Disease-protected major histocompatibility complex Ea-transgenic non-obese diabetic (NOD) mice show interleukin-4 production not seen in susceptible Ea-transgenic and non-transgenic NOD mice.

The non-obese diabetic (NOD) mouse is an animal model for insulin-dependent diabetes that has many similarities to the human disease. NOD mice transgenic for the Ea gene, allowing expression of the E molecule, are protected from diabetes and rarely develop insulitis. An Ea transgene mutated in the promoter region, (DeltaY) lacks E expression on most B cells, thymic medullary epithelium and primary antigen-presenting cells, and confers no protection whatsoever. We have used these transgenic NOD mice, together with non-transgenic NOD mice, to study the correlation of E expression and production of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma). We show that protected E-transgenic NOD mice have elevated levels of IL-4 compared with non-transgenic mice, both in the thymus and in the periphery. However, susceptible DeltaY-transgenic mice have elevated thymic IL-4 levels, but express almost as little IL-4 as non-transgenic NOD mice in the periphery. This drop in peripheral IL-4 production seen in DeltaY-transgenic mice thus correlates with the decreased E expression in the periphery of DeltaY-transgenic NOD mice. In contrast, there were no differences in IFN-gamma production between the three NOD lines. We suggest that Ea-transgenic NOD mice have E-selected regulatory T cells producing IL-4, which are subsequently activated by E-expressing primary antigen-presenting cells in the periphery. This activation would then be instrumental for the E-mediated protection from disease in NOD mice. Such a process would explain the total absence of protection in DeltaY-transgenic NOD mice, despite their widespread E expression.     

Insulin autoantibodies in non-obese diabetic (NOD) mice.

Anti-insulin autoantibodies were detected in NOD mice using ELISA. The antibodies were detected as early as at 5 weeks of age, long before onset of clinically overt diabetes, especially in diabetes-prone female mice. The anti-insulin specificity was verified by passage on affinity chromatography insulin columns and demonstration that the anti-insulin activity was located on the F(ab')2 region of the immunoglobulins. The presence of anti-insulin antibodies in prediabetic NOD mice provides a unique possibility for studying their significance and their eventual pathogenic role in the development of insulin-dependent diabetes.     

Absence of significant Th2 response in diabetes-prone non-obese diabetic (NOD) mice.

Accumulating evidence suggests that Th1 T cells play a pivotal role in the development of autoimmune diabetes. Conversely, promoting a Th2 response inhibits disease progression. However, it has not been determined whether Th2 cells are regulatory T cells that fail at the time of diabetes development in naive non-diabetic NOD mice. Therefore, in order to evaluate cytokine secretion by spleen and islet infiltrating T cells in NOD mice at different stages of the autoimmune process, we developed an ELISPOT assay that detects IL-2, IL-4, and interferon-gamma (IFN-gamma) secretion in vitro at the single-cell level. We showed that, whatever the age considered, IFN-gamma is predominantly secreted, and that no IL-4-secreting cells are detected in the islets of male and female NOD mice. Spleen cells from 8-week-old female NOD mice, which include regulatory suppressor T cells, do not secrete IL-4, either upon presentation of islet cell antigens in vitro, or after transfer in vivo, but do secrete IFN-gamma. IFN-gamma secretion by T cells from diabetic mice results from CD4 but not CD8 T cells in transfer experiments into NOD/severe combined immunodeficient (SCID) recipients. These results suggest that (i) detection of regulatory CD4 T cells in NOD mice is not paralleled by a Th2 response; (ii) beta cell destruction does not depend on a switch from a Th2 to a Th1-type response; and (iii) CD8 T cells do not participate in induction of diabetes by secreting IFN-gamma.     

Rapamycin prevents the onset of insulin-dependent diabetes mellitus (IDDM) in NOD mice.

The effect of the immunosuppressive agent rapamycin (RAPA) was assessed in the non-obese diabetic (NOD) mouse which is an autoimmune model of IDDM. RAPA was prepared in a vehicle of 8% cremophor EL/2% ethanol and investigated in two studies. NOD/MrK female mice (six per group, study no. 1; 10 per group, study no. 2) were dosed three times per week p.o. by gavage from 56 to 170 days of age (study no. 1) or from 64 to 176 days of age (study no. 2). Mice treated with RAPA at 0.6 mg/kg, 6 mg/kg, or 12 mg/kg maintained normal plasma glucose through 170 or 176 days of age with 10%, 0%, and 0% incidence of diabetes respectively. In contrast, naive, vehicle-treated, or RAPA 0.06 mg/kg-treated mice exhibited elevated plasma glucose and disease incidence typical for female NOD mice. Mice which became diabetic had elevated levels of beta-hydroxybutyrate, triglycerides and cholesterol. These plasma lipid concentrations were positively correlated with the duration of hyperglycaemia (r = 0.85, 0.87 and 0.84 respectively). Outside of its ability to prevent diabetes, RAPA itself did not affect the lipid profile of the mice. Intervention therapy with RAPA was ineffective at reversing the course of disease after IDDM onset under these experimental conditions. Finally, we report here that prophylactic treatment with RAPA was able to protect against IDDM development in some RAPA-treated mice 41 weeks after cessation of treatment. These data show that orally administered RAPA is effective in preventing onset of disease in the NOD mouse, a relevant model of autoimmune type I diabetes in man.     

Accumulation ofIodine Labeled Interleukin-2 in the Pancreas of NOD Mice

Interleukin-2 (IL-2) is an important cytokine in the autoimmune process proceeding Type 1 diabetes. Our aim was to investigate, in two previously used animal models, the NOD mouse and the BB/W rat, the in vivo tissue distribution of radio-labeled IL-2. If the radio-labeled IL-2 accumulated significantly in the pancreas compared to surrounding organs it could allow imaging of lymphocyte infiltration of the islets of Langerhans by scintigraphic methods. IL-2 was labeled enzymatically with¹²⁵Iodine. Radio-labeled IL-2 was injected iv in prediabetic NOD mice, diabetic NOD mice and Balb/c mice in the first animal model and in BB rats in the second model. Animals were sacrificed at different time points and the activity in different organs was measured. It was found that the mean activity in the pancreas in both diabetic and prediabetic NOD mice was significantly higher compared to pancreas from Balb/c mice (P< 0.001 and P=0.005, respectively). However, the mean activity in the pancreas was at the lower range of the surrounding organs in both animal models, thereby excluding the possibility of imaging the autoimmune process by scintigraphic methods. It is concluded that radio-labeled IL-2 did accumulate significantly in the pancreas of NOD mice compared to control mice but there is a need to develop new techniques in order to visualize the localized activity.     

H-Y responses of non-obese diabetic (NOD) mice

Female non-obese diabetic (NOD) mice were tested for their ability to make responses to the male-specific (H-Y) transplantation antigen. In vivo assessment of this ability was made using skin graft rejection. A proportion (60%) spontaneously rejected NOD male tail skin by 80 days post-transplantation. The detection of the generation of H-Y-specific cytotoxic T cells, following in vivo priming and secondary in vitro restimulation, was carried out using a conventional 51Cr release assay. Female NOD mice primed either by skin grafting, intraperitoneal (i.p.) or footpad (f.p.) injection of male NOD spleen cells could be induced to make anti-H-Y cytotoxic responses, but not every immunized mouse responded. The nature of the H-Y-reactive T cells was investigated further by the in vitro isolation of T-cell clones of which some were H-Y specific.     

Spontaneous autoimmune thyroiditis in NOD.H-2h4 mice

NOD.H-2h4 mice, which express I-Ak on the NOD genetic background, spontaneously develop autoimmune thyroiditis (SAT) and anti-mouse thyroglobulin (MTg) autoantibodies. The incidence of SAT is nearly 100% in mice of both sexes 6-8 weeks after administration of 0.05% NaI in the drinking water. After reaching maximum severity, inflammation is chronic over the next 3-4 months. All mice that develop thyroid lesions also produce MTg-specific IgG1 and IgG2b autoantibodies. Thyroid lesions and anti-MTg autoantibodies did not develop in CBA/J (H-2(k)) or NOD.SWR(H-2(q)) mice after administration of NaI water. Both CD4(+)and CD8(+)T cells are involved in the initial development of SAT. Depletion of CD4(+), but not CD8(+), T cells after thyroid lesions have developed also markedly reduced SAT severity, indicating that CD4(+)T cells are required for both developing and maintaining SAT. Analysis of cytokine gene expression indicated that both Th1 and Th2 cytokines were expressed in thyroids of NOD.H-2h4 mice with SAT. Th1 and proinflammatory cytokines were maximally expressed 4-6 weeks after mice began receiving NaI water, while Th2 cytokine gene expression was greatest at 8-15 weeks, when lesions had reached maximal severity and were in the chronic phase. TGF-beta was highly expressed in NOD.H-2h4 thyroids, irrespective of whether the mice had received NaI water or had thyroid lesions.     

Immunobiology of DC in NOD mice.

NOD mice spontaneously develop diabetes between 15 and 20 weeks of age, which is preceded by insulitis characterized by the infiltration of lymphocytes. Dendritic cells (DC) are among the first cells to infiltrate the islet and they have been implicated in the pathogenesis of the disease. Our work has been concerned with the detailed characterization of four distinct DC populations in NOD mice: two derived from bone marrow (BM) cells cultured in either granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4) or GM-CSF alone and two from the spleen of Flt3 ligand (Flt3L) -treated mice, isolated on the basis of CD8alpha expression. Phenotypic and functional differences between these DC subsets in NOD mice have been identified. In addition, we obtained a lower yield of NOD BM-derived DC and they expressed higher levels of cell-surface CD40 and IL-12 p40 mRNA than BM-derived DC from the diabetes-resistant strain, B10.BR. We have also investigated the ability of these DC populations to modulate the development and progression of diabetes in NOD mice.     

Poly(I∶C)加重碘过量诱发的NOD鼠慢性淋巴细胞性甲状腺炎

目的:探讨poly(I∶C)作为病毒类似物对碘过量所致NOD鼠慢性淋巴细胞性甲状腺炎的影响。方法:将32只雌性NOD鼠,随机分为4组,每组8只,分别为:(1)对照组;(2)碘过量组;(3)poly(I∶C)组;(4)碘过量联合poly(I∶C)组。作用9周后处死动物:观测体重、甲状腺重量及其解剖形态;采用放射免疫分析法检测血清中甲状腺激素(T4)水平;HE染色观察甲状腺组织形态变化,TUNEL技术检测甲状腺组织细胞凋亡情况,定量PCR法检测凋亡相关基因(TRAIL、TRAIL-sR1)和炎症相关基因(ICAM-1、CXCL10)mRNA表达。结果:碘过量组较对照组、Poly(I∶C)组甲状腺绝对重量及相对重量均明显增加(P0.01);血清总T4水平下降(P0.05);炎症破坏和细胞凋亡明显加强;上调TRAIL、TRAIL-sR1、ICAM-1、CXCL10mRNA表达(P0.05)。碘过量联合poly(I∶C)组较单纯碘过量组甲状腺绝对重量及相对重量均进一步增加;血清总T4水平显著下降(P0.05);炎症进一步加重,破坏程度IV级所占比例升至50.0%;凋亡细胞数量明显增加,进一步上调TRAIL、TRAIL-sR1、ICAM-1、CXCL10mRNA表达(P0.05)。Poly(I∶C)组各值与对照组趋势一致(P0.05)。结论:Poly(I∶C)加重碘过量所致慢性淋巴细胞性甲状腺炎发病程度,其作用途径与增加淋巴细胞浸润和甲状腺细胞凋亡有关。     

Analysis of mercury-induced immune activation in nonobese diabetic (NOD) mice

In susceptible mice, the heavy metal ion mercury is able to induce a strong immune activation, which resembles a T helper 2 (Th2) type of immune response and is characterized by a polyclonal B cell activation, formation of high levels of IgG1 and IgE antibodies, production of autoantibodies of different specificities and development of renal IgG deposits. In the present study, we analysed the in vivo effects of mercury in nonobese diabetic (NOD) mice, which is believed to develop a spontaneous Th1 cell-mediated autoimmune diabetes similar to type 1 diabetes in humans. Three weeks of treatment with mercury induced a strong Th2 like immune/autoimmune response in NOD mice. This response was characterized by an intensive increase in splenic IgG1 antibody secreting cells, a marked elevation in serum IgE levels, a substantial increase in splenic IL-4 mRNA, but a significant decrease in splenic IFN-gamma mRNA. Mercury-induced IgG1 antibodies were mainly against ssDNA, TNP and thyroglobulin, but not against nucleolar antigen. Moreover, mercury-injected NOD mice developed high titres of IgG1 deposits in the kidney glomeruli. We further tested if the generated Th2 response could interfere with the development of insulitis and diabetes in NOD mice. We found that three weeks of treatment with mercury was also able to significantly suppress the development of insulitis and postpone the onset of diabetes in these mice. Thus, mercury-induced immune activation can counter-regulate the Th1 cell-mediated autoimmune responses and confer a partial protection against autoimmune diabetes in NOD mice.     

Accelerated diabetes in non-obese diabetic (NOD) mice differing in incidence of spontaneous disease.

The NOD mouse is an established model of autoimmune diabetes mellitus. Various lines of NOD mice differ in their incidence of spontaneous diabetes, e.g. 93% of female NOD/Lt mice compared with 46% of female NOD/Wehi mice develop diabetes by 250 days. These two lines were studied under conditions which greatly accelerate the onset of hyperglycaemia. It was hoped that their responses to these manipulations would reveal characteristic differences which would increase our understanding of diabetes resistance in the low incidence NOD/Wehi line. One dose of 300 mg/kg of cyclophosphamide (CP) produced hyperglycaemia in 50% of NOD mice within 2 weeks in both lines. They were also equally susceptible to diabetes induced by splenocyte transfer at 21 days of age from prediabetic 150-day-old NOD/Lt or NOD/Wehi females. Five daily 40 mg/kg doses of streptozotocin (STZ) resulted in a severity of diabetes in the NOD mice greater than in C57BL or SJL/mice. While the incidence and severity of diabetes induced in the two NOD lines were similar, this appeared to be principally due to sensitivity to the toxic effects of STZ rather than its ability to exacerbate autoimmune beta cell destruction. It has previously been shown that it is possible to prevent diabetes in susceptible NOD mice with simple, relatively benign therapies and here we show that it is possible to induce diabetes in resistant animals at a rate indistinguishable from fully predisposed individuals. It therefore appears that the prediabetic NOD mouse is poised in an immunologically precarious state with the onset of disease being highly dependent on factors which exacerbate or moderate autoimmune destruction.     

Inhibition of autoimmune diabetes in NOD mice with serum from streptococcal preparation (OK-432)-injected mice.

We have recently reported that systemic and chronic administration of recombinant tumour necrosis factor alpha (TNF-alpha), as well as streptococcal preparation (OK-432), inhibits development of insulin-dependent diabetes mellitus (IDDM) in NOD mice and BB rats, models of IDDM. In this study we examined whether serum containing endogenous TNF induced by OK-432 injection could inhibit IDDM in NOD mice. Treatment twice a week from 4 weeks of age with OK-432-injected mouse serum, which contained endogenous TNF (75U), but not IL-1, IL-2 and interferon-gamma (IFN-gamma) activity, reduced the intensity of insulitis and significantly inhibited the cumulative incidence of diabetes by 28 weeks of age in NOD mice, as compared with the incidence in non-treated mice (P less than 0.01) and in mice treated with control serum (P less than 0.02). This inhibitory effect of the serum was diminished, although not significantly, by neutralization of serum TNF activity with anti-mouse TNF antibody. In the mice treated with the serum from OK-432-injected mice, Thy-1.2+ or CD8+ spleen cells decreased (P less than 0.01) and surface-Ig+ (S-Ig+) cells increased (P less than 0.05), whereas the proliferative response of spleen cells to concanavalin A (P less than 0.01) and lipopolysaccharide (P less than 0.05) increased. The results indicate that the inhibition by OK-432 treatment of IDDM in NOD mice was partially mediated by serum factors including endogenous TNF.     

Dosimetric study of the new Intersource125 iodine seed.

The use of low energy photon emitters for brachytherapy applications, as in the treatment of the prostate or of eye tumors, has significantly increased these last few years. New seed models for 125I have been recently introduced. The aim of this study is to determine the dosimetric parameters as recommended by the AAPM in the TG43 formalism for a new iodine seed design: the InterSource125 (Furnished by IBt, Seneffe, Belgium). Measurements are made with LiF thermoluminescent dosimeters (size of 1 mm3) in solid water phantoms to obtain the dose constant, the radial dose function, and the anisotropy function. The TLDs were calibrated at 6 MV and an energy correction factor of 1.41 has been applied. The same dose parameters are also obtained by Monte Carlo calculations (MCNP4B) in solid water and in liquid water. The radial function was measured at 1, 1.5, 2, 3, 4, 5, 6, and 7 cm and calculated between 0.3 and 7 cm. The anisotropy functions were measured at 2, 3, and 5 cm and calculated between 0.3 and 7 cm. The calculated and the measured TG43 functions for solid water are in excellent agreement. We have then calculated these functions in liquid water to obtain the dosimetric information for clinical applications as per TG43 recommendations. In WTI, the calculated dose rate constant is 0.98+/-1% and the measured value is 1.03 +/- 7 %. The calculated value for water is 1.02+/- 1 %. In conclusion, the dosimetric functions for the new iodine seed InterSource125 have been determined. They are quite different from the data of the well-known model 6711 from Amersham due to the absence of silver in the new seed. The characteristics are very similar to those of model 6702.     

~(125)I标记重组融合蛋白dTMP-GH在小鼠体内分布的实验研究

目的研究重组融合蛋白dTMP-GH在小鼠体内的分布情况,明确其是否具有靶向分布特点。方法实验室制备dTMP-GH重组融合蛋白;用放射性125I标记融合蛋白dTMP-GH后,按100μg/kg的标准小鼠尾静脉注射125I-dTMP-GH,分别于给药后5 min、15 min、30 min、1 h、2 h、4 h、8 h、12 h、24 h取心、肝、脾、肾、股骨、甲状腺等进行放射性计数。结果制备得到纯度大于98%的重组融合蛋白dTMP-GH;125I标记率为71.53%,放化纯度为96.53%,比活度为0.22 MBq/μl;尾静脉注射125I标记的dTMPGH后30 min股骨的放射性计数占到注射总量的10%,并随时间推移经肝脏和肾脏代谢。结论融合蛋白dTMP-GH经尾静脉注射后在小鼠体内主要分布于骨髓,具有骨髓组织偏向分布特点。     

Experimental study on the effects of chronic iodine excess on thyroid function, structure, and autoimmunity in autoimmune-prone NOD.H-2h4 mice

Iodine is an essential component of thyroid hormones; high intake may lead to thyroid disease. Epidemiologic researches have shown that exposure to iodine may be involved in the onset and development of autoimmune thyroiditis. Iodine may induce hypothyroidism in patients with autoimmune thyroiditis in a dose-dependent manner. The aim of the present study was to investigate the effects of dosages of iodine on thyroid autoimmunity, morphology, structure, and function in autoimmune-prone animals. NOD.H-2h4 mice were randomly divided into normal iodine, 5-fold, 10-fold, 100-fold, 1,000-fold iodine excess group, anesthetized by diethyl ether and bleed from eye socket vein at 4, 8, 16, and 24 weeks after the commencement of experiment. Iodine and thyroid hormone levels in the thyroid tissue and sera, serum thyroglobulin antibody levels, as well as thyroid gland histological appearance were measured. Excessive iodine caused thyroid goiter, and there was a positive correlation between the thyroid weight and the dosage of iodine (r = 0.64–0.92, P < 0.01). Iodine overdose ultrastructurally damaged thyroid epithelial cells in a dose-dependent manner. The incidence of thyroiditis, as well as the degree of lymphocytic infiltration in the thyroid gradually increased as the dosage increased (r = 0.87–0.98, P ≤ 0.05). Excessive iodine intake might induce goiter, lead to thyroiditis, worsen lymphocytic infiltration, as well as damage to the thyroid follicular structure in a dose-dependent manner in autoimmune-prone NOD.H-2h4 mice.     

β2-Glycoprotein I-dependent and -independent anticardiolipin antibody in non-obese diabetic (NOD) mice

In the present study we investigate whether or not anticardiolipin antibody (aCL) is produced in NOD mice, which is a representative animal model of insulin-dependent diabetes mellitus (IDDM). We found that IgG class aCL appeared in 6.9% of non-diabetic NOD mice at weeks 5–15. The rates increased with age to 31.6% at weeks 16–25 and 71.9% at weeks 26–35. In addition, the titre and incidence of aCL were higher in diabetic mice than in non-diabetic mice. It was also found that aCL in NOD mice involved β₂-glycoprotein I (β₂-GPI)-dependent and -independent aCL, when β₂-GPI was added to the aCL assay system. The IgG subclass of both β₂-GPI-dependent and -independent aCL belonged exclusively to IgG2a. In addition, immunohistochemical studies revealed the predominant accumulation of IgG2a- or IgG3-positive B lymphocytes within insulitis. These results suggest that the autoimmunity in NOD mice may thus be associated with Th1 predominant immunological response. In conclusion, aCL with multiple antigenic specificity were produced in NOD mice along with the development of insulitis and diabetes. NOD mice should thus be added to the list of animal models possessing antiphospholipid antibody.     

Busulfan produces efficient human cell engraftment in NOD/LtSz-Scid IL2Rgamma(null) mice

Xenografting immunodeficient mice after low-dose irradiation has been used as a surrogate human hematopoietic stem cell (HSC) assay; however, irradiation requires strict and meticulous animal support and can produce significant mortality rates, limiting the usefulness of this model. In this work, we examined the use of parenteral busulfan as an alternative conditioning agent. Busulfan led to dose-dependent human HSC engraftment in NOD/LtSz-scid/IL2Rgamma(null) mice, with marked improvement in survival rates. Terminally differentiated B and T lymphocytes made up most of the human CD45+ cells observed during the initial 5 weeks post-transplant when unselected cord blood (CB) products were infused, suggesting derivation from existing mature elements rather than HSCs. Beyond 5 weeks, CD34+-enriched products produced and sustained superior engraftment rates compared with unselected grafts (CB CD34+, 65.8% +/- 5.35%, vs. whole CB, 4.27% +/- 0.67%, at 24 weeks). CB CD34+ group achieved significantly higher levels of engraftment than mobilized CD34+-enriched peripheral blood (PB CD34+). At 8 weeks, all leukocyte subsets were detected, yet human red blood cells (RBCs) were not observed. Transfused human red cells persisted in the chimeric mice for up to 3 days; an accompanying rise in total bilirubin suggested hemolysis as a contributing factor to their clearance. Recipient mouse-derived human HSCs had the capacity to form erythroid colonies in vitro at various time points post-transplant in the presence of human transferrin (Tf). When human Tf was administered singly or in combination with anti-CD122 antibody and human cytokines, up to 0.1% human RBCs were detectable in the peripheral blood. This long evasive model should prove valuable for the study of human erythroid cells.     

Specific autoantibody slows the rapid plasma clearance of 125I-SS-B (La) in mice.

In BALB/c mice the plasma clearance of intravenously (i.v.) injected 125I-SS-B (La) (T 1/2 = 3 min) is markedly delayed when complexed in vitro to specific autoantibody (T 1/2 = 60 min) and is associated with diminished hepatic and renal uptake. The in vivo behaviour of 11S 125I-SS-B-IgG-anti-SS-B complexes was similar to that of 20-30S 125I-heat-aggregated IgG. The presence of anti-SS-B antibodies in systemic lupus erythematosus and Sjögren's syndrome could similarly result in persistence of SS-B containing immune complexes and provide a mechanism which may perpetuate autoimmunity.