Therapeutic effects of IL-6 and BAFF fusion protein eukaryotic expression plasmid on animal model of Sj?gren′s syndrome / 中华微生物学和免疫学杂志

Objective:To investigate the effects of a eukaryotic expression plasmid for IL-6 and B-cell activating factor (BAFF) fusion protein on the histopathological changes in salivary and lacrimal glands of non-obese diabetic (NOD) mice with Sj?gren′s syndrome and to elucidate the possible therapeutic mechanism of IL-6/BAFF fusion protein eukaryotic expression plasmid in NOD mice.Methods:The eukaryotic expression plasmid for IL-6/BAFF fusion protein was constructed. After transfecting CHO cells with the plasmid, the expression of IL-6/BAFF fusion protein was detected by Western blot. BALB/c mice were injected with the plasmid every two weeks for three times and the titers of anti-IL-6 and anti-BAFF antibodies were measured by ELISA. Twenty-one NOD mice were randomly divided into three groups (control group, empty vector group and therapy group) by numerical table method. The mice in the therapy group were injected with the IL-6/BAFF fusion protein eukaryotic expression plasmid once a week for six times and the mice in the empty vector group were injected with empty plasmid. The levels of anti-IL-6 and anti-BAFF antibodies as well as cytokines (IL-6, BAFF, INF-γ, IL-10 and IL-17A) in mouse serum samples were detected by ELISA. The proportions of Th17, Treg, Th1 and Th2 cells in mouse splenocytes were measured by flow cytometry. Focal lymphocyte infiltration and pathological changes in the lacrimal and salivary glands of mice were observed under light microscopy after HE staining.Results:The eukaryotic expression plasmid for IL-6/BAFF fusion protein increased the levels of anti-IL-6 and anti-BAFF antibodies in the serum of BALB/c mice ( P<0.05). The levels of anti-IL-6 and anti-BAFF antibodies in the serum of NOD mice in the therapy group increased ( P<0.01), while the expression of IL-6, BAFF, INF-γ, IL-10 and IL-17A in NOD mice in the therapy group was lower than that in the control group and the empty vector group ( P<0.05). The percentages of Treg and Th2 cells in the splenocytes of NOD mice increased after treatment ( P<0.05). Moreover, the eukaryotic expression plasmid for IL-6/BAFF fusion protein significantly improved the irregular size and morphology of glandular vesicles in the lacrimal and salivary glands, reduced the ductal dilatation and decreased the focal lymphocyte infiltration in NOD mice. Conclusions:The eukaryotic expression plasmid for IL-6/BAFF fusion protein induced the production of anti-IL-6 and anti-BAFF antibodies, decreased the expression of inflammatory cytokines, regulated the balance of Th17/Treg and Th1/Th2 cells, improved the irregular alveolar structure and ductal dilation in the lacrimal and salivary glands and reduced the focal lymphocyte infiltration in NOD mice. This study showed that eukaryotic expression plasmid for IL-6/BAFF fusion protein might serve as a potential target for therapeutic targeting of T and B cells.

Effect of topical injection of cyclosporine A on saliva secretion and inflammation in the submandibular gland of non-obese diabetic mice / 北京大学学报(医学版)

OBJECTIVE@#To investigate the effects of topical administration of cyclosporine A (CsA) on salivary secretion and inflammation of the submandibular glands in non-obese diabetic (NOD) mice.@*METHODS@#Female NOD mice, 21 aged 14 weeks and 18 aged 21 weeks were selected and randomly divided into low-dose group, high-dose group and control group on average. CsA was injected into submandibular glands. One week later the saliva stimulated by pilocarpine was collected and measured. The submandibular glands were collected to make paraffin sections. The lymphocyte infiltration in submandi-bular gland was observed by microscope after hematoxylin-eosin (HE) staining. The number of lymphocyte infiltration foci was counted to calculate the focus sore and the ratio of lymphocyte infiltration area to total gland area was figured up by Leica image analysis system. The expressions of inflammatory cytokines tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-4 (IL-4), IL-13, IL-17F, IL22 and IL-23a in the submandibular glands of the NOD mice were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell apoptosis in the submandibular gland was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The levels of serum creatinine (Scr), blood urea nitrogen (BUN), uric acid (UA), alanine aminotransferase (ALT), aspertate aminotransferase (AST), alkaline phosphatase (ALP), albumin (ALB) and γ-glutamyl transferase (GGT) were measured by automatic biochemical analyzer to evaluate liver and kidney functions.@*RESULTS@#After topical injection of CsA in the submandibular gland, the stimulated salivary flow rate of the 14- and 21-week-old NOD mice significantly increased compared with the control group (P < 0.01 or P < 0.05), and the number and area of lymphocyte infiltration foci in the 14-week-old NOD mice low-dose group significantly decreased compared with the control group (P < 0.01). Low and high dose of CsA had similar effects on reducing inflammation and improving salivary secretion. The overall level of inflammatory cytokines in the submandibular gland did not decrease significantly. The number of cell apoptosis of submandibular gland in the NOD mice treated with CsA decreased compared with the control group, but there was no statistically significant difference. Topical injection of CsA had no adverse effect on liver and kidney function in the NOD mice.@*CONCLUSION@#Topical injection of CsA can reduce lymphocyte infiltration in submandibular gland of NOD mice and improve salivary secretion.

Mechanism of immunotolerence induced by bone mesenchymal stem cell co-transplanted with islets through regulating Tfh in NOD mice / 中华肝胆外科杂志

Objective To study the regulation mechanism of bone mesenchymal stem cell (MSC)combined co-translation of islets in differentiation of Follicular Helper T cell (Tfh),and its roll on immunotolerence induction in non-obese diabetic (NOD) mice transplantation model.Methods The NOD mice were divided into 4 groups:Group A with islet transplantation alone;Group B with MSC co-transplantation with islets (MSC:0.5 × 106);Group C with MSC co-transplantation with islets (MSC:2 × 106);Group D with MSC co-transplantation with islets (MSC:3 × 106).ELISA was used to test the expression level of diabetes autoantibody GAD65Ab and IAA.Tfh cell count was detected by FACS.Results The survival time of transplantation groups was much longer in MSCs co-transplantation group than islet-alone group;the level of GAD65Ab,IAA and Tfh cell count were much lower in MSCs co-transplantation group than islet-alone group.Conclusion MSC may protect the islet transplants by regulating the Tfh cell differentiation.

Modelos experimentais parao estudo do diabetes tipo 1: [revisão] / Animal models for type 1 diabetes studies: [review]

Os modelos animais de diabetes têm sido usados extensivamente na obtenção do esclarecimento sobre esta doença. O objetivo deste estudo foi realizar uma revisão bibliográfica sobre os principais modelos experimentais para o estudo do diabetes mellitus. Dentre os modelos experimentais para o estudo do diabetes, existem os modelos induzidos quimicamente por aloxana e streptozotocina, sendo que a dose utilizada depende da espécie do animal e do seu peso. Além disso, existem dois excelentes modelos de diabetes espontâneo: os ratos BB (Biobreading) e os camundongos NOD (Non ObeseDiabetic). Os camundongos NOD são o modelo mais estudado de doença espontânea auto-imune órgão-específico em todo o mundo. As razões para a preferência deste modelo incluem um genoma bem definido, maior quantidade de reagentes monoclonais para a análise de componentes do sistema imune e um custo razoavelmente baixo, comparado com a utilização de ratos. Estes camundongos exibem autoimunidade espontânea com destruição das ilhotas pancreáticas, de forma semelhante à observada em humanos. A destruição auto-imune é caracterizada por insulite e infiltrado leucocitárionas ilhotas pancreáticas. Esta infiltração é composta predominantemente por células dendríticas, macrófagos, por células TCD4, TCD8 e células B. Os fatores ambientais em conjunto com a genética, claramente modificam a incidência do diabetes tipo 1 nos modelos experimentais espontâneos. A suscetibilidade destes camundongos é poligênica e ambiental, enfatizando condições de habitação, sanitárias, dietéticas e de gênero. A incidência de diabetes em camundongos NOD é aproximadamente quatro vezes maior em fêmeas do que em machos. As informações obtidas através deste excelente modelo animal podem ser relevantes para O entendimento do processo da doença nos humanos.

The animal models of diabetes have been used extensively in obtaining the information on this disease. The objective of this study was a literature review on the main experimental models for the study of diabetes mellitus. Among the experimental models for the study of diabetes, the models are chemically induced by aloxan and streptozotocin, and the dose used depends on the species of the animal and its weight. Also, there are two excellent models of spontaneous diabetes: the BB rats (Biobreading) and NOD mice (Non Obese Diabetic). The NOD mice are the most studied model of spontaneous self immune disease-specific body in the world. The reasons for the preference genome of this model include a well-defined, greater quantity of monoclonal reagents for the analysis of components of the immune system and a reasonably low cost, compared with the use of rats. These mice exhibit spontaneous autoimmunity with destruction of pancreatic is lets, in a manner similar to that seen in humans. The auto-immune destruction is characterized by insulite in pancreatic is lets. This infiltration is composed predominantly of dendritic cells, macrophages, CD4 T cells, CD8 cells and B. The environmental factors together with the genetics, clearly alter the incidence of type 1 diabetes in experimental models spontaneous. The susceptibility of these mice is genetics and environment, emphasizing adequate housing, health, diet and gender. The incidence of diabetes in NOD mice is about four times higher in females than in males. Information obtained through this excellent animal model may be relevant to the understanding of the process of the disease in humans.

Preliminary study of alleviating new onset diabetic NOD mice by a DPP-Ⅳ inhibitor / 中华内分泌代谢杂志

To investigate the effect of NVP-DPP728, a DPP-Ⅳ inhibitor on new-onset diabetes and the autoimmune response in non-obese diabetic ( NOD ) mice. Diabetes could be reversed in 75% of NVP-DPP728 treated 20 NOD mice. In these 15 mice with remission, insulitis scores were significantly lower than those of the control group. The percentage of Tregs was increased in the thymus and celiac lymph nodes, plasma TGF-β1 and GLP-1 were also significantly increased ( P＜0. 01 ). NVP-DPP728 treatment may reverse new-onset diabetes in NOD mice by reducing insulitis and increasing Tregs.

IL-10 Plasmid DNA Delivery in NOD Mice for the Prevention of Autoimmune Pancreatic Beta Cell Destruction / 대한내분비학회지

BACKGROUND: Recently, we have reported that biodegradable poly [-(4-aminobutyl)-L-glycolic acid] (PAGA) can condense and protect plasmid DNA from DNase I. In this study, we investigated whether the systemic administration of pCAGGS mouse IL-10 (mIL-10) expression plasmid complexed with PAGA can reduce the development of insulitis in non-obese diabetic (NOD) mice. METHODS: PAGA/mIL-10 plasmid complexes were stable for more than 60 minutes, but the naked DNA was destroyed within 10 minutes by DNase I. The PAGA/DNA complexes were injected into the tail vein of 3 week-old NOD mice. RESULTS: Serum mIL-10 level peaked at 5 days after injection, could be detected for more than 7 weeks. The prevalence of severe insulitis at 12 week-old NOD mice was markedly reduced by the intravenous injection of PAGA/DNA complex (15.7%) compared to that of naked DNA injection (34.5%) and non-treated controls (90.9%). In conclusion, systemic administration of pCAGGS mIL-10 plasmid/PAGA complexes can reduce the severity of insulitis in NOD mice. CONCLUSION: The study presents the PAGA/DNA complex has the potential for the application of the prevention of autoimmune diabetes mellitus.