Our studies demonstrate for the first time in vivo the pathogenic consequences of deregulated Activin-A expression in the lung, document novel aspects of Activin-A biology that provide mechanistic explanation for the observed phenotype, link Activin-A to ALI/ARDS pathophysiology, and provide the rationale for therapeutic targeting of Activin-A in these disorders.

Pulmonary fibrosis is a common feature of a large group of lung diseases. The molecular mechanisms underlying pulmonary fibrosis and the key macromolecules involved are not fully understood yet. In an effort to better understand aspects of pulmonary fibrosis, the established bleomycin injection model in mice was used and the focus of the present study was on integrin-linked kinase (ILK) expression. ILK is an intracellular protein involved in the regulation of integrin-mediated processes. In fibrosis, ILK has been examined in the kidney and in the liver where it mediates epithelial to mesenchymal transition (EMT) and hepatic stellate cell activation, respectively. However, information on ILK's involvement in lung fibrosis is missing. In order to examine ILK's role in pulmonary fibrosis, we used both an in vivo and an in vitro approach. In vivo, the bleomycin model was used in order to examine ILK's expression and localization in the fibrotic lung. In vitro, transforming growth factor-β1 was used to induce fibrotic characteristics and EMT in alveolar epithelial cells. ILK's role in alveolar EMT was studied by siRNA. Our results demonstrate that in the animal model used, ILK exhibits a decrease in expression at early stages of the fibrotic process and that a specific subset of fibroblasts is expressing ILK. The in vitro experiments suggested that ILK is not directly involved in E-cadherin downregulation and initiation of EMT (as is the case in renal fibrosis) but is involved in upregulation of vimentin. These results suggest that ILK is involved in lung fibrosis in a tissue-specific manner and raise the possibility to use it as a specific therapeutic target for lung fibrosis in the future.

Using the rat beta-cell RIN-5AH insulinoma line as a means for studying insulin-dependent diabetes mellitus (IDDM), it is shown that interleukin-1 (IL-1) induces beta-cell damage initiated by early apoptotic signals. This action is demonstrated by DNA fragmentation, as assessed by specific BrdU labeling, surface expression of Fas and nitric oxide (NO) production. In addition, the interplay between NO and Fas is shown, while scanning electron microscopy (SEM) confirms apoptosis by revealing the degree and type of cellular damage which, in the case of IL-1alpha, can be reversed by an inhibitor to NO synthesis. Apoptosis is also reconfirmed by transmission electron microscopy (TEM) by observing condensed nuclear chromatin after IL-1 exposure. Thus, treatment of insulinoma cells with IL-1alpha and IL-1beta seems to initiate a number of signals, including PKC activation as published previously, that ultimately lead to beta-cell destruction. Each IL-1 isoform, however, definitely follows a different pathway of action.

The isolation of human antibodies against muscle acetylcholine receptor (AChR), the autoantigen involved in myasthenia gravis (MG), is important for the development of therapeutically useful reagents. Monovalent antibody fragments from monoclonal antibodies against the main immunogenic region (MIR) of AChR protect the receptor from the destructive activity of MG autoantibodies. Human anti-AChR a-subunit antibody fragments with therapeutic potential have been isolated using phage display antibody libraries. An alternative approach for obtaining human mAb has been provided by the development of humanized mice. In this report, we show that immunization of transgenic mouse strains with the extracellular domain of the human AChR a-subunit results in antibody responses and isolation of hybridomas producing human mAb. Four specific IgM mAb were isolated and analyzed. mAb170 recognized the native receptor the best and was capable of inducing AChR antigenic modulation, suggesting its specificity for a pathogenic epitope. Moreover, the recombinant antigen-binding (Fab) fragment of this mAb competed with an anti-MIR mAb, revealing that its antigenic determinant lies in or near the MIR. Finally, Fab170 was able to compete with MG autoantibodies and protect the AChR against antigenic modulation induced by MG sera. This approach will be useful for isolating additional mAb with therapeutic potential against the other AChR subunits.