Miriam Corti

Connective tissue growth factor mRNA expression is upregulated in bleomycin-induced lung fibrosis

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.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-beta1 (TGF-beta1) response element. Because TGF-beta1 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-beta1. 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-beta1 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-beta1 because fibroblasts from untreated BALB/c mouse lung upregulated their CTGF message when treated with TGF-beta1 in vitro. These data demonstrate that CTGF is expressed in lung fibroblasts and may play a role in the pathogenesis of lung fibrosis.

Dexamethasone activates expression of the PDGF-α receptor and induces lung fibroblast proliferation

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.

Antibody to Ricin A Chain Hinders Intracellular Routing of Toxin and Protects Cells Even after Toxin Has Been Internalized

Background Mechanisms 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 Findings We 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 Findings We 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.

Hypoxia induces selective SAPK/JNK-2-AP-1 pathway activation in the nucleus tractus solitarii of the conscious rat.

Abstract : In the nucleus tractus solitarii, NMDA glutamate receptors are critical to the hypoxic ventilatory response. However, the signal transduction pathways underlying the hypoxic ventilatory response remain undefined. To assess the effect of a moderate hypoxic stimulus (10% O2) on tyrosine phosphorylation of proteins in the nucleus tractus solitarii, tissue lysates were harvested by repeated punch sampling at 0, 1, 10, and 60 min of hypoxia and examined for the presence of phosphorylated tyrosine residues by immunoblotting. Time‐dependent phosphotyrosine increases occurred in proteins migrating at regions corresponding to molecular masses of 38‐42, 50, 55, and 60 kDa, which were attenuated by pretreatment with the NMDA receptor channel blocker, MK‐801. As extracellular signal‐regulated kinase (Erk) and stress‐activated protein kinase/c‐Jun N‐terminal kinase (SAPK/JNK) phosphorylation may induce Fos and Jun gene transcription and activator protein‐1 (AP‐1) DNA binding, the activation of Erk1, Erk2, p38, and SAPK/JNK was examined in the nucleus tractus solitarii and neocortex during hypoxia and following administration of MK‐801. Hypoxia enhanced Erk1, Erk2, and p38 activity in the cortex, but not in the nucleus tractus solitarii. Increased phosphorylation of SEK1 and SAPK/JNK‐2 occurred in the nucleus tractus solitarii during hypoxia, whereas both SAPK/JNK‐1 and SAPK/JNK‐2 were recruited in cortex. MK‐801 attenuated hypoxia‐induced SEK1, SAPK/JNK‐2, and AP‐1 binding in the nucleus tractus solitarii, and the widespread activation of all MAP kinases in the cortex was also attenuated. We conclude that in conscious rats, a moderate hypoxic stimulus elicits NMDA‐dependent widespread mitogenactivated protein kinase activation in cortex, but selective SAPK/JNK‐2 and AP‐1 activation in the nucleus tractus solitarii, thereby suggesting a functional role for the SAPK/JNK‐2‐AP‐1 pathway.

Inhibition of Protein-Protein Interaction of HER2-EGFR and HER2–HER3 by a Rationally Designed Peptidomimetic

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 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.

Anti-HIV Double Variable Domain Immunoglobulins Binding Both gp41 and gp120 for Targeted Delivery of Immunoconjugates

Background Anti-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. Methods Synthetic 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. Findings The 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. Conclusions DVD-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.

Horizontal transmission of Candida albicans and evidence of a vaccine response in mice colonized with the fungus.

Disseminated candidiasis is the third leading nosocomial blood stream infection in the United States and is often fatal. We previously showed that disseminated candidiasis was preventable in normal mice by immunization with either a glycopeptide or a peptide synthetic vaccine, both of which were Candida albicans cell wall derived. A weakness of these studies is that, unlike humans, mice do not have a C. albicans GI flora and they lack Candida serum antibodies. We examined the influence of C. albicans GI tract colonization and serum antibodies on mouse vaccination responses to the peptide, Fba, derived from fructose bisphosphate aldolase which has cytosolic and cell wall distributions in the fungus. We evaluated the effect of live C. albicans in drinking water and antimicrobial agents on establishment of Candida colonization of the mouse GI tract. Body mass, C. albicans in feces, and fungal-specific serum antibodies were monitored longitudinally. Unexpectedly, C. albicans colonization occurred in mice that received only antibiotics in their drinking water, provided that the mice were housed in the same room as intentionally colonized mice. The fungal strain in unintentionally colonized mice appeared identical to the strain used for intentional GI-tract colonization. This is the first report of horizontal transmission and spontaneous C. albicans colonization in mice. Importantly, many Candida-colonized mice developed serum fungal-specific antibodies. Despite the GI-tract colonization and presence of serum antibodies, the animals made antibodies in response to the Fba immunogen. This mouse model has potential for elucidating C. albicans horizontal transmission and for exploring factors that induce host defense against disseminated candidiasis. Furthermore, a combined protracted GI-tract colonization with Candida and the possibility of serum antibody responses to the presence of the fungus makes this an attractive mouse model for testing the efficacy of vaccines designed to prevent human disseminated candidiasis.

Role of Fc in antibody-mediated protection from ricin toxin.

We have studied the role of the antibody (Ab) Fc region in mediating protection from ricin toxicity. We compared the in vitro and in vivo effects of intact Ig and of Fab fragments derived from two different neutralizing Ab preparations, one monoclonal, the other polyclonal. Consistent results were obtained from each, showing little difference between Ig and Fab in terms of antigen binding and in vitro neutralization, but with relatively large differences in protection of animals. We also studied whether importing Ab into the cell by Fc receptors enhanced the intracellular neutralization of ricin toxin. We found that the imported Ab was found in the ER and Golgi, a compartment traversed by ricin, as it traffics through the cell, but intracellular Ab did not contribute to the neutralization of ricin. These results indicate that the Fc region of antibody is important for in vivo protection, although the mechanism of enhanced protection by intact Ig does not appear to operate at the single cell level. When using xenogeneic antibodies, the diminished immunogenicity of Fab/F(ab’)2 preparations should be balanced against possible loss of protective efficacy.

Influences of gender, development, pregnancy and ethanol consumption on the hematotoxicity of inhaled 10 ppm benzene

The hematotoxic effects of benzene in both humans and animals are well documented. Current estimates concerning the risks associated with benzene exposure are usually based on adult, male cohort studies; however, there are indications that females may respond differently than males to benzene and that fetuses may respond differently than adults. Another factor to be considered in risk estimates is the impact of personal habits. In experimental animals, ethanol consumption is known to increase the hematotoxicity of benzene; therefore, alcohol consumption may also alter the potential risk of individuals exposed to benzene. To address some of the factors that may confound risk estimates for benzene exposure, a series of experiments were performed. Age-matched male as well as pregnant and virgin female Swiss Webster mice were exposed to 10 ppm benzene for 6 h a day over 10 consecutive days (days 6 through 15 of gestation for the pregnant females). Half of the animals also received 5% ethanol in the drinking water during this period. On day 11, bone marrow cells from the adults and liver cells from the fetuses were assayed for the numbers of erythroid colony-forming units (CFU-e). CFU-e assays were also performed on bone marrow cells isolated from 6-week postpartum dams exposed during gestation and from in utero-exposed 6-week old males and females. Gender differences were clearly observed in the responses to the various exposure protocols. Depressions in CFU-e numbers were only seen in male mice while elevations in CFU-e numbers were only seen in female mice. Male mice exposed as adults for 10 days to benzene (B), ethanol (E) or benzene+ ethanol (B+E) exhibited depressed CFU-e levels as did male fetal mice exposed to B in utero. In addition, adult male mice which had been exposed in utero to either B or to E individually displayed depressed CFU-e levels. In contrast, none of the groups of female mice exhibited any depressions in CFU-e numbers after any of the exposures. Elevations in CFU-e numbers were observed among pregnant females exposed to E and among adult females exposed to B+E in utero. In summary, a majority (6/9) of the exposure protocols produced depressions in the CFU-e numbers of male mice, whereas a majority (7/9) of the exposure protocols produced no changes in the CFU-e numbers of female mice. Those changes that were observed in females consisted of elevations of CFU-e numbers. These results suggest that the male erythron is more susceptible than the female erythron to the hematotoxicants benzene and ethanol, regardless of whether exposures occur in utero or during adulthood.

Abstract 1227: Modulation of EGF-receptors signaling by small peptidomimetic molecules in breast cancer cells

Protein-protein interactions play a crucial role in regulating many biological processes as well as in disease pathology. Understanding the structural basis and modulating protein-protein interactions has a tremendous impact in developing new therapeutic strategies for many human diseases. Human epidermal growth factor receptor family of proteins (EGFR, HER2, 3 and 4) plays an important role in cell growth and proliferation. Aberrant interactions of EGFR family of proteins lead to tumor growth and metastasis. HER2, a member of EGFR family of proteins is overexpressed in approximately 25% of breast cancers. Deregulation of HER2 signaling pathways and overexpression of HER2 is known to occur in many cancers. HER2 is known to form heterodimers and is a major therapeutic target in breast cancer treatment. Formation of HER2 receptor heterodimerization with other EGFR family of proteins such as EGFR, HER3 is essential for intracellular kinase activation and is the basis for exploring direct inhibition of EGFR activation by blocking dimerization for therapeutic purposes. We have designed several small peptidomimetics to inhibit HER2-mediated signaling for cell growth. One of such peptidomimetics, Compound 5 (Arg-[3-amino-naphthyl-propionic acid]-Phe) designed, exhibited antiproliferative activity with IC50 values in the nM range against HER2 overexpressing breast cancer cell lines, SKBR-3 and BT-474. Binding studies using Florescence Microscopy, ELISA, Circular Dichroism (CD) and Surface Plasmon Resonance (SPR) confirmed that compound 5 binds specifically to HER2-extracellular domain. SPR studies revealed that compound 5 specifically binds to domain IV of HER2 extracellular region. Also, Compound 5 inhibited HER2-HER3 as well as HER2-EGFR heterodimerization evaluated by SPR. Pathhunter and proximity ligation assays confirmed the inhibition of HER2 heterodimerization with EGFR and HER3 by compound 5. Western blots revealed that compound 5 blocks the transphosphorylation of HER2 protein. Based on these observations small peptidomimetic inhibitor molecules can be potential therapeutic agents in disabling HER2 mediated heterodimerization. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1227. doi:1538-7445.AM2012-1227