Molecular mechanisms involved in activity of h7C10, a humanized monoclonal antibody, to IGF-1 receptor.

IGF-1 receptor (IGF-1R) plays a key role in the development of numerous tumors. Blockade of IGF-1R axis using monoclonal antibodies constitutes an interesting approach to inhibit tumor growth. We have previously shown that h7C10, a humanized anti-IGF-1R Mab, exhibited potent antitumor activity in vivo. However, mechanisms of action of h7C10 are still unknown. Here, we showed that h7C10 inhibited IGF-1-induced IGF-1R phosphorylation in a dose-dependent manner. Also, h7C10 abolished IGF-1-induced activation of PI3K/AKT and MAPK pathways. Cell cycle progression and colony formation were affected in the presence of h7C10 probably because of the inhibition of IGF-1-induced cyclin D1 and E expression. In addition, we demonstrated that h7C10 induced a rapid IGF-1R internalization leading to an accumulation into cytoplasm resulting in receptor degradation. Using lysosome and proteasome inhibitors, we observed that the IGF-1R alpha- and beta-chains could follow different degradation routes. Thus, we demonstrated that antitumoral properties of h7C10 are the result of IGF-1-induced cell signaling inhibition and down-regulation of IGF-1R level suggesting that h7C10 could be a candidate for therapeutic applications.

Direct bacterial protein PAMP recognition by human NK cells involves TLRs and triggers alpha-defensin production.

Although human CD56(+)CD3(-) natural killer (NK) cells participate in immune responses against microorganisms, their capacity to directly recognize and be activated by pathogens remains unclear. These cells encode members of the Toll-like receptor (TLR) family, involved in innate cell activation on recognition of pathogen-associated molecular patterns (PAMPs). We therefore evaluated whether the 2 bacterial protein PAMPs, the outer membrane protein A from Klebsiella pneumoniae (KpOmpA) and flagellin, which signal through TLR2 and TLR5, respectively, may directly stimulate human NK cells. These proteins induce interferon-gamma (IFN-gamma) production by NK cells and synergize with interleukin-2 (IL-2) and proinflammatory cytokines in PAMP-induced activation. Similar results were obtained using CD56(+)CD3(+) (NKR-expressing) T cells. NK cells from TLR2(-/-) mice fail to respond to KpOmpA, demonstrating TLR involvement in this effect. Defensins are antimicrobial peptides expressed mainly by epithelial cells and neutrophils that disrupt the bacterial membrane, leading to pathogen death. We show that NK cells and NKR-expressing T cells constitutively express alpha-defensins and that KpOmpA and flagellin rapidly induce their release. These data demonstrate for the first time that highly purified NK cells directly recognize and respond to pathogen components through TLRs and evidence defensins as a novel and direct cytotoxic pathway involved in NK cell-mediated protection against microorganisms.

The immunogenicity, protective efficacy and safety of BBG2Na, a subunit respiratory syncytial virus (RSV) vaccine candidate, against RSV-B.

Respiratory syncytial virus (RSV) is divided into subgroups A and B, based primarily on variation within the G glycoprotein. A safe vaccine that protects against both would be the ideal. BBG2Na is a recombinant subunit RSV vaccine candidate derived in part from the G protein of RSV-A. Interestingly, BBG2Na formulated in alum protected against RSV-B challenge at early time points following vaccination in mice. Over 6 months, however, BBG2Na-induced immunogenicity and protective efficacy progressively diminished, such that few animals were considered protected at the end. To study the safety of BBG2Na relative to RSV-B challenge, we established a novel enhanced immunopathology mouse model. We confirmed that RSV-B challenge of formalin-inactivated RSV-A (FI-RSV-A)-immunized BALB/c mice results in enhanced pulmonary pathology. Therefore, this phenomenon is neither subgroup-specific nor dependent on a previously incriminated Th epitope in the RSV-A G protein. In stark contrast, BBG2Na did not induce any signs of enhanced pulmonary pathology. In conclusion, our data indicate that BBG2Na, formulated in alum, induces safe and protective immune responses against RSV-B challenge in mice. However, the duration of protective immunity will probably be insufficient to prevent RSV-B infection for the duration of the RSV epidemic season.

Measurement of nuclear factor-kappa B translocation on lipopolysaccharide-activated human dendritic cells by confocal microscopy and flow cytometry.

BACKGROUND: Nuclear factor kappa B (NF-kappaB) is a ubiquitously expressed transcription factor that regulates cytokine and immunoglobulin (Ig) gene expression. In most cell types, the inactive p50/p65 NF-kappaB heterodimer is located in the cytoplasm, complexed to its IkappaB inhibitory unit. Stimulation of cells by various reagents such as bacterial endotoxin or cytokines leads to a dissociation of NF-kappaB from IkappaB and a rapid translocation of free NF-kappaB to the nucleus. The aim of this article is to define optimal conditions for the measurement of NF-kappaB translocation by both confocal microscopy and flow cytometry. METHODS: Four commercial anti-NF-kappaB antibodies were evaluated by confocal microscopy, after using two methods of fixation and permeabilization of the cells. These antibodies were examined further by flow cytometry on purified nuclei. RESULTS: Paraformaldehyde-methanol treatment of dendritic cells is a good combination to visualize NF-kappaB translocation by confocal microscopy. Three of the four antibodies tested gave good results on nonactivated and on lipopolysaccharide (LPS)-activated dendritic cells. The measurement of NF-kappaB translocation by flow cytometry on purified nuclei is a quick and sensitive method. Only one of the four evaluated antibodies showed a significant difference between nonactivated and activated cells. CONCLUSIONS; Microscopy and flow cytometry are quick and reproducible methods to measure NF-kappaB translocation and can be adapted to identify new molecules that activate dendritic cells.