Dimeric and polymeric IgA, but not monomeric IgA, enhance the production of IL-6 by human renal mesangial cells

Depositions of IgA in the renal glomerular mesangial area are a hallmark of IgA nephropathy, and are thought to be crucial for the onset of inflammation processes in IgA nephropathy. In this report we show that human mesangial cells (MC) in vitro bind IgA and that binding of IgA enhances the production of IL-6 by MC. Furthermore we show that the size of IgA is crucial in its capability to enhance IL-6 production. Monomeric IgA does not affect basic IL-6 production, whereas dimeric and polymeric IgA enhance IL-6 production up to 3- to 9-fold respectively. Additional studies demonstrate that enhanced IL-6 production by MC is not accompanied by increased proliferation of human mesangial cells, a finding which is distinct from that found with rat mesangial cells. Taken together, these fmdings suggest that deposition of dimeric and polymeric IgA in the mesangial area of human kidneys in IgA nephropathy may amplify local inflammation

Selection and characterisation of a phage-displayed human antibody (Fab) reactive to the lung resistance-related major vault protein

The major vault protein is the main component on multimeric vault particles, that are likely to play an essential role in normal cell physiology and to be associated with multidrug resistance of tumour cells. In order to unravel the function of vaults and their putative contribution to multidrug resistance, specific antibodies are invaluable tools. Until now, only conventional major vault protein-reactive murine monoclonal antibodies have been generated, that are most suitable for immunohistochemical analyses. The phage display method allows for selection of human antibody fragments with potential use in clinical applications. Furthermore, cDNA sequences encoding selected antibody fragments are readily identified, facilitating various molecular targeting approaches. In order to obtain such human Fab fragments recognising major vault protein we used a large non-immunized human Fab fragment phage library. Phages displaying major vault protein-reactive Fabs were obtained through several rounds of selection on major vault protein-coated immunotubes and subsequent amplification in TG1 E coli bacteria. Eventually, one major vault protein-reactive clone was selected and further examined. The anti-major vault protein Fab was found suitable for immunohistochemical and Western blot analysis of tumour cell lines and human tissues. BIAcore analysis showed that the binding affinity of the major vault protein-reactive clone almost equalled that of the murine anti-major vault protein Mabs. The cDNA sequence of this human Fab may be exploited to generate an intrabody for major vault protein-knock out studies. Thus, this human Fab fragment should provide a valuable tool in elucidating the contribution(s) of major vault protein/vaults to normal physiology and cellular drug resistance mechanisms

Recruitment of the Major Vault Protein by InlK: A Listeria monocytogenes Strategy to Avoid Autophagy

L. monocytogenes is a facultative intracellular bacterium responsible for listeriosis. It is able to invade, survive and replicate in phagocytic and non-phagocytic cells. The infectious process at the cellular level has been extensively studied and many virulence factors have been identified. Yet, the role of InlK, a member of the internalin family specific to L. monocytogenes, remains unknown. Here, we first show using deletion analysis and in vivo infection, that InlK is a bona fide virulence factor, poorly expressed in vitro and well expressed in vivo, and that it is anchored to the bacterial surface by sortase A. We then demonstrate by a yeast two hybrid screen using InlK as a bait, validated by pulldown experiments and immunofluorescence analysis that intracytosolic bacteria via an interaction with the protein InlK interact with the Major Vault Protein (MVP), the main component of cytoplasmic ribonucleoproteic particules named vaults. Although vaults have been implicated in several cellular processes, their role has remained elusive. Our analysis demonstrates that MVP recruitment disguises intracytosolic bacteria from autophagic recognition, leading to an increased survival rate of InlK over-expressing bacteria compared to InlK− bacteria. Together these results reveal that MVP is hijacked by L. monocytogenes in order to counteract the autophagy process, a finding that could have major implications in deciphering the cellular role of vault particles

Diminished expression of multidrug resistance-associated protein 1 (MRP1) in bronchial epithelium of COPD patients

Cigarette smoke is the principal risk factor for chronic obstructive pulmonary disease (COPD). Multidrug resistance proteins, such as multidrug resistance-associated protein-1 (MRP1), P-glycoprotein (P-gp), and lung resistance-related protein (LRP), may protect against oxidative stress and toxic compounds generated by cigarette smoking. Expression of MRP1, P-gp, and LRP was evaluated in bronchial epithelium of two study groups of COPD patients and their controls and was associated with disease status and smoking history. In study group 1, MRP1, but not P-gp and LRP expression, was lower (p=0.029) in normal bronchial epithelium of COPD patients (n=11) compared to healthy controls (n=8). MRP1 expression was high in squamous metaplastic epithelium. When including expression in squamous metaplastic cells, MRP1 was still lower in total bronchial epithelium in the COPD group (p=0.038). In study group 2, expression of MRP1, but not of P-gp and LRP, was lower (p=0.047) in lung tissue of (very) severe COPD (n=10) vs mild to moderate COPD (n=9) patients. In conclusion, MRP1 expression was lower in bronchial biopsies of COPD patients than of healthy controls and was also lower in patients with severe COPD than with mild/moderate COPD. Our findings indicate that diminished MRP1 expression in normal bronchial epithelium is associated with COPD. The exact role in COPD pathogenesis is to be revealed by further functional studies