Influence of mouse genetic background on hAPP transgene-induced brain amyloidosis and inflammatory response to beta-amyloid protein

Brain inflammation is a hallmark of Alzheimer's Disease (AD) neuropathology. This involves microglial activation which has been well characterized around fibrillar plaques in humans and in transgenic mouse models. Plaques are initiated by the accumulation of Abeta1-42 (beta-amyloid) which is a highly aggregating, neurotoxic protein derived from alternative processing of APP (amyloid precursor protein). Genetic factors involved in influencing the intensity of an inflammatory response have been shown clearly in other pathologies but not in AD. Therefore, an in vitro model of microglial activation by Abeta1-42, was developed using mouse strains having a low (A/J) or high (C57BL/6) inflammatory response. Interferon-gamma-primed C57BL/6 microglia underwent morphological changes from resting (ramified) morphology to activated (ameboid) morphology, increases in nitric oxide (NO) production, and very high levels of tumor necrosis factor alpha (TNF-alpha) when stimulated with Abeta1-42. On the other hand, A/J microglia underwent few morphological changes, had increased levels of NO and had minimal increases in TNF-alpha levels when submitted to the same treatment. Thus, microglial activation to Abeta1-42 was influenced by the magnitude in inflammatory response which may be determined by genetic factors. The timing of an inflammatory response in the process of Abeta deposition is still under debate. Therefore, to define the kinetics of the inflammatory response in brain amyloidogenesis, TgCRND8 mice which overexpress hAPP and aggressively develop amyloidosis, were used to characterize the evolution of diffuse and fibrillar plaque formation and gliosis. Histopathological analysis revealed diffuse plaques as early as 12 weeks of age. Fibrillar (senile) plaques and microgliosis were seen at 13 weeks of age whereas astrocytic clustering began at 14--15 weeks of age. Microglial activation was found to be correlated strongly to Abeta deposition and fibri

IL-2 triggers specific signaling pathways in human NKT cells leading to the production of pro- and anti-inflammatory cytokines.

International audienceNKT cells belong to a conserved T lymphocyte subgroup that has been implicated in the regulation of various immune responses, including responses to viruses, bacteria, and parasites. They express a semi-invariant TCR that recognizes glycolipids presented by the nonpolymorphic MHC class I-like molecule CD1d, and upon activation, they produce various pro- and anti-inflammatory cytokines. Recent studies have shed light on the nature of glycolipids and the environmental signals that may influence the production of cytokines by NKT cells and thus, modulate the immune response. To better understand the regulation mechanisms of NKT cells, we explored their behavior following activation by IL-2 and investigated the signaling pathways and biological responses triggered. We demonstrated that IL-2 activates not only STAT3 and -5 and the PI-3K and ERK-2 pathways as in all IL-2 responder cells but also STAT4 as in NK cells and the p38 MAPK pathway as in alphabeta T cells. We also showed that STAT6 is activated by IL-2 in NKT cells. Moreover, IL-2 induces the production of IFN-gamma and IL-4. The ability of IL-2 to induce pro- and anti-inflammatory cytokine production, in addition to proliferation, could open new therapeutic approaches for use in combination with molecules that activate NKT cells through TCR activation

Use of a generic LC-MS/MS assay to characterize atypical PK profile of a biotherapeutic monoclonal antibody

Background: The fully human monoclonal antibody mAb123 which binds to and neutralizes chemokine motif ligand-21 (CCL21) displays a faster clearance in cynomolgus monkey compared to typical IgG kinetics. Two variants of LC-MS/MS assays were developed to better understand this rapid clearance, previously established by ELISA. Results: Excellent correlation of LC-MS/MS PK data with ELISA data confirmed the rapid clearance of mAb123, indicating that this is an intrinsic property of the molecule. In one study, two animals tested positive for anti-mAb123 antibodies, in one animal these antibodies resulted in lower serum concentrations of mAb123. The LC-MS/MS assay indicates that the underlying reason for this apparent rapid disappearance of mAb123 from serum is due to catabolism, rather than interference of anti-mAb123 antibodies in the ELISA assay. Conclusions: The data illustrate that in cases of unexpected results from ligand binding assays, application of orthogonal bioanalytical techniques such as LC-MS/MS can add value

How the bioanalyst plays a key role in interdisciplinary project teams in the development of biotherapeutics – a reflection of the European Bioanalysis Forum

The role of the bioanalyst within project teams developing biotherapeutics, is not well-understood. Thus, the goal of this manuscript is to enhance the understanding of bioanalytical needs from the different project team functions. The ideas presented here come from an EBF (European Bioanalysis Forum) topic team assigned by the EBF steering committee to elucidate the challenges of total and free macromolecule quantification. This topic team started their activities in 2011 following up on the publication of the White paper by Lee et al (1) and held a break-out session at the EBF Open Symposium in Barcelona in November 2011 where all speakers agreed on the importance of inter-disciplinary communication between bioanalysts and data end-user in bringing forward understanding of the challenges faced by both sides(2)