta-subunit appendages assist in the formation of alpha-rings and dimerization of half-proteasomes, but the process involved in the assembly of beta-rings is poorly understood. Here, we clarify the mechanism of beta-ring formation on alpha-rings by characterizing assembly intermediates accumulated in cells depleted of each beta-subunit. Starting from beta2, incorporation of beta-subunits occurs in an orderly manner dependent on the propeptides of beta2 and beta5, and the C-terminal tail of beta2. Unexpectedly, hUmp1, a chaperone functioning at the final assembly step, is incorporated as early as beta2 and is required for the structural integrity of early assembly intermediates. We propose a model in which beta-ring formation is assisted by both intramolecular and extrinsic chaperones, whose roles are partially different between yeast and mammals.