Cloning and immunobiochemical analyses on recombinant chymopapain allergen Cari p 2 showing pollen-fruit cross-reaction.

Papaya is reported to trigger food and respiratory allergy. Here, we identified chymopapain Cari p 2 as an allergen that can sensitize atopic individuals through fruit consumption followed by respiratory hazards through pollen exposure. Recombinant Cari p 2 displayed IgE-reactivity with 78% of papaya allergic sera. rCari p 2 also displayed allergenic activity through basophil degranulation. rCari p 2 is correctly folded and showed irreversible denaturation in the melting curve. rCari p 2 displayed IgE-cross-reactivity with homologous cysteine proteases from kiwi and pineapple. Cari p 2 transcript was also detected in papaya pulps. rCari p 2 was resistant to pepsin digestion and retained IgE-reactivity after 60 minutes of pepsin digestion. In mouse model, rCari p 2 was found to elicit inflammatory responses in the lung and gastrointestinal epithelium. Hence, Cari p 2 is a newly characterized allergen with diagnostic and immunotherapeutic potential for managing allergic disorders in papaya sensitized individuals.

[Chemical modification of chymopapain with monomethoxypolyethylene glycol and its effect on enzymic activity and antigenicity].

OBJECTIVE: To study the effect of chemical modification of chymopapain with monomethoxypolyethylene glycol on enzymic activity and antigenicity. METHODS: Under the substrate protecting and non-substrate protecting, the chymopapain (Cp) was modified with two types of mPEG derivatives mPEG1 and mPEG2. The average ratio of modified-NH2 was tested by trinitrobenzenesulfonic acid (TNBS) method, the enzymic activity was tested with macromolecular casein and ATEE as substrates, the antigenicity of modified enzyme was determined by ELISA method. RESULTS: (1) Both mPEG1 and mPEG2 can reduce and eliminate antigenicity of Cp, the mPEG2 was better than mPEG1. (2) The enzymic activities of modified Cp were reduced, the enzymic activities of mPEG1-modified Cp were higher than that of mPEG2-modified Cp (especially macromolecular protein as its substrate). (3) The enzymic activities in present of ATEE were obviously higher than that in absent of substrate. CONCLUSION: When mPEG1 as the modifier and in present of ATEE, the antigenicity of Cp was completely eliminated, and the enzymic activities were still higher.

[Preparation and purification of polyclonal antibody against chymopapain].

OBJECTIVE: To prepare and purify polyclonal antibody against chymopapain, and to make a foundation for establishing an immunossay for chymopapain. METHODS: New Zealand rabbit was immunized with chymopapain. Antiserum was purified by Protein A and analyzed by ELISA. RESULTS: The titer of the antiserum obtained in this experiment by ELISA was up to 1:380000 and the purity was proved to be high by SDS-PAGE.

[Construction and identification of anti-chymopapain scFv phage display library].

AIM: To construct phage display library of anti-chymopapain scFv. METHODS: V(H) and V(L) gene repertoires were amplified from splenocyte mRNA by RT-PCR and joined by a (Gly(4)ser)3 linker to obtain scFv genes. The scFv genes were then cloned into phagemid pFAB5C to construct phage display library. Affinity selection and ELISA were used to identify specific phage antibody to chymopapain. RESULTS: After 4 rounds of panning, high affinity scFv was obtained. CONCLUSION: Phage display library of anti-chymopapain scFv was successfully constructed, and scFv with binding ability to chymopapain was obtained.

[Study of preparation methods of polyclonal antibody of chymopapain].

OBJECTIVE: To study the preparation methods of polyclonal antibody of chymopapain with higher titre. METHODS: Three types of antigens-native chymopapain, chymopapain inactivated by H2O2 and inactivated by iodoacetic acid were injected into different male rabbits by hypodermic injections and by intravenous injection. The blood samples were obtained from veins in ear to test antisera after the 3rd injection, the used methods were Western blotting and ELISA. The data was analyzed by the SAS system. RESULTS: The antisera titre of two inactivated antigens were obviously higher than that of nature antigen (P < 0.05), while there was no significant difference between the two inactivated antigens. Intravenous injection can obviously increase the ability of immunoresponse and raise antisera titre rapidly. CONCLUSION: The inactivated antigens have better immunogenicity than native chymopapain. As compared with iodoacetic acid, using H2O2 was an economical and effective method. The combination method of hypodermic injections and intravenous injection can raise antisera titre effectively and shorten the period of antibody preparation.

[The preparation and identification of rabbit's antibody of chymopapain].

Chymopapain, one of the four cysteine proteinases of papaya latex, has milk clotting and proteolytic activity. It is mainly used to treat prolapsed intervertebral discs. This paper introduced the preparation of polyclonal antibody of partially purified chymopapain and the identification of the antibody specificity. The polyclonal antibody provides a basis for the further researches and applications of chymopapain.

Immunoglobulin E antibodies to papaya proteinases and their relevance to chemonucleolysis.

STUDY DESIGN: Levels of four papaya cysteine proteinases were determined in Chymodiactin, a pharmaceutical preparation of chymopapain (EC 3.4.22.6) used in chemonucleolysis for the treatment of sciatica. Twelve sera known to contain immunoglobulin E antibodies to Chymodiactin were assayed for immunoglobulin E antibodies to these enzymes. OBJECTIVES: The goal of the study was to determine what contribution each of the four proteinases makes to the allergic response that occasionally occurs during injection of a damaged intervertebral disc with chymopapain preparations. SUMMARY OF BACKGROUND DATA: The occurrence of an allergic reaction during chemonucleolysis implies prior sensitization to components of the injected enzyme solution. The latex of the unripe fruit of the papaya plant Carica papaya, from which chymopapain is purified, contains another three immunologically distinct cysteine proteinases: 1) caricain (EC 3.4.22.30), 2) glycyl endopeptidase (EC 3.4.22.25), and 3) papain (EC 3.4.22.2). METHODS: A dot-blot immunoassay was developed to quantify each enzyme in Chymodiactin. Total serum immunoglobulin E levels and specific immunoglobulin E antibody levels to each of the four papaya cysteine proteinases were assayed by an enzyme-linked immunoassay in 12 sera containing immunoglobulin E antibodies to Chymodiactin. RESULTS: Chymodiactin contained 70% chymopapain, 20% caricain, 4% glycyl endopeptidase, and 0.1% papain. Immunoglobulin E antibodies to all four proteinases were found in most of the 12 sera, but in varying proportions. Antibodies to glycyl endopeptidase were predominant in eight sera, and the mean amounts of immunoglobulin E directed against each protein were: glycyl endopeptidase, 4.21 IU/ml; caricain, 2.9 IU/ml; chymopapain, 1.97 IU/ml; and papain, 1.39 IU/ml. Total serum immunoglobulin E levels showed little correlation with immunoglobulin E responses to Chymodiactin. CONCLUSIONS: The results suggested that removal of glycyl endopeptidase and caricain from pharmaceutical preparations of chymopapain may help reduce the incidence of allergic reactions during chemonucleolysis.

Immunohistochemical study of chymopapain injected into the rabbit intervertebral disc using anti-chymopapain antibody.

This study was undertaken to investigate the morphological action of chymopapain (CP) in intervertebral discs. Of 20 mature Japanese white rabbits, 19 lumbar intervertebral discs were used for electron microscopic examination, and 36 discs, for immunohistochemical examination. Discs from 3 hours to 8 weeks after injection were observed by light and electron microscopy using anti-CP rabbit antibody labeled with horseradish peroxidase. Using Kagami's DMF-dehydration, two types of fine fibers were observed in the extracellular matrix of normal nucleus pulposus, in addition to collagen fiber. After injection of CP, the thinner fibers disappeared, while the thicker fibers remained. The injected CP spread from the nucleus pulposus to the annulus fibrosus within several hours and remained as long as 4 weeks. By immunoelectron microscopy, positive granules were divided into Type 1 (lacking relationship to collagen fibers) and Type 2 (adhering to or surrounding collagen fibers). The thinner fibers appear to be proteoglycan monomer and are the target for CP.

Residual chymopapain activity after chemonucleolysis in normal intervertebral discs in dogs.

Studies were carried out to demonstrate residual chymopapain activity in intervertebral discs after chemonucleolysis; protease assay, enzyme-linked immunosorbent assay, and immunohistochemical localization of the chymopapain in the disc tissue were done. Chymopapain, one milligram per level, was injected into the normal lumbar intervertebral discs of adult mongrel dogs and the discs were excised after two weeks. Proteolytically active chymopapain was still present in the extract of intervertebral disc at this time. The proteolytic activity was decreased by sulfhydryl inhibitors but not by inhibitors of metalloproteases or serine proteases. Protease and enzyme-linked immunosorbent assays showed that 0.60 +/- 0.48 per cent and 0.49 +/- 0.38 per cent of the original dose was present two weeks after the injection. Chymopapain was shown by immunohistochemical staining to be diffusely located throughout the extracellular matrix of the anulus fibrosus and the nucleus pulposus. Some cells, located mainly in the inner portion of the anulus, contained vacuoles filled with immunoreactive product.

[Sensitization to chymopapain in patients treated with chemonucleolysis]. / Sensibilización a la quimopapaína en pacientes tratados con quimionucleólisis.

BACKGROUND: Chemonucleolysis (intradisk administration of chymopapain) is a procedure to treat intervertebral disk hernia. Recently, its use has been questioned due to the development of anaphylactic reactions in patients sensitized to chymopapain. The prevalence of sensitization to chymopapain has been evaluated before and after chemonucleolysis, and the possibility to establish risk groups through the allergy history has been assessed. METHODS: 104 consecutive patients who were candidates to chemonucleolysis were evaluated with an allergy questionnaire, cutaneous tests to aeroallergens and to chymopapain, and chymopapain-specific IgE. The two latter tests were repeated one month after chemonucleolysis. RESULTS: Only 2 patients (1.9%) showed evidence of chymopapain sensitization before the procedure. Sixteen patients (16%) were sensitized after chemonucleolysis. None of the possible risk factors evaluated in the allergy questionnaire (atopy, drug allergy, papaya occupational exposure or use of additives, cosmetics or drugs containing papaine) were significantly related with the risk of sensitization to chymopapain. CONCLUSIONS: The prevalence of chymopapain sensitization in the study group was low. The allergy questionnaire (atopy, drug allergy, use of papaya, occupational history did not identify sensitized patients. Cutaneous tests and specific IgE are the best method to detect chymopapain sensitization. The remarkable rate of sensitization after chemonucleolysis may partially limit the usefulness of the procedure.

Prevention of chymopapain anaphylaxis by screening chemonucleolysis candidates with cutaneous chymopapain testing.

With chymopapain at a concentration of 10 mg/ml, the authors skin tested 540 chemonucleolysis candidates; six were positive, and 534 were negative. None of the positive patients received therapeutic injections of chymopapain. There were no instances of unequivocal anaphylaxis to chymopapain in the patients with negative skin tests treated with chymopapain. When this 0% incidence of systemic reactions in skin test negative patients is compared with the historical rate of 1%, this difference is statistically significant (p less than 0.05). Restriction of chymopapain treatment to patients with negative prick tests can reduce the incidence of systemic reactions.

[Skin tests and chymopapain-specific immunoglobulins E after chemonucleolysis]. / Tests cutanés et immunoglobulines E spécifiques à la chymopapaïne après chimionucléolyse.

Skin tests (prick tests) with chymopapain were performed on 3 groups of patients: 75 patients awaiting chemonucleolysis with chymopapain (group I), 42 of these 75 patients 2-3 weeks after chemonucleolysis (group II), and 60 atopic patients suffering from asthma and/or rhinitis with positive skin tests to at least one of the airborne allergens (group III). A positive skin test was found in one patient of group I (1.33%), one patient of group III (1.7%) and 11 patients of group II (26.2%). Thus, sensitization to chymopapain is not more frequent among atopic patients, and chemonucleolysis has a highly significant (P less than 0.001) sensitizing effect. Chymopapain-specific IgE's were found in one out of 75 patients (1.33%) before, and in 14 out of 45 patients (31%) after chemonucleolysis; the difference was significant (P less than 0.001). Concordance between skin tests and specific IgE's reached the 74% level. Our results are consistent with those of the literature. They show that prick tests with a 10 mg/ml solution of chymopapain constitute, for the time being, a simple, reliable and cheap method for detecting subjects at risk.

Iatrogenic pseudoanaphylaxis.

A nurse exposed accidentally to chymopapain by ocular exposure was treated vigorously for chymopapain anaphylaxis. Retrospective analysis of the case indicates that there was no evidence of IgE antibody against chymopapain, and the clinical events were inconsistent with anaphylaxis and could be explained by a vasovagal reaction and the cardiorespiratory effects of repeated intravenous epinephrine. Our assessment is that the nurse is currently in a state of good health; however, she did not accept our absence of allergic disease diagnosis and has sought "clinical ecology therapy." Although no litigation in this case has arisen, the legal implications of this case report are reviewed.

[Immunologic methods of study for evaluating the risk of anaphylactic reactions in intradisk chymopapain injections]. / Immunologische Untersuchungsmethoden zur Risikoabschätzung einer anaphylaktischen Reaktion bei der intradiskalen Chymopapain-Injektion.

According to results available to date, skin tests are of limited value in determining the risk of anaphylactic shock. By identifying specific IgE antibodies, e.g. via the Chymo-FAST test or the corresponding RAST, the risk of anaphylactic shock of 0.2%-1% in the total group of patients can be reduced to 0.05% in patients in whom the test was negative, whereas in test-positive patients the risk is above 60%. Previous intolerance reactions among the remaining 0.05% are probably due to a non-testable pseudoallergic reaction, just like the 6% mild late reactions after the injection. For the remaining risk of 0.05% in IgE-negative patients drug prophylaxis with antihistamines or if necessary even steroids will continue to be mandatory. Examination of histamine release from leucocytes, which can be conducted in very few centres only, may possibly be able to clarify in future to what extent reinjection is permissible if a disc on a different level is affected, besides other scientific problems.

Monoclonal antibodies to the two most basic papaya proteinases.

The proteinases from Carica papaya include papain, isoenzymes of chymopapain and two proteinases A and B distinguished by their unusually high pI. The identity of one of the most basic proteinases has been questioned. The present report describes the preparation and characterisation of two monoclonal antibodies that react specifically with papaya proteinases A and B respectively and a third that identifies a common structural feature found in papain and proteinase A.

Evaluation of papain/chymopapain cross allergenicity.

Recent clinical evidence suggests that papain and chymopapain may share common allergenicity. Patients that become sensitized to papain may subsequently experience an allergic reaction when they are exposed to chymopapain. This study demonstrates a cross antigenicity between the proteolytic enzyme preparations papain and chymopapain. Serum samples from six patients who demonstrated 4+ skin reactions to papain also revealed positive RAST ratios to both papain and chymopapain. In addition, serum samples from 12 clinically nonreactive patients who had discolysis with chymopapain demonstrated positive RAST results to papain as well as to chymopapain.

Pretreatment serum levels of IgE to chymopapain in reactive patients.

Discolysis (chemonucleolysis) with chymopapain, the proteolytic enzyme, is currently being used in the treatment of herniated lumbar discs. Of the patients receiving treatment, approximately 1% experience an anaphylactic reaction. This reaction is thought to be mediated by IgE antibodies to chymopapain. Because of this, it may be possible to predict potential reactors using in vitro testing. In the current study, five out of seven patients who experienced allergic anaphylaxis during discolysis demonstrated high IgE serum levels to chymopapain before treatment.