Maintaining or breaking CD8+ T-cell tolerance to beta islet cell antigens: lessons from transgenic mouse models.
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Analysis of the mouse CSF-1 gene promoter in a transgenic mouse model.
CSF-1 stimulates monocyte and osteoclast populations. However, the molecular mechanisms involved in regulating CSF-1 gene expression are unclear. To identify regulatory regions that control normal CSF-1 gene expression, a -774/+183-bp fragment of the murine CSF-1 promoter was analyzed in vitro and in vivo. Transcriptional activity was high in cultured osteoblasts that express CSF-1 mRNA compared to ARH-77 B cells that lack CSF-1 gene expression. Transient transfection of osteoblasts with promoter deletion constructs showed that the -774-bp fragment conferred the highest transcriptional activity and contained activator and repressor sequences. To assess the ability of the CSF-1 promoter to confer normal tissue expression of CSF-1, transgenic mice containing the -774/+183-bp region driving the E. coli beta-galactosidase (lacZ) reporter gene were generated. beta-Gal analysis of whole tissue extracts showed transgene expression in all tissues tested except liver and kidney. At the cellular level, the pattern of beta-gal expression in the spleen, thymus, bone, lung, and testes of adult transgenic mice mimicked normal endogenous CSF-1 mRNA expression in non-transgenic littermates detected by in situ hybridization. This region also directed appropriate transgene expression to sites in other tissues known to synthesize CSF-1, with the exception of the liver and kidney. These findings indicate that the -774-bp fragment contains cis-acting elements sufficient to direct CSF-1 gene expression in many tissues. CSF-1 promoter/lacZ mice may be useful for studying the transcriptional mechanisms involved in regulating CSF-1 gene expression in tissues throughout development.
Animals↗
Transgenic mouse models for prostate cancer. Identification of an androgen-dependent promoter and creation and characterization of the long probasin promoter-Large T antigen (LPB-Tag) model.
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Androgen-Binding Protein↗
P2X7 mediates superoxide production in primary microglia and is up-regulated in a transgenic mouse model of Alzheimer's disease.
Primary rat microglia stimulated with either ATP or 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) release copious amounts of superoxide (O(2)(-)*). ATP and BzATP stimulate O(2)(-)* production through purinergic receptors, primarily the P2X(7) receptor. O(2)(-)* is produced through the activation of the NADPH oxidase. Although both p42/44 MAPK and p38 MAPK were activated rapidly in cells stimulated with BzATP, only pharmacological inhibition of p38 MAPK attenuated O(2)(-)* production. Furthermore, an inhibitor of phosphatidylinositol 3-kinase attenuated O(2)(-)* production to a greater extent than an inhibitor of p38 MAPK. Both ATP and BzATP stimulated microglia-induced cortical cell death indicating this pathway may contribute to neurodegeneration. Consistent with this hypothesis, P2X(7) receptor was specifically up-regulated around beta-amyloid plaques in a mouse model of Alzheimer's disease (Tg2576).
Adenosine Triphosphate↗
Activation of programmed cell death markers in ventral horn motor neurons during early presymptomatic stages of amyotrophic lateral sclerosis in a transgenic mouse model.
The identification of the pathogenic mechanism of selective motor neuron (MN) death in amyotrophic lateral sclerosis (ALS) may lead to the development of new therapies to halt or slow the disease course. A novel, MN-specific, Fas-mediated programmed cell death (PCD) pathway has been reported in MNs which involves the activation of p38 MAP kinase (phospho-p38) and neuronal nitric oxide synthase (nNOS). PCD was found to be exacerbated in MNs expressing ALS-linked superoxide dismutase (SOD) mutations. Because this MN-specific pathway was investigated in vitro, we performed an in vivo study to evaluate its potential involvement in MN loss in the lumbar region of spinal cord of mutant SOD transgenic (G93A) mice. Compared to nontransgenic littermates, we found significant increases in the numbers of immunopositive ventral horn MNs of G93A mice as young as 60 days of age for several constituents of this putative PCD pathway, including phospho-p38, nNOS, phospho-ASK1 MAP kinase kinase, and active caspase-3. This study provides in vivo evidence of an MN-specific PCD pathway that may be a pathogenic mechanism of ALS and may be activated very early in the disease process, well before clinical symptoms are evident (200 days). These findings suggest that early diagnosis and therapeutic intervention may be critical for the successful treatment of the disease. These enzymes may provide new markers for earlier diagnosis of ALS and new molecular targets for therapeutic intervention.
Age Factors↗
Transgenic mouse model of intestine-specific mucosal injury and repair.
Most studies of injury and repair to mucosal tissue have used nonspecific mediators to induce injury. Damage to the mucosal epithelium resulting from chemical or radiation treatment associated with cancer therapy may fall into this category of injury. When such treatments are applied, it is generally not possible to predict or control the extent of possible injury. This fact makes analysis of inductive and reparative processes difficult. In addition, the role of the immune system in the etiology and subsequent healing of mucosal tissue following cancer therapy with or without bone marrow transplantation remains unclear. To study tissue- and antigen-specific immune damage of intestinal mucosal tissue, we generated transgenic mice that express a nominal antigen exclusively in intestinal epithelial cells. The transfer of antigen-specific CD8 T cells with concomitant virus infection resulted in the destruction of intestinal epithelial cells and disease. The destructive phase in some cases was followed by complete recovery and tolerance induction. This model will provide a system that can be regulated for analysis of the mediators of mucosa-specific tissue damage and repair.
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Identification of memory B cells using a novel transgenic mouse model.
Memory B cells help to protect the host from invading pathogens by maintaining persistent levels of Ag-specific serum Ab and generating rapid Ab responses upon re-exposure to Ag. Unambiguous identification of memory B cells has been a major obstacle to furthering our knowledge concerning both the development of B cell memory and secondary Ab responses due to an absence of specific cell surface markers. Germinal centers (GCs) are thought to be the major site of Ig hypermutation and Ag-driven selection of memory B cells. To develop a model that would identify GC-derived memory B cells, we generated transgenic mice that expressed cre recombinase in a GC-specific fashion. Interbreeding these mice with the cre-reporter strain, ROSA26R, produced progeny in which beta-galactosidase (beta-gal) was permanently expressed in B cells of the GC-memory pathway. Analysis following immunization with (4-hydroxy-3-nitrophenyl)acetyl coupled to chicken gamma globulin showed that long-lived beta-gal+ B cells exclusively contained somatically mutated lambda1 V regions and were capable of producing Ag-specific Ab-forming cell (AFC) responses that were >100-fold higher than those afforded by beta-gal- B cells following adoptive transfer to naive hosts. Secondary challenge of immune mice showed that only approximately 20% of secondary AFCs expressed beta-gal. Interestingly, we found that somatic hypermutation of rearranged lambda1 V regions within secondary AFCs showed a strong correlation with beta-gal expression, suggesting that nonmutated B cells contribute significantly to secondary Ab responses. This model should provide useful insights into memory B cell development, maintenance, and differentiation following immunization or pathogenic infection.
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Defective tumor necrosis factor-alpha-dependent control of astrocyte glutamate release in a transgenic mouse model of Alzheimer disease.
The cytokine tumor necrosis factor-alpha (TNFalpha) induces Ca2+-dependent glutamate release from astrocytes via the downstream action of prostaglandin (PG) E2. By this process, astrocytes may participate in intercellular communication and neuromodulation. Acute inflammation in vitro, induced by adding reactive microglia to astrocyte cultures, enhances TNFalpha production and amplifies glutamate release, switching the pathway into a neurodamaging cascade (Bezzi, P., Domercq, M., Brambilla, L., Galli, R., Schols, D., De Clercq, E., Vescovi, A., Bagetta, G., Kollias, G., Meldolesi, J., and Volterra, A. (2001) Nat. Neurosci. 4, 702-710). Because glial inflammation is a component of Alzheimer disease (AD) and TNFalpha is overexpressed in AD brains, we investigated possible alterations of the cytokine-dependent pathway in PDAPP mice, a transgenic model of AD. Glutamate release was measured in acute hippocampal and cerebellar slices from mice at early (4-month-old) and late (12-month-old) disease stages in comparison with age-matched controls. Surprisingly, TNFalpha-evoked glutamate release, normal in 4-month-old PDAPP mice, was dramatically reduced in the hippocampus of 12-month-old animals. This defect correlated with the presence of numerous beta-amyloid deposits and hypertrophic astrocytes. In contrast, release was normal in cerebellum, a region devoid of beta-amyloid deposition and astrocytosis. The Ca2+-dependent process by which TNFalpha evokes glutamate release in acute slices is distinct from synaptic release and displays properties identical to those observed in cultured astrocytes, notably PG dependence. However, prostaglandin E2 induced normal glutamate release responses in 12-month-old PDAPP mice, suggesting that the pathology-associated defect involves the TNFalpha-dependent control of secretion rather than the secretory process itself. Reduced expression of DENN/MADD, a mediator of TNFalpha-PG coupling, might account for the defect. Alteration of this neuromodulatory astrocytic pathway is described here for the first time in relation to Alzheimer disease.
Alzheimer Disease↗
Definition of the human N-myc promoter region during development in a transgenic mouse model.
The N-myc oncogene directs organogenesis, and gene amplification is associated with aggressive forms of neuroblastoma, a common malignant tumor in children. N-myc is expressed in fetal epithelium, and expression decreases markedly postnatally. To localize sequences responsible for directing expression, we have analyzed the human N-myc promoter. We noted previously that N-myc promoter regions 5' to exon 1 directed reporter gene expression in all cell lines, including those without detectable N-myc transcripts. However, when promoter constructs included 3' exon 1 and the 5' portion of intron 1, reporter activity was detected only when there was expression of the endogenous gene. To determine the role of this "tissue-specific region" in directing expression during development, we generated transgenic mice carrying N-myc promoter lacZ minigenes that contained 5' N-myc promoter elements alone or the promoter linked to the 3' exon 1/5' intron 1 tissue-specific region. Animals lacking the tissue-specific exon 1/intron 1 region showed beta-galactosidase expression in the CNS, but expression was not observed in other organs in which endogenously derived N-myc transcripts were seen. Within the CNS, transgene expression was seen mainly in the olfactory system and was not observed in other areas in which expression of the murine gene has been noted. In contrast, no transgene expression was observed in any of the animals carrying the tissue-specific exon 1/intron 1 region. Thus, sequences that direct expression within the olfactory system were contained within our 5' promoter transgene, whereas sequences that guide the ubiquitous expression of N-myc during organogenesis lie outside the regions studied here. Finally, the exon 1/intron 1 region seems to act in a dominant fashion to repress expression in the CNS from the immediate 5' N-myc promoter.
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Inhibition of carcinogenesis in transgenic mouse models over-expressing 15-lipoxygenase in the vascular wall under the control of murine preproendothelin-1 promoter.
Oxygenases are a family of enzymes that dioxygenate unsaturated fatty acids, thus initiating membrane oxidation and signaling molecule synthesis. The lipoxygenases (LOs), a family of lipid-peroxidizing enzymes that induce structural and metabolic changes in the cell in a number of pathophysiological conditions, belong to the oxygenases family. This class of enzymes has several subgroups, named 5-, 8-, 12- and 15-LOs, and these LO-isoforms are capable of oxygenating arachidonic and linoleic acid. 15-LOs were reported to play an inhibitory role in tumor angiogenesis and, consequently, they slow down carcinogenesis. It has been suggested that its anti-carcinogenic effect is conferred by promoting cell differentiation and apoptosis. Using transgenic mice that over-express 15-LO-1 in endothelial cells under the regulation of the murine preproendothelin-1 promoter, we studied its effect on tumor and metastasis growth. We found that 15-LO-1 inhibited tumor and metastasis growth in the transgenic mice in two different models of cancer (mammary gland and Lewis lung carcinoma). This inhibition was concomitant with a higher number of apoptotic cells in the metastases of the transgenic mice and with a complicated network of multiple small blood vessels. This finding targets 15-LO as a new candidate in the treatment of carcinogenesis.
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An HSV-TK transgenic mouse model to evaluate elimination of fibroblasts for fibrosis therapy.
Pathological fibroproliferation after tissue injury is harmful and may lead to organ dysfunction. Unfortunately, fibroproliferative diseases remain intractable to current therapeutic strategies. Thus, new therapeutic approaches are needed. One possible approach is to promote resolution of physiological fibroproliferation that follows injury before it becomes pathological by activating apoptosis selectively in fibrotic lesions. However, it is not known whether selective elimination of fibroblasts will prevent fibrosis or impede repair or worsen injury by eliminating topographic signals essential to organ reconstitution. To address this question, a tractable in vivo model system is needed in which fibroblasts can be targeted to undergo apoptosis at a chosen time and place. We developed transgenic mice expressing HSV-TK from the type I collagen promoter to determine whether selective elimination of fibroblasts actively forming fibrotic lesions is an effective therapeutic strategy for fibroproliferative disorders. The transgene renders fibroblasts actively forming fibrotic tissue susceptible to ganciclovir. To validate the transgenic model we examined whether administration of ganciclovir prevents the development of fibrosis in sponges implanted subcutaneously in the backs of the transgenic mice. We demonstrate that fibroblasts/myofibroblasts isolated from sponges express HSV-TK protein and are selectively ablated by ganciclovir in vitro. In adult transgenic mice, ganciclovir treatment attenuated the development of fibrotic tissue in the sponges both biochemically and histologically. We conclude that this transgenic model system is an ideal approach to determine whether targeted ablation of fibroblasts is an effective therapeutic strategy for fibrotic diseases.
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Presenilin mutations in familial Alzheimer disease and transgenic mouse models accelerate neuronal lysosomal pathology.
The neuronal lysosomal system is a major degradative pathway, induced by cell stress and closely linked to Alzheimer disease (AD) and other neurodegenerative diseases. Here, we show that mutations of presenilin (PS) 1 and 2, which cause familial early-onset AD (FAD), induce more severe lysosomal system neuropathology in humans than does sporadic AD (SAD). Cathepsin D and B levels were higher in PS-FAD neocortex than in SAD and, unlike neurons in SAD, expressed higher levels of the cation-independent mannose-6-phosphate receptor. Lysosomal pathology was also evident in more populations of neurons in PS-FAD brains, including the less vulnerable neurons in laminae II and IV and affected neurons contained high numbers of hydrolase-positive vesicular compartments with a broader range of abnormal morphology. In transgenic mice expressing mutant amyloid precursor protein (APPswe), introducing mutant PSI significantly upregulated the lysosomal system in neocortical and hippocampal neurons. This upregulation, though milder in severity, resembled that seen in human PS-FAD. Accumulation of hydrolases in dystrophic neurites in senile plaques was particularly strong, suggesting that amyloid deposition may be a stimulus for local mobilization of the lysosomal system. PS1 mice lacking the APPswe transgene also had a mild lysosomal response in some neuronal populations, which was not seen in the APPswe mice. Our findings suggest that presenilin mutations have amyloid-independent effects on the lysosomal system, which are synergistic with the lysosomal system pathology that is associated with beta-amyloid.
Aged↗
Lack of plasminogen activator inhibitor-1 effect in a transgenic mouse model of metastatic melanoma.
Tumor cell invasion and metastasis is a complex, multistep process that is postulated to require degradation of extracellular matrix at several steps. Urokinase-type plasminogen activator (uPA) is expressed on the cell surface of B16 murine melanoma cells and is thought to contribute to the pericellular proteolysis necessary for tumor cell migration. In vitro modification of B16 melanoma cell surface uPA activity has been shown to alter the invasive and metastatic potential of these murine melanoma cells in vivo. Plasminogen activator inhibitor-1 (PAI-1), a rapid inhibitor of both uPA and tissue-type plasminogen activator (tPA) is the major physiologic regulator of plasminogen activator activity. To test the role of host PAI-1 in the invasive and metastatic capacity of B16 melanoma cells we analyzed local tumor growth and pulmonary metastasis in transgenic mice engineered to overexpress murine PAI-1 in multiple tissues including lung, and in mice completely deficient in PAI-1. No significant difference in the number of pulmonary metastases was observed after intravenous inoculation of tumor cells into PAI-1-overexpressing and PAI-1-deficient mice when compared with wild-type controls. Similarly, in a spontaneous metastasis model, PAI-1-overexpressing and PAI-1-deficient mice demonstrated no difference in primary tumor size or overall survival. These data demonstrate that wide variations of host PAI-1 expression, from complete absence to marked overexpression, does not significantly influence the metastatic potential of B16 melanoma cells in a murine model.
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Transgenic mouse models of neurodegenerative disease caused by CAG/polyglutamine expansions.
CAG/polyglutamine expansion is the mutational mechanism that causes a number of late-onset neurodegenerative diseases. Expanded CAG repeats are unstable: they vary in size between tissues and change in size upon transmission from parent to offspring. These mutations are thought to impart a dominant gain of function to the proteins in which they are located. Recent reports describing the first mouse models of these diseases promise to shed light on the molecular mechanisms underlying CAG-repeat instability, the pathways by which polyglutamine expansion causes cell death and the factors that determine the specificity of the neurodegeneration.
Animals↗
Apolipoprotein E dose-dependent modulation of beta-amyloid deposition in a transgenic mouse model of Alzheimer's disease.
Susceptibility to the development of Alzheimer's disease (AD) is increased for individuals harboring one or more apolipoprotein E4 (apoE4) alleles. Even though several isoform-specific effects of apoE have been identified, the relationship between biochemical function and risk factor assessment remains unknown. Our previous studies have demonstrated that there is an equilibrium between cerebral spinal fluid (CSF) and plasma beta-amyloid (Abeta) and that amyloid plaques can modify this equilibrium. Trafficking of soluble central nervous system (CNS) Abeta is a very dynamic system that almost certainly is modulated by Abeta-binding proteins. Altered trafficking of the Abeta peptide might have a dramatic consequence as to whether the peptide is metabolized or begins to deposit within the brain. To gain a better understanding of the molecular mechanisms by which apoE influences AD pathogenesis and/or Abeta trafficking, we developed PDAPP transgenic mice that express different levels of human apoE3. Analysis of the soluble CNS pools of Abeta in young mice showed an apoE3 dose-dependent decrease in Abeta levels (E3-/- > E3-/- > E3+/+). In addition to the dose-dependent effects on soluble Abeta, by 15 mo of age there were highly significant differences in the amount of deposited Abeta between the genotypes (E3-/- > E3-/- > E3+/+). These data indicate that apoE3 provides a dose-dependent protective effect against Abeta deposition. This study suggests that increasing human apoE levels in brain might be a possible therapeutic target for preventing AD.
Alzheimer Disease↗
Comparative effects of lovastatin on mammary and prostate oncogenesis in transgenic mouse models.
The effects of lovastatin, a potent inhibitor of HMG CoA reductase, on experimental mammary and prostate oncogenesis, were studied in vitro and in vivo. Lovastatin inhibited cell growth in vitro in a dose-dependent manner for both mammary and prostate cancer cell lines, which was associated with p53-independent apoptosis. Flow cytometric analyses of lovastatin-treated mammary and prostate cancer cells demonstrated cell-cycle G(1) arrest, as well as decreases in S and G(2)/M fractions. p21(Waf1) and p27(Kip1) were induced by lovastatin in both types of cancer cells. Gene expression profiling of cells treated with lovastatin, however, was remarkable for a paucity of transcriptional changes induced by lovastatin. Treatment with lovastatin for 4 weeks did inhibit the formation of pre-neoplastic mammary intraepithelial neoplasias (MIN) in vivo, but not invasive carcinomas in the C3(1)/SV40 TAg transgenic model of mammary cancer. The decreased multiplicity of MIN lesions was associated with increased levels of apoptosis in these lesions. However, cell proliferation in the mammary lesions was not significantly different between lovastatin-treated and control mice 1 day after lovastatin treatment. In female mice treated with lovastatin for 12 weeks, there was a tendency for reduced tumor volume, which did not reach statistical significance. However, lovastatin did not suppress any lesion formation in the prostate of C3(1)/SV40 TAg transgenic male mice. Our results suggest that as lovastatin exerts an inhibitory effect on the development of early mammary lesions of mammary carcinogenesis, this compound may be useful for the chemoprevention of mammary cancer and might have utility as an adjuvant in breast cancer therapy. The chemopreventive effects of lovastatin in vivo, however, may be tissue-specific.
Animals↗
A transgenic mouse model for monitoring endoplasmic reticulum stress.
Endoplasmic reticulum (ER) stress is caused by the accumulation of unfolded proteins in the ER lumen, and is associated with vascular and neurodegenerative diseases. Although the connection between ER stress and some disease-related proteins has been studied using animal models of these diseases, no in vivo data concerning ER stress are available. Here we report a new method for monitoring ER stress in vivo, based on XBP-1 mRNA splicing by inositol requiring-1 (IRE-1) during ER stress. The stress indicator was constructed by fusing XBP-1 and venus, a variant of green fluorescent protein. During stress, the spliced indicator mRNA is translated into an XBP-1-venus fusion protein, which can be detected by its fluorescence. We used transgenic animals expressing the ER stress indicator to show that it can be used to monitor physiological and pathological ER stress in vivo.
Animals↗
The effects of a human IgM monoclonal anticardiolipin antibody on pregnancy in a transgenic mouse model.
The clinical features of antiphospholipid (or Hughes') syndrome (APS) are most commonly seen in individuals who have raised levels of IgG anticardiolipin antibodies. Most murine models of the syndrome have involved the administration of such antibodies to normal mice. However, APS can occur in the presence of raised levels of serum IgM anticardiolipin antibodies alone. The present study was designed to see if an IgM monoclonal antibody can induce changes in mice similar to those seen in human APS. This antibody, BH1, has previously been derived from a patient with primary APS. In its ligand-binding and idiotypic characteristics it is representative of antiphospholipid antibodies (aPL) found in the serum of patients with APS. In order to minimise the immune response to human IgM, we used transgenic mice (F15) which express, and are predicted to be tolerant of, human immunoglobulin mu chains. The features of APS may develop more readily in individuals who have an existing autoimmune disorder, such as systemic lupus erythematosus (SLE). We therefore crossed these transgenic mice with New Zealand Black (NZB, SLE-prone) mice, and used the progeny (F15 x NZB/F1) in our experiments. Twenty-four F15 x NZB/F1 mice were given BH1, or a control IgM antibody, (A5566) immediately preceding and then three times during pregnancy. There was a reduction in the mean number of foetuses in animals given BH1 compared with those given A5566 (8.6 vs 11.0; P < 0.05), and a similar reduction in mean total foetal weight per pregnancy (9.05 vs 12.73 g; P < 0.05). Two mice showed a marked reduction in platelet count. No evidence of thrombosis was detected macroscopically or histologically. Our results show a lower incidence of APS-type features compared to previous studies in which mice have been administered aPL. This may be because BH1 is an IgM antibody. Nevertheless, the data support the concept that IgM aPL of particular ligand-binding specificities may have a direct pathogenetic role in certain cases of human APS.
Animals↗