幽门螺杆菌感染过程中特异性抗体消长规律的研究

目的研究幽门螺杆菌(Helicobacter pylori,H.py)感染Balb/c小鼠后多种抗原特异性抗体应答的消长特征,评估可用于血清学分析的抗原种类与检测窗口期。方法 70只Balb/c小鼠分为感染组和对照组,ELISA法检测血清中CagA、UreB、HpaA、KatA特异性IgG水平,Real-time PCR法检测胃组织H.py定植数量。结果小鼠感染组胃组织H.py定植数量显著高于对照组(P0.01),且感染组小鼠胃组织中H.py定植数量较为恒定,拷贝数维持在106左右。在H.py感染的16周内,4种抗原特异性IgG水平存在不同的消长规律和阳性应答窗口期,其中CagA抗体阳性窗口期为4~12周,UreB抗体阳性窗口期为6~9周,HpaA抗体阳性窗口期为2~16周,KatA抗体阳性窗口期为5~6周和11~12周。结论由于不同抗原特异性IgG应答存在不同消长规律,可能影响H.py感染的血清学检测的准确性。     

抗脊髓灰质炎病毒Ⅱ型Sabin株D抗原单克隆抗体的研制

目的应用杂交瘤技术制备抗脊髓灰质炎病毒Ⅱ型Sabin株D抗原的单克隆抗体。方法用脊髓灰质炎病毒Ⅱ型Sabin株D抗原免疫雌性Balbc小鼠,取其致敏脾脏淋巴细胞与SP20小鼠骨髓瘤细胞融合。间接ELISA法筛选阳性克隆,制备腹水。检测McAb效价、抗体亚类和杂交瘤细胞核型。结果获得4株阳性杂瘤细胞株,选择一株制备腹水,ELISA效价为1∶2.4×106,中和试验效价1∶1.3×104,蛋白浓度为27.2mgmL。McAb与Ⅱ型Sabin株C抗原(ⅡC)、Ⅰ型Sabin株及Ⅲ型Phizer株D抗原(ⅠD、ⅢD)不发生交叉反应,具有高度特异性。结论成功的制备了针对脊髓灰质炎病毒Ⅱ型Sabin株D抗原的McAb,为进一步研究IPV效力检测奠定了基础。     

旋毛虫新生幼虫T668重组抗原对小鼠的保护性免疫

目的研究旋毛虫新生幼虫T668重组抗原的免疫原性,制备基因工程疫苗。方法以旋毛虫新生幼虫期特异性基因T668在大肠杆菌中的表达蛋白为抗原免疫小鼠,每间隔10d免疫1次,共免疫3次。末次免疫后10d,每只小鼠攻击感染200条旋毛虫感染性肌幼虫,感染后5周用消化法检查肌幼虫(ML)负荷。结果T668重组抗原免疫组肌幼虫减虫率明显高于佐剂组和对照组。结论T668重组抗原能诱导小鼠产生一定程度的保护性免疫,且可激发特异性体液免疫。     

治疗和未治疗小鼠日本血吸虫病免疫应答的动态观察

本文报道定期检测治疗和未治疗日本血吸虫病小鼠循环抗原(CSA)、特异性抗体、循环免疫复合物(CIC)以及嗜酸性粒细胞(EOS)的结果。实验表明 CSA 于感染后2周的阳性检出率(ELISA 双抗体法)为40%,4周时全部呈现阳性反应。抗虫卵抗体在感染后3周阳性率为10%,6周时全部检出。结果表明检测 CSA 有早期诊断之价值。CIC 峰见于感染后7～10周,此时 EOS绝对计数亦最高。上述指标在感染后13周明显下降,但在未治疗鼠直至感染后12～30天 CSA逐渐转阴,而特异性抗体仍持续呈现阳性反应,用 ELISA 检测 CSA 在疗效考核上较用 IHA 检测抗虫卵抗体的方法为优。     

巨细胞病毒单克隆抗体的制备及其初步应用

用CMV AD-169株感染自制人胚肺纤维母细胞，制备抗原，锣疲BALB／c小鼠，．取其脾细胞与Sp2／0-Ag14细胞融合，建立了一株稳定分泌抗HCMV特异性McAb的3B0杂交瘤细胞株。3B0McAb经鉴定属于IgG3亚娄。用间接免疫荧光法和间接酶免疫法证实为抗CMV特异性抗体。用微量细胞培养中和试验证实3B0McAb在加有豚鼠血清后可阻止HcMV感染后细胞病变的出现。3B0McAb经间接免疲荧光法检测中，晚期孕妇羊水细胞中HCMV抗原，在68例标本中有一例为阳性，阳性率为1．47％。该法方便，快速可为在胎儿时期诊断先天性HCMV感染提供依据。     

用重组SAG1抗原检测弓形虫抗体

目的探讨重组SAG1抗原对弓形虫IgG和IgM抗体的检测效果。方法用rSAG1作抗原建立免疫印迹方法（rSAG1-WB）,与玻片虫体过氧化物酶免疫染色试验（TSHE）平行检测不同来源血清。结果15例病原学检查阳性小鼠血清和5例免疫兔血清的IgG抗体均为阳性,30例正常小鼠血清和10例正常兔血清均未出现阳性反应。rSAG1-WB检测可疑弓形虫病患者血清阳性率为60．3％（38／63）,献血员血清阳性率为6％（3／50）,与TSHE检测结果（65．1％和4％）均无统计学差异（P〉0．05）。1例IgM强阳性血清和13例IgM弱阳性血清在Western—blot检测中分别出现相应的强阳性与弱阳性反应,50例献血员血清均未出现IgM阳性反应,结果与TSHE一致。结论rSAG1-WB检测弓形虫IgG和IgM抗体均具有高度的敏感性和特异性．与TSHE的符合率高。     

日本血吸虫感染宿主的免疫抑制现象

本文报告用巨噬细胞移动抑制试验,溶血空斑试验及T细胞辅助活力测定观察小鼠感染日本血吸虫后对植物血凝素、血吸虫成虫抗原、羊红细胞及半抗原-载体(TNP-童虫)免疫应答的抑制现象。MMIT的结果表明:对PHA的应答于感染后28天出现抑制;对血吸虫成虫抗原的应答于感染后2周内正常,第3周开始出现抑制,14周后又基本恢复正常。溶血空斑试验结果表明,感染后头3周对SRBC的应答水平递增,第3周达最高水平,第4周开始应答逐渐下降。不同H-2的纯系小鼠感染日本血吸虫后对SRBC的应答规律基本相似,但于感染后同一时间内不同H-2的小鼠应答水平随小鼠品系的不同而不同。T细胞辅助活力测定的结果表明:小鼠感染后3周内对TNP-童虫的应答逐渐增加,第3周最高,之后逐渐下降。上述结果提示感染宿主存在免疫应答的抑制现象。这种抑制现象不仅表现在T细胞对促有丝分裂因子PHA和血吸虫成虫抗原的应答上;也表现在B细胞对胸腺依赖抗原SRBC的应答上,以及在T-B细胞协作产生抗半抗原抗体时,T细胞辅助应答也受到抑制的影响。     

D抗原遮蔽现象致新生儿RhD抗原假阴性的鉴定与分析

目的对1例RHD阴性产妇产下的RHD阳性新生儿因D抗原遮蔽现象造成血清学假阴性的进一步鉴定与分析。方法采用血清学方法检测新生儿及其父母双方ABO及Rh血型抗原,对患儿血样进行直接抗球蛋白试验、游离抗体试验及放散试验,对患儿母亲血清进行抗体鉴定,对患儿红细胞热放散洗涤后再做D抗原鉴定。PCR扩增患儿Rh基因的上下游盒子并电泳分析产物。结果新生儿血型初次定型为A、d Ccee,患儿母亲血型为A、dccee,患儿父亲为O、DCCee;新生儿直接抗球蛋白试验阳性、游离抗体试验阳性且抗体特异性为抗-D抗体、放散试验阳性且抗体特异性为抗-D抗体;患儿红细胞热放散洗涤后与试剂抗-D发生凝集为RhD阳性;PCR产物电泳发现同时存在上游盒子、下游盒子及杂交盒子,为RHD+/RhD-杂合型。结论因母亲体内高效价的抗体进入患儿体内后完全封闭了新生儿红细胞上的D抗原,造成其红细胞上没有多余的D位点与血清试剂结合,产生假阴性现象。     

单克隆抗体竞争性ELISA在日本血吸虫病诊断中的应用

本文应用单克隆抗体ⅢD10竞争性ELISA,对369例粪检阳性日本血吸虫病患者(慢性316例,急性54例),检测血清内抗CCA 抗体水平,并与环卵沉淀试验和间接血凝试验对比。结果表明,C-ELISA 的敏感性为 97.75～100％,高于 CCPT(90.27％,p<0.05)。而正常人无假阳性,与肝吸虫、肺吸虫和包虫感染的突叉反应依次为1.39％、1/18和0。特异性明显高于COPT和IHA。本试验快速、简便、重复性好。     

巨细胞病毒单克隆抗体的制备及其初步应用

用CMV AD-169株感染自制人胚肺纤维母细胞,制备抗原,免疫BALB/c小鼠,取其牌细胞与Sp2/0-Ag14细胞融合,建立了一株稳定分泌抗HCMV特异性McAb的3B_6杂交瘤细胞株。3B_6McAb经鉴定属于IgG_3亚类。用间接免疫荧光法和间接酶免疫法证实为抗CMV特异性抗体。用微量细胞培养中和试验证实3B_6McAb在加有豚鼠血清后可阻止HCMV感染后细胞病变的出现。3B_6 McAb经间接免疫荧光法检测中,晚期孕妇羊水细胞中HCMV抗原,在68例标本中有一例为阳性,阳性率为1.47％。该法方便,快速可为在胎儿时期诊断先天性HCMV感染提供依据。     

The acquisition and loss of antigen-specific cellular immune responsiveness in acute and chronic schistosomiasis in man

To characterize the development and evolution of cellular immune responsiveness in individuals infected with the parasite Schistosoma mansoni, we studied fifteen patients with acute, subacute and chronic schistosomiasis. Lymphocytes from the three acutely infected patients responded vigorously to schistosome antigens in an in vitro blastogenic assay. By contrast, cells from nine chronically infected individuals were essentially unreactive to these same antigens. Patients infected for an intermediate period of time (9 months) generated responses between those of acute and chronic patients.

The diminished responsiveness of chronically infected individuals was specific for schistosome antigens and did not extend to humoral immune responses. Following treatment of the infection with niridazole, these patients temporarily regained responsiveness to schistosome antigens.

From these data we speculate that during the course of this parasitic helminth infection there develops a progressive and specific modulation of antigen recognition and proliferation by lymphocytes to schistosome antigens, and that such diminished immune reactivity may be important in maintaining the unique biological relationship which exists between a host and its parasites.

     

Single- or mixed-sex Schistosoma japonicum infections of intermediate host snails in hilly areas of Anhui,China

Schistosomiasis japonicum is one of the most serious communicable diseases, and the transmission of the parasite is dependent of its complex life cycle on which many factors can have an impact. Multiple infections comprising both male and female schistosome within snail intermediate hosts, for example, would facilitate parasite transmission. However, no research on Schistosoma japonicum communities in field-collected Oncomelania hupensis hupensis in relation to schistosome sex has been reported. Therefore, snail survey was performed in a hilly region of Anhui, China, and single- or mixed-sex schistosome infections of snails were detected with final host mouse infection. A total of 8,563 snails were sampled in the field, and 67 were identified with schistosome infections. Of these infected snails, 46 were selected for final host infection. From this, 21 snails were infected with female schistosome, 23 with males and 2 with both males and females. More worms were recovered for snails with mixed-sex infections than with single-sex infection and for snails with male schistosome infection than with female infection (P?<?0.001). The observed frequency of mixed-sex infections of snails was significantly higher than would be expected if randomly distributed (P?<?0.01). The ratio male/female of schistosome infections in snails was nearly equal and up to 95.65 % (44/46) of infected snails were single-sex infection. Schistosome infections in snails collected from the hilly area of Anhui Province were not randomly distributed but over-dispersed.     

Circulating antigens in schistosomiasis: detection of 31/32-kDa proteins in sera from patients infected with Schistosoma japonicum,S. mansoni,S. haematobium,or S. intercalatum

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect 31/32-kDa schistosome proteins as circulating antigens in sera from schistosomiasis patients. A monoclonal antibody was used as a capture antibody and rabbit antiserum raised against purified 31/32-kDa proteins was the detecting antibody. Positive results were obtained with patients infected with Schistosoma japonicum (88%; n=69), S. mansoni (80%; n=56), S. haematobium (100%; n=40), or S. intercalatum (94%; n=65). Sera from uninfected Chinese and African individuals and from Chinese patients with trichinosis, cysticercosis, or paragonimiasis did not react in the assay. This ELISA appears to be valuable in diagnosing infections by all major human schistosome species.     

Effect of oltipraz on the susceptibility of adultSchistosoma mansoni to killing by mouse peritoneal exudate cells

Incubation of the adultSchistosoma mansoni with the anti-schistosomal compound oltipraz (OPZ) (40 nM) resulted in a significant decrease in schistosome-reduced glutathione (GSH), a thiol compound which may have a role in protection against oxidant-mediated damage. A significant proportion (20–47%) of worms treated with OPZ became susceptible to in vitro killing by zymosan-stimulated peritoneal exudate cells from mice infected withS. mansoni or inoculated with Bacillus Calmette Guérin (BCG). Killing of the worms was partially inhibited by the addition to the assay system of exogenous glutathione peroxidase with GSH but not by superoxide dismutase. These results suggested that killing of parasites exposed to the drug was partly mediated by cell-generated hydrogen peroxide. They indicate also that depletion of schistosome GSH levels could render the parasites susceptible to killing by oxidative mechanisms, and suggest that there is potential in exploiting schistosome oxidant defense systems and reactive oxygen byproducts in the treatment of schistosomiasis. Inhibition of schistosome oxidant defense systems with drugs may render the parasites susceptible to killing by reactive oxygen byproducts of effector cells.     

Murine Intestinal Humoral Responses in Chronic Schistosoma mansoni Infections

Specific and non-specific production of immunoglobulins (Ig) by the intestinal mucosa was examined in mice infected with the human blood fluke Schistosoma mansoni. Ileal and colonic mucosal tissue samples were cultured for 2 days, the medium replaced and the culture continued for a further 2 days. Ig concentrations and specific antibodies to soluble schistosome egg antigens in culture supernatants were estimated by isotype-specific ELISA. Cultured mucosae from control mice produced little IgG, but significant amounts of IgA and IgM on prolonged culture. IgG concentrations were increased in infected animals, mainly in the initial culture period, indicative of systemic, rather than local origins. By contrast, significantly increased local production of IgA and IgM occurred after the start of egg deposition in the intestinal mucosae. Although specific anti-egg antibodies of the IgG and IgM class were detected, none of the local IgA response was specific for schistosome eggs. We conclude that specific intestinal immune responses to schistosome eggs reflect systemic responses, whereas locally increased IgA production is largely non-specific. This pattern of response is likely to be related to the prior systemic exposure to schistosome eggs, which results in polyclonal local B-cell activation, but fails to trigger an antigen-specific IgA mucosal response.     

Cutaneous basophil hypersensitivity and inhibited macrophage migration in guinea-pigs with schistosomiasis

In guinea-pigs infected with schistosomes, delayed cutaneous reactions rich in basophils (CBH) were found to characterize skin test responses to schistosome egg antigens. In addition, strong contact hypersensitivity-like skin eruptions with large basophil infiltrates resulted from skin penetration challenge by live cercariae (larvae) in these animals. Oedema and diminished basophil granule staining were noted around schistosomula which had penetrated the skin of sensitized animals. CBH responses to egg antigens and to live cercarial challenges were also noted after immunization with a single injection of dead cercariae.

Using peritoneal exudates from guinea-pigs immunized with dead cercariae or infected with schistosomes, direct macrophage migration inhibition with schistosome antigens was found only in animals with infections. Thus, CBH correlated with intradermal exposure to schistosome cercarial antigens, while MMI correlated with live infections. It is suggested that cutaneous basophil responses may play a role in protection from re-infection with schistosomes, and that dead cercarial vaccines might stimulate this beneficial response, without immunizing for potentially harmful granulomatous hypersensitivity.

     

Circulating antigens in schistosomiasis: detection of 31/32-kDa proteins in sera from patients infected with Schistosoma japonicum , S. mansoni , S. haematobium , or S. intercalatum

 A sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect 31/32-kDa schistosome proteins as circulating antigens in sera from schistosomiasis patients. A monoclonal antibody was used as a capture antibody and rabbit antiserum raised against purified 31/32-kDa proteins was the detecting antibody. Positive results were obtained with patients infected with Schistosoma japonicum (88%; n=69), S. mansoni (80%; n=56), S. haematobium (100%; n=40), or S. intercalatum (94%; n=65). Sera from uninfected Chinese and African individuals and from Chinese patients with trichinosis, cysticercosis, or paragonimiasis did not react in the assay. This ELISA appears to be valuable in diagnosing infections by all major human schistosome species. Received: 23 February 1995 / Accepted: 2 May 1995     

Immunoblot analysis of Schistosoma mansoni antigens with sera of schistosomiasis patients: diagnostic potential of an adult schistosome polypeptide.

We compared the reaction in immunoblots of sera obtained from patients with parasitologically proven S. mansoni infections, with a suspected history of schistosomiasis infection, or with unrelated parasitic diseases. Several polypeptides from adult S. mansoni reacted with the schistosomiasis patients' sera in a heterogeneous manner. However, a component of approximately 31 kilo daltons (kD) reacted with all schistosomiasis sera and with several sera of suspected schistosomiasis cases. No reaction was ever observed with sera of patients harbouring other parasites. Thus, the polypeptide has potential diagnostic value. The use of sera of patients with recent infections demonstrated that: the earliest time of antibody formation against the 31 kD component was approximately 40 days post infection, the reaction with this polypeptide in immunoblots was exceptionally strong and antibodies directed against other schistosome proteins were barely detectable at this time. Identical results were obtained with sera of experimentally infected mice. The 31 kD component was present in parasites of either sex. It was apparently not a glycoprotein. Evidence suggests that the 31 kD polypeptide may originate from the schistosome gut.     

Behavior of Biozzi high and low responder mice upon infection with Schistosoma mansoni

Mice genetically selected for high (Ab/H) or low (Ab/L) humoral antibody responses were infected with Schistosoma mansoni in order to assess the role of antibodies in innate and acquired immunity to this parasite. AbH mice produced higher levels of humoral antibodies to schistosome antigens, but were more susceptible to infection than Ab/L mice. This was shown by the higher number of parasites recovered from Ab/H mice, by the larger size of the parasites themselves, by the number of schistosome eggs and their rate of deposition in the host liver. In addition, Ab/L mice could develop an acquired resistance to schistosome re-infection which was as good as, or possibly even better than the resistance developed by Ab/H mice. These findings suggest that humoral antibodies per se may not play a critical role in schistosome immunity, and at the same time call attention to the possible importance of macrophages in determining the results observed.