In vitro and in vivo antifungal properties of cysteine proteinase inhibitor from green kiwifruit.

BACKGROUND: Higher plants possess several mechanisms of defense against plant pathogens. Proteins actively synthesized in response to those stresses are called defense-related proteins which, among others, include certain protease inhibitors. It is of particular relevance to investigate plant natural defense mechanisms for pathogen control which include cystatins-specific inhibitors of cysteine proteases. RESULTS: In this study, a cysteine proteinase inhibitor (CPI), 11 kDa in size, was purified from green kiwifruit to homogeneity. Immuno-tissue print results indicated that CPI is most abundant in the outer layer of pericarp, near the peel, and the inner most part of the pulp-sites where it could act as a natural barrier against pathogens entering the fruit. The purified protein (15 µmol L(-1)) showed antifungal activity against two phytopathogenic fungi (Alternaria radicina and Botrytis cinerea) by inhibiting fungal spore germination. In vivo, CPI (10 µmol L(-1)) was able to prevent artificial infection of apple and carrot with spore suspension of B. cinerea and A. radicina, respectively. It also exerted activity on both intracellular and fermentation fluid proteinases. CONCLUSION: Identification and characterization of plant defense molecules is the first step towards creation of improved methods for pathogen control based on naturally occurring molecules.

In vitro stimulation of Balb/c and C57 BL/6 splenocytes by a recombinantly produced banana lectin isoform results in both a proliferation of T cells and an increased secretion of interferon-gamma.

Lectins are widely used in many types of assay but some lectins such as banana lectin (BanLec) are recognised as potent immunostimulators. Although BanLec's structure and binding characteristics are now familiar, its immunostimulatory potential has not yet been fully explored. The synthesis by recombinant technology of a BanLec isoform (rBanLec) whose binding properties are similar to its natural counterpart has made it possible to overcome the twin problems of natural BanLec's microheterogeneity and low availability. This study's aim is to explore the immunostimulatory potential of rBanLec in the murine model. Analyses of the responses of Balb/c- and C57 BL/6-originated splenocytes to in vitro rBanLec stimulation were performed to examine the dependency of rBanLec's immunostimulatory potential upon the splenocytes' genetic background. It is shown that the responses of Balb/c- and C57 BL/6-originated splenocytes to rBanLec stimulation differ both qualitatively and in intensity. The hallmarks of the induced responses are T lymphocyte proliferation and intensive interferon-gamma secretion. Both phenomena are more marked in Balb/c-originated cultures; Balb/c-originated lymphocytes produce interleukin (IL)-4 and IL-10 following rBanLec stimulation. Our results demonstrate that any responses to rBanLec stimulation are highly dependent upon genetic background; they suggest that genetic background must be an important consideration in any further investigations using animal models or when exploring rBanLec's potential human applications.

Design and modifications of allergens for improving specific immunotherapy.

Specific immunotherapy is a clinically effective causative treatment for allergic conditions. However, the reagents used for immunotherapy are crude extracts, prepared from natural sources, and as such may cause potential life-threatening anaphylactic side effects. Our increasing knowledge of mechanisms that lead to allergy and of the mechanisms of successful immunotherapy have introduced new ideas for the treatment of allergic diseases. Techniques of biochemistry and molecular biology have made it feasible to design novel therapeutic approaches for improved and safer forms of allergen-specific immunotherapy. The purpose of this review is to examine different approaches in designing novel allergen formulations for specific immunotherapy. These include chemically modified forms of allergens, genetically modified allergens (hypoallergens), hybrid allergens and allergens bearing immunostimulatory molecules, or adjuvants, which foster Th 1 immune responses. There is great interest in newly designed allergens and their derivatives to improve the efficacy and safety of allergen immunotherapy. A better understanding of immunological mechanisms and further clinical trials utilizing new allergen formulations are needed.

Quantification of the thaumatin-like kiwi allergen by a monoclonal antibody-based ELISA.

Thaumatin-like proteins (TLPs) have been established as a new family of fruit and pollen allergens. The aim of this study was to develop a two-site ELISA for the quantification of the thaumatin-like kiwi allergen (Act d 2) in kiwifruit extracts and kiwifruit-containing food products. Genomic DNA (gDNA) of Act d 2 was amplified and the deduced amino acid sequence was determined to obtain a primary structure. Act d 2 purified from kiwifruit extract by HPLC was identified by Edman degradation and MS. Balb/c mice were immunized with Act d 2 for the production of mAbs by hybridoma technology. The optimized ELISA measured Act d 2 concentrations ranging from 0.2 to 9.0 ng/mL, with intra- and interassay coefficients of variation of 3.65 and 10.44%, respectively. The developed ELISA is a useful method for the quantification of the thaumatin-like kiwi allergen in kiwifruit extracts as well as the allergen level in kiwifruit-containing food products. It may be a helpful analytical tool for the evaluation of the stability (integrity) of fruit allergen extracts for in vitro diagnosis.

Development of sandwich enzyme-linked immunosorbent assay for determination of tetanus toxoid concentration.

According to the recommendation of the World Health Organization (WHO), the use of an in vivo test for measuring of the potency of tetanus toxoid vaccine (TTdV) is still unavoidable, but the establishment of a convenient in vitro test would significantly improve the work in this field. A sandwich enzyme-linked immunosorbent assay (sELISA) was developed for a rapid and sensitive quantification of tetanus toxoid (TTd). We produced four monoclonal antibodies (MAbs) designated 41, 51, 62, and 71 that reacted with TTd and recognized different antigenic determinants on TTd. We also used two of these antibodies for developing a sELISA, with MoAb 71 as an immobilized and MoAb 51 as a capture antibody. The measurement range of this assay was from 31-1000 ng/mL and the minimum detection limit for TTd was 31 ng/mL. This high sensitivity of this sELISA and its good reproducibility suggest that the developed method could be reliably used to estimate the concentration of TTd, which could be easily extrapolated to the estimation of vaccine potency.

Isolation and biochemical characterization of a thaumatin-like kiwi allergen.

BACKGROUND: Kiwi fruit allergy, as well as its association with hypersensitivity to other foods and to pollen, has been extensively reported in the last few years. Several IgE-binding components have been detected in kiwi extract, but only one 30- kd allergen has been isolated; it was identified as actinidin (Act c 1). Recently, we have reported a 24-kd kiwi protein to be a potential major allergen in a group of patients with oral allergy syndrome (OAS). OBJECTIVE: The aim of this study was to purify and characterize the 24-kd kiwi allergen biochemically. METHODS: Seven polysensitized patients with OAS to kiwi were used in this study. The kiwi allergen was isolated by using a combination of gel permeation, ion exchange, and immobilized metal ion affinity chromatography. Its biochemical characterization included determination of its isoelectric point, molecular weight, N-terminal sequencing, concanavalin A -binding ability, digestibility in simulated gastric fluid, and antifungal activity. Western blotting, 2-dimensional PAGE immunoblotting, and skin prick tests were performed to characterize the isolated protein immunochemically. RESULTS: All 7 patients recognized the isolated 24-kd kiwi protein as an allergen. The isolated protein consisted of 2 isoforms with isoelectric points of 9.4 and 9.5 migrated as one protein band of 20 kd after SDS-PAGE under nonreducing conditions or at 24 kd under reducing conditions. The partial N-terminal sequence revealed that it is a thaumatin-like protein (TLP) with concanavalin A -binding ability. The protein showed antifungal activity toward Saccharomyces carlsbergensis, and Candida albicans. The protein was degraded by the simulated gastric fluid within 1 minute. Both isoforms bound IgE from a pool of sera in a 2-dimensional PAGE immunoblot. The TLP elicited positive skin prick test responses in 4 (80 %) of 5 patients with OAS. CONCLUSION: This study reported isolation and full characterization of a new kiwi allergen, TLP (isoelectric points of 9.4 and 9.5 and molecular weight of 24 kd), which belongs to the family of pathogenesis-related proteins. The isolated protein expressed antifungal activity toward S carlsbergensis and C albicans.