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Reslizumab and mepolizumab are recently approved monoclonal antibodies for the treatment of severe (uncontrolled) eosinophilic asthma. Both are effective in neutralizing the function of interleukin-5 (IL-5). This study is the first to compare the binding affinity and in vitro potency of both antibodies in head-to-head assays. Two assays assessed binding affinity (using the equilibrium dissociation constant [KD]) of each drug for human IL-5. In the Biacore surface plasmon resonance assay, the association constant (kon) values for human IL-5 for reslizumab and mepolizumab were 3.93 × 106 and 1.83 × 105, respectively. The dissociation constant (koff) values were 4.29 × 10−4 and 2.14 × 10−4, respectively. Calculated KD values for human IL-5 for reslizumab and mepolizumab were 109 and 1,170 pM, respectively, representing an approximately 11-fold stronger binding affinity with reslizumab. In the Kinetic Exclusion Assay, the kon values for human IL-5 for reslizumab and mepolizumab were 3.17 × 106 and 1.32 × 105, respectively. The koff values were 1.36 × 10−5 and 1.48 × 10−5, respectively. Measured KD values for human IL-5 for reslizumab and mepolizumab were 4.3 and 112 pM, respectively, representing an approximately 26-fold stronger binding affinity for reslizumab. A human-IL-5-dependent cell proliferation assay was developed to assess in vitro potency, based on a human cell line selected for enhanced surface expression of IL-5 receptor-alpha and consistent proliferation response to IL-5. The concentration at which 50% inhibition occurred (IC50) was determined for both antibodies. Reslizumab and mepolizumab inhibited IL-5-dependent cell proliferation, with IC50 values of approximately 91.1 and 286.5 pM, respectively, representing on average 3.1-fold higher potency with reslizumab. In conclusion, comparative assays show that reslizumab has higher affinity binding for and in vitro potency against human IL-5 compared with mepolizumab. However, these results do not take into consideration the different methods of administration of reslizumab and mepolizumab.

SummaryThe cytokine TWEAK and its cognate receptor Fn14 are members of the TNF/TNFR superfamily and are upregulated in tumors. We found that Fn14, when expressed in tumors, causes cachexia and that antibodies against Fn14 dramatically extended lifespan by inhibiting tumor-induced weight loss although having only moderate inhibitory effects on tumor growth. Anti-Fn14 antibodies prevented tumor-induced inflammation and loss of fat and muscle mass. Fn14 signaling in the tumor, rather than host, is responsible for inducing this cachexia because tumors in Fn14- and TWEAK-deficient hosts developed cachexia that was comparable to that of wild-type mice. These results extend the role of Fn14 in wound repair and muscle development to involvement in the etiology of cachexia and indicate that Fn14 antibodies may be a promising approach to treat cachexia, thereby extending lifespan and improving quality of life for cancer patients.

Rotavirus is a ubiquitous double-stranded RNA virus responsible for most cases of infantile gastroenteritis. It infects pancreatic islets in vitro and is implicated as a trigger of autoimmune destruction of islet beta cells leading to type 1 diabetes, but pancreatic pathology secondary to rotavirus infection in vivo has not been documented. To address this issue, we inoculated 3 week-old C57Bl/6 mice at weaning with rhesus rotavirus, which is closely related to human rotaviruses and known to infect mouse islets in vitro. Virus was quantified in tissues by culture-isolation and enzyme-linked immunosorbent assay. A requirement for viral double stranded RNA was investigated in toll-like receptor 3 (TLR3)-deficient mice. Cell proliferation and apoptosis, and insulin expression, were analyzed by immunohistochemistry. Following rotavirus inoculation by gavage, two phases of mild, transient hyperglycemia were observed beginning after 2 and 8 days. In the first phase, widespread apoptosis of pancreatic cells was associated with a decrease in pancreas mass and insulin production, without detectable virus in the pancreas. These effects were mimicked by injection of the double-stranded RNA mimic, polyinosinic-polycytidylic acid, and were TLR3-dependent. By the second phase, the pancreas had regenerated but islets were smaller than normal and viral antigen was then detected in the pancreas for several days. These findings directly demonstrate pathogenic effects of rotavirus infection on the pancreas in vivo, mediated initially by the interaction of rotavirus double-stranded RNA with TLR3.

Mitochondria form intricate networks through fission and fusion events. Here, we identify mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51, respectively) anchored in the mitochondrial outer membrane. MiD49/51 form foci and rings around mitochondria similar to the fission mediator dynamin-related protein 1 (Drp1). MiD49/51 directly recruit Drp1 to the mitochondrial surface, whereas their knockdown reduces Drp1 association, leading to unopposed fusion. Overexpression of MiD49/51 seems to sequester Drp1 from functioning at mitochondria and cause fused tubules to associate with actin. Thus, MiD49/51 are new mediators of mitochondrial division affecting Drp1 action at mitochondria.

Measles virus (MeV) infection is characterized by the formation of multinuclear giant cells (MGC). We report that beta interferon (IFN-β) production is amplified in vitro by the formation of virus-induced MGC derived from human epithelial cells or mature conventional dendritic cells. Both fusion and IFN-β response amplification were inhibited in a dose-dependent way by a fusion-inhibitory peptide after MeV infection of epithelial cells. This effect was observed at both low and high multiplicities of infection. While in the absence of virus replication, the cell-cell fusion mediated by MeV H/F glycoproteins did not activate any IFN-α/β production, an amplified IFN-β response was observed when H/F-induced MGC were infected with a nonfusogenic recombinant chimerical virus. Time lapse microscopy studies revealed that MeV-infected MGC from epithelial cells have a highly dynamic behavior and an unexpected long life span. Following cell-cell fusion, both of the RIG-I and IFN-β gene deficiencies were trans complemented to induce IFN-β production. Production of IFN-β and IFN-α was also observed in MeV-infected immature dendritic cells (iDC) and mature dendritic cells (mDC). In contrast to iDC, MeV infection of mDC induced MGC, which produced enhanced amounts of IFN-α/β. The amplification of IFN-β production was associated with a sustained nuclear localization of IFN regulatory factor 3 (IRF-3) in MeV-induced MGC derived from both epithelial cells and mDC, while the IRF-7 up-regulation was poorly sensitive to the fusion process. Therefore, MeV-induced cell-cell fusion amplifies IFN-α/β production in infected cells, and this indicates that MGC contribute to the antiviral immune response.

Proper mitochondrial distribution is crucial for cell function. In Drosophila, mitochondrial transport is facilitated by Miro and Milton, which regulate mitochondrial attachment to microtubules via kinesin heavy chain. Mammals contain two sequence orthologs of Milton however, they have been ascribed various functions in intracellular transport. In this report, we show that the human Miltons target to mitochondria irrespective of whether they are linked to GFP at their C- or N-termini. Their ectopic expression induces the formation of extended mitochondrial tubules as well as large bulbous-like mitochondria with narrow tubular membrane necks that connect them to the mitochondrial mass. The mitochondrial extensions appear highly dynamic and their formation relies on the presence of microtubules. Using the photoswitchable fluorescent protein Dendra2 targeted to the mitochondrial matrix, we found that the mitochondrial extensions and bulbous mitochondria are fused with neighboring regions of the network. Truncation analysis of huMilton1 revealed that the N-terminal region, inclusive of the coiled-coil segment could localize to microtubules, suggesting that Milton attachment to kinesin occurs independent of Miro or mitochondrial attachment. In addition, we show that the huMiltons have the capacity to self-interact and can also facilitate mitochondrial recruitment of a cytosolic Miro mutant. We conclude that the human Miltons are important mediators of the mitochondrial trafficking machinery.