【作者】 乔海灵；

【导师】 董子明；

【作者基本信息】 郑州大学 ， 病理学与病理生理学， 2005， 博士

【摘要】 青霉素类抗生素临床应用广泛,但其过敏反应发生率居各类药物之首,尤其是过敏性休克危害严重。青霉素过敏反应机制及诊断迄今尚未明了,仍为目前研究热点之一。 体液免疫和细胞免疫都参与青霉素过敏反应。IgE长期以来被认为是过敏反应的关键,目前国内主要通过皮肤过敏试验(皮试)检测IgE,但由于受病人年龄、皮试剂、阳性结果判断标准等因素的影响,皮试阳性率相差较大,且存在假阳性和假阴性,尤为严重的是皮试即可发生过敏休克甚至死亡。放射过敏原吸附试验(RAST)是一种敏感性、特异性均较高的体外检测IgE的试验方法,在国外广泛使用于青霉素过敏反应的诊断,国内除本室外,未见其他报道。 IgG与过敏反应的关系至今尚未明确,其在过敏反应中的作用存在着两种截然不同的观点,即介导作用和抑制作用。这可能与IgG的不同亚型有关,IgG可分为4种亚型:IgG1、IgG2、IgG3、IgG4,其中IgG1、IgG2、IgG4与过敏反应更为密切。有观点认为,IgG1可分为致敏型和非致敏型两种抗体,IgG2可与不同的受体(FcγRIIA和FcγRIIB)结合,而使其在不同的过敏反应中可能表现出不同的作用。关于IgG4,近年来争议甚多,有人认为是一种封闭型抗体,也有人认为是一种类似IgE的介导型抗体。IgG在过敏反应中的双重作用,取决于不同的过敏原、不同的过敏阶段及不同的过敏体质,由此可见IgG与过敏反应关系的复杂性。更多还原

【Abstract】 Penicillins are the most widely used antibiotics for common infectious diseases, and they are the most common causes of drug-induced, IgE-mediated hypersensitivity and the most studied so far. The mechanism and diagnosis of penicillins allergy were not cleared although they were studied for many years, so they were still the most important questions for investigation.The allergic reactions to penicillins include immediate IgE-mediated reactions such as anaphylactic shock, maculopapular rashes, and allergic contact dermatitis, et al. Both cellular and humoral responses are involved in eliciting the reactions. In our country, the skin test has been used for a long time to diagnose penicillins allergy. However, there were some serious problems in skin test, such as false positive or negative reactions, and sometimes anaphylactic shock can threat the life. The most widely-used and safe method is measurement of specific IgE antibodies to penicillins by different methods, of which the most common is radioallergosorbent test (RAST). There were no other reports about the RAST in China except our laboratory.The relationship between allergy and IgG is not clear. There are two viewpoints about IgG: blocking and mediating antibody. These may be related to IgG subclasses. IgG can be classified four subclasses, IgG1, IgG2, IgG3 and IgG4. IgG1 can be divided into anaphylactic IgGl and non-anaphylactic IgGl. IgG2a and IgG2b produce different biological effect by binding the different point on the mast cell surface, such as FcγRIIA and FcγRIIB. There are controversies about IgG4 in allergy. Someone considered that IgG4 blocked allergic reactions, others had demonstrated that IgG4 acted as anaphylactic antibody like IgE. IgG antibodies are adouble-edged sword in allergy, which may beresulted from different antigen, different time of allergy and different subjects, et al.Cellular immunity is activated by forming TCR(T cell receptors)-MHC-peptide complex to stimulate T cells. T cell of Th type produce large quantities of interleukin(IL)-4, IL-5, IL-10, IL-13, IFN-γ and TNF-α, which play an important role in allergy. The Th1/Th2 balance is regulated by different cytokines, which associated with IgE level. Some opinions suggest allergy is due to Thl/Th2 skewing that derived from interplay between genes and environment. That is, atopy is characterized by some loci contain major genes influencing hypersensitivity reactions to certain allergen (gene print hypothesis). Drug allergy is the same.We conducted this study to investigate the levels of specific IgE and IgG antibodies, cytokines(IL-4, IL-10 and IL-13) in the sera of allergic patients, and then we evaluated the genetic polymorphism of related genes(IL-4, IL-13, et al) by PCR-RFLP. Materials and Methods1. Patients with allergic history The study of antibody specificity included 650 patients with allergy history (374 male, 276 female; mean age: 28.38±14.65 years) who were recruited from Zhengzhou University, the first affiliated hospital of Zhengzhou University, et al. The study included patients with positive skin test (n=404) and patients with typical clinical symptoms after penicillins administration when skin test was negative (n=246). Among patients with allergic history, subjects with allergy to penicillin G (PG), ampicillin (AMP) amoxicillin (AX), and others were 180,29,18, and 19, respectively.2. Preparation for antigen paper discs First, we prepared major or minor determinant antigenic discs of PG, phenoxomethylpenicillin(PV), AMP, AX, respectively, then they were compared with the paper discs made by Australia. The major or minor determinants of penicillins including in the study were: BPO-PLL and BPA-PLL, PVO-PLL and PVA-PLL, APO-PLL and APA-PLL, AXO-PLL and AXA-PLL, respectively.3. Detemination of specific IgE antibodies RAST was used to determine the specific IgE in the sera of 650 patients with penicillins allergy.4. Detemination of IgG antibodies Enzyme linked immunosorbent assay (ELISA) was used to examine 8 kinds of specific IgG to different antigenic determinants in serum (BPO, BPA, APO, APA, AXO, AXA, PVO, PVA) among 249 patients with penicillins allergy.5. Detemination of cytokines The levels of IL-4, IL-10 and IL-13 in serum of allergic patients were determined by ELISA.6. Genetic polymorphism A restriction endonuclease fragment length polymorphism (RFLP) of a polymerase chain reaction (PCR) product was used for analysis of the IL-4C-589T, IL-4RαQ576R, IL-13 R130Q and FcεR I β polymorphism.7. Statistical analysis The analyses were performed by using the SPSS (10.0) statisticalprogram. The measurement data were analysed with t-test, ANOVA, Mann-Whitney U testand Kruskal-wallis test. The analysises of correlation were performed with Spearman test.The enumeration data were analysed with chi-square test. A value of P<0.05 wasconsidered statistically significant.Results1. Specific IgE antibodies and penicillins allergy1.1 Methodology Compared with the discs made by Australia, the paper discs made by our lab were sensitive, specific and stable. There were no statistical difference between them (P>0.05).1.2 The positive rate of specific IgE antibodies The positive rate of specific IgE antibodies in 650 patients was 55.69% (362). There was no difference between patients with allergic history and those with positive skin test. The same were true among different ages(<18, 18-59, >60 years) , gender, and allergic symptoms(anaphylactic shock, urticaria and other symptoms).1.3 Kinds of specific IgE antibodies Among 8 kinds of IgE antibodies to different determinants, the positive rate of BPO-IgE was the highest (24.15%), and the second was BPA-IgE antibody (21.54%), and that of APA-IgE antibody was the lowest (13.23%). Among 4 kinds of IgE antibodies to different drugs, the IgE positive rate of PG was the highest (34.31%), and the second was AX (30.77%), and that of AMP was the lowest (24.62%).The positive rate in patients with only one specific IgE antibody screened was 24.15%, but that in patients with all 8 specific IgE antibodies examined was 55.69%. There were no difference between the IgE positive rates to 5 kinds of determinants(BPO, PVA, BPA, AXA and AXO) and that of all of 8 kinds(P>0.05). The results indicated that the total positive rate of IgE antibodies increased as the kinds of determinants increasing, and 5 kinds of determinants were enough to determine the antibodies in allergic patients.1.4 Specificity and crossreaction In the positive IgE patients, there were 129(35.64%) patients with only one kind of positive IgE to determinant, and there were 135(37.29%) cases whose sera were positive only to one drug. The positive rate of specific antiodies was significantly higher than that of crossreaction (P<0.05). The cross positive rates of BPO, BPA, APO, APA, AXO, AXA, PVO and PVA with others were 37.85%, 30.11%, 30.39%, 27.90%, 24.86%, 21.82%, 25.41% and 33.98%, respectively. The cross positiverates of BP, AMP, AX and PV with others were 50.28%, 43.65%, 38.67% and 45.86%, respectively. The results showed that partial crossreaction existed in penicillins allergy, and the specific reaction played an important role in the reactions. 1.5 The skin test and RAST In 103 patients with immediate positive skin test, when the degrees of skin test were +, 2+, 3+ and 4+, the positive rates of specific IgE were 44.19%, 63.33%, 90.91% and 100%, respectively(P<0.05). And there was a significant difference among the 4 groups when we increased the the positive criterion of skin test (P<0.05). The data showed that there was a correlating relationship between the specific IgE antibodies and the degree of skin test.2. Specific IgG antibodies and penicillins allergy2.1 The positive rate of specific IgG antibodiesExcept BPA-IgG, 7 kinds of antigenic determinants IgG levels were higher than that in control group (P<0.05). The positive rate of specific IgG in 249 patients was 58.63% (146/249), and the positive rate of IgG in patients with negative skin test (66.15%) was significantly higher than that of IgG in those with positive skin test (50.42%) (PO.05).2.2 Kinds of specific IgG antibodiesAmong 8 kinds of antigenic determinants, the positive rate of BPO-IgG was the highest (33.73%), followed by AXO-IgG (16.47%), and that of BPA-IgG was the lowest (0.80%). The positive rates of IgG to major and minor antigenic determinants were 56.22% (140) and 8.84% (22), respectively (P<0.01). The major antigenic determinants to IgG probably play an important role in the process of penicillins allergic reaction.2.3 Specificity and crossreactionIn the positive IgG patients, there were 103(70.55%) patients with only one kind of positive IgG to determinants, and there were 107 cases (73.29%) whose sera were positive only to one drug. The cross positive rates of BPO, BPA, APO, APA, AXO, AXA, PVO and PVA with others were 15.75%, 0.80%, 14.38%, 0.68%, 18.49%, 8.22%, 11.64% and 2.74%, respectively. The cross positive rates of BP, AMP, AX and PV with others were 15.75%, 15.75%, 23.97% and 14.38%, respectively. The total cross positive rate of IgG was 26.71%. The positive rate of specific antibodies was significantly higher than that of crossreaction. The results showed that partial crossreaction existing in penicillins allergy.3. IgE and IgG antibodies in penicillins allergy 3.1 IgE and IgG antibidiesThere were 77 patients whose IgE and IgG were both positive among 249 patients, and the total positive rate was 84.74% when we determined IgE and IgG simultaneously.There was no correlation between positive rate of IgG and IgE (P>0.05). In the present study, we had shown a significant correlation between IgE and IgG of BPO(r=0.254, P<0.05) and AXO(r = -0.152, P<0.05). The positive rates of IgG in patients with positive and negative IgE were 46.53%% and 53.47% (P>0.05), and the positive rate of IgE in patients with positive and negative IgG were 54.11% and 63.11% (P>0.05), respectively. The results suggested that detection of IgE and IgG together could find more penicillins allergic subjects. 3.2 Kinds of antigen determinantsIf only to detect IgG, there was no difference in the positive rates of IgG between 3 kinds determinants examined and 8 kinds determinants examined, between 3 kinds drugs examined and 4 kinds drugs examined. If to detect IgG and IgE together, there was no difference in the positive rates between 5 kinds determinants examined and 8 kinds determinants examined, between 3 kinds drugs examined and 4 kinds drugs examined. The results suggested that the positive rate of penicillins allergy had a growing and steady trend with the kind increasing of antigen determinants or drugs. 4. IL-4, IL-10 and IL-13 in penicillins allergy4.1 Penicillins allergy and cytokines like IL-4, IL-10, IL-13Compared with control subjects, there were significantly higher levels of IL-4, IL-13 and lower IL-10 in allergic patients with positive specific IgE (P<0.01). The level of IL-4 in allergic patients with negative specific IgE was lower than that in control group (p<0.05). The result showed that the increasing of IL-4, IL-13 and decreasing of IL-10 could be related to the positive specific IgE, and the decreasing of IL-4 was associated with the negative specific IgE.4.2 Associations between the degree of positive skin test and cytokinesThere was significant difference of IL-4, IL-13 level between positive group and control group(P<0.05), which suggested that IL-4, IL-13 levels in serum of patients were growing with the increase of severe degree of skin test.4.3 Associations between specific IgE and cytokinesAccording to the different kinds of specific IgE, the allergic patients with positive specific IgE were divided into three groups with 1, 2, 3 or more kinds positive specific IgE, respectively. More kinds of positive specific IgE, more higher levels of IL-4, IL-13 in the sera of patients(P<0.01). It showed that there was a growing trend of IL-4, IL-13 levels with the increased number of positive specific IgE.4.4 Associations between specific IgE and cytokinesAmong patients with positive specific IgE, there was a significant association between IL-4 and IL-13 levels (P<0.01), and the associations also existed between the two kinds of cytokines and many kinds of specific IgE (P<0.01). 4.5 Associations between specific IgG and cytokinesThere was no difference in levels of IL-4, IL-13 between patients with positive and negative specific IgG. Compared with patients with negative IgG, IL-10 level was significantly lower in patients with positive IgG. 5. Allergic reaction and genetic polymorphismThe IL-4Ra*Q576 allele was significantly increased in patients with penicillins allergy compared with control subjects (P<0.01). Furthermore, the IL-4Ra*Q576 allele was strongly associated with increased serum specific BPO-, PVA- or APA-IgE levels in patients with positive specific IgE (P<0.05). Significant distributions of E237G genotype were also observed between patients with positive BPA-, PVO- or APO-IgE and control group(P<0.05). Conclusions1. Compared with reference discs, the penicillins determinant PLL paper discs prepared by our lab are sensitive, specific and stable.2. There are antibodies to minor determinants in penicillins allergic patients. As major determinants, the minor determinants play an important role in penicillins allergy.3. Not only IgE but also IgG is involved in the development of penicillins allergy, and the positive rate increases when we determine both two kinds of antibodies in allergic patients at the same time. The total positive rate of IgE and IgG antibodies increase according to the enhanced kind of determinant. There is a correlating relationship between the specific IgE antibodies and the degree of skin test.4. There are partial crossreaction in penicillins allergy, and the specific reaction is important in penicillins allergy.5. The IL-4Ra Q576R polymorphism may involve in the development of penicillins allergy, and through modulating specific serum IgE levels. The FcεR I βE237G genotype is associated with the increasing of BPA-, PVO-, APO-IgE antibodies.更多还原