Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by the self-destruction of insulin-producing β cells. Recently, studies have revealed that neutrophils contribute to the early pathological injury to the pancreas, predominantly via the formation of neutrophil extracellular traps (NETs). To determine whether early intervention targeting NETs with staphylococcal nuclease (SNase) can delay the onset of T1DM, non-obese diabetic (NOD) mice were orally administered recombinant Lactococcus lactis (L. lactis) expressing SNase. The results showed that NETs were effectively disrupted by SNase both in vivo and in vitro, leading to a significant decrease in neutrophil-derived circulating free DNA (cf-DNA/NETs), neutrophil elastase (NE), and protease 3 (PR3) in the serum compared with the controls. In addition, SNase effectively regulated the blood glucose levels of NOD mice, and the onset of diabetes was postponed with reduced mortality and morbidity. Recombinant L. lactis also ameliorated inflammation in NOD mice, as evidenced by the remarkable increase in IL-4 and reductions in TNF-α and CRP. Moreover, HE staining results showed that L. lactis expressing SNase exerted protective effects on pancreatic islets and relieved inflammation of the small intestine in NOD mice. Hence, the present study indicates that the oral delivery of SNase by L. lactis can effectively prevent T1DM, ameliorate inflammation, and contribute to immunomodulatory balance in NOD mice.

Recent studies have investigated the potential of type 1 diabetes mellitus–related autoantigens, such as heat shock protein 60, to induce immunological tolerance or to suppress the immune response. A functional 24-residue peptide derived from heat shock protein 60 (P277) has shown anti-type 1 diabetes mellitus potential in experimental animals and in clinical studies, but it also carries a potential atherogenic effect. In this study, we have modified P277 to retain an anti-type 1 diabetes mellitus effect and minimize the atherogenic potential by replacing the P277 B epitope with another diabetes-associated autoantigen, insulinoma antigen-2 (IA-2), to create the fusion peptide IA-2-P2. In streptozotocin-induced diabetic C57BL/6J mice, the IA-2-P2 peptide displayed similar anti-diabetic effects to the control P277 peptide. Also, the IA-2-P2 peptide did not show atherogenic activity in a rabbit model. Our findings indicate the potential of IA-2-P2 as a promising vaccine against type 1 diabetes mellitus.

In order to study the relationship between Staphylococcal nuclease(SNase) and diabetes mellitus, genetic engineering bacteria E. coli BL21/pET28a-His-SNase was constructed, the expression of soluble extracellular protein SNase was induced and the a preliminary research was made on it. An expression vector pET28a-His-SNase plasmid containing the His-SNase gene was constructed and transformed into E. coli BL21 (DE3) competent cells. The protein was induced by lactose and purified by ultrasound destruction and Ni-affinity chromatography, respectively. It was then analyzed by SDS-PAGE and Western blot. The enzymatic properties for SNase has been preliminary studied as well. Results indicated that the purity of the correctly expressed fusion protein His-SNase was over 85%. SNase showed good activity within a wide range of pH and good heat resistance. This experiment might be a foundation work for the further study on the relationship between SNase and with diabetes.

Human heat shock protein 60 (Hsp60), is an endogenous β-cells autoantigen, it could postpone the onset of insulitis and sooner type 1 diabetes mellitus. P277 is one of Hsp65 determinants at position 437-469 of amino acids cascaded. Meanwhile, it's already well-known that there were several better anti-diabetic B epitopes, such as insulinoma antigen-2 (IA-2). Currently, fusion protein IA2P2 has constructed in order to enhance its pharmacological efficacy. In addition, added homologous bacterial-derived Hsp65 and His tag were beneficial to protein immunogenicity and purification separately. So, finally we examined a fusion protein His-Hsp65-6IA2P2 could regulate Th2 immune response and reduce natural diabetic incidence in NOD mice. We constructed two express vector pET28a-His-Hsp65-6P277 and pET28a-His-Hsp65-6IA2P2. After purification, we observed that triple intranasal administration of these two fusion protein in 4-week-old NOD mice maintained normal blood glucose and weight, with a lower diabetic or insulitis incidence. Consistent with induced splenic T cells proliferation and tolerance, His-Hsp65-6IA2P2-treated mice performed reduced IFN-γ and increased IL-10 level. In conclusion, we suggested that fusion protein His-Hsp65-6IA2P2 could be reconstructed and purified successively. Furthermore, nasal administration of this fusion protein could rebalance T cells population and prevent T1DM.

中性粒细胞是非特异性免疫系统中的重要一员,介导吞噬和杀菌作用.中性粒细胞胞外诱捕网(Neutrophil extracellular traps,NETs)是中性粒细胞受到刺激后释放的一种纤维网状结构.近年来,NETs作为中性粒细胞一种全新的机制在自身免疫性疾病上的研究备受关注.NETs在固有免疫中主要利用其特殊的网状结构捕获病原微生物并对其消化加工.内源性和外源性抗原的修饰是中性粒细胞胞外诱捕网主要特点,可产生多种新的自身抗原.诱捕网中组蛋白瓜氨酸化已确定参与多种自身免疫病,并且患者体内存在异常的NETs,并与疾病严重程度呈正相关.文章结合近年来的文献对NETs的形成、NETs参与自身免疫性疾病的发生发展以及探索其应用于临床研究的可能机制分别进行了综述.

Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease characterized by the destruction of insulin-secreting β cells upon autoreactive T cell attack. Oral administration of autoantigens is an attractive approach to treating T1DM, but an effective carrier should be used in order to protect antigens. Lactococcus lactis, a safe engineering strain, was used for this task in the present study. Two recombinant L. lactis expressing protein HSP65-6IA2P2 were used and be investigated the effects and mechanisms against T1DM in NOD mice. Our findings demonstrate that recombinant L. lactis strains can successfully both deliver antigens to intestinal mucosa and maintain the epitopes for a long time in NOD mice. Oral administration of recombinant L. lactis could prevent hyperglycemia, improve glucose tolerance, and reduce insulitis by inhibiting antigen-specific proliferation of T cells, augmenting regulatory immune reactions, and balancing ratios of Th17/Tregs and Th1/Th2. These results prove that orally administrated L. lactis expressing HSP65-6IA2P2 is an effective approach for the prevention of T1DM in NOD mice.

构建融合蛋白His-Hsp65-6IA2P2基因工程表达菌E.coli BL21 (DE3),诱导其表达可溶性胞外蛋白,并进行初步免疫学研究.构建pET28a-His-Hsp65-6IA2P2质粒载体,电转化E.coli BL21(DE3),乳糖诱导表达及镍柱亲和层析等纯化步骤后,SDS-PAGE和BandScan进行检测融合蛋白.最后将此融合蛋白鼻粘膜免疫NOD小鼠,观察其生理特征.融合蛋白His-Hsp65-6IA2P2正确表达,纯化后融合蛋白纯度可达95.2％.免疫动物后能够有效维持正常的体重与血糖.本实验构建了His-Hsp65-6IA2P2基因工程表达菌,成功表达了具有预防治疗及改善1型糖尿病病症潜力的融合蛋白,为进一步探究其药效奠定了基础.

To study the hypoglycemic effect and mechanisms of vaccine HSP65-6P277 protein on type 1 diabetic mice induced by streptozotocin( STZ),Lactococcus lactis live vector was used to express the vaccine protein. The mouse model of type 1 diabetes mellitus were established by intraperitoneal injections of multiple low dose STZ( MLD-STZ) and divided into three groups. The inducible expression strain L. lactis NZ9000 pCYT ∶ HSP65-6P277,constitutive expression strain L. lactis NZ9000 pHJ ∶ HSP65-6P277 and control strains L. lactis NZ9000 pCYT ∶ Nuc were respectively immunized to diabetic mice orally once daily( 2 × 109CFU,200 μL) in the first week and once every week in the following weeks lasting for 16 weeks. Fasting blood glucose levels were measured once a month on blood samples taken from orbital venous plexus. Four months later,the mice were sacrificed. Spleen cell proliferation experiment was performed. IL-10 and IFN-γ cytokine levels of spleen cells and serum IgG levels were assayed using ELISA kits. The results showed that pCYT ∶ HSP65-6P277 group and pHJ ∶ HSP65-6P277 group have low blood glucose( P 0. 05),and maintain normal body weight,compared with control group( pCYT ∶ Nuc). On the other hand,cytokine IFN-γ levels were significantly lower( P 0. 05) in spleen cells supernatant and spleen cell proliferation was reduced against HSP65 and P277( P 0. 05),but serum IgG levels were not significantly different( P 0. 05).

IA-2-P2是由来自P277多肽的T表位与胰岛素瘤相关蛋白2(IA-2)的B表位组合而成的新疫苗肽,为验证其对1型糖尿病的治疗效果,采用多次小剂量注射链脲佐菌素(STZ)诱导C57BL/6雄性小鼠建立1型糖尿病模型,成模2w后,sc 100 μg多肽进行免疫治疗,间隔3 w免疫,共6次,每3 w收集被免疫动物的血清进行生化分析,测定血糖浓度和IgG抗体滴度并进行体重监测.结果显示IA-2-P2多肽能在一定程度上降低血糖,与对照组相比,IA-2-P2组终末平均血糖下降38％;并能维持小鼠的体重的稳定;多肽治疗能延长小鼠生存时间,在32 w观测期间,IA-2-P2组小鼠生存率达70％,较对照组40％的生存率有显著差异(P＜0.05);并且具有免疫调节作用,ELISA结果显示多肽可以有效引起免疫应答产生,与对照组相比,抗体水平有显著差异(P＜0.05).

Acinetobacter baumannii had emerged as a major pathogen causing hospital infections. With the increase of multidrug-resistant Acinetobacter baumannii(MDRAB), there is a clear need to seek therapeutic approaches. Antibacterial agents for MDRAB infections were mainly introduced in this article. Combination of antibiotics was still the effective method. New peptide antibiotics such as Human β-defensin 2,(LLKK)2C and [E4K]alyteserin-1c could hopefully be ideal antibacterial agents with advantages of potent antibacterial activity and little side effects.