Design and development of the ''all-welded-and- brazed ''instrumentation ports for NERVA REACTORS

Design and development of all welded and brazed instrumentation ports for nuclear engine for rocket vehicle applications reactor

Transport of anti-IL-6 antigen binding fragments into cartilage and the effects of injury

The efficacy of biological therapeutics against cartilage degradation in osteoarthritis is restricted by the limited transport of macromolecules through the dense, avascular extracellular matrix. The availability of biologics to cell surface and matrix targets is limited by steric hindrance of the matrix, and the microstructure of matrix itself can be dramatically altered by joint injury and the subsequent inflammatory response. We studied the transport into cartilage of a 48 kDa anti-IL-6 antigen binding fragment (Fab) using an in vitro model of joint injury to quantify the transport of Fab fragments into normal and mechanically injured cartilage. The anti-IL-6 Fab was able to diffuse throughout the depth of the tissue, suggesting that Fab fragments can have the desired property of achieving local delivery to targets within cartilage, unlike full-sized antibodies which are too large to penetrate beyond the cartilage surface. Uptake of the anti-IL-6 Fab was significantly increased following mechanical injury, and an additional increase in uptake was observed in response to combined treatment with TNFα and mechanical injury, a model used to mimic the inflammatory response following joint injury. These results suggest that joint trauma leading to cartilage degradation can further alter the transport of such therapeutics and similar-sized macromolecules.National Institute of Arthritis and Musculoskeletal and Skin Diseases (U.S.) (Grant AR45779)National Institute of Arthritis and Musculoskeletal and Skin Diseases (U.S.) (Grant AR60331)Janssen Pharmaceutical Ltd. (Research and Development Grant

Surrogate Antibodies That Specifically Bind and Neutralize CCL17 But Not CCL22

The chemokines CCL17 (TARC) and CCL22 (MDC) function through the same receptor, CCR4, but have been proposed to differentially affect the immune response. To better understand the role of the individual ligands, a panel of rat anti-mouse CCL17 surrogate antibodies was generated that can be used to differentiate CCL17 and CCL22 function in vitro and in vivo. We have successfully identified a panel of neutralizing antibodies by screening hybridomas for the ability to inhibit CCL17-mediated calcium mobilization. Chemotaxis in response to CCL17 is also inhibited, providing further evidence that the antibodies in this panel are antagonistic. Using a recombinant cell line expressing human CCR4, we show that the antibodies block ?-arrestin recruitment as evidence that the antibodies are specifically blocking CCL17 signaling through CCR4. The antibodies within this panel inhibit calcium mobilization with varying potency in the calcium flux assay, having apparent IC50 ranging from approximately 1 to >400?ng/mL. Although both CCL17 and CCL22 function through CCR4, only a single antibody was identified as having detectable binding to CCL22. This panel of CCL17-specific antibodies provides tools that can be used to differentiate CCL17 and CCL22 function through CCR4 interaction in vitro and in vivo.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140164/1/mab.2012.0112.pd

Transport of anti-IL-6 antigen binding fragments into cartilage and the effects of injury

The efficacy of biological therapeutics against cartilage degradation in osteoarthritis is restricted by the limited transport of macromolecules through the dense, avascular extracellular matrix. The availability of biologics to cell surface and matrix targets is limited by steric hindrance of the matrix, and the microstructure of matrix itself can be dramatically altered by joint injury and the subsequent inflammatory response. We studied the transport into cartilage of a 48 kDa anti-IL-6 antigen binding fragment (Fab) using an in vitro model of joint injury to quantify the transport of Fab fragments into normal and mechanically injured cartilage. The anti-IL-6 Fab was able to diffuse throughout the depth of the tissue, suggesting that Fab fragments can have the desired property of achieving local delivery to targets within cartilage, unlike full-sized antibodies which are too large to penetrate beyond the cartilage surface. Uptake of the anti-IL-6 Fab was significantly increased following mechanical injury, and an additional increase in uptake was observed in response to combined treatment with TNFα and mechanical injury, a model used to mimic the inflammatory response following joint injury. These results suggest that joint trauma leading to cartilage degradation can further alter the transport of such therapeutics and similar-sized macromolecules

Engagement of two distinct binding domains on CCL17 is required for signaling through CCR4 and establishment of localized inflammatory conditions in the lung.

CCL17 (TARC) function can be completely abolished by mAbs that block either one of two distinct sites required for CCR4 signaling. This chemokine is elevated in sera of asthma patients and is responsible for establishing inflammatory sites through CCR4-mediated recruitment of immune cells. CCL17 shares the GPCR CCR4, with CCL22 (MDC) but these two chemokines differentially affect the immune response. To better understand chemokine mediated effects through CCR4, we have generated chimeric anti-mouse CCL17 surrogate antibodies that inhibit function of this ligand in vitro and in vivo. The affinities of the surrogate antibodies for CCL17 range from 685 pM for B225 to 4.9 nM for B202. One antibody, B202, also exhibits weak binding to CCL22 (KD∼2 µM) and no binding to CCL22 is detectable with the second antibody, B225. In vitro, both antibodies inhibit CCL17-mediated calcium mobilization, β-arrestin recruitment and chemotaxis; B202 can also partially inhibit CCL22-mediated β-arrestin recruitment. Both B202 and B225 antibodies neutralize CCL17 in vivo as demonstrated by reduction of methacholine-induced airway hyperreactivity in the A. fumigatus model of asthma. That both antibodies block CCL17 function but only B202 shows any inhibition of CCL22 function suggests that they bind CCL17 at different sites. Competition binding studies confirm that these two antibodies recognize unique epitopes that are non-overlapping despite the small size of CCL17. Taking into consideration the data from both the functional and binding studies, we propose that effective engagement of CCR4 by CCL17 involves two distinct binding domains and interaction with both is required for signaling