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Biomedical subjects

E Fattori

Publications and source records attributed to E Fattori.

At least 19 recordsLinked to original sources
PubMed1999

Development of animal models for adeno-associated virus site-specific integration.

The adeno-associated virus (AAV) is unique in its ability to target viral DNA integration to a defined region of human chromosome 19 (AAVS1). Since AAVS1 sequences are not conserved in a rodent's genome, no animal model is currently available to study AAV-mediated site-specific integration. We describe here the generation of transgenic rats and mice that carry the AAVS1 3.5-kb DNA fragment. To test the response of the transgenic animals to Rep-mediated targeting, primary cultures of mouse fibroblasts, rat hepatocytes, and fibroblasts were infected with wild-type wt AAV. PCR amplification of the inverted terminal repeat (ITR)-AAVS1 junction revealed that the AAV genome integrated into the AAVS1 site in fibroblasts and hepatocytes. Integration in rat fibroblasts was also observed upon transfection of a plasmid containing the rep gene under the control of the p5 and p19 promoters and a dicistronic cassette carrying the green fluorescent protein (GFP) and neomycin (neo) resistance gene between the ITRs of AAV. The localization of the GFP-Neo sequence in the AAVS1 region was determined by Southern blot and FISH analysis. Lastly, AAV genomic DNA integration into the AAVS1 site in vivo was assessed by virus injection into the quadriceps muscle of transgenic rats and mice. Rep-mediated targeting to the AAVS1 site was detected in several injected animals. These results indicate that the transgenic lines are proficient for Rep-mediated targeting. These animals should allow further characterization of the molecular aspects of site-specific integration and testing of the efficacy of targeted integration of AAV recombinant vectors designed for human gene therapy.

Animals↗
PubMed1998

Targeted integration of adeno-associated virus-derived plasmids in transfected human cells.

Adeno-associated virus (AAV) integrates its genomic DNA into a defined region of human chromosome 19 (AAVS1). The specificity of integration is dependent on the presence of the inverted terminal repeats (ITR) and on expression of the rep gene. To develop vectors capable of targeting the insertion of a selected DNA sequence into a specific location of human chromosome, we determined whether the rep gene can mediate site-specific integration when cloned outside of an ITR-flanked transgene cassette. HeLa and Huh-7 cells were transfected with a plasmid containing the rep gene, as well as the green fluorescent protein (GFP) and neomycin (neo) resistance gene inserted between the ITRs of AAV. Southern blot analysis of individual clones detected Rep-mediated site-specific integration of the ITR-flanked DNA in 25% and 12% of the HeLa and Huh-7 clones, respectively. The localization of the GFP-Neo sequence on chromosome 19 also was confirmed by fluorescent in situ hybridization analysis of the transfected HeLa clones. Sequence analysis of the ITR-AAVS1 junction of one of the transfected Huh-7 clones indicated that the insertion of the ITR DNA fragment had occurred at nucleotide 1003. These results have implications for the development of AAV-derived vectors capable of directing the site-specific integration of a gene of interest.

Base Sequence↗
PubMed1998

Interleukin 6 is required for the development of collagen-induced arthritis.

Interleukin-6 (IL-6) is overproduced in the joints of patients with rheumatoid arthritis (RA) and, based on its multiple stimulatory effects on cells of the immune system and on vascular endothelia, osteoclasts, and synovial fibroblasts, is believed to participate in the development and clinical manifestations of this disease. In this study we have analysed the effect of ablating cytokine production in two mouse models of arthritis: collagen-induced arthritis (CIA) in DBA/1J mice and the inflammatory polyarthritis of tumor necrosis factor alpha (TNF-alpha) transgenic mice. IL-6 was ablated by intercrossing an IL-6 null mutation into both arthritis-susceptible genetic backgrounds and disease development was monitored by measuring clinical, histological, and biochemical parameters. Two opposite responses were observed; while arthritis in TNF-alpha transgenic mice was not affected by inactivation of the IL-6 gene, DBA/1J, IL-6(-/-) mice were completely protected from CIA, accompanied by a reduced antibody response to type II collagen and the absence of inflammatory cells and tissue damage in knee joints. These results are discussed in the light of the present knowledge of cytokine networks in chronic inflammatory disorders and suggest that IL-6 receptor antagonists might be beneficial for the treatment of RA.

Animals↗
PubMed1998

Impaired Stat3 activation following localized inflammatory stimulus in IL-6-deficient mice.

Interleukin 6 (IL-6) and related gp130-signalling cytokines rapidly activate latent cytoplasmic Stat transcription factors and these are believed to play pivotal roles in the expression of downstream cytokine-responsive genes. We have previously shown in IL-6-deficient (-/-) mice that IL-6 is absolutely required for the transcriptional induction of acute phase response (APR) genes in the liver following localized tissue damage caused by subcutaneous injection of turpentine oil, but is not required when the inflammatory stimulus is administered systemically by intraperitoneal injection of bacterial lipopolysaccharide (LPS). In this paper we show that Stat3 is the only Stat factor induced in liver tissue upon localized inflammatory stimuli, and that its activation is virtually absent in IL-6 deficient mice. During LPS-induced inflammation both Stat1 and Stat3 are activated, and only minor kinetic alterations are detected in IL-6-/- mice. These defects are not due to altered intracellular signal transduction, since they could be complemented by injection of recombinant cytokines. These results establish a direct causal relationship in vivo between Stat activation and acute phase gene expression and define unique functions of IL-6 in Stat3 activation upon localized inflammation.

Acute-Phase Proteins↗
PubMed1997

Soluble IL-6 receptor leads to a paracrine modulation of the IL-6-induced hepatic acute phase response in double transgenic mice.

There is a growing number of soluble agonistic (IL-6, ciliary neurotropic factor, IL-11, and glia-derived neurotropic factor receptors) and antagonistic (IL-1 and TNF receptors) receptor proteins, modulating the biological functions of their cognate ligands. The physiologic role of these receptor molecules in vivo is unclear. In particular, it is not known how the specificity of function of soluble receptors after release into the blood stream is maintained. We addressed this question by studying the function of the soluble IL-6R (sIL-6R) at the cellular level in the liver. We have generated double transgenic mice coexpressing human sIL-6R and human IL-6 in the liver and have analyzed the expression patterns by in situ hybridization. The expression of the human sIL-6R, driven by the phosphoenolpyruvate carboxykinase promoter, is located mainly in periportal areas, whereas human IL-6 under the control of the metallothionein promoter is uniformly expressed throughout the liver. We show here by in situ hybridization that acute phase protein gene expression induced by human IL-6 and human sIL-6R correlated with the periportal expression of sIL-6R, indicating that sIL-6R acts mainly in an area where it is generated. We conclude that in a concentration-dependent manner, at low concentrations of sIL-6R, there is a predominantly paracrine action at the site of its generation, whereas at higher concentrations of the sIL-6R there are both local and systemic effects.

Acute-Phase Proteins↗
PubMed1997

Extramedullary expansion of hematopoietic progenitor cells in interleukin (IL)-6-sIL-6R double transgenic mice.

Soluble cytokine receptors modulate the activity of their cognate ligands. Interleukin (IL)-6 in association with the soluble IL-6 receptor (sIL-6R) can activate cells expressing the gp130 signal transducer lacking the specific IL-6R. To investigate the function of the IL-6-sIL-6R complex in vivo and to discriminate the function of the IL-6-sIL-6R complex from the function of IL-6 alone, we have established a transgenic mouse model. Double-transgenic mice coexpressing IL-6 and sIL-6R were generated and compared with IL-6 and sIL-6R single-transgenic mice. The main phenotype found in IL-6-sIL-6R mice was a dramatic increase of extramedullary hematopoietic progenitor cells in liver and spleen but not in the bone marrow. In IL-6 single-transgenic mice and sIL-6R single-transgenic mice no such effects were observed. The high numbers of hematopoietic progenitor cells were reflected by a strong increase of peripheral blood cell numbers. Therefore, activators of the gp130 signal transducer like the IL-6-IL-6R complex may represent most powerful stimulators for extramedullary hematopoietic progenitor cells. gp130 activators may become important for the expansion of hematopoietic progenitor cells in vivo and in vitro.

Animals↗
PubMed1997

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.

Adolescent↗
PubMed1997

Differential effects of IL-6 on systemic and central production of TNF: a study with IL-6-deficient mice.

Interleukin 6 (IL-6) is known to inhibit the synthesis of tumour necrosis factor (TNF) in vitro and in vivo. In this study we investigated the possible role of IL-6 as an endogenous inhibitor of TNF production in the brain or in the periphery using IL-6-deficient mice or administering recombinant human IL-6 (rhIL-6). When IL-6-deficient mice were injected intracerebroventricularly (i.c.v.) with lipopolysaccaride (LPS), no differences were observed in the production of TNF in the brain, while in the periphery (serum or spleen) TNF levels were markedly increased (about four-fold). When normal mice were injected i.c.v. with a combination of LPS and rhIL-6, inhibition of TNF production was only slight (about 20%), while IL-6 had a stronger effect (> 80% inhibition) in the periphery. Co-administration of soluble IL-6 receptor (sIL-6R) did not enhance the effect of IL-6 on brain TNF, so this refractoriness cannot be attributed to a lack of IL-6 receptors. Interestingly, IL-6 potently inhibited LPS-induced TNF production by macrophagic cells but not by a microglial cell clone, suggesting that the defective response to IL-6 of the brain lies within the responsiveness TNF producing cells to IL-6. It thus appears that the TNF-inhibitory role of IL-6 is confined to the periphery.

Animals↗
PubMed1996

Overexpression of interleukin-6 in the central nervous system of transgenic mice increases central but not systemic proinflammatory cytokine production.

Production of inflammatory cytokines, including tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-6 (IL-6), in the brain is increased in various diseases. To investigate the relationships between the effect of overproduction of IL-6 in the brain on central and peripheral production of TNF, IL-1 beta and IL-6 itself, we used transgenic mice (NSE-hIL-6) where neuronal human IL-6 expression under the control of the neuronal specific enolase promoter results in astrocytosis and gliosis. These mice had higher cerebral endogenous IL-6 (12-fold), IL-1 beta (12-fold) and TNF (4-fold) production measured in brain homogenates after intracerebroventricular (i.c.v.) injection of 2.5 micrograms LPS, lipopolysaccharide (LPS) than wild-type mice (no TNF or IL-1 were detectable in saline-injected NSE or control mice). Cerebral cytokines production was also increased in NSE-hIL-6 mice treated i.p. with LPS doses that do not normally induce cytokines in the brain. The induction of peripheral (serum or spleen) TNF, IL-1 beta or IL-6 was the same in all these experiments in NSE-hIL-6 and wild-type mice. Furthermore, using microglial cell clone pretreated in vitro with IL-6, we noted an increase in LPS-induced TNF and IL-6 production and proliferation of pretreated cells than control. This study indicates that overproduction of IL-6 in the central nervous system (CNS) may ultimately result in increased central production of inflammatory cytokines, probably due to increased proliferation and activation of the cells which produce cytokine in the CNS.

Animals↗
PubMed1995

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.

Animals↗
PubMed1995

Loss of heterozygosity of imprinted genes in SV40 t/T antigen-induced hepatocellular carcinomas.

Expression of the chromosomally linked Insulin-like Growth Factor II (IGF-II) and H19 genes is regulated by parental imprinting during development, since the maternally inherited IGF-II and the paternally inherited H19 alleles are inactive in fetal tissues. Here we show that expression of IGF-II and H19 genes is activated in transgenic mice during SV40 Tag-induced hepatocarcinogenesis and that imprinting of both genes is conserved in the liver tumors. Allelic imbalances of IGF-II and H19 genes and other chromosome 7 markers were detected in one third (13/39) of the hepatocellular carcinomas analysed. A strong bias on the allele retained in the neoplasms was observed, since underrepresentation or complete loss of maternal chromosome 7 was recognised in 12/13 cases. High levels of IGF-II mRNA were expressed by all carcinomas with relative excess of paternal chromosome 7 alleles and suppressed H19 expression was found in the neoplasms lacking the maternal alleles. Overall the results indicate that expression of imprinted genes is involved in progression of experimental liver tumors and suggest that the murine chromosome 7, whose loss may possibly cause the inactivation of a growth-inhibitory gene, is preferentially retained as paternal copy in the liver tumors because of parental imprinting of IGF-II gene.

Alleles↗
PubMed1995

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 Castleman's 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.

Animals↗
PubMed1994

Defective inflammatory response in interleukin 6-deficient mice.

Systemic and localized inflammation elicit a number of host responses which include fever, cachexia, hypoglycemia, and major changes in the concentration of liver plasma proteins. Interleukin 6 (IL-6) is considered an important mediator of the inflammatory response, together with IL-1 and tumor necrosis factor alpha (TNF-alpha). The purpose of this study was to unequivocally determine the role of IL-6 in these phenomena making use of IL-6-deficient mice that we have recently generated by gene targeting. We report here that in the absence of IL-6, mice are unable to mount a normal inflammatory response to localized tissue damage generated by turpentine injection. The induction of acute phase proteins is dramatically reduced, mice do not lose body weight and only suffer from mild anorexia and hypoglycemia. In contrast, when systemic inflammation is elicited through the injection of bacterial lipopolysaccharide (LPS), these parameters are altered to the same extent both in wild-type and IL-6-deficient mice, demonstrating that under these conditions IL-6 function is dispensable. Moreover, we show that LPS-treated IL-6-deficient mice produce three times more TNF-alpha than wild-type controls, suggesting that increased TNF-alpha production might be one of the compensatory mechanisms through which a normal response to LPS is achieved in the absence of IL-6. We also show that corticosterone is normally induced in IL-6-deficient mice, demonstrating that IL-6 is not required for the activation of the hypothalamic-pituitary-adrenal axis. Our results reinforce the idea that different patterns of cytokines are involved in systemic and localized tissue damage, and identify IL-6 as an essential mediator of the inflammatory response to localized inflammation.

Acute-Phase Reaction↗
PubMed1994

Development of progressive kidney damage and myeloma kidney in interleukin-6 transgenic mice.

Interleukin-6 (IL-6) is a pleiotropic cytokine that has been postulated as playing a role in the pathogenesis of multiple myeloma, chronic autoimmune diseases, and alcoholic liver cirrhosis. We generated transgenic mice carrying a fusion between the mouse metallothionein-I (MT-I) gene promoter and the human IL-6 cDNA. MT-I/IL-6 transgenics express IL-6 constitutively in the liver and secrete the cytokine in the blood. They show initially activation of acute-phase response genes and accumulation of alpha 2- and beta-globulins in the plasma, which is followed by polyclonal hypergammaglobulinemia. MT-I/IL-6 transgenics die between 12 to 20 weeks of age. Histologic examination of transgenic animals at different ages and after necropsy showed, as expected from previous studies of IL-6 disregulation in vivo, an increase in the number of megakaryocytes in the spleen and bone marrow and, at later stages, IgG plasmacytosis in the spleen, lymph nodes, and thymus. However, no plasma cell infiltration was detected in other organs. The distinguishing feature of MT-I/IL-6 transgenics is the development of a progressive kidney pathology, in which the initial membranous glomerulonephritis is followed by focal glomerulosclerosis and finally by extensive tubular damage that reproduces the damage observed in patients at terminal stages of multiple myeloma (myeloma kidney). The pathogenetic role of IL-6 overproduction and of the resulting serum protein overload in the kidney damage is discussed.

Acute-Phase Proteins↗
PubMed1994

Interleukin-6 deficient mice are protected from bone loss caused by estrogen depletion.

Interleukin-6 (IL-6) is a multifunctional cytokine whose circulating levels are under physiological conditions below detection, but whose production is rapidly and strongly induced by several pathological and inflammatory stimuli. IL-6 has been implicated in a number of cell functions connected to immunity and hematopoiesis. Recently, it has been proposed to act as a stimulator of osteoclast formation and activity, in particular following estrogen depletion. The purpose of this study was to gain additional insights into the role of IL-6 during development, as well as in physiological and pathological conditions. We report here that IL-6 deficient mice generated by gene targeting are viable and do not present any evident phenotypic abnormality. However, analysis of bone metabolism revealed a specific bone phenotype. IL-6 deficient female mice have a normal amount of trabecular bone, but higher rates of bone turnover than control littermates. Estrogen deficiency induced by ovariectomy causes in wild type animals a significant loss of bone mass together with an increase in bone turnover rates. Strikingly, ovariectomy does not induce any change in either bone mass or bone remodeling rates in the IL-6 deficient mice. These findings indicate that IL-6 plays an important role in the local regulation of bone turnover and, at least in mice, appears to be essential for the bone loss caused by estrogen deficiency.

Animals↗
PubMed1993

Inducible formation of liver tumors in transgenic mice.

Transgenic mice have been generated with an inducible SV40 t/T antigen construct with the aim of analysing the early changes that take place in the course of liver tumorigenesis. The strictly liver-specific human C-reactive protein (CRP) gene promoter was chosen for the control of the transgene expression because this promoter can be turned on transiently by injection of bacterial lipopolysaccharide. Among 10 independently derived CRP-Tag mouse lines five showed inducible expression of the CRP-Tag transgene in liver. However, only one had a tight control of the transgene with virtually no expression under physiological conditions and high levels of Tag expression after stimulation. Females of this line were used to analyse the progression of liver alterations upon repeated induction of the t/T antigen for different lengths of time. The first signs of transgene-induced liver alterations could be monitored by the activation of the marker enzyme gamma-glutamyltranspeptidase 30 days after the start of the induction program. After 90 days hepatocellular carcinomas were already detectable. Thus, CRP-Tag mice constitute an excellent system to analyse the sequential events that take place during liver carcinogenesis.

Animals↗
PubMed1992

Serine hydroxymethyltransferase: origin of substrate specificity.

All forms of serine hydroxymethyltransferase, for which a primary structure is known, have five threonine residues near the active-site lysyl residue (K229) that forms the internal aldimine with pyridoxal phosphate. For Escherichia coli serine hydroxymethyltransferase each of these threonine residues has been changed to an alanine residue. The resulting five mutant enzymes were purified and characterized with respect to kinetic and spectral properties. The mutant enzymes T224A and T227A showed no significant changes in kinetic and spectral properties compared to the wild-type enzyme. The T225A and T230A enzymes exhibited differences in Km and kcat values but exhibited the same spectral properties as the wild-type enzyme. The four threonine residues at positions 224, 225, 227, and 230 do not play a critical role in the mechanism of the enzyme. The T226A enzyme had nearly normal affinity for substrates and coenzymes but had only 3% of the catalytic activity of the wild-type enzyme. The spectrum of the T226A enzyme in the presence of amino acid substrates showed a large absorption maximum at 343 nm with only a small absorption band at 425 nm, unlike the wild-type enzyme whose enzyme-substrate complexes absorb at 425 nm. Rapid reaction studies showed that when amino acid substrates and substrate analogues were added to the T226A enzyme, the internal aldimine absorbing at 422 nm was rapidly converted to a complex absorbing at 343 nm in a second-order process. This was followed by a very slow first-order formation of a complex absorbing at 425 nm.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗