霍乱毒素B亚单位胰岛素混合蛋白抗糖尿病作用

目的观察口服不同剂量霍乱毒紊B亚单位（CrB）与胰岛紊（insulin）混合蛋白疫苗的免疫耐受及预防1型糖尿病作用，为探讨社区糖尿病干预方案提供依据。方法3周龄雌性NOD小鼠60只，随机分为磷酸缓冲液（PBs）阴性对照、1mg胰岛紊阳性对照、CTB＋insulin100μg，CTB＋insulin 1mg等4组。从4周龄起，每周灌胃2次～12周龄；12周龄起，每周1次动态监测小鼠尿糖、血糖，至26周龄，观察糖尿病的发生，比较各组糖尿病发病率。结果与磷酸缓冲液（PBs）组比较，CTB＋insulin100μg。CTB＋insulin 1mg能够减少NOD小鼠糖尿病的发生，降低小鼠糖尿病的累积发病率。至鼠龄26周时，PBS组、CTB＋insulin100μg，CTB＋insulin1mg组和1mg胰岛素组的累积发病率分别为100％，60％，60％和40％，与阴性对照组比较，其他3组的累积发病率差异均有统计学意义（均P〈0．05）；2个试验组与阳性对照组的累积生存率及生存分布的差异无统计学意义。结论CTB＋insulin混合蛋白疫苗能够降低NOD小鼠糖尿病累积发病率，提高小鼠生存率；CTB＋insulin1mg组的预防作用优于100μg组。     

霍乱毒素B亚单位与志贺毒素2B亚单位融合表达及抗原性检测

目的 克隆表达出血性大肠埃希菌(EHEC)O157:H7志贺毒素2B亚单位(Stx2B)与霍乱毒素B亚单位(CTB)的融合蛋白(CTB-Stx2B),并检测其抗原性和与神经节苷脂(GM1)结合能力.方法 设计引物扩增融合蛋白CTB-Stx2B的编码基因ctb-stx2b,T-A克隆测序验证后克隆入原核表达质粒pET30a(+)C,构建表达质粒pET30a(+)-ctb-stx2b,转化E.coliBL21(DE3),IPTG诱导表达、纯化,获得目的蛋白CTB-Stx2B,SDS-PAGE和Western-blot检测其抗原性和形成五聚体的能力;GM1-ELISA法检测其与GM1结合能力.结果 扩增出约750 bp的目的片段,测序鉴定与设计序列一致;原核表达质粒pET30a(+)-ctb-stx2b转化E.coliBL21(DE3)后,经酶切和PCR扩增鉴定正确;IPTG诱导后SDS-PAGE初步测定CTB-Stx2B单体的相对分子质量(MR)约为20×103;Western-blot检测CTB-Stx2B能与CTB单克隆抗体结合,纯化产物经复性后大多以五聚体形式存在;ELISA检测CTB-Stx2B具有结合GM1活性.结论 成功克隆、表达了融合蛋白CTB-Stx2B;表达的蛋白具有良好的CTB抗原性和GM1结合能力.     

霍乱毒素B亚单位与志贺毒素2B亚单位融合表达及抗原性检测

目的 克隆表达出血性大肠埃希菌(EHEC)O157:H7志贺毒素2B亚单位(Stx2B)与霍乱毒素B亚单位(CTB)的融合蛋白(CTB-Stx2B),并检测其抗原性和与神经节苷脂(GM1)结合能力.方法 设计引物扩增融合蛋白CTB-Stx2B的编码基因ctb-stx2b,T-A克隆测序验证后克隆入原核表达质粒pET30a(+)C,构建表达质粒pET30a(+)-ctb-stx2b,转化E.coliBL21(DE3),IPTG诱导表达、纯化,获得目的蛋白CTB-Stx2B,SDS-PAGE和Western-blot检测其抗原性和形成五聚体的能力;GM1-ELISA法检测其与GM1结合能力.结果 扩增出约750 bp的目的片段,测序鉴定与设计序列一致;原核表达质粒pET30a(+)-ctb-stx2b转化E.coliBL21(DE3)后,经酶切和PCR扩增鉴定正确;IPTG诱导后SDS-PAGE初步测定CTB-Stx2B单体的相对分子质量(MR)约为20×103;Western-blot检测CTB-Stx2B能与CTB单克隆抗体结合,纯化产物经复性后大多以五聚体形式存在;ELISA检测CTB-Stx2B具有结合GM1活性.结论 成功克隆、表达了融合蛋白CTB-Stx2B;表达的蛋白具有良好的CTB抗原性和GM1结合能力.     

非糖尿病成人的胰岛素原水平与心脏病有强联系

分析 14 5 6名年龄较大的非糖尿病成人的资料 ,探明胰岛素、胰岛素原、C-肽水平与 CHD患病率的关系。其中 1/4的男、女患 CHD,它们的胰岛素原水平明显高于无 CHD的该年龄男、女。妇女的 C-肽及胰岛素水平与妇女的 CHD危险直接有关 ,与男子这两水平无关系。结果 :仅胰岛素原水平     

霍乱毒素B亚单位作为免疫佐剂的研究进展

霍乱肠毒素（CT）是霍乱弧菌重要的毒力因子,内含A和B两个亚单位。A亚单位（CTA）为毒性亚基,B亚单位（CTB）不具毒性。CTB因其独特的生理功能已成为重要的佐剂之一,近年来备受各国学者重视。深入探究CTB的免疫调节机制有助于更好地设计疫苗并优化其效果,此文将对CTB作为免疫佐剂在疫苗研究中的进展进行综述。     

胰岛素强化治疗对新诊断成年人隐匿性自身免疫性糖尿病患者胰岛素原的临床观察

目的 观察胰岛素强化治疗对新诊断成年人隐匿性自身免疫性糖尿病(LADA)患者胰岛素原的影响.方法 中青年(年龄20～54岁)发病新诊断LADA的患者42例,且所有入选患者空腹血糖≥11.1 mmol/L,随机分为强化治疗组22例和普通治疗组20例.结果 强化治疗组治疗15 d餐后2 h胰岛素原与治疗前餐后2 h胰岛素原相比明显降低,同时也明显低于普通治疗组治疗15 d餐后2 h胰岛素原,差异有统计学意义(P＜0.01).结论 对新诊断LADA患者行胰岛素强化治疗15 d可使胰岛B细胞功能恢复到较佳状态.     

糖尿病怎样进行抗炎治疗

现在认为2型糖尿病是一种自身免疫和慢性炎症性疾病。在降糖、降脂、降体重等综合治疗下,还需同时进行抗炎、抗凝、抗胰岛素抵抗的治疗。才能有力改善糖代谢,提高胰岛素敏感性,预防糖尿病并发症。     

基于霍乱毒素B亚单位结构功能分析的霍乱弧菌基因分型

目的:描述和分类自然发生的霍乱毒素B亚单位DNA和蛋白序列的多态性,及时提供潜在有用的霍乱诊断、疫苗研制和流行病学信息。方法:采用生物信息频谱分析平台,同时包括Clustal W,MEGA和PyMOL分析法,对245个霍乱毒素B亚单位DNA和蛋白序列进行结构和功能及基因分型特性的研究。结果:所分析的霍乱毒素B亚单位的103个氨基酸区域有共同的信息频谱特征,其主峰特征为F(0.3333,19),显示了霍乱菌株的霍乱毒素B亚单位的共同作用模式。霍乱毒素B亚单位蛋白的T68I氨基酸残基关键变异明显增加了F(0.3333)的特征峰高,可能对"人类"作用的模式有较大影响。经过系统聚类分析,发现了19个霍乱毒素B亚单位DNA序列多态。基于以上结果和流行病学资料,提出了19个霍乱弧菌的霍乱毒素B亚单位基因型。霍乱弧菌古典生物型参考株(569B)的霍乱毒素B亚单位基因型为1a(CTBgenotype 1a)。霍乱弧菌埃尔托生物型参考株(N16961)和霍乱弧菌O139血清群的参考株(4260B)的霍乱毒素B亚单位基因型都为2a(CTB genotype 2a)。结论:以上内容可对霍乱毒素B亚单位DNA和蛋白序列的多态性提供更好的理解;提供了对霍乱毒素B亚单位分子变异监测的一个技术和方案;可为今后预防和处置霍乱弧菌的感染和流行,进一步扩展了鉴别潜在的免疫、治疗和诊断的分子靶标的可能性。     

成人隐匿性自身免疫性糖尿病合并糖尿病酮症一例

人隐匿性自身免疫性糖尿病(LADA)多发生于成年人。LADA临床上常表现为类似1型糖尿病的症状,常合并酮症,易被误诊。我国LADA患病率在全球处于较高水平。目前,自身抗体检测已成为诊断LADA的重要指标。如果代谢状态良好,治疗可以考虑使用磺脲类以外的其他口服药物。且应尽早使用胰岛素治疗。在临床工作中,早期确诊、尽早使用恰当的药物降糖,对患者减少并发症,提高生存质量非常重要。     

Plant-based cholera toxin B subunit-insulin/GAD fusion pro-teins suppress the development of autoimmune diabetes

Summary Insulin-dependent diabetes mellitus (IDDM) is a T lymphocyte mediated organ-specific autoimmune disease in which pancreatic islet inflammation (insulitis) destroys the insulin-producing β-cells leading to a permanent increase in blood sugar levels (hyperglycemia). Principal self antigens recognized by the T-cells are glutamic acid decarboxylase (GAD) and insulin (INS), with GAD being the initiating antigen. Oral administration of pancreatic self antigens generate partial suppression of both insulitis and hyperglycemia symptoms. To expedite targeting of self antigens to the mucosal immune system, the 11.6 kDa non-toxic cholera toxin B subunit protein was genetically conjugated to proinsulin or GAD65 and expressed in potato tuber tissues. Oral immunization with transformed tuber tissues containing CTB-insulin or CTB-GAD65 fusion proteins resulted in suppression of insulitis and clinical diabetes symptoms (hyperglycemia) in prediabetic NOD mice. Immunized NOD mice generated significant levels of serum and intestinal anti-INS and anti-GAD IgG and IgA antibodies. At 30 weeks after immunization, all the unimmunized mice were hyperglycemic. However, 50% of the mice fed CTB-INS and 40% fed CTB-GAD65 remained free of hyperglycemia.     

The effect of cholera toxin and cholera toxin B subunit on the nasal mucosal membrane.

The effects of the self-adjuvanting substances, cholera toxin (CT) and cholera toxin B subunit (CTB), on rabbit nasal mucosal membrane, were investigated by using Ussing chambers. The control nasal mucosa (lateral wall), isolated from rabbits and mounted in the chamber, showed transepithelial potential difference, short-circuit current and conductance of -10 mV, 200 microA cm-2 and 20 mS cm-2, respectively. These parameters were compared with mucosa isolated from human inferior conchae, showing that rabbit nasal mucosa may be usable to understand effects on human mucosa. When the mucosa was exposed to various concentrations of CTB and CT, the short-circuit current and conductance of the mucosa increased with increasing concentration. CTB showed gradual increase in the short-circuit current when added in the same molar concentration as the B subunit contained in CT, which caused drastic changes by increasing the current to infinite value. Furthermore, the total amount of transepithelially fluxed CTB, which occurred rapidly after addition to the mucosal side of the chambers, increased with increasing CTB concentration. On the other hand, less flux was observed after addition of CT. These changes could be blocked by addition of ganglioside GM1. This demonstrates that the effect of CTB on the rabbit mucosal membrane are different from those of CT, although both CT and CTB act specifically on the membrane via the CTB receptor, ganglioside GM1.     

Plant-based cholera toxin B subunit-insulin/GAD fusion pro-teins suppress the development of autoimmune diabetes

Insulin-dependent diabetes mellitus (IDDM) is a T lymphocyte mediated organ-specific autoimmune disease in which pancreatic islet inflammation (insulitis) destroys the insulin-producing β-cells leading to a permanent increase in blood sugar levels (hyperglycemia). Principal self antigens recognized by the T-cells are glutamic acid decarboxylase (GAD) and insulin (INS), with GAD being the initiating antigen. Oral administration of pancreatic self antigens generate partial suppression of both insulitis and hyperglycemia symptoms. To expedite targeting of self antigens to the mucosal immune system, the 11.6 kDa non-toxic cholera toxin B subunit protein was genetically conjugated to proinsulin or GAD65 and expressed in potato tuber tissues. Oral immunization with transformed tuber tissues containing CTB-insulin or CTB-GAD65 fusion proteins resulted in suppression of insulitis and clinical diabetes symptoms (hyperglycemia) in prediabetic NOD mice. Immunized NOD mice generated significant levels of serum and intestinal anti-INS and anti-GAD IgG and IgA antibodies. At 30 weeks after immunization, all the unimmunized mice were hyperglycemic. However, 50% of the mice fed CTB-INS and 40% fed CTB-GAD65 remained free of hyperglycemia.     

Preparation and preclinical evaluation of experimental group B streptococcus type III polysaccharide-cholera toxin B subunit conjugate vaccine for intranasal immunization

Streptococcus group B (GBS) is usually carried asymptomatically in the vaginal tract of women and can be transferred to the newborn during parturition. Serum antibodies to the capsular polysaccharide (CPS) can prevent invasive diseases, whereas immunity acting at the mucosal surface may be more important to inhibit the mucosal colonization of GBS and thus the risk of infection for the newborn. We prepared different GBS type III CPS-protein conjugate vaccines and evaluated their systemic and mucosal immunogenicity in mice. GBS type III CPS was conjugated to tetanus toxoid (TT) or recombinant cholera toxin B subunit (rCTB) either directly or to rCTB indirectly via TT. The conjugation was performed by different methods: (1) CPS was coupled to TT with 1-ethyl-3 (3-dimethylaminopropyl)-carbodiimide (EDAC), using adipic acid dihydrazide (ADH) as a spacer; (2) CPS was conjugated with rCTB using reductive amination; or, (3) N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) was used to bind rCTB to the TT of the CPS-TT conjugate. Mice were immunized with these conjugates or purified CPS by subcutaneous (s.c.) and intranasal (i. n.) routes. Antibodies to GBS III in serum, lungs and vagina were measured with ELISA. All of the CPS-protein conjugates were superior to unconjugated CPS in eliciting CPS-specific immune responses in serum and mucosal tissue extracts. The conjugates, when administrated s.c., induced only IgG responses in serum, lung and vagina, while i.n. vaccination also elicited IgA responses in the lungs and vagina. The CPS-TT conjugate administrated i.n. induced a strong serum IgG, but only a weak mucosal IgA response, while the CPS-rCTB conjugate elicited high IgG as well as IgA antibodies in the lungs after i.n. immunization. GBS III CPS-TT conjugated with rCTB produced a strong systemic and local anti-CPSIII response after i.n. administration. Co-administration of CT as adjuvant enhanced the anti-CPS systemic and mucosal immune responses further after i.n. administration with the CPS conjugates. These findings indicate that: (i) i.n. immunization with GBS CPS-protein conjugates was more effective than s.c immunization for stimulating serum as well as mucosal immune responses; (ii) rCTB as a carrier protein for GBS III CPS could markedly improve the mucosal immune response; and (iii) the experimental GBS type III CPS conjugates containing rCTB should be investigated as mucosal vaccine to prevent GBS infection in humans.     

Effect of pre-existing immunity for systemic and mucosal immune responses to intranasal immunization with group B Streptococcus type III capsular polysaccharide-cholera toxin B subunit conjugate

The effects of priming with a group B Streptococcus type III capsular polysaccharide (GBS CPS III)-recombinant cholera toxin B subunit (rCTB) conjugate, purified GBS CPS III or rCTB alone on the systemic and mucosal immune responses to CPS III after intranasal (i.n.) immunization were investigated in mice. Priming with purified GBS CPS III followed by boosting with GBS CPS III-rCTB conjugate or priming with the conjugate followed by boosting with free CPS induced comparable levels of specific IgG and IgA in both serum and in lungs and vagina. However, i.n. immunization comprising both priming and boosting with conjugate was superior to priming with CPS and boosting with conjugate or the reverse, especially with regard to inducing mucosal IgA anti-CPS responses. All the immunization schemes, except priming and boosting with free CPS, induced high and similar levels of IgG1 in serum. In contrast, mice primed with free CPS III and then boosted with CPS III-rCTB conjugate by the i.n. route failed to produce significant levels of IgG2a, IgG2b and IgG3 in serum, at difference from mice primed with the conjugate and boosted with either conjugate or free CPS. Pre-immunization with rCTB either i.n. or i.p. did not suppress specific serum IgG responses induced by GBS CPS III-rCTB conjugate intranasally, but did inhibit serum and especially mucosal IgA responses. Our findings suggest that priming with CPS affects the distribution of IgG subclasses to GBS CPS and that pre-existing anti-carrier rCTB immunity can have an inhibitory effect on mucosal immune responses elicited by the conjugate vaccine given by the i.n. route.     

Cross-protection against influenza B type virus infection by intranasal inoculation of the HA vaccines combined with cholera toxin B subunit

The relationship between the antibody responses to various influenza B type virus HA vaccines and protection against live B virus infection was investigated in Balb/c mice which had been inoculated intranasally with a combination of the HA vaccines and B subunit of cholera toxin (CTB) 4 weeks previously. The inoculation of HA vaccine, prepared from B/Ibaraki/2/85 (B/Ibaraki), B/Nagasaki/1/87 (B/Nagasaki) or B/Aichi/5/88 (B/Aichi) viruses, combined with CTB induced high levels of both nasal IgA and serum HI antibodies to any of B/Ibaraki, B/Nagasaki and B/Aichi viral antigens. Simultaneous inoculation of each CTB-combined HA vaccine provided complete protection against B/Ibaraki virus infection which is demonstrated by both rapid clearance of pulmonary virus and complete survival. On the other hand, the inoculation of HA vaccine prepared from B/Yamagata/16/88 (B/Yamagata) virus together with CTB induced only a low level of nasal IgA antibodies, cross-reactive to B/Ibaraki, B/Nagasaki and B/Aichi viral antigens and protected only partially against B/Ibaraki virus challenge. The involvement of the B type virus-specific immunity in this protection was suggested by the absence of protection against B/Ibaraki virus infection in mice previously inoculated with both A/PR/8/34 (H1N1) virus HA vaccine and CTB. These results suggest that antibodies to various influenza B viruses are cross-reactive to each B type virus antigens and that cross-protection against B virus infection could be conferred depending on the degree of B type virus cross-reactive immunity including secretory IgA antibodies.     

Effectiveness of cholera toxin B subunit as an adjuvant for nasal influenza vaccination despite pre-existing immunity to CTB

In a previous paper, it was shown that cholera toxin B subunit (CTB) augments the production of protective antibodies to influenza virus when CTB is inoculated intranasally into Balb/c mice together with influenza HA vaccine¹. The present study was designed to determine whether the effectiveness of CTB as a potent adjuvant for nasal vaccination could be limited by pre-existing immunity to CTB. Mice were sensitized by intranasal inoculation of either 1 or 0.05 μg of CTB 2, 4 and/or 6 weeks before nasal vaccination. They were then vaccinated by either a single inoculation of vaccine together with 1 μg of CTB or a two-dose regimen, composed of a primary inoculation of vaccine together with 0.05 μg of CTB and a subsequent inoculation of vaccine alone¹⁻³. Levels of nasal IgA antibodies to CTB increased with the increase of the dose of CTB and the frequency of CTB-inoculation. Pre-existing immunity to CTB, however, did not significantly reduce the levels of both nasal IgA and serum haemagglutination-inhibiting (HI) antibodies to influenza virus and did not change the ability of the vaccinated mice to resist viral challenge. These results suggest that a relatively low dose of CTB could be inoculated repeatedly into animals as an adjuvant for nasal vaccination.