| Penicillins are highly effective, low toxicity and inexpensive antibiotics. However, allergy induced by penicillins has the highest incidence in all allergic response among numerous medicines, in which some serious response can cause allergic shock or even death. The rised total serum IgE and specific IgE levels were the characteristic in allergic reactions to penicillins. In the previous studies, it was generally believed that certain allergic reactions to penicillin belong to TypeⅠhypersensitive reaction that is mediated by specific IgE antibody. The specific IgE plays the important role in allergic reactions. Along with the progress of research, it was found that T cells were also involved in both immediate reaction and non-immediate reaction in penicillin allergy. At present, penicillin intradermal testing is a well-established method to evaluate current penicillin allergic status in China. The advantages of the method are fast, sensitive, reproducible, and inexpensive. The disadvantage is the high incidences of false positive and false negative due to individual variation, the varied components of testing medicines and the differed standards defining positive response. The extreme cases include shock and death in highly responsive patients while undergoing the skin test. It has attracted great attention worldwide to develop more reliable in vitro tests for penicillin allergy which may replace the skin tests. Penicillin-specific antibodies are highly responsible to penicillin allergy. The specific antibodies were secreted from B lymphocytes, and the secreting process is modulated by T lymphocyte and its subgroups. Allergy to penicillins was not only induced through TypeⅠhypersensitivity reactions, but also other hypersensitive reactions which are modulated by multiple cytokines.By studying the association between penicillin allergy and the immune moleculars, it has been found that many factors are involved in allergic reactions. However, it is still underdiscovered that genetic variation of the candidate genes and their roles in penicillins allergy. The current study focuses on the cytokines of interleukin 12 (IL-12) and interleukin-18 (IL-18), which are highly responded to immune diseasesIn the present study, the case-control studies were conducted to examine the serum levels of 8 kinds of penicillin specific IgE by RAST, to measure serum levels of IL-12 and IL-18 cytokines by ELISA, to analyze the IL-18 gene and IL-12 gene polymorphisms by single nucleotide polymorphisms (SNPs) in the subjects. Interleukin-8 receptor (IL-8R) was selected based on its differential expression between penicillin allergic subjects and normal controls. Its expressions in peripheral white blood cells were measured at protein and mRNA levels by Realtime RT-PCR and Western blotting methods in the subjects. To provide the solid references in preventing, determining, and treating penicillin allergy, the relationship between the specific antibodies and the expressions of these cytokines were studied and the roles of IL-12, IL-18, and IL-8R in penicillin allergy were revealed.PartⅠAssociation between specific IgE and penicillin allergyObjective:To evaluate RAST method in the diagnosis of penicillin allergy, analyze the cross-allergic reactions to penicillins, and study the importance of side chain structure in the cross-allergic reactions.Methods:The major and minor antigenic determinant papers were prepared linking with four drugs (penicillin G, PG; penicillin V, PV; amoxicillin, AX and ampicillin, AMP). The major and minor antigenic determinants of PG, PV, AMP, and AX are BPO-PLL, BPA-PLL, PVO-PLL, PVA-PLL, APO-PLL, APA-PLL, AXO-PLL, and AXA-PLL respectively. Total 614 of health volunteers and 606 of individuals with positive penicillin allergic history were assigned into normal control and allergic patient groups respectively. The specific IgE levels to the 8 antigens mentioned above were measured using RAST assay. The mean±2.33SD of the control group was defined as the standard value limit. The patients who had higher level of IgE were defined as IgE positive. The allergic patients were subgrouped based on the skin test, allergic symptoms and the occurring time of the symptoms after penicillin application and skin test, age, and gender. The IgE levels and the rate of IgE positive between the subgroups were compared.Results:There was no significant difference in sensitivity between the our lab made paper and reference paper (P<0.05). The detection rate of IgE positive was increased from 41.9% to 63.7% by using major and minor antigen determinants compared with using major antigen determinant using alone. BPO-IgE, PVO-IgE, BPA-IgE and PVA-IgE levels were increased with age of allergic patients and not associated with gender. The levels of BPO-IgE and BPA-IgE were related to the patient’s skin test results.The levels of specific IgE against penicillin G and penicillin V were associated with dyspnea in allergic patients. The positive skin test and the allergic symptoms were associated with the specific IgE level. Penicillin G has the highest risk in the four penicillins.The specific IgE levels tended to decline over time in penicillin allergic patients.Conclusions:The test paper for penicillin antigens made by our laboratory has good sensitivity, specificity and stability in detecting serum IgE levels in penicillin allergic patients. The detection rate of specific IgE was increased by using major and minor antigen determinant. The specific IgE against penicillin G was more closely associated with positive skin test than the other three penicillins. The specific IgE levels in penicillin allergic patients tended to decreased over time. PartⅡThe association between penicillins allergy and serum levels of IL-18 and IL-12Objective:To explore the roles of IL-18 and IL-12 in penicillins allergy by comparing the serum levels of IL-18 and IL-12 in normal and penicillin allergic patientsMethods:Based on the diagnostic principles for penicillin allergy, the allergic subjects were defined as the individuals with positive skin test or negative skin test but positive allergic symptoms. The serum levels of IL-18 and IL-12 in normal and penicillin allergic patients were measured by ELISA.Results:Serum levels of IL-18 and IL-12 in penicillin allergic patients were higher than those in normal subjects (102.8 pg/ml vs.67.9 pg/ml,15.8 ng/ml vs.13.5 ng/ml, P<0.05). IL-18 level in patients with symptoms such as shock, dyspnea, and urticaria was higher than the normal (P<0.05) subjects. The levels of IL-18 in positive skin test individuals without clinical symptoms patients was similar with the normal subjects (P>0.05). IL-12 level in patients with negative skin test but positive symptoms after penicillin application was higher than the positive skin test patients (P<0.05). There was a positive correlation between IL-12 and IL-18 in patients and normal subjects. Serum levels of IL-12 and IL-18 had no significant difference between rapid and delayed type hypersensitivity of penicillin. There was not significantly difference between serum IL-12 and IL-18 levels and the number of different type positive specific IgE in allergic subjects (P<0.05).Conclusions:The serum levels of IL-18 and IL-12 in penicillin allergic patients are higher than the normal subjects. Their levels are associated with penicillin allergy occurring. The serum levels of IL-18 and IL-12 are associated with clinical symptoms and the positive skin test respectively. The types of clinical allergic symptoms are not related to serum levels of IL-18 and IL-12. There is a positive correlation between IL-12 and IL-18 level both in penicillin allergic subjects and in the normal controls. PartⅢCorrelation between IL-18 and IL-12 genes polymorphisms and penicillin allergyObjective: To explore the correlation between IL-18 and IL-12 gene polymorphisms and penicillin allergy, Single nucleotide polymorphisms (SNPs) of IL-18 and IL-12 genes in the patients of penicillin allergy were detected by polymerase chain reaction with sequence-specific primer (PCR-SSP).Methods:We chosed -607A/C and -137G/C sites in the promoter region of IL-18 gene,-6415(CTCTAA/GC) site in the promoter region of IL-12 gene and 10841(C/A) site in 3’uncode terminal region (3’UTR) of IL-12 gene, and designed the specific PCR primers according to each gene polymorphisms site (-607A and -607C,-137G and -137C,-6415C and -6415G,10841C and 10841A). The 3’terminal basyl of each primer was matched to the corresponding SNP site. The paired specific primers (607R, 137R,-6415F,10841R) and positive control primers (607F,137F,-6415R,10841F) were also designed. Genomic DNA of blood monocytes were extracted from the penicillin allergic patients and the control subjects The target cDNAs were synthesized and being as the templates for DNA amplification in PCR.The PCR products were verified through agarose gel electrophoresis to identify whether the specific primers could produce the target DNA products. Then the positive or negative amplified products through the specified primers could demonstrate potential basyls of the SNP. Otherwise, some sample PCR products were selected to be sequenced randomly, in order to verify the accuracy of the PCR-SSP.Results:1. All of the specific PCR primers were verified for detecting the corresponding SNP site. And the sequencing results also confirmed the result of PCR-SSP for detecting IL-18--607A/C, IL-18--137G/C, IL-12--6415(CTCTAA/GC), and IL-12-10841(C/A) site.2. In both allergic patients and normal subjects, four genetic sites of IL-18--607A/C/, IL-18--137G/C, IL-12--6415(CTCTAA/GC), IL-12-10841 (C/A) met Hardy-Weinberg equilibrium test. 3. At IL-18 -607A/C position, compared with the control group, there was a significantly higher frequency of the -607AA genotype (15.5% vs 9.0%, P<0.01) in patient group. The A allele occurred more frequently in patients group than that in the controls (42.1% vs.32.5% , P<0.05). At IL-18 -607A/C position, compared with the control group, there was a significantly lower frequency of the-137GG genotype and G allele (69.3% vs 81.3%, P<0.01; 84.2% vs 90.5%, P<0.01) in patient groups. Moreover, significant differences in the allele and genotype frequencies of IL-18 -607 and -137 polymorphism were not found in all patient subgroups with positive skin test, immediate reactions, non-immediate reactions, and allergic symptoms.4. At IL-12B-6415 position, compared with the control group, there was a significantly higher frequency of the -6415GC/GC genotype and GC allele (37.6% vs 18.8%, P<0.01; 53.9% vs 47.7%, P<0.05) in patient group. At IL-12B 10841 position, compared with the control group, there was a significantly higher frequency of the CC genotype (35.4% vs 22.5%, P<0.05) in patient group. Moreover, significant differences in the allele and genotype frequencies of IL-12B-6415 polymorphism were not found in each patient subgroup with immediate reactions, non-immediate reactions, and allergic symptoms. And compared with other subgroups of allergic symptoms patients, there was a significantly higher frequency of the IL-12B 10841CC genotype and C allele in patients with urticaria (40.4% vs 22.5%, P<0.05,62.9% vs 50.4%, P<0.05).5. With SHEsis software, no linkage disequilibrium was observed between two genetic sites of IL-18-607A/C and -137G/C (D’=0.223). And no linkage disequilibrium was found between two genetic sites of IL-12B-6415 CTCTAA/ GC and 10841 C/A(D’=0.203).6. With SHEsis software, in four haplotypes of IL-18 composed of two polymorphic genetic sites of 607A/C and -137G/C, haplotype of -607A/-137C occurred more frequently in allergic patients than that in controls. While haplotype of-607C/-137G in allergic patients was lower frequency than that in controls. In four haplotypes of IL-12B composed of two polymorphic genetic sites of -6415 CTCTAA/GC and 10841C/A, haplotype of -6415GC/10841C appeared more frequently in allergic patients than that in controls (P<0.01). While haplotype of-6415CTCTAA/10841A in allergic patients was lower frequency than that in controls.7. The levels of IL-18 in the three genotype groups of IL-18 -607A/C or -137 G/C polymorphism were significantly higher in patients than that in the controls (P<0.01). Moreover, there was significantly higher serum IL-18 level in patients with -137 GC and -137 CC genotypes than those with GG genotype (P<0.05). However, there was no significantly different serum IL-18 level among the three genotype groups of IL-18 -607A/C (P>0.05).8. There was no significantly different serum IL-18 levels in the three genotype groups of IL-12B CTCTAA/GC or 10841 C/A polymorphism, (P>0.05).Conclusions:1. Allergic reaction to penicillins associates with genetic polymorphisms of IL-18-607A/C, IL-18-137G/C and IL-12B-6415(CTCTAA/GC), IL-12B 10841 (C/A).2. The polymorphism of IL-18 -607A/C or -137 G/C is not associated with the results of skin test, allergic type and allergic symptoms.3. The polymorphism of IL-12 -6415(CTCTAA/GC) is not associated with the result of skin test, allergic type and allergic symptoms. However, IL-12B 10841 (C/A) site CC genotype is associated with urticaria in penicillin allergic patients.4. There is no linkage disequilibrium between two genetic sites of IL-18-607A/C and-137G/C and between IL-12B -6415 CTCTAA/GC and 10841 C/A.5. IL-18-607A/-137C haplotype and IL-12B-6415GC/10841C are associated with allergic reaction to penicillins.6. In penicillin-allergic patients, the -137 position plays an important role in regulating IL-18 expression. However, the polymorphism of IL-18 -607A/C has no effect on the serum IL-18 level, and there is no association between the two polymorphism of IL-12B and serum IL-12 levels. Part IV Differential gene expression in penicillin allergic patients and the roles of IL-8R in penicillin allergyObjective: To screen the differential expression genes between penicillin allergic patients and normal control subjects by oligonucleotide microarray analysis, select the allergic associated cytokines, and discover the expression and roles of these molecules in penicillin allergy.Methods:Blood samples were drawn from both the penicillin allergic and normal subjects. The total RNA was extracted from the blood cells. Oligonucleotide microarray analysis was used to detect the differential gene expression between the allergic and normal subjects. IL-8R mRNA and protein expression were detected using realtime RT-PCR and Western blotting methods.Results:1.3294 genes (1731 genes were up regulated,1563 genes were down regulated) were obtained from the oligonucleotide microarray analysis between penicillin allergic and normal subjects. Of which,338 genes were associated with immunity. And the expression of IL-8R was obviously higher in penicillin allergic patients than in control subjects (Ratio of diffscore was 3.49)2. Compared with the control group, the mRNA and protein expression of IL-8RA were significantly higher in penicillin allergic patients, especially in dyspnea group and urticaria sub-groups of penicillin allergic patients with positive skin test. And the mRNA and protein expression of IL-8RB were not significantly different among the patients with allergic symptoms and the control subjects.3. Compared with the control group, the mRNA and protein expression of IL-8RA were significantly higher in non-immediate reaction (NIR) group of penicillin allergic patients, but there was no significant difference of IL-8RB expression between control subjects and IR or NIR group of penicillin allergic patients.Conclusions:1.3294 genes obtained from the oligonucleotide microarray analysis between penicillin allergic patients and normal control subjects provides abundant candidate genes for studying the mechanisms of penicillin allergy.2. The higher expression of IL-8RA in penicillin allergic patients might be responsible for the dyspnea or urticaria allergic symptoms and non-immediate reaction in the penicillin allergic patients. IL-8RB does not play important role in penicillin allergy. |
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