Early detection of PrPres in BSE-infected bovine PrP transgenic mice

Summary.  Transgenic mouse lines expressing different levels of the bovine prion protein gene (boPrP^C) were generated. Upon infection with BSE prions, all transgenic lines tested exhibited characteristics of the bovine disease. Typical CNS spongiform degeneration was observed by histopathology and presence of PrP^res could be detected both by Western blot and immunohistochemistry (IHC) assays, confirming for this model the absence of an interspecies barrier to BSE infection. Differences in incubation times post-inoculation depend upon the expression level of boPrP^C and the amount of prions in the inoculum. In the absence of clinical signs, pathognomonic markers of disease could be detected as early as 150 or 196 days post-inoculation by IHC and Western blot analysis, respectively. This result indicates that prion infectivity in experimental mouse bioassays can be measured earlier by assessing immunologically the presence of PrP^res in brains from inoculated animals. Although these transgenic mice were also susceptible to sheep scrapie prion infection, the extent of incubation times was considerably longer and PrP^res was detected in only 70 % of inoculated mice. Interestingly, transgenic mice-propagated sheep scrapie prions displayed distinct biochemical properties when compared to both the original sheep scrapie and transgenic mouse-propagated BSE inoculum. Received November 16, 2002; accepted November 18, 2002     

Age-dependent induction of congophilic neurofibrillary tau inclusions in tau transgenic mice

Intraneuronal filamentous tau inclusions such as neurofibrillary tangles (NFTs) are neuropathological hallmarks of Alzheimer's disease (AD) and related sporadic and familial tauopathies. NFTs identical to those found in AD brains have also been detected in the hippocampus and entorhinal cortex of cognitively normal individuals as they age. To recapitulate age-induced NFT formation in a mouse model, we examined 12- to 24-month-old transgenic (Tg) mice overexpressing the smallest human brain tau isoform. These Tg mice develop congophilic tau inclusions in several brain regions including the hippocampus, amygdala, and entorhinal cortex. NFT-like inclusions were first detected in Tg mice at 18 to 20 months of age and they were detected by histochemical dyes that bind specifically to crossed beta-pleated sheet structures (eg, Congo red, Thioflavin S). Moreover, ultrastructurally these lesions contained straight tau filaments comprised of both mouse and human tau proteins but not other cytoskeletal proteins (eg, neurofilaments, microtubules). Isolated tau filaments were also recovered from detergent-insoluble tau fractions and insoluble tau proteins accumulated in brain in an age-dependent manner. Thus, overexpression of the smallest human brain tau isoform resulted in late onset and age-dependent formation of congophilic tau inclusions with properties similar to those in the tangles of human tauopathies, thereby implicating aging in the pathogenesis of fibrous tau inclusions.     

Early axonopathy preceding neurofibrillary tangles in mutant tau transgenic mice

Neurodegenerative diseases characterized by brain and spinal cord involvement often show widespread accumulations of tau aggregates. We have generated a transgenic mouse line (Tg30tau) expressing in the forebrain and the spinal cord a human tau protein bearing two pathogenic mutations (P301S and G272V). These mice developed age-dependent brain and hippocampal atrophy, central and peripheral axonopathy, progressive motor impairment with neurogenic muscle atrophy, and neurofibrillary tangles and had decreased survival. Axonal spheroids and axonal atrophy developed early before neurofibrillary tangles. Neurofibrillary inclusions developed in neurons at 3 months and were of two types, suggestive of a selective vulnerability of neurons to form different types of fibrillary aggregates. A first type of tau-positive neurofibrillary tangles, more abundant in the forebrain, were composed of ribbon-like 19-nm-wide filaments and twisted paired helical filaments. A second type of tau and neurofilament-positive neurofibrillary tangles, more abundant in the spinal cord and the brainstem, were composed of 10-nm-wide neurofilaments and straight 19-nm filaments. Unbiased stereological analysis indicated that total number of pyramidal neurons and density of neurons in the lumbar spinal cord were not reduced up to 12 months in Tg30tau mice. This Tg30tau model thus provides evidence that axonopathy precedes tangle formation and that both lesions can be dissociated from overt neuronal loss in selected brain areas but not from neuronal dysfunction.     

Prolactin potentiates transforming growth factor alpha induction of mammary neoplasia in transgenic mice

Prolactin influences mammary development and carcinogenesis through endocrine and autocrine/paracrine mechanisms. In virgin female mice, pro-lactin overexpression under control of a mammary selective nonhormonally responsive promoter, neu-related lipocalin, results in estrogen receptor alpha (ERalpha)-positive and ERalpha-negative adenocarcinomas. However, disease in vivo occurs in the context of dysregulation of multiple pathways. In this study, we investigated the ability of prolactin to modulate carcinogenesis when co-expressed with the potent oncogene transforming growth factor alpha (TGFalpha) in bitransgenic mice. Prolactin and TGFalpha cooperated to reduce dramatically the latency of mammary macrocyst development, the principal lesion type induced by TGFalpha. In combination, prolactin and TGFalpha also increased the incidence and reduced the latency of other preneoplastic lesions and increased cellular turnover in structurally normal alveoli and ducts compared with single transgenic females. Bitransgenic glands contained higher levels of phosphorylated ERK1/2 compared with single TGFalpha transgenic glands, suggesting that this kinase may be a point of signaling crosstalk. Furthermore, transgenic prolactin also reversed the decrease in ERalpha induced by neu-related lipocalin-TGFalpha. Our findings demonstrate that locally produced prolactin can strikingly potentiate the carcinogenic actions of another oncogene and modify ovarian hormone responsiveness, suggesting that prolactin signaling may be a potential therapeutic target.     

Involvement of IL-10 in induction of autoimmune hemolytic anemia in anti-erythrocyte Ig transgenic mice

Activation of peritoneal B-1 cells triggers autoimmune anemia in anti- erythrocyte Ig transgenic mice (HL mice). Numbers of peritoneal B-1 cells and Ig-producing cells were negligible in the T cell-deficient HL mice generated by the cross with RAG-2-/- mice (RAG-2-/- x HL mice). Proliferation and activation of B-1 cells in RAG-2-/- x HL mice were recovered by fetal thymus transfer, indicating involvement of T cells in B-1 cell-mediated autoimmune hemolytic anemia. Involvement of T cells in proliferation and activation of B-1 cells could be by-passed by administration of lipopolysaccharide (LPS), IL-5 or IL-10 to RAG-2-/- x HL mice. Administration of LPS elevated the serum IL-10 level in HL, RAG-2-/- x HL and normal mice. Proliferation and activation of B-1 cells were blocked by an anti-IL-10 antibody in conventionally bred as well as LPS-treated HL mice. Taken together, IL-10 plays a pivotal role in activation of peritoneal B-1 cells.      

Early temporal short-term memory deficits in double transgenic APP/PS1 mice

We tested single APP (Tg2576) transgenic, PS1 (PS1dE9) transgenic, and double APP/PS1 transgenic mice at 3 and 6 months of age on the acquisition of a hippocampal-dependent operant “differential reinforcement of low rate schedule” (DRL) paradigm. In this task mice are required to wait for at least 10 seconds (DRL-10s) between 2 consecutive nose poke responses. Our data showed that while single APP and PS1 transgene expression did not affect DRL learning and performance, mice expressing double APP/PS1 transgenes were impaired in the acquisition of DRL-10s at 6 months, but not at 3 months of age. The same impaired double transgenic mice, however, were perfectly capable of normal acquisition of signaled DRL-10s (SDRL-10s) task, a hippocampal-independent task, wherein mice were required to emit responses when the end of the 10-second delay was signaled by a lighting of the chamber. The age-dependent and early deficits of APP/PS1 mice suggest that the appetitive DRL paradigm is sensitive to the amyloid pathology present in double APP/PS1 mice, and that this mouse line represents a good model with which to study the efficacy of therapeutic strategies against Alzheimer's disease.     

Quantitative analysis of antigen for the induction of tolerance in carcinoembryonic antigen transgenic mice.

In order to analyse the amounts of antigen in the thymus for the induction of tolerance, several carcinoembryonic antigen (CEA) transgenic lines were established which expressed human CEA antigen with different amounts. The chimeric KSN nude mice transplanted with the thymus of the B601 line (in which CEA mRNA and CEA protein could be detected in various tissues) to kidney capsule showed tolerance to human CEA. On the other hand, the chimeric KSN nude mice transplanted with the thymus of the B602 or BC60 line (in which neither CEA mRNA nor CEA protein could be detected by Northern blot analysis and flow cytometry analysis) or normal C57BL/6 (B6) did not develop the tolerance to human CEA. However, the chimeric KSN nude mice transplanted simultaneously with thymus of the B6 and spleen of the B601 line became tolerant to human CEA antigen. In the case of systemic immunization with cells which had CEA antigen, the B601 line was tolerant to human CEA. Surprisingly, the B602 and BC60 lines were also tolerant to CEA molecule. These results indicate that not only the antigen present in the thymus but also the antigen which flows from the peripheral organs to the thymus may be necessary for the induction of CEA tolerance.     

alpha4 integrin-dependent eotaxin induction of bronchial hyperresponsiveness and eosinophil migration in interleukin-5 transgenic mice

We investigated the roles of eosinophil infiltration and activation induced by the eosinophil-selective chemokine eotaxin, and of the expression of eosinophil alpha4 and beta2 integrins in causing bronchial hyperresponsiveness (BHR) in interleukin (IL)-5 CBA/Ca transgenic mice. These mice did not show BHR, despite the presence of some eosinophils in the lungs. Intratracheal mouse recombinant eotaxin (3 micrograms) did not induce BHR in wild-type mice. In IL-5 transgenic mice, eotaxin (3 and 5 micrograms) increased responsiveness at 24 h and increased eosinophils in bronchoalveolar lavage (BAL) fluid by 9.4- and 14-fold by 24 h, respectively, together with augmentation of eosinophil peroxidase activity and eosinophil infiltration in the airway submucosa. Using flow cytometry, the expression of alpha4, CD11b, and CD18 was upregulated in BAL, but not in blood, eosinophils. A rat anti-alpha4 antibody inhibited eotaxin-induced BHR and eosinophil migration and activation, but an anti-CD11b antibody had no significant effects on BHR. A combination of both antibodies was more effective. IL-5 and eotaxin synergize in the induction of BHR and airway eosinophilia, effects that are dependent on the induction of eosinophil alpha4 integrin. Expression of BHR depends on the recruitment and activation of eosinophils.     

B cells are important as antigen presenting cells for induction of MHC-restricted arthritis in transgenic mice

Rheumatoid arthritis and its animal model, collagen-induced arthritis, are known as a T and B cell dependent disease. To analyze the role of B cells in arthritis, we generated B cell deficient (microMT) mice carrying HLA-DQ8 as transgene, Abetao.DQ8.micromt mice. HLA-DQ8 transgenic mice (Abetao.DQ8) are susceptible to collagen induced arthritis, an animal model for inflammatory arthritis. Deletion of IgM gene led to the absence of B cells while T cells were comparable to Abetao.DQ8 mice. Arthritis and autoantibodies was completely abrogated in B cell deficient DQ8 mice. T cell response and proinflammatory cytokine production in response to type II collagen and its derived peptides in vitro was significantly decreased despite an increased number of Mac-1 positive cells in DQ8.micromt mice compared to DQ8 mice suggesting B cells could be important for antigen presentation as well. In vitro substitution of B cells from wild type mice restored the response in DQ8.micromt mice. B cells could also present CII-derived peptides to antigen-specific DQ8-restricted hybridomas reinforcing the role of B cells in presentation of antigens to T cells. The data suggest that B cells can be involved in pathogenesis of arthritis by producing autoantibodies and antigen presentation.     

Polycystic kidney disease in SBM transgenic mice: role of c-myc in disease induction and progression.

SBM mouse is a unique transgenic model of polycystic kidney disease (PKD) produced by dysregulation of c-myc in the kidneys. Our previous demonstration that c-myc is overexpressed in human autosomal polycystic kidney disease (ADPKD) prompted us to investigate the pathogenetic role of c-myc in the induction and progression of the cystogenic phenotype in our mouse model. In young SBM kidneys, c-myc was two- to threefold increased with persistent expression levels into adulthood, an age when c-myc is normally undetectable. In situ hybridization analysis of the c-myc transgene demonstrated intense signal specifically overlying glomerular and tubular epithelium of developing cysts in fetal and young kidneys. Increased expression of c-myc correlated with the initiation and progression of the PKD phenotype as evidenced by early tubular and glomerular cysts at E16.5. Cyst number and size increased with age, with co-development of glomerular and tubular epithelial hyperplasia. Consistently, the mean renal proliferative index was increased approximately 5- to 20-fold in noncystic and cystic tubules of newborn SBM animals compared with littermate controls. Similarly, in fetal and newborn kidneys the tubular apoptotic indices were increased approximately three- to ninefold over controls. Both proliferation and apoptotic rates in cystic tubules approached levels in developing tubules from the normal nephrogenic zone. We conclude that the pathogenesis of PKD hinges on a critical imbalance in c-myc regulation of the opposing processes of cell proliferation and apoptosis, recapitulating the cellular phenomena in developing fetal kidney.     

Requirement of IL-5 for induction of autoimmune hemolytic anemia in anti-red blood cell autoantibody transgenic mice

IL-5, IL-10 and lipopolysaccharide (LPS) are known to activateB-1 cells in vivo in normal mice and anti-red blood cell autoantibodytransgenic mice (HL mice). To assess the exact role of IL-5in proliferation and activation of peritoneal B-1 cells, weanalyzed IL-5 receptor chain-deficient HL (IL-5R^–/–x HL) mice generated by the cross between IL-5R^–/–and HL mice. In IL-5R^–/– x HL mice, Ig-producingB-1 cells in the peritoneal cavity were negligible, althoughthe total number of B-1 cells in the peritoneal cavity wereas many as 30% of that in HL mice. Moreover, LPS- or IL-10-induceddifferentiation of B-1 cells into antibody-producing cells wasseverely impaired in IL-5R^–/– x HL mice. We alsoused in vivo 5-bromo-2'-deoxyuridine labeling to estimate theproliferation of B-1 cells in IL-5R^–/– mice. Theabsence of IL-5R did not affect spontaneous proliferation ofperitoneal B-1 cells. However, induced proliferation of peritorealB-1 cells by oral administration of LPS was markedly impairedin IL-5R^–/– mice. These results suggest that IL-5is required for activation-associated proliferation of B-1 cellsbut not for their spontaneous proliferation and support theidea that IL-5 plays an important role on the induction of autoantibodyproduction from B-1 cells.     

The induction of Toll-like receptor tolerance enhances rather than suppresses HIV-1 gene expression in transgenic mice

Microbial-induced proinflammatory pathways are thought to play a key role in the activation of human immunodeficiency virus type 1 (HIV-1) gene expression. The induction of Toll-like receptor (TLR) tolerance leads to a complex reprogramming in the pattern of inflammatory gene expression and down-modulates tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1, and IL-6 production. Using transgenic (Tg) mice that incorporate the entire HIV-1 genome, including the long-terminal repeat, we have previously demonstrated that a number of different TLR ligands induce HIV-1 gene expression in cultured splenocytes as well as purified antigen-presenting cell populations. Here, we have used this model to determine the effect of TLR-mediated tolerance as an approach to inhibiting microbial-induced viral gene expression in vivo. Unexpectedly, Tg splenocytes and macrophages, rendered tolerant in vitro to TLR2, TLR4, and TLR9 ligands as assessed by proinflammatory cytokine secretion and nuclear factor-kappaB activation, showed enhanced HIV-1 p24 production. A similar enhancement was observed in splenocytes tolerized and then challenged with heterologous TLR ligands. Moreover, TLR2- and TLR4-homotolerized mice demonstrated significantly increased plasma p24 production in vivo despite lower levels of TNF-alpha. Together, these results demonstrate that HIV-1 expression is enhanced in TLR-reprogrammed host cells, possibly reflecting a mechanism used by the virus to escape the effects of microbial-induced tolerance during natural infection in vivo.     

Evidence for the lack of base-change and small-deletion mutation induction by trichloroethylene in lacZ transgenic mice.

Trichloroethylene (TCE) is a widely used industrial solvent employed mainly for degreasing and cold-cleaning metal parts. It is also used for dry cleaning, and in the production of a number of chemical products. It has been shown to induce liver and lung tumors in rodents, and have a variety of positive and negative results using in vitro and in vivo mutagenicity tests. In order to assist in the interpretation of the mechanism of carcinogenicity, TCE was tested for the ability to induce gene mutations and small deletions using the lacZ transgenic mouse model (MutaMouse). Male and female animals were exposed by inhalation to 0, 203, 1153, and 3141 ppm TCE, 6 h per day for 12 days. 14 and 60 days following the last exposure, animals were sacrificed and the mutation frequency in bone marrow, kidney, spleen, liver, lung, and testicular germ cells determined. The results of this study indicate that TCE did not induce base-change or small-deletion mutations as detected in this assay in any of the tissues examined. Environ. Mol. Mutagen. 34: 190-194, 1999. Published 1999 Wiley-Liss, Inc.     

Human antibody expression in transgenic mice

Human antibody repertoires can be created in transgenic mice following the introduction of human immunoglobulin heavy and light chain genes in their germline configuration. Transgene constructs or transloci have been obtained by plasmid assembly, cloning in yeast artificial chromosomes, and the use of chromosome fragments. Translocus integration and maintenance in transgenic mouse strains has been achieved by pronuclear DNA injection into oocytes and various transfection methods using embryonic stem cells. The human DNA segments rearrange faithfully in the mouse and produce extensive V(D)J combinations. Specific human monoclonal antibodies of high affinity for use in therapeutic applications have been produced from these translocus mice.     

Antisense RNA production in transgenic mice

There are many reports of antisense inhibition of gene expression in cultured cells. We have generated four strains of transgenic mice expressing antisense hypoxanthine guanine phosphoribosyltransferase (HPRT) RNA in brain, or heart and liver, or all three organs. In the brain of one strain, the level of antisense RNA in the different brain regions roughly correlates with the degree of inhibition of the native HPRT mRNA in those same regions. Despite this decrease of up to 60% of endogenous HPRT mRNA, no reproducible reduction in HPRT activity has been observed. Possible reasons for the differences between the effectiveness of antisense inhibition in cultured cells and transgenic animals are discussed.     

Auto-reactive B cells in transgenic mice

In order to understand how the natural occurrence of autoreactive B cells is controlled in normal individuals, and how self reactive B cells can escape this control during diverse clinical situations, many different transgenic mice have been generated expressing self reactive antibodies. In this review, we focus our attention on disease-associated self reactive transgenic models which show the variety of the tolerization mechanisms. The same transgenic lines are also used to analyse the effects of the autoimmune genetic background on the self reactive B cell fate, as well as to study the influence of infectious agents on the behaviour of the auto-reactive transgenic B cells.     

SUMO-1 immunoreactivity co-localizes with phospho-Tau in APP transgenic mice but not in mutant Tau transgenic mice

Agmatine, an endogenous ligand of imidazoline receptors, was employed to screen the effect on insulin resistance in rats induced by a diet containing 60% fructose. Single intravenous (i.v.) injection of agmatine sulfate for 30min decreased the plasma glucose concentrations in a dose-dependent manner from 0.5mg/kg to 3mg/kg in rats received 4-week fructose-rich chow without an alteration of systolic blood pressure. The plasma glucose lowering action of agmatine (1mg/kg, i.v.) was abolished by the pretreatment with BU224 (1mg/kg, i.v.) at sufficient dosage to block I(2)-imidazoline receptors. In addition, the value of glucose-insulin index, the areas under the curve of glucose and insulin during the intraperitoneal glucose tolerance test, showing an index of in vivo insulin sensitivity was reversed by the same treatment with agmatine in fructose-rich chow-fed rats; this action was also blocked by BU224. Our results suggest that activation of I(2)-imidazoline receptor to improve insulin action on glucose disposal can be considered for targeting glucose metabolism under insulin-resistant state.     

SUMO-1 immunoreactivity co-localizes with phospho-Tau in APP transgenic mice but not in mutant Tau transgenic mice

Sumoylation is a post-translational modification process that is supposed to be implicated in the pathogenesis of several neurodegenerative diseases. Recently, the microtubule-associated protein Tau was identified as a target for sumoylation in the analysis of the transfected cells. We investigated the localization of SUMO-1 protein in APP transgenic mice and mutant Tau transgenic mice, and found that SUMO-1 immunoreactivity was co-localized with phosphorylated Tau aggregates in amyloid plaques of APP transgenic mice. By contrast, no SUMO-1 immunoreactivity was observed in phosphorylated Tau aggregates of mutant Tau transgenic mice. The contribution of sumoylation to the neurodegeneration in Alzheimer's disease will be further elucidated via the analysis of APP transgenics.