Evaluation Of The Immunosensitizing Potential Of Low Molecule Weight Compounds Using Basophil Activation Test Newly-established In Preclinical Study

Drug-induced type I hypersensitivity is a specific IgE-mediated hyperimmune response when the drug stimulate the body,its main clinical manifestations include dermatitis,urticaria,bronchial asthma,angioneurotic edema,food allergy,gastrointestinal allergy,even anaphylactic shock and death.Type I hypersensitivity also knowns as immediate hypersensitivity reactions(IHRs).Preclinical prediction of IHRs induced by drug is an essential part of the development of new drugs.Due to the lack of an ideal preclinical experimental model,it is not yet possible to accurately predict the allergenicity of drugs.In particular,the accurate prediction of the sensitization of low molecule weight compounds(LMWC)is a difficult problem to resolve in short period.From this reason,it is necessary to establish an effective model to predict the immunosensitizing of LMWC.Basophils are the least white blood cells in normal peripheral blood,but they are the most important effector cells of IHRs.At present,the basophil activation test(BAT)has been widely used clinically to diagnose allergic diseases(including allergies caused by food,venom,pollen,and drugs).Its basic principle is to use flow cytometry to detect changes of activation markers in basophils surface after the basophil is activated to diagnose and study allergic diseases.Compared with other diagnostic methods,BAT just needs a small amount of peripheral blood samples and is incubated with allergens in vitro.It has advantages of high safety,short time-consuming,specific sensitivity,mature technology,and simple operation.However,the research and application of BAT in preclinical is still not completed.The clinical BAT method was used to establish BAT model in sensitized animals,which was expected to form a evaluation sensitization technology of new drug that could be combined with preclinical long-term toxicity test to improve preclinical prediction of LMWC sensitization capability and the level of safety evaluation of new drugs in China.The high expression of IgE in BALB/c mice has been widely used in the construction of animal models for allergic reactions and the study of TH2-type immune responses.At the same time,it was considered that rats are the most commonly used for long-term toxicity studies in the preclinical safety evaluation of drugs,and BN rats are also high-impact immunoglobulin(especially IgE)strains,were often used to study immune responses.It is also one of the main animal models that can be used to study IHRs.This project intended to establish the BAT model of BALB/c mice and BN rats.Penicillin G(PG),which often induces IHRs in clinical,as a positive tool drug to simulate the hapten hypothesis that LMWC sensitizes and stimulates the mechanism of IHRs in vivo.The hapten PG and the macroprotein carrier were conjugated in vitro to form the whole antigen.BALB/c mice and BN rats were sensitized with PG conjugated BSA(PG/BSA),the peripheral blood of sensitized animal was inoculated PG conjugated OVA(PG/OVA)in vitro,flow cytometry was used to detect the expression of CD63 in order to verify the effectiveness of LMWC sensitization.The first part of the experiment:The number of basophils in mice was determined by flow cytometry(CD45 APClow/IgE FITChigh).Meanwhile,The blood of BALB/c mice was taken from canthus to make peripheral blood smears,stained by Wright-Giemsa.The morphology and counts of basophils in mice were observed under microscope.Compared the changes of basophils morphology and quantity in control and sensitized(Ovalbumin,OVA)mice.Compare the differences between flow cytometry and traditional blood smear counts,and calculate the correlation between the two.CD45 APClow/IgE FITChigh was used a Flow-Scheme to identify mice basophils,anti-IgE was used as a positive control to incubate with basophils in vitro to observe the expression of CD63,and optimized incubation time in vitro.Heparin completely anticoagulant peripheral blood of OVA sensitized mice incubated with OVA in vitro,the expression of CD63 on basophils was detected by flow cytometry to establish the BAT of mice,verification of sensitized mice in BAT by passive cutaneous anaphylaxis(PCA).In order to verify the accuracy of mice BAT in the prediction of LMWC sensitization,PG that can clinically cause IHRs.Firstly,the BALB/c mice were sensitized by PG/BSA,and the sensitized mice peripheral blood was incubated with PG/OVA in vitro to stimulate mice basophils,then observe the expression of CD63 molecule.(1)Flow cytometry showed that the numbers of(CD45 APClow/IgE FITChigh)basophils in control and sensitized mice were 0.59±0.14%and 0.54±.018%respectively.There was no significant difference(P>0.05).Morphological of basophils in blood smear showed that the numbers of dark purple black basophilic granules were found in the cytoplasm,often covering the nucleus.Most of the nuclei have lobulation,which is not easy to observe.The obvious morphological changes were not observed between control and sensitized group.Blood smear cell counts showed that the number of basophils in control group and sensitized group about 0.60 ± 0.42%and 0.70±0.27%,respectively,with no significant difference(P>0.05).The t test of the two counting methods showed no significant difference(P>0.05),the correlation coefficient of the two counting methods was counted is 0.51,suggesting that CD45 APClow/IgE FITChigh can be as markers to identify mice basophils.(2)Anti-IgE can stimulate the expression of CD63 on basophils and the optimal incubation time is 15 min in vitro,indicating that the CD63 can be as activiation marker of basophil in mice BAT.(3)OVA incubated with blood from OVA-sensitized mice,the percentage of CD63 expression on basophils was 59.70±37.93%,which was significantly increase than PBS stimulation(2.15±1.13%P<0.05).(4)The blood of PG/BSA sensitized mice was incubated with PG/OVA in vitro,CD63 expression on basophils was 62.74±41.05%,which was significantly higher than PBS control group(2.73±1.40%P<0.05).This part of the experiment successfully established mice BAT and this can be used to detect the allergenicity of LMWC.The second part:The number of basophils was measured by flow cytometry(CD45 APClow/IgE FITChigh)to determine the basophilic granulocyte population in the peripheral blood of rats.At the same time,Wright-Giemsa stained blood smears,microscopically observed morphological changes of basophils in control and sensitized rats,and counted the relative number of basophils.Compared of control and OVA-sensitized BN rat basophils morphology and quantity changes.The results of the two counting methods was compared.To detect the expression of CD63 on the surface of basophils in BN rats by flow cytometry,heparin completely anticoagulated peripheral blood from OVA sensitized rat incubated with OVA in vitro,verification of sensitized mice in BAT by PCA.In addition,BN rats were sensitized with PG/BSA and challenged with PG/OVA in vitro to observe the expression of CD63 molecule.(1)Flow cytometry showed that the number of basophils in control and sensitized rats was 0.24 ±0.11%and 0.25 ±0.07%,respectively.No significant difference was found(P>0.05).The morphology of rat blood smears showed no obvious morphological changes in the sensitized and control groups.Blood smear cell counts showed that the number of basophils in the control and sensitized groups accounted for approximately 0.3±0.24%and 0.4±0.37%,respectively.There was no statistical difference between the two groups(P>0.05).The t test of the two counting methods showed no significant difference(P>0.05),and the correlation coefficient was R=0.61,suggesting that CD45 APClow/IgE FITChigh can be as markers to identify rat basophils.(2)After incubation with OVA in peripheral blood of OVA-sensitized rats,the increase of CD63 on basophils was 35.00±23.90%,which was more than that of PBS in vitro(1.78±1.08%%P<0.05).(3)After PG/OVA was incubated with peripheral blood of PG/BSA sensitized rats,the expression of CD63 on basophils was 31.10±20.04%,which was significantly higher than that of PBS control group(2.74±2.10%P<0.05).This part of the experiment successfully established rat BAT and this can be used to detect the allergenicity of LMWC.In summary,when the basophils in peripheral blood from sensitized mice and rats were again exposed to the same antigen in vitro,flow cytometry could detect a significant increase of CD63 in basophil,demonstrating mouse and rat BAT models were successfully built.Using two models can effectively detect the sensitization potential of PG,suggesting that this model can be used to evaluate the allergenicity of LMWC.