Asbestos-induced autoimmunity in C57BL/6 mice

Environmental impacts on autoimmunity have significant public health implications. Epidemiological studies have shown associations between exposure to airborne silicates, such as crystalline silica or asbestos, and autoimmunity, but the etiology remains unclear. The purpose of this study was to test the hypothesis that asbestos could lead to a specific pattern of autoantibodies and pathology indicative of systemic autoimmune disease (SAID). Female C57Bl/6 mice were instilled intratracheally with 2 doses x 60 microg/mouse of amphibole asbestos (tremolite), wollastonite (a non-fibrogenic control fiber), or saline alone. Serum samples were collected and urine was checked for protein bi-weekly for 7 months. By 26 weeks, the asbestos-instilled animals had a significantly higher frequency of positive anti-nuclear antibody (ANA) tests compared to wollastonite and saline groups. The majority of positive ANAs showed homogeneous or combined homogeneous/speckled patterns, and tested positive for antibodies to dsDNA and SSA/Ro 52. Serum isotyping showed no significant changes in IgM, IgA, or IgG subclasses. However, there was an overall decrease in the mean IgG serum concentration in asbestos-instilled mice. IgG immune complex deposition was demonstrated in the kidneys of asbestos-instilled mice, with evidence of glomerular and tubule abnormalities suggestive of glomerulonephritis. Flow cytometry demonstrated moderate changes in the percentages of CD25+ T-suppressor cells and B1a B-cells in the superficial cervical lymph nodes of the asbestos-instilled mice. These data demonstrate that asbestos leads to immunologic changes consistent with the development of autoimmunity. This study provides a non-autoimmune prone murine model for use in future elucidation of mechanisms involved in asbestos-induced autoimmune disease.     

Punta Toro virus infection of C57BL/6J mice: a model for phlebovirus-induced disease

Punta Toro virus infections of inbred strains of mice have been characterized and evaluated as a model in which to study various aspects of the host response to phlebovirus infections and the requirements for protective immunity. The Adames strain of Punta Toro virus was found to be strongly hepatotropic and lymphotropic and the outcome of infection was largely a function of age. C57BL/6J mice of less than 5 weeks of age uniformly developed fulminant hepatocellular necrosis with mean survival times of 4.2 days. Resistance to lethal infection increased with age such that greater than 95% of 8-week-old mice survived challenge. The kinetics of viremia, antibody production, and hematological changes in 4- and 8-week animals indicated that the survival of the older animals is related to their ability to delay virus replication and the development of hepatic lesions during the initial 48 h of infection and their ability to terminate virus replication and clear virus from the circulation 4 to 5 days after infection. The mechanisms responsible for this resistance were studied using anti-interferon serum, immunosuppression, and passive immunization.     

昆明种小鼠与C57BL/6J小鼠磷酸二酯酶β亚单位编码基因pde6b序列特点的比较分析

目的:分段克隆昆明种小鼠磷酸二酯酶(PDE) β亚单位编码基因pde6b CDS序列全长,分析比较昆明种小鼠与C57BL/6J小鼠PDE β亚单位编码基因pde6b序列的差异.方法:设计覆盖pde6b CDS区的引物序列,通过逆转录-聚合酶链式反应扩增并克隆入载体pMD18-T中,转化大肠杆菌,酶切鉴定后测序.通过生物信息检索、序列拼接并应用生物信息学相关软件进行序列分析.结果:克隆了野生型昆明种小鼠的pde6b CDS全长.昆明种小鼠与C57BL/6J小鼠pde6b CDS区(NM_008806)相比较存在如下不同:昆明种小鼠第706位碱基为A,而数据库中序列NM_008806为G;第1149位碱基前者为T,后者为C.昆明种小鼠与C57BL/6J小鼠pde6b编码的蛋白序列差异并不明显,仅有第236位氨基酸残基为甘氨酸(G)突变成丝氨酸(S),为相对保守性替换关系.结论:克隆了昆明种小鼠pde6b CDS 区全长序列;pde6b基因在不同种属小鼠之间编码序列存在差异,表现出多样性特点,但蛋白序列保守性较高.     

Benzo(a)pyrene inhibits ovulation in C57BL/6N mice

Successful female reproductive function requires follicle growth, ovulation, and formation of the corpus luteum. Treatment of C57BL/6N mice with a single intraperitoneal injection of benzo(a)pyrene in doses ranging from 1 to 500 mg/kg produced a dose- and time-dependent decrease in the number of corpora lutea. This effect on the number of corpora lutea is most pronounced at 1 week after treatment, with a threshold of about 1 mg/kg, and an ED50 of 1.6 mg/kg. By 2 weeks after treatment partial recovery of follicle growth and ovulation occurred, as indicated by an increase in the ED50 to 20 mg/kg. Complete recovery of normal corpora lutea number occurs in mice treated with less than 100 mg/kg by 3 weeks after treatment, with little change in the ED50 noted between 3 and 4 weeks post-treatment, 78 mg/kg at both times. Mice treated with 100 or 500 mg/kg did not recover normal corpora lutea number over the course of this experiment. These data indicate that acute exposure to benzo(a)pyrene, and perhaps other polycyclic aromatic hydrocarbons, may have a transient adverse effect on follicle growth, ovulation, or formation of corpora lutea. A consequence of this effect, transient infertility, has been observed previously when exploring the effect of polycyclic aromatic hydrocarbons on murine reproduction.     

Collagen-induced arthritis in B10.RIII mice (H-2r): identification of an arthritogenic T-cell determinant.

Susceptibility to collagen-induced arthritis (CIA), a murine model of autoimmune arthritis, is strongly linked to only two major histocompatibility complex (MHC) haplotypes, H-2q and H-2r. In order to identify the determinants of type II collagen (CII) required to induce arthritis in H-2r-bearing mice, B10.RIII mice were immunized with bovine, chick or human CII. Only bovine CII induced significant arthritis and autoantibodies. When the major CNBr peptides of bovine collagen were isolated and used for immunization, only mice immunized with CB8, representing CII 403-551, developed arthritis. To identify immunogenic epitope(s) within CB8, a panel of synthetic peptides representing overlapping sequences of the bovine peptide was generated. When each peptide was cultured with T cells from B10.RIII mice immunized with CII, one peptide, representing CII 430-466, contained a major T-cell epitope. By using an in vitro lymphokine production assay, the T-cell epitope was further narrowed to CII 442-456. These findings suggest that a T-cell determinant important for the initiation of arthritis in B10.RIII (H-2r) mice is located within a 15 amino acid sequence, residues 442-456 of bovine CII.     

Phenotype conversion from rheumatoid arthritis to systemic lupus erythematosus by introduction of Yaa mutation into FcγRIIB‐deficient C57BL/6 mice

We previously established an IgG Fc receptor IIB (FcγRIIB)‐deficient C57BL/6 (B6)‐congenic mouse strain (KO1), which spontaneously develops rheumatoid arthritis (RA), but not systemic lupus erythematosus (SLE). Here, we show that when Y chromosome‐linked autoimmune acceleration (Yaa) mutation was introduced in KO1 strain (KO1.Yaa), the majority of KO1.Yaa mice did not develop RA, but instead did develop SLE. This phenotype conversion did not depend on autoantibody specificity, since KO1.Yaa mice, compared with KO1, showed a marked increase in serum levels of both lupus‐related and RA‐related autoantibodies. The increase in frequencies of CD69⁺ activated B cells and T cells, and the spontaneous splenic GC formation with T follicular helper cell generation were manifest early in life of KO1.Yaa, but not KO1 and B6.Yaa, mice. Activated CD4⁺ T cells from KO1.Yaa mice showed upregulated production of IL‐21 and IL‐10, compared with the finding in KO1 mice, indicating the possibility that this aberrant cytokine milieu relates to the disease phenotype conversion. Thus, our model is useful to clarify the shared and the disease‐specific mechanisms underlying the clinically distinct systemic autoimmune diseases RA and SLE.     

The superior olivary complex in C57BL/6 mice

The cellular and cytoarchitectural features of the lateral superior olive, the medial superior olive, the superior paraolivary nucleus and the medial, lateral and ventral nuclei of the trapezoid body are described in C57BL/6 mice using Nissl, Bodian and Golgi techniques. Principal, spindle and marginal cells are present in a well-defined lateral superior olive. The dendrites of these cells run primarily within rostrocaudal sheets as in the cat. The principal cells of the medial nucleus of the trapezoid body are similar to the principal cells in the cat. Large multipolar cells characterize the lateral nucleus of the trapezoid body and bipolar cells with a medial-lateral orientation are found in the medial superior olive. The largest neurons are found in the superior paraolivary nucleus and the lateral superior olive, and the medial and ventral nuclei of the trapezoid body. While brain weight and neuronal packing density change with development, the characteristic location of cell groups and the shape and Nissl-staining pattern of neurons in the youngest brains examined were essentially unchanged in the adult mice, although dendritic maturation had occurred. The homologies of the C57BL/6 superior olivary complex nuclei with the same areas described in other mouse strains, rat and cat are discussed. This study expands our understanding of the organization of the superior olivary complex in an inbred strain of Mus musculus and relates it to other species. The data about changes occurring during postnatal maturation may aid in the interpretation of behavioral and physiological studies of neonatal plasticity of the auditory system.     

Behavioral changes in aging female C57BL/6 mice

Using a range of tests we have studied alterations in behavior with advancing age in female C57BL/6 (of Jackson origin), the golden standard on which most genetically engineered mice are back-crossed. In parallel, growth and survival data were collected. In a protected environment the 90% and 75% cohort survival age was 20 and 25 months, respectively, and the 50% cohort survival was 32 months. In mice, body weight increases continuously until 15-20 months of age, while in advanced age whole body weight drops. The body mass loss in senescence is associated with emergence of other aged phenotype features such as kyphosis, balding and loss of fur-color.Our behavioral data show that aging modulates certain aspects of basic behavior in a continuous manner, like explorative and locomotor activities. Advanced age associates with an acceleration of behavioral impairments evident in most of the tests used, including motor skill acquisition and memory consolidation. However, certain domains of mouse behavior were well preserved also in advanced age such as thermal noxious threshold and working memory as assessed by an object recognition task. The decreased drive to explore is suggested to be a key factor underlying many aspects of reduced performance including cognitive capacity during aging. Behavioral aging affects genetically closely related individuals housed under strictly standardized conditions differentially (Collier, T.J., Coleman, P.D., 1991. Divergence of biological and chronological aging: evidence from rodent studies. Neurobiol. Aging, 12, 685-693; Ingram, D.K., 1988. Motor performance variability during aging in rodents. Assessment of reliability and validity of individual differences. Ann. N.Y. Acad. Sci., 515, 70-96). Consistent with this a subpopulation of the 28-month-old mice showed an explorative activity similar to young-adult mice and a significantly stronger preference for a novel object than aged mice with a less explorative behavior. Thus, subtle environmental factors and epigenetic modifications may be important modulators of aging.     

Hunger and satiety in genetically obese mice (C57BL/6J-ob/ob)

To determine the mechanism for hyperphagia in genetically obese mice (C57BL/6J-ob/ob), several experiments were conducted on the ability of these mice to respond to caloric deficits and surpluses. Presentation of food or sugar reduces subsequent operant licking in both obese and lean mice. When given sugar solutions, evaporated milk, or sweetened non-fat milk, both obese and lean mice reduce food intake to compensate for the calories obtained from the solutions. These findings indicate that genetically obese mice respond normally to caloric surpluses. Obese mice respond to food deprivation (caloric deficit) by increasing subsequent food intake but they do so more slowly than controls.     

2型糖尿病神经病理性痛C57BL/6J小鼠模型的建立

目的利用胃饲酒精和腹腔注射链脲佐菌素(streptozotocin,STZ)建立2型糖尿病神经病理性痛C57BL/6J小鼠模型。方法 80只C57BL/6J小鼠随机分为:对照组(n=15)和实验组(n=65)。对照组每日胃饲生理盐水,12周后单次空腹腹腔注射柠檬酸缓冲液;实验组每日胃饲酒精12周诱导胰岛素抵抗,继以不同剂量链脲佐菌素(40 mg/kg、50 mg/kg、60 mg/kg)腹腔注射1次,于不同时间点分别测体重、血糖、血胰岛素浓度,计算胰岛素抵抗指数、机械缩足阈值和热缩足潜伏期的变化。结果连续胃饲酒精12周后,实验组小鼠体重明显增加,空腹胰岛素浓度升高,胰岛素抵抗指数升高,血糖未升高,机械缩足阈值和热缩足潜伏期无变化。注射STZ后,40 mg/kg剂量组血糖升高但不能长期维持,60 mg/kg剂量组血糖较高,死亡率高;50 mg/kg剂量组血糖中度升高且相对稳定,胰岛素浓度和胰岛素敏感性均降低,其机械缩足阈值和热缩足潜伏期均低于基础值和对照组(P0.05)。结论连续酒精胃饲12周后联合腹腔注射STZ50 mg/kg可以建立理想的2型糖尿病神经病理性痛小鼠模型。     

CD3+ T cells in severe combined immunodeficiency (scid) mice. IV. Graft-vs.-host resistance of H-2d scid mice to intravenous injection of allogeneic H-2b (C57BL/6) spleen cells.

Intraperitoneal injection of 2 x 10(7) nonfractionated spleen cells (SC) from C57BL/6 (B6, H-2b) mice into completely allogeneic immunodeficient H-2d scid mice induced clinical and histological signs of acute graft-vs.-host disease (GVHD), with all transplanted severe combined immunodeficiency (scid) mice dying in the 3rd week post-transfer. In contrast four out of five scid mice survived for greater than 7 weeks after intravenous (i.v.) injections of equal numbers of B6 SC. Intravenously allotransplanted scid mice analyzed in the 8th week post-transfer had engrafted donor-type CD4+ and CD8+ T cells in the spleens but showed no clinical or histological evidence of GVHD. i.v. injection of 10(7) or 10(6)O B6 SC engrafted allogeneic T cells in spleens of scid recipients; in contrast, i.v. injection of 10(5) nonfractionated B6 SC or 3 x 10(5) cell sorter-purified, naive or anti-H-2d-primed splenic CD4+ or CD8+ B6 T cells led to rejection by young scid recipient mice. B6 T cells engrafted into spleens of scid mice after i.v. injection showed proliferative anti-host alloreactivity in vitro. No cytotoxic reactivity against host-type alloantigens was found in standard 4-h 51Cr-release assays. These data demonstrate that allogeneic T cells injected i.v. into immunodeficient scid mice are partially tolerized against host-type alloantigens.     

Simvastatin enhances hippocampal long-term potentiation in C57BL/6 mice

Statins inhibit 3-hydroxy-3-methylglutaryl CoA reductase, the rate-limiting enzyme in the cholesterol biosynthetic pathway, and they are widely used to control plasma cholesterol levels and prevent cardiovascular disease. However, emerging evidence indicates that the beneficial effects of statins extend to the CNS. Statins have been shown to improve the outcome of stroke and traumatic brain injury, and statin use has been associated with a reduced prevalence of Alzheimer's disease (AD) and dementia. However, prospective studies with statins in AD have produced mixed results. Recently, we reported that simvastatin, a widely used statin in humans, enhances learning and memory in non-transgenic mice as well as in transgenic mice with AD-like pathology on a mixed genetic background. However, the cellular and molecular mechanisms underlying the beneficial effects of simvastatin on learning and memory remain elusive. The present study was undertaken to investigate the effect of acute simvastatin treatment on hippocampal long-term potentiation (LTP), a cellular model of learning and memory, in brain slices from C57BL/6 mice. Our results demonstrate that a prolonged in vitro simvastatin treatment for 2–4 h, but not a short-term 20-min exposure, significantly increases the magnitude of LTP at CA3–CA1 synapses without altering basal synaptic transmission or the paired-pulse facilitation ratio in hippocampal slices. Furthermore, we show that phosphorylation of Akt (protein kinase B) is increased significantly in the CA1 region following 2-hour treatment with simvastatin, and that inhibition of Akt phosphorylation suppresses the simvastatin-induced enhancement of LTP. These findings suggest activation of Akt as a molecular pathway for augmented hippocampal LTP by simvastatin treatment, and implicate enhancement of hippocampal LTP as a potential cellular mechanism underlying the beneficial effects of simvastatin on cognitive function.     

The Y chromosome from autoimmune BXSB/MpJ mice induces a lupus-like syndrome in (NZW x C57BL/6)F1 male mice, but not in C57BL/6 male mice

The Y chromosome of the BXSB mouse has been shown to be responsible for the acceleration of lupus-like autoimmune syndrome in inbred BXSB mice and in their F1 hybrids with NZB or NZW mice. To further define the role of this as yet unidentified gene linked to the BXSB Y chromosome, designated Yaa (Y chromosome-linked autoimmune acceleration), the Y chromosome was transferred from the BXSB strain to nonautoimmune C57BL/6 (B6) mice. The effect of the Yaa gene on the autoantibody formation and the development of glomerulonephritis was investigated in B6 mice and in their F1 hybrids with NZW mice. The presence of the BXSB Y chromosome was not able to induce significant autoimmune responses in B6 mice. However, (NZW x B6)F1 males bearing the BXSB Y chromosome developed a severe lupus-like autoimmune syndrome, as documented by the production of anti-DNA antibodies and gp70-anti-gp70 immune complexes and the development of lethal lupus nephritis. Both sexes of (NZW x B6)F1 hybrids without the BXSB Y chromosome were essentially normal. Our results suggest that (a) the BXSB Y chromosome by itself is not sufficient to initiate autoimmune responses in nonautoimmune B6 mice, and (b) it is able to induce autoimmune responses in mice potentially capable of developing the disease, but whose autosomal abnormality by itself is not sufficient to develop autoimmune diseases.     

Immunosuppression after injection of Friend virus into C57BL/6 mice of different ages

Injection with Friend virus (FV) causes immunosuppression in young and old C57BL/6 mice, i.e. it occurs whether or not the virus replicates very briefly or for a long period. There are only minor age-related differences in the extent of immunosuppression, except that suppression appears to persist somewhat longer in old than in young animals.     

Memory lymphocytes of young and old C57BL/6 mice express high levels of class I major histocompatibility complex (H-2 Kb) protein

Memory lymphocytes play a central role in the secondary immune response. The concentration of memory lymphocytes increases with age. A high level of cell surface CD44 is a marker of memory lymphocytes compared to na?ve lymphocytes which express a low level of CD44. Major histocompatibility complex (MHC) class I protein expression also increases with age. To explore a possible correlation between the expression of CD44 and MHC class I protein (Kb), peripheral blood lymphocytes from 27 C57BL/6 mice ranging in age from 3 months to 33 months were isolated by Ficoll-Hypaque gradient centrifugation. Single and double indirect immunofluorescence assays were then performed with rat IgG anti-CD44 and/or mouse IgG anti-Kb as first antibodies, and phycoerythrin (PE) labeled goat anti-rat IgG and/or fluorescein (FITC) labeled goat anti-mouse IgG as second antibodies. Cells were then analyzed by using a FACScan flow cytometer. As expected, the percentage of lymphocytes expressing high levels of CD44 (memory cells) increased significantly with age and the expression of Kb increased significantly with age. Interestingly, the expression of Kb in lymphocytes expressing high levels of CD44 (memory cells) was 72% more than in cells expressing low levels of CD44 (naive cells) regardless of age.     

Cytogenetic studies of ethyl acrylate using C57BL/6 mice

The clastogenicity of ethyl acrylate (EA) was examined in vivo by injecting i.p. five male C57BL/6 mice per dose group with either 125, 250, 500 or 1000 mg/kg EA dissolved in saline. Controls received solvent only. Acrylamide (100 mg/kg), because of its similarity in structure and mode of action to EA, was used as a positive control. Twenty-four hours after injection, the animals were anesthetized and the spleens aseptically removed. Splenocytes were isolated on density gradients and cultured with concanavalin A to stimulate cell division. In half the cultures bromodeoxyuridine was added at 21 h for analysis of chromosome aberrations (CAs) in first division cells and sister chromatid exchange (SCE) in second division cells. In the remaining cultures cytochalasin B was added to produce binucleated cells for scoring of micronuclei (MN). There was no significant increase in SCE or CAs at any of the doses of EA examined. At the highest dose examined (1000 mg/kg), EA did cause a small but significant increase in binucleated cell MN. Acrylamide caused an increase in MN and SCEs in splenocytes. Because others have found EA to be clastogenic in vitro, isolated splenocytes were exposed to a wide range of concentrations of EA during the G0 stage of the cell cycle or 23 h after mitogen stimulation during the late G1 or early S phase of the cell cycle. Although EA was toxic for both exposure regimens, significant increases in chromatid-type aberrations were found only when the target cells were treated 23 h after mitogenic stimulation. No statistically significant increase in SCE frequency was found after either treatment regimen. These data suggest that EA is only clastogenic at near toxic concentrations during a specific stage of the cell cycle.     

Specific recognition and rejection of the H-2-deficient cell line LR.4 By C57BL/6J mice

The humoral immune response developed by C57BL/6J mice against the β₂-microglobulin (β₂m) and major histocompatibility complex (MHC) class I- and class II-deficient cell variant of L5178Y, LR.4, is strain specific, is not linked to a given haplotype and involves at least one antigenic determinant expressed on the cell membrane. Anti-LR.4 antibodies can be detected in the serum and ascitic fluid of tumour-bearing animals, and in the serum of mice immunized with mitomycin C (MC)-treated cells. In vitro, cytotoxic T lymphocytes (CTL) cannot be induced under different experimental conditions. However, recognition and lysis of LR.4 axe mediated by an antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism in which natural killer (NK) cells extracted from the spleen of resistant or susceptible strains are the effector cells. The NK cells responsible for ADCC against LR.4 are not inducible with polyinosinic-polycytidylic acid (poly(I:C)) and could represent a subset that is not detectable by conventional assays. In conclusion, the incapacity of BALB/c and possibly of other strains of mice to reject LR.4 is determined by the failure to mount a humoral immune response.     

Slow disease progression in a C57BL/6 pten-deficient mouse model of prostate cancer

Prostate-specific deletion of Pten in mice has been reported to recapitulate histological progression of human prostate cancer. To improve on this model, we introduced the conditional ROSA26 luciferase reporter allele to monitor prostate cancer progression via bioluminescence imaging and extensively backcrossed mice onto the albino C57BL/6 genetic background to address variability in tumor kinetics and to enhance imaging sensitivity. Bioluminescence signal increased rapidly in Pten(p-/-) mice from 3 to 11 weeks, but was much slower from 11 to 52 weeks. Changes in bioluminescence signal were correlated with epithelial proliferation. Magnetic resonance imaging revealed progressive increases in prostate volume, which were attributed to excessive fluid retention in the anterior prostate and to expansion of the stroma. Development of invasive prostate cancer in 52-week-old Pten(p-/-) mice was rare, indicating that disease progression was slowed relative to that in previous reports. Tumors in these mice exhibited a spontaneous inflammatory phenotype and were rapidly infiltrated by myeloid-derived suppressor cells. Although Pten(p-/-) tumors responded to androgen withdrawal, they failed to exhibit relapsed growth for up to 1 year. Taken together, these data identify a mild prostate cancer phenotype in C57BL/6 prostate-specific Pten-deficient mice, reflecting effects of the C57BL/6 genetic background on cancer progression. This model provides a platform for noninvasive assessment of how genetic and environmental risk factors may affect disease progression.