Previous attempts to culture mouse alveolar type II (ATII) cells have been hampered by limited purity and cell recovery. We have now obtained culturable ATII cells from female C57BL/6 mice at a purity of 92% +/- 3 (mean +/- SD; n = 20), with viabilities of 96% +/- 2 and total yields of 5.1 +/- 0.7 X 10(6) cells per mouse. Crude lung cell suspensions were prepared by intratracheal instillation of Dispase and agarose followed by mechanical disaggregation of the lungs. Crude cell suspensions were purified by negative selection using a biotinylated-antibody, streptavidin-coated biomagnetic particle system. Cell purities were determined by Pap staining and confirmed ultrastructurally. Purified ATII cells were cultured on fibronectin-coated chamber slides and maintained for up to 5 days in DMEM with 10% fetal bovine serum. Cultures exhibited minimal contamination by Clara cells, mesenchymal cells, or endothelial cells, and the epithelial nature of the cultures was confirmed by positive cytokeratin staining in at least 97% of the cells through day 5. Day 3 cultures demonstrated osmium tetroxide/tannic acid-stained granules consistent with lamellar bodies in 76% +/- 3.6 of the cells. The cultures displayed features distinct from those previously described for adult rat ATII cells, including irregularly-shaped cells and the formation of numerous cytoplasmic projections in direct contact with other cells. These studies indicate that excellent yields of highly purified, culturable ATII cells can be obtained from genetically defined mice. These techniques may provide powerful new models for the study of parenchymal lung disease in vitro.

Connective tissue growth factor (CTGF) is a newly described 38-kDa peptide mitogen for fibroblasts and a promoter of connective tissue deposition in the skin. The CTGF gene promotor contains a transforming growth factor-β1 (TGF-β1) response element. Because TGF-β1 expression is upregulated in several models of fibroproliferative lung disease, we asked whether CTGF is also upregulated in a murine lung fibrosis model and whether CTGF could mediate some of the fibrogenic effects associated with TGF-β1. A portion of the rat CTGF gene was cloned and used to show that primary isolates of both murine and human lung fibroblasts express CTGF mRNA in vitro. There was a greater than twofold increase in CTGF expression in both human and murine lung fibroblasts 2, 4, and 24 h after the addition of TGF-β1 in vitro. A bleomycin-sensitive mouse strain (C57BL/6) and a bleomycin-resistant mouse strain (BALB/c) were given bleomycin, a known lung fibrogenic agent. CTGF mRNA expression was upregulated in the sensitive, but not in the resistant, mouse strain after administration of bleomycin. In vivo differences in the CTGF expression between the two mouse strains were not due to an inherent inability of BALB/c lung fibroblasts to respond to TGF-β1 because fibroblasts from untreated BALB/c mouse lung upregulated their CTGF message when treated with TGF-β1 in vitro. These data demonstrate that CTGF is expressed in lung fibroblasts and may play a role in the pathogenesis of lung fibrosis.

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BackgroundMechanisms of antibody-mediated neutralization are of much interest. For plant and bacterial A-B toxins, A chain mediates toxicity and B chain binds target cells. It is generally accepted and taught that antibody (Ab) neutralizes by preventing toxin binding to cells. Yet for some toxins, ricin included, anti-A chain Abs afford greater protection than anti-B. The mechanism(s) whereby Abs to the A chain neutralize toxins are not understood.Methodology/Principal FindingsWe use quantitative confocal imaging, neutralization assays, and other techniques to study how anti-A chain Abs function to protect cells. Without Ab, ricin enters cells and penetrates to the endoplasmic reticulum within 15 min. Within 45–60 min, ricin entering and being expelled from cells reaches equilibrium. These results are consistent with previous observations, and support the validity of our novel methodology. The addition of neutralizing Ab causes ricin accumulation at the cell surface, delays internalization, and postpones retrograde transport of ricin. Ab binds ricin for >6hr as they traffic together through the cell. Ab protects cells even when administered hours after exposure.Conclusions/Key FindingsWe demonstrate the dynamic nature of the interaction between the host cell and toxin, and how Ab can alter the balance in favor of the cell. Ab blocks ricin’s entry into cells, hinders its intracellular routing, and can protect even after ricin is present in the target organelle, providing evidence that the major site of neutralization is intracellular. These data add toxins to the list of pathogenic agents that can be neutralized intracellularly and explain the in vivo efficacy of delayed administration of anti-toxin Abs. The results encourage the use of post-exposure passive Ab therapy, and show the importance of the A chain as a target of Abs.

BackgroundAnti-HIV immunoconjugates targeted to the HIV envelope protein may be used to eradicate the latent reservoir of HIV infection using activate-and-purge protocols. Previous studies have identified the two target epitopes most effective for the delivery of cytotoxic immunoconjugates the CD4-binding site of gp120, and the hairpin loop of gp41. Here we construct and test tetravalent double variable domain immunoglobulin molecules (DVD-Igs) that bind to both epitopes.MethodsSynthetic genes that encode DVD-Igs utilizing V-domains derived from human anti-gp120 and anti-gp41 Abs were designed and expressed in 293F cells. A series of constructs tested different inter-V-linker domains and orientations of the two V domains. Antibodies were tested for binding to recombinant Ag and native Env expressed on infected cells, for neutralization of infectious HIV, and for their ability to deliver cytotoxic immunoconjugates to infected cells.FindingsThe outer V-domain was the major determinant of binding and functional activity of the DVD-Ig. Function of the inner V-domain and bifunctional binding required at least 15 AA in the inter-V-domain linker. A molecular model showing the spatial orientation of the two epitopes is consistent with this observation. Linkers that incorporated helical domains (A[EAAAK]nA) resulted in more effective DVD-Igs than those based solely on flexible domains ([GGGGS]n). In general, the DVD-Igs outperformed the less effective parental antibody and equaled the activity of the more effective. The ability of the DVD-Igs to deliver cytotoxic immunoconjugates in the absence of soluble CD4 was improved over that of either parent.ConclusionsDVD-Igs can be designed that bind to both gp120 and gp41 on the HIV envelope. DVD-Igs are effective in delivering cytotoxic immunoconjugates. The optimal design of these DVD-Igs, in which both domains are fully functional, has not yet been achieved.

Protein-protein interactions (PPI) play a crucial role in many biological processes, and modulation of PPI using small molecules to target hot spots has therapeutic value. As a model system we will use PPI of human epidermal growth factor receptors (EGFRs). Among the four EGFRs, EGFR-HER2 and HER2–HER3 are well known in cancer. We have designed a small molecule that is targeted to modulate HER2-mediated signaling. Our approach is novel because the small molecule designed disrupts dimerization not only of EGFR-HER2 but also of HER2–HER3. In the present study we have shown, using surface plasmon resonance (SPR) analysis, that a peptidomimetic, compound 5, binds specifically to HER2 protein extracellular domain and disrupts the dimerization of EGFRs. To evaluate the effect of compound 5 on HER2 signaling in vitro, Western blot and PathHunter assays were used. Results indicated that compound 5 inhibits the phosphorylation of HER2 kinase domain and inhibits the heterodimerization in a dose-dependent manner. Molecular modeling methods were used to model the PPI of HER2–HER3 heterodimer.

Corticosteroids (CSs) are commonly used for anti-inflammatory therapy in asthma and in interstitial lung diseases. In attempting to understand the mechanisms through which CSs control cell proliferation, we have carried out experiments to test the effects of dexamethasone (Dex) on the growth of lung fibroblasts. Using mouse 3T3 fibroblasts as well as early-passage rat lung fibroblasts (RLFs), we show that the quiescent cells in 1% serum or in serum-free media proliferate significantly in response to the addition of 10−7 to 10−9 M Dex. Increases as high as fourfold in cell numbers were recorded for the RLFs after 48 h in culture. A polyclonal antibody to the AB isoform of human platelet-derived growth factor (PDGF) blocked the proliferative response. As expected, the fibroblasts produced primarily PDGF-A chain, and the RLFs exhibited few PDGF-α receptors (PDGF-Rα), the receptor type necessary for binding the AA isoform. Accordingly, we determined that Dex upregulated PDGF-Rα mRNA and protein. Therefore, we can postulate that Dex-induced fibroblast proliferation is mediated, at least in part, by PDGF-AA, which binds to the PDGF-Rα.

Essential parts of epitopes have been identified on rubella virion envelope glycoprotein E1, by scanning with overlapping octapeptides situated between amino acids 243-286 in a previously determined antigenic domain.