Loss of serum response factor in keratinocytes results in hyperproliferative skin disease in mice.

The transcription factor serum response factor (SRF) plays a crucial role in the development of several organs. However, its role in the skin has not been explored. Here, we show that keratinocytes in normal human and mouse skin expressed high levels of SRF but that SRF expression was strongly downregulated in the hyperproliferative epidermis of wounded and psoriatic skin. Keratinocyte-specific deletion within the mouse SRF locus during embryonic development caused edema and skin blistering, and all animals died in utero. Postnatal loss of mouse SRF in keratinocytes resulted in the development of psoriasis-like skin lesions. These lesions were characterized by inflammation, hyperproliferation, and abnormal differentiation of keratinocytes as well as by disruption of the actin cytoskeleton. Ultrastructural analysis revealed markedly reduced cell-cell and cell-matrix contacts and loss of cell compaction in all epidermal layers. siRNA-mediated knockdown of SRF in primary human keratinocytes revealed that the cytoskeletal abnormalities and adhesion defects were a direct consequence of the loss of SRF. In contrast, the hyperproliferation observed in vivo was an indirect effect that was most likely a consequence of the inflammation. These results reveal that loss of SRF disrupts epidermal homeostasis and strongly suggest its involvement in the pathogenesis of hyperproliferative skin diseases, including psoriasis.

Viral superantigen-induced negative selection of TCR transgenic CD4+ CD8+ thymocytes depends on activation, but not proliferation.

T-cell negative selection, a process by which intrathymic immunological tolerance is induced, involves the apoptosis-mediated clonal deletion of potentially autoreactive T cells. Although different experimental approaches suggest that this process is triggered as the result of activation-mediated cell death, the signal transduction pathways underlying this process is not fully understood. In the present report we have used an in vitro system to analyze the cell activation and proliferation requirements for the deletion of viral superantigen (SAg)-reactive Vbeta8.1 T-cell receptor (TCR) transgenic (TG) thymocytes. Our results indicate that in vitro negative selection of viral SAg-reactive CD4+ CD8+ thymocytes is dependent on thymocyte activation but does not require the proliferation of the negatively signaled thymocytes.

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.

Gene therapy regenerates protein expression in cone photoreceptors in Rpe65(R91W/R91W) mice.

Cone photoreceptors mediate visual acuity under daylight conditions, so loss of cone-mediated central vision of course dramatically affects the quality of life of patients suffering from retinal degeneration. Therefore, promoting cone survival has become the goal of many ocular therapies and defining the stage of degeneration that still allows cell rescue is of prime importance. Using the Rpe65(R91W/R91W) mouse, which carries a mutation in the Rpe65 gene leading to progressive photoreceptor degeneration in both patients and mice, we defined stages of retinal degeneration that still allow cone rescue. We evaluated the therapeutic window within which cones can be rescued, using a subretinal injection of a lentiviral vector driving expression of RPE65 in the Rpe65(R91W/R91W) mice. Surprisingly, when applied to adult mice (1 month) this treatment not only stalls or slows cone degeneration but, actually, induces cone-specific protein expression that was previously absent. Before the intervention only part of the cones (40% of the number found in wild-type animals) in the Rpe65(R91W/R91W) mice expressed cone transducin (GNAT2); this fraction increased to 64% after treatment. Correct S-opsin localization is also recovered in the transduced region. In consequence these results represent an extended therapeutic window compared to the Rpe65(-/-) mice, implying that patients suffering from missense mutations might also benefit from a prolonged therapeutic window. Moreover, cones are not only rescued during the course of the degeneration, but can actually recover their initial status, meaning that a proportion of altered cones in chromophore deficiency-related disease can be rehabilitated even though they are severely affected.

Association of T-zone reticular networks and conduits with ectopic lymphoid tissues in mice and humans.

Ectopic or tertiary lymphoid tissues (TLTs) are often induced at sites of chronic inflammation. They typically contain various hematopoietic cell types, high endothelial venules, and follicular dendritic cells; and are organized in lymph node-like structures. Although fibroblastic stromal cells may play a role in TLT induction and persistence, they have remained poorly defined. Herein, we report that TLTs arising during inflammation in mice and humans in a variety of tissues (eg, pancreas, kidney, liver, and salivary gland) contain stromal cell networks consisting of podoplanin(+) T-zone fibroblastic reticular cells (TRCs), distinct from follicular dendritic cells. Similar to lymph nodes, TRCs were present throughout T-cell-rich areas and had dendritic cells associated with them. They expressed lymphotoxin (LT) β receptor (LTβR), produced CCL21, and formed a functional conduit system. In rat insulin promoter-CXCL13-transgenic pancreas, the maintenance of TRC networks and conduits was partially dependent on LTβR and on lymphoid tissue inducer cells expressing LTβR ligands. In conclusion, TRCs and conduits are hallmarks of secondary lymphoid organs and of well-developed TLTs, in both mice and humans, and are likely to act as important scaffold and organizer cells of the T-cell-rich zone.

The inhibition of fat cell proliferation by n-3 fatty acids in dietary obese mice.

ABSTRACT: BACKGROUND: Long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) of marine origin exert multiple beneficial effects on health. Our previous study in mice showed that reduction of adiposity by LC n-3 PUFA was associated with both, a shift in adipose tissue metabolism and a decrease in tissue cellularity. The aim of this study was to further characterize the effects of LC n-3 PUFA on fat cell proliferation and differentiation in obese mice. METHODS: A model of inducible and reversible lipoatrophy (aP2-Cre-ERT2 PPARgammaL2/L2 mice) was used, in which the death of mature adipocytes could be achieved by a selective ablation of peroxisome proliferator-activated receptor gamma in response to i.p. injection of tamoxifen. Before the injection, obesity was induced in male mice by 8-week-feeding a corn oil-based high-fat diet (cHF) and, subsequently, mice were randomly assigned (day 0) to one of the following groups: (i) mice injected by corn-oil-vehicle only, i.e."control" mice, and fed cHF; (ii) mice injected by tamoxifen in corn oil, i.e. "mutant" mice, fed cHF; (iii) control mice fed cHF diet with 15% of dietary lipids replaced by LC n-3 PUFA concentrate (cHF+F); and (iv) mutant mice fed cHF+F. Blood and tissue samples were collected at days 14 and 42. RESULTS: Mutant mice achieved a maximum weight loss within 10 days post-injection, followed by a compensatory body weight gain, which was significantly faster in the cHF as compared with the cHF+F mutant mice. Also in control mice, body weight gain was depressed in response to dietary LC n-3 PUFA. At day 42, body weights in all groups stabilized, with no significant differences in adipocyte size between the groups, although body weight and adiposity was lower in the cHF+F as compared with the cHF mice, with a stronger effect in the mutant than in control mice. Gene expression analysis documented depression of adipocyte maturation during the reconstitution of adipose tissue in the cHF+F mutant mice. CONCLUSION: Dietary LC n-3 PUFA could reduce both hypertrophy and hyperplasia of fat cells in vivo. Results are in agreement with the involvement of fat cell turnover in control of adiposity.

Scnn1 sodium channel gene family in genetically engineered mice.

The amiloride-sensitive epithelial sodium channel is the limiting step in salt absorption. In mice, this channel is composed of three subunits (alpha, beta, and gamma), which are encoded by different genes (Scnn1a, Scnn1b, and Scnn1c, respectively). The functions of these genes were recently investigated in transgenic (knockout) experiments, and the absence of any subunit led to perinatal lethality. More defined phenotypes have been obtained by introducing specific mutations or using transgenic rescue experiments. In this report, these approaches are summarized and a current gene-targeting strategy to obtain conditional inactivation of the channel is illustrated. This latter approach will be indispensable for the investigation of channel function in a wide variety of organ systems.

Two-kidney, one clip and one-kidney, one clip hypertension in mice.

The mouse remains the animal of choice in transgenic experiments, creating a need for methods of evaluating the physiology of genetically modified animals. We have established and characterized two murine models of renovascular hypertension known as the two-kidney, one clip and one-kidney, one clip models. The appropriate size of the clip lumen needed to induce high blood pressure was determined to be 0.12 mm. Clips with a lumen of 0.11 mm induced a high percentage of renal infarction, and clips with a 0.13-mm opening did not produce hypertension. Four weeks after clipping, two-kidney, one clip hypertensive mice exhibited blood pressure approximately 20 mm Hg higher than their sham-operated controls. After a similar period, this increase reached almost 35 mm Hg in the one-kidney, one clip model. Depending on the model, mice develop either renin-dependent or renin-independent hypertension. Both models are characterized by the development of cardiovascular hypertrophy.

Intraperikaryal neurofilamentous accumulations in a subset of retinal ganglion cells in aged mice that express a human neurofilament gene.

Neurofilamentous changes in select groups of neurons are associated with the degenerative changes of many human age-related neurodegenerative diseases. To examine the possible effects of aging on the neuronal cytoskeleton containing human proteins, the retinas of transgenic mice expressing the gene for the human middle-sized neurofilament triplet were investigated at 3 or 12 months of age. Transgenic mice developed tangle-like neurofilamentous accumulations in a subset of retinal ganglion cells at 12 months of age. These neurofilamentous accumulations, which also involved endogenous neurofilament proteins, were present in the perikarya and proximal processes of large ganglion cells and were predominantly located in peripheral retina. The presence of the human protein may thus confer vulnerability of the cytoskeleton to age-related alterations in this specific retinal cell type and may serve as a model for similar cellular changes associated with Alzheimer's disease and glaucoma.

Conditional transgenic expression of fibroblast growth factor 9 in the adult mouse heart reduces heart failure mortality after myocardial infarction

BACKGROUND: Fibroblast growth factor 9 (FGF9) is secreted from bone marrow cells, which have been shown to improve systolic function after myocardial infarction (MI) in a clinical trial. FGF9 promotes cardiac vascularization during embryonic development but is only weakly expressed in the adult heart. METHODS AND RESULTS: We used a tetracycline-responsive binary transgene system based on the α-myosin heavy chain promoter to test whether conditional expression of FGF9 in the adult myocardium supports adaptation after MI. In sham-operated mice, transgenic FGF9 stimulated left ventricular hypertrophy with microvessel expansion and preserved systolic and diastolic function. After coronary artery ligation, transgenic FGF9 enhanced hypertrophy of the noninfarcted left ventricular myocardium with increased microvessel density, reduced interstitial fibrosis, attenuated fetal gene expression, and improved systolic function. Heart failure mortality after MI was markedly reduced by transgenic FGF9, whereas rupture rates were not affected. Adenoviral FGF9 gene transfer after MI similarly promoted left ventricular hypertrophy with improved systolic function and reduced heart failure mortality. Mechanistically, FGF9 stimulated proliferation and network formation of endothelial cells but induced no direct hypertrophic effects in neonatal or adult rat cardiomyocytes in vitro. FGF9-stimulated endothelial cell supernatants, however, induced cardiomyocyte hypertrophy via paracrine release of bone morphogenetic protein 6. In accord with this observation, expression of bone morphogenetic protein 6 and phosphorylation of its downstream targets SMAD1/5 were increased in the myocardium of FGF9 transgenic mice. CONCLUSIONS: Conditional expression of FGF9 promotes myocardial vascularization and hypertrophy with enhanced systolic function and reduced heart failure mortality after MI. These observations suggest a previously unrecognized therapeutic potential for FGF9 after MI.

Prime-boost vaccination with plasmid and adenovirus gene vaccines control HER2/neu+ metastatic breast cancer in mice.

INTRODUCTION: Once metastasis has occurred, the possibility of completely curing breast cancer is unlikely, particularly for the 30 to 40% of cancers overexpressing the gene for HER2/neu. A vaccine targeting p185, the protein product of the HER2/neu gene, could have therapeutic application by controlling the growth and metastasis of highly aggressive HER2/neu+ cells. The purpose of this study was to determine the effectiveness of two gene vaccines targeting HER2/neu in preventive and therapeutic tumor models. METHODS: The mouse breast cancer cell line A2L2, which expresses the gene for rat HER2/neu and hence p185, was injected into the mammary fat pad of mice as a model of solid tumor growth or was injected intravenously as a model of lung metastasis. SINCP-neu, a plasmid containing Sindbis virus genes and the gene for rat HER2/neu, and Adeno-neu, an E1,E2a-deleted adenovirus also containing the gene for rat HER2/neu, were tested as preventive and therapeutic vaccines. RESULTS: Vaccination with SINCP-neu or Adeno-neu before tumor challenge with A2L2 cells significantly inhibited the growth of the cells injected into the mammary fat or intravenously. Vaccination 2 days after tumor challenge with either vaccine was ineffective in both tumor models. However, therapeutic vaccination in a prime-boost protocol with SINCP-neu followed by Adeno-neu significantly prolonged the overall survival rate of mice injected intravenously with the tumor cells. Naive mice vaccinated using the same prime-boost protocol demonstrated a strong serum immunoglobulin G response and p185-specific cellular immunity, as shown by the results of ELISPOT (enzyme-linked immunospot) analysis for IFNgamma. CONCLUSION: We report herein that vaccination of mice with a plasmid gene vaccine and an adenovirus gene vaccine, each containing the gene for HER2/neu, prevented growth of a HER2/neu-expressing breast cancer cell line injected into the mammary fat pad or intravenously. Sequential administration of the vaccines in a prime-boost protocol was therapeutically effective when tumor cells were injected intravenously before the vaccination. The vaccines induced high levels of both cellular and humoral immunity as determined by in vitro assessment. These findings indicate that clinical evaluation of these vaccines, particularly when used sequentially in a prime-boost protocol, is justified.

Cyan fluorescent protein expression in ganglion and amacrine cells in a thy1-CFP transgenic mouse retina.

PURPOSE: To characterize cyan fluorescent protein (CFP) expression in the retina of the thy1-CFP (B6.Cg-Tg(Thy1-CFP)23Jrs/J) transgenic mouse line. METHODS: CFP expression was characterized using morphometric methods and immunohistochemistry with antibodies to neurofilament light (NF-L), neuronal nuclei (NeuN), POU-domain protein (Brn3a) and calretinin, which immunolabel ganglion cells, and syntaxin 1 (HPC-1), glutamate decarboxylase 67 (GAD(67)), GABA plasma membrane transporter-1 (GAT-1), and choline acetyltransferase (ChAT), which immunolabel amacrine cells. RESULTS: CFP was extensively expressed in the inner retina, primarily in the inner plexiform layer (IPL), ganglion cell layer (GCL), nerve fiber layer, and optic nerve. CFP fluorescent cell bodies were in all retinal regions and their processes ramified in all laminae of the IPL. Some small, weakly CFP fluorescent somata were in the inner nuclear layer (INL). CFP-containing somata in the GCL ranged from 6 to 20 microm in diameter, and they had a density of 2636+/-347 cells/mm2 at 1.5 mm from the optic nerve head. Immunohistochemical studies demonstrated colocalization of CFP with the ganglion cell markers NF-L, NeuN, Brn3a, and calretinin. Immunohistochemistry with antibodies to HPC-1, GAD(67), GAT-1, and ChAT indicated that the small, weakly fluorescent CFP cells in the INL and GCL were cholinergic amacrine cells. CONCLUSIONS: The total number and density of CFP-fluorescent cells in the GCL were within the range of previous estimates of the total number of ganglion cells in the C57BL/6J line. Together these findings suggest that most ganglion cells in the thy1-CFP mouse line 23 express CFP. In conclusion, the thy1-CFP mouse line is highly useful for studies requiring the identification of ganglion cells.

Neoplastic Transformation of T Lymphocytes through Transgenic Expression of a Virus Host Modification Protein

Virus host evasion genes are ready-made tools for gene manipulation and therapy. In this work we have assessed the impact in vivo of the evasion gene A238L of the African Swine Fever Virus, a gene which inhibits transcription mediated by both NF-κB and NFAT. The A238L gene has been selectively expressed in mouse T lymphocytes using tissue specific promoter, enhancer and locus control region sequences for CD2. The resulting two independently derived transgenic mice expressed the transgene and developed a metastasic, angiogenic and transplantable CD4(+)CD8(+)CD69(-) lymphoma. The CD4(+)CD8(+)CD69(-) cells also grew vigorously in vitro. The absence of CD69 from the tumour cells suggests that they were derived from T cells at a stage prior to positive selection. In contrast, transgenic mice similarly expressing a mutant A238L, solely inhibiting transcription mediated by NF-κB, were indistinguishable from wild type mice. Expression of Rag1, Rag2, TCRβ-V8.2, CD25, FoxP3, Bcl3, Bcl2 l14, Myc, IL-2, NFAT1 and Itk, by purified CD4(+)CD8(+)CD69(-) thymocytes from A238L transgenic mice was consistent with the phenotype. Similarly evaluated expression profiles of CD4(+)CD8(+) CD69(-) thymocytes from the mutant A238L transgenic mice were comparable to those of wild type mice. These features, together with the demonstration of (mono-)oligoclonality, suggest a transgene-NFAT-dependent transformation yielding a lymphoma with a phenotype reminiscent of some acute lymphoblastic lymphomas.

Human growth hormone presented by K14hGH-transgenic skin grafts induces a strong immune response but no graft rejection

Although immune responses leading to rejection of transplantable tumours have been well studied, requirements for epithelial tumour rejection are unclear. Here, we use human growth hormone (hGH) expressed in epithelial cells (skin keratinocytes) as a model neo-self antigen to investigate the consequences of antigen presentation from epithelial cells. Mice transgenic for hGH driven from the keratin 14 promoter express hGH in skin keratinocytes. This hGH-transgenic skin is not rejected by syngeneic non-transgenic recipients, although an antibody response to hGH develops in grafted animals. Systemic immunization of graft recipients with hGH peptides, or local administration of stimulatory anti-CD40 antibody, induces temporary macroscopic graft inflammation, and an obvious dermal infiltrate of inflammatory cells, but not graft rejection. These results suggest that a neo-self antigen expressed in somatic cells in skin can induce an immune response that can be enhanced further by induction of specific immunity systemically or non-specific immunity locally. However, immune responses do not always lead to rejection, despite induction of local inflammatory changes. Therefore, in vitro immune responses and in vivo delayed type hypersensitivity are not surrogate markers for immune responses effective against epithelial cells expressing neoantigens.