Members of the interleukin‐6 cytokine family, including leukemia inhibitory factor (LIF), signal through gp130. The neuroprotective role of gp130 activation has been widely demonstrated in both CNS and PNS, but the mechanism by which this is accomplished is not well established. We investigated temporal and cell‐specific activation of signaling pathways induced by LIF in the mature mouse retina. Intravitreal injection of LIF preserved photoreceptor function and prevented photoreceptor cell death from light‐induced oxidative damage in a dose‐dependent manner (2 days post‐injection). A therapeutic dose of LIF induced rapid and sustained activation of signal transducer and activator of transcription (STAT) 3. Activated STAT3 was localized to all the retinal neurons and glial cells, including photoreceptors. Activation of extracellular signal‐regulated kinase 1 and 2 was robust but transient in Müller glial cells, and undetectable at the time of light exposure. Akt was not activated by LIF. We also show that at the time of neuroprotection, STAT3 but not extracellular signal‐regulated kinase 1 and 2 or the Akt pathways was active in LIF‐treated retinas, and activated STAT3 was clearly localized in transcriptionally active areas of photoreceptor nuclei. Our data suggest that photoreceptor protection in response to LIF can be directly mediated by activation of STAT3 in photoreceptors.

Background: FBXL5 is required for proper regulation of cellular iron homeostasis. Results: FBXL5 contains a structurally characterized hemerythrin-like domain. Conclusion:The atypical features of the hemerythrin-like domain facilitate its role as a metabolic sensor. Significance: FBXL5 is the only identified mammalian protein containing a hemerythrin-like domain. Resolution of the structure of the domain provides mechanistic insights into its iron and oxygen responsiveness.

Considerable progress has been made increasing productivity of cell cultures to meet the rapidly growing demand for antibody biopharmaceuticals through increased cell densities and longer culture times. This in turn has dramatically increased the burden of process and product related impurities on the purification processes. In addition, current trends in the biopharmaceutical industry point toward both increased productivity and targeting smaller patient populations for new indications. Taken together, these developments are driving the industry to explore alternative separation technologies as a future manufacturing strategy. Clarification technologies well established in other industries, such as flocculation and precipitation are increasingly considered as a viable solution to address this bottleneck in antibody processes. However, several technical issues need to be fully addressed including suitability as a platform application, robustness, process cost, toxicity, and clearance. This review will focus on recent efforts to incorporate new generation clarification technologies for mammalian cell cultures producing monoclonal antibodies as well as challenges to their implementation supported by a case study.

Preconditioning with moderate oxidative stress (e.g., moderate bright light or mild hypoxia) can induce changes in retinal tissue that protect photoreceptors from a subsequent dose of lethal oxidative stress. The mechanism underlying this induced protection is likely a general mechanism of endogenous protection which has been demonstrated in heart and brain using ischemia and reperfusion. While multiple factors like bFGF, CNTF, LIF and BDNF have been hypothesized to play a role in preconditioning induced endogenous neuroprotection, it has not yet been demonstrated which factors or receptors are playing an essential role. Using quantitative PCR techniques we provide evidence that in the retina, LIFR activating cytokines leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1) and cardiotrophin like cytokine (CLC) are strongly up regulated in response to preconditioning with bright cyclic light leading to robust activation of signal transducer and activator of transcription-3 (STAT3) in a time-dependent manner. Further, we found that blocking LIFR activation during preconditioning using a LIFR antagonist (LIF05) attenuated the induced STAT3 activation and also resulted in reduced preconditioning-induced protection of the retinal photoreceptors. These data demonstrate that LIFR and its ligands play an essential role in endogenous neuroprotective mechanisms triggered by preconditioning-induced stress.

To be administered to patients, therapeutic monoclonal antibodies must have very high purity, with process related impurities like host-cell proteins (HCPs) and DNA reduced to <100 ppm and <10 ppb, respectively, relative to desired product. Traditionally, Protein-A chromatography as a capture step has been the work horse for clearing a large proportion of these impurities. However, remaining levels of process and product related impurities still present significant challenges on the development of polishing steps further downstream. In this study, we have incorporated high throughput screening to evaluate three areas of separation: (i) Harvest treatment; (ii) Protein-A Chromatography; and (iii) Low pH Viral Inactivation. Precipitation with low pH treatment of cell culture harvest resulted in selective removal of impurities while manipulating the pH of wash buffers used in Protein-A chromatography and incorporating wash additives that disrupt various modes of protein-protein interaction resulted in further and more pronounced reduction in impurity levels. In addition, our study also demonstrate that optimizing the neutralization pH post Protein-A elution can result in selective removal of impurities. When applied over multiple mAbs, this optimization method proved to be very robust and the strategy provides a new and improved purification process that reduces process related impurities like HCPs and DNA to drug substance specifications with just one chromatography column and open avenues for significant decrease in operating costs in monoclonal antibody purification.

Retinal degenerations are a class of neurodegenerative disorders that ultimately lead to blindness due to the death of retinal photoreceptors. In most cases, death is the result of long-term exposure to environmental, inflammatory, and genetic insults. In age-related macular degeneration, significant vision loss may take up to 70 -80 years to develop. The protracted time to develop blindness suggests that retinal neurons have an endogenous mechanism for protection from chronic injury. Previous studies have shown that endogenous protective mechanisms can be induced by preconditioning animals with sublethal bright cyclic light. Such preconditioning can protect photoreceptors from a subsequent damaging insult and is thought to be accomplished through induced expression of protective factors. Some of the factors shown to be associated with protection bind and activate the signal transducing receptor gp130. To determine whether stress-induced endogenous protection of photoreceptors requires gp130, we generated conditional gp130 knockout (KO) mice with the Cre/lox system and used light-preconditioning to induce neuroprotection in these mice. Functional and morphological analyses demonstrated that the retina-specific gp130 KO impaired preconditioning-induced endogenous protection. Photoreceptor-specific gp130 KO mice had reduced protection, although the Mü ller cell KO mice did not, thus gp130-induced protection was restricted to photoreceptors. Using an animal model of retinitis pigmentosa, we found that the photoreceptor-specific gp130 KO increased sensitivity to genetically induced photoreceptor cell death, demonstrating that gp130 activation in photoreceptors had a general protective role independent of whether stress was caused by light or genetic mutations.IL6 signal transducing receptor ͉ neuroprotection ͉ conditional gp130 knockout ͉ inherited retinal degeneration ͉ light damage T he signal-transducing receptor gp130 is a common receptor for the IL-6 family of cytokines, such as ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), and cardiotrophin-like cytokine (CLC). Use of these cytokines has been shown to be neuroprotective in both the central and peripheral nervous systems, suggesting that this receptor and its signal transduction pathways may be important for the design of neuroprotective therapeutics (1-4).Up-regulation of protective cytokines by acute or chronic stress has been observed in several studies. Mild light stress has been shown to induce endogenous protection of photoreceptors from a subsequent light damage, and protection coincided with prolonged up-regulation of neurotrophic factors (5). bFGF mRNA expression was up-regulated in mice homozygous for the rd1 mutation in the PDE6b gene (a model of retinitis pigmentosa) (6). Elevated levels of bFGF and/or CNTF expression in the retina were also found in light-induced as well as inherited models of photoreceptor degeneration in both mice and rats (7,8). LIF and CLC were also up-regulated by stress from constant light exposure in ...

Background:The hemerythrin-like domain of FBXL5 (FBXL5-Hr) governs its stability. Results: The ability of FBXL5-Hr to sense iron and oxygen availability is restricted within cells and is mediated by distinct conformations. Conclusion: FBXL5-Hr employs different mechanisms to signal changes in iron and oxygen availability. Significance: This study provides new mechanistic insights into the maintenance of iron homeostasis.

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