Domenico Lazzaro

Interleukin 6 causes growth impairment in transgenic mice through a decrease in insulin-like growth factor-I. A model for stunted growth in children with chronic inflammation.

Stunted growth is a major complication of chronic inflammation and recurrent infections in children. Systemic juvenile rheumatoid arthritis is a chronic inflammatory disorder characterized by markedly elevated circulating levels of IL-6 and stunted growth. In this study we found that NSE/hIL-6 transgenic mouse lines expressing high levels of circulating IL-6 since early after birth presented a reduced growth rate that led to mice 50-70% the size of nontransgenic littermates. Administration of a monoclonal antibody to the murine IL-6 receptor partially reverted the growth defect. In NSE/hIL-6 transgenic mice, circulating IGF-I levels were significantly lower than those of nontransgenic littermates; on the contrary, the distribution of growth hormone pituitary cells, as well as circulating growth hormone levels, were normal. Treatment of nontransgenic mice of the same strain with IL-6 resulted in a significant decrease in IGF-I levels. Moreover, in patients with systemic juvenile rheumatoid arthritis, circulating IL-6 levels were negatively correlated with IGF-I levels. Our findings suggest that IL-6-mediated decrease in IGF-I production represents a major mechanism by which chronic inflammation affects growth.

Lymphoproliferative disorder and imbalanced T-helper response in C/EBP beta-deficient mice.

C/EBP beta is considered a key element of interleukin‐6 (IL‐6) signalling as well as an important transcriptional regulator of the IL‐6 gene itself. We describe here how mice lacking C/EBP beta develop a pathology similar to mice overexpressing IL‐6 and nearly identical to multicentric Castlemans disease in human patients, with marked splenomegaly, peripheral lymphadenopathy and enhanced haemopoiesis. Humoral, innate and cellular immunity are also profoundly distorted, as shown by the defective activation of splenic macrophages, the strong impairement of IL‐12 production, the increased susceptibility to Candida albicans infection and the altered T‐helper function. Our data show that C/EBP beta is crucial for the correct functional regulation and homeostatic control of haemopoietic and lymphoid compartments.

IL-6 Expression in Neurons of Transgenic Mice Causes Reactive Astrocytosis and Increase in Ramified Microglial Cells but no Neuronal Damage

Growing evidence suggests that aberrant production of inflammatory cytokines within the central nervous system (CNS) contributes to the development of pathological conditions. To test the cause—effect relationship between the overproduction of interleukin‐6 (IL‐6) in the CNS and the onset of neuropathological changes, we have generated transgenic mice in which human IL‐6 expression has been targeted to the neurons by using the rat neuron‐specific enolase promoter. These mice develop reactive astrocytosis and an increase in ramified microglial cells but do not show histological or behavioural signs of neuron damage at the light microscope level. We thus conclude that a constant release of human IL‐6 by neuronal subpopulations in mice is sufficient to activate cells potentially capable of modulating the local immune response, but at the same time is compatible with normal neuron functions.

Coexpression of IL-6 and soluble IL-6R causes nodular regenerative hyperplasia and adenomas of the liver

Studies with tumor necrosis factor p55 receptor‐ and interleukin‐6 (IL‐6)‐deficient mice have shown that IL‐6 is required for hepatocyte proliferation and reconstitution of the liver mass after partial hepatectomy. The biological activities of IL‐6 are potentiated when this cytokine binds soluble forms of its specific receptor subunit (sIL‐6R) and the resulting complex interacts with the transmembrane signaling chain gp130. We show here that double transgenic mice expressing high levels of both human IL‐6 and sIL‐6R under the control of liver‐specific promoters spontaneously develop nodules of hepatocellular hyperplasia around periportal spaces and present signs of sustained hepatocyte proliferation. The resulting picture is identical to that of human nodular regenerative hyperplasia, a condition frequently associated with immunological and myeloproliferative disorders. In high expressors, hyperplastic lesions progress with time into discrete liver adenomas. These data strongly suggest that the IL‐6/sIL‐6R complex is both a primary stimulus to hepatocyte proliferation and a pathogenic factor of hepatocellular transformation.

Helper-dependent adenovirus for the gene therapy of proliferative retinopathies: stable gene transfer, regulated gene expression and therapeutic efficacy

Ocular neovascular disorders, such as diabetic retinopathy and age‐related macular degeneration, are the principal causes of blindness in developed countries. Current treatments are of limited efficacy, whereas a therapy based on intraocular gene transfer of angiostatic factors represents a promising alternative. For the first time we have explored the potential of helper‐dependent adenovirus (HD‐Ad), the last generation of Ad vectors, in the therapy of retinal neovascularization.

Preventive Vaccination with Telomerase Controls Tumor Growth in Genetically Engineered and Carcinogen-Induced Mouse Models of Cancer

The telomerase reverse transcriptase, TERT, is an attractive target for human cancer vaccination because its expression is reactivated in a conspicuous fraction of human tumors. Genetic vaccination with murine telomerase (mTERT) could break immune tolerance in different mouse strains and resulted in the induction of both CD4+ and CD8+ telomerase-specific T cells. The mTERT-derived immunodominant epitopes recognized by CD8+ T cells were further defined in these mouse strains and used to track immune responses. Antitumor efficacy of telomerase-based vaccination was investigated in two cancer models closely resembling human diseases: the TRAMP transgenic mice for prostate cancer and a carcinogen-induced model for colon cancer. TERT overexpression in tumor lesions was shown in both models by immunohistochemistry, thus reinforcing the similarity of these tumors to their human counterparts. Repeated immunizations with mTERT-encoding DNA resulted in a significant delay of tumor formation and progression in both the prostate cancer and the colon cancer models. Moreover, evaluation of the intratumoral infiltrate revealed the presence of telomerase-specific T cells in vaccinated mice. The safety of vaccination was confirmed by the absence of histomorphologic changes on postnecropsy analysis of several organs and lack of adverse effects on blood cell counts. These results indicate that TERT vaccination can elicit antigen-specific immunosurveillance and imply this antigen as a potential candidate for preventive cancer vaccines.

Generation of infectious and transmissible virions from a GB virus B full-length consensus clone in tamarins

The strong similarity between GB virus B (GBV-B) and hepatitis C virus (HCV) makes tamarins infected by GBV-B an acceptable surrogate animal model for HCV infection. Even more attractive, for drug discovery purposes, is the idea of constructing chimeric viruses by inserting HCV genes of interest into a GBV-B genome frame. To accomplish this, infectious cDNA clones of both viruses must be available. The characterization of several HCV molecular clones capable of infecting chimpanzees has been published, whereas only one infectious GBV-B clone inducing hepatitis in tamarins has been reported so far. Here we describe the infection of tamarins by intrahepatic injection of RNA transcribed from a genomic GBV-B clone (FL-3) and transmission of the disease from infected to naive tamarins via serum inoculation. The disease resulting from both direct and secondary infection was characterized for viral RNA titre and hepatitis parameters as well as for viral RNA distribution in the hepatic tissue. Host humoral immune response to GBV-B antigens was also monitored. The progression of the disease was compared to that induced by intravenous injection of different amounts of the non-recombinant virus.

Revealing New Mouse Epicardial Cell Markers through Transcriptomics

Background The epicardium has key functions during myocardial development, by contributing to the formation of coronary endothelial and smooth muscle cells, cardiac fibroblasts, and potentially cardiomyocytes. The epicardium plays a morphogenetic role by emitting signals to promote and maintain cardiomyocyte proliferation. In a regenerative context, the adult epicardium might comprise a progenitor cell population that can be induced to contribute to cardiac repair. Although some genes involved in epicardial function have been identified, a detailed molecular profile of epicardial gene expression has not been available. Methodology Using laser capture microscopy, we isolated the epicardial layer from the adult murine heart before or after cardiac infarction in wildtype mice and mice expressing a transgenic IGF-1 propeptide (mIGF-1) that enhances cardiac repair, and analyzed the transcription profile using DNA microarrays. Principal Findings Expression of epithelial genes such as basonuclin, dermokine, and glycoprotein M6A are highly enriched in the epicardial layer, which maintains expression of selected embryonic genes involved in epicardial development in mIGF-1 transgenic hearts. After myocardial infarct, a subset of differentially expressed genes are down-regulated in the epicardium representing an epicardium-specific signature that responds to injury. Conclusion This study presents the description of the murine epicardial transcriptome obtained from snap frozen tissues, providing essential information for further analysis of this important cardiac cell layer.

Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer

The EphA2 receptor tyrosine kinase is overexpressed in a variety of human epithelial cancers and is a determinant of malignant cellular behavior in pancreatic adenocarcinoma cells. Moreover, it is expressed in tumor endothelium and its activation promotes angiogenesis. To better clarify the therapeutic potential of monoclonal antibodies (mAbs) directed to the EphA2 receptor, we generated a large number of mAbs by differential screening of phage-Ab libraries by oligonucleotide microarray technology and implemented a strategy for the rapid identification of antibodies with the desired properties. We selected two high-affinity and highly specific EphA2 monoclonal antibodies with different in vitro properties on the human pancreatic tumor cell line MiaPaCa2. One is a potent EphA2-agonistic antibody, IgG25, that promotes receptor endocytosis and subsequent degradation, and the second is a ligand antagonist, IgG28, that blocks the binding to ephrin A1 and is cross-reactive with the mouse EphA2 receptor. We measured the effect of antibody treatment on the growth of MiaPaCa2 cells orthotopically transplanted in nude mice. Both IgG25 and IgG28 had strong antitumor and antimetastatic efficacy. In vivo treatment with IgG25 determined the reduction of the EphA2 protein levels in the tumor and the phosphorylation of FAK on Tyr576 while administration of IgG28 caused a decrease in tumor vascularization as measured by immunohistochemical analysis of CD31 in tumor sections. These data show that in a pancreatic cancer model comparable therapeutic efficacy is obtained either by promoting receptor degradation or by blocking receptor activation.

A therapeutic cancer vaccine targeting carcinoembryonic antigen in intestinal carcinomas.

A genetic vaccine platform based on DNA electroporation (DNA-EP) and adenovirus (Ad) was used to generate immune response against human carcinoembryonic antigen (CEA) and antitumor effects in murine models with spontaneous tumors arising in an orthotopic location. CEA transgenic (CEA.Tg) mice treated with the carcinogen 1,2-dimethylhydrazine developed CEA-overexpressing tumors that resembled human sporadic colorectal cancer. APC1638N/CEA hybrid mice, generated by crossing mice carrying the adenomatous polyposis coli (Apc1638N) gene mutation with CEA.Tg mice, are representative of human familial polyposis and develop polyps that overexpress the antigen. In both models, the DNA-EP/Ad vaccine succeeded in breaking immune tolerance and achieved significant antitumor effects in therapeutic settings. Our data suggest that genetic vaccines targeting CEA may be feasible strategies against gut tumors that overexpress the antigen. In addition, these models are powerful systems for evaluating antigen-specific tumor immunity and assessing therapeutic vaccine strategies for human colorectal cancer.