Identification of T-cell stimulating antigens from Giardia lamblia by using Giardia-specific T-cell hybridomas.

T-cell immune response plays an important role in controlling Giardia lamblia infections. Little is known about the G. lamblia-specific antigens that stimulate a cell-mediated immune response. The aim of the present study was to identify T-cell stimulating G. lamblia antigens. For this purpose, we generated a group of Giardia-specific T-cell hybridomas (2F9, 4D5, 6D10, 8B9, 9B10, 10F7 and 10G5). Hybridomas were screened for reactivity with G. lamblia protein extract by the CTLL bioassay. These T-cell hybridomas did not exhibit any significant activation either in the absence of G. lamblia protein extract or in the presence of irrelevant antigen (hen white egg lysozyme). To further characterize the T-cell hybridomas generated, we selected three hybridomas (10G5, 4D5 and 9B10). Giardia lamblia proteins of 90-110, 65-77 and 40-64 kDa showed T-cell stimulating activity for the hybridomas 10G5, 4D5 and 9B10, respectively, in a concentration-dependent manner. Protein extract obtained from different G. lamblia strains (GS/M-83-H7, WB C6 and a clinical isolate (YJJ)) stimulated all T-cell hybridomas, indicating that T-cell-stimulating antigens are expressed among different G. lamblia strains. In conclusion, we identified T-cell stimulating G. lamblia antigens by using Giardia-specific T-cell hybridomas. To our knowledge, these hybridomas are the first-described T-cell hybridomas specific for G. lamblia.

Isolation and properties of AMP deaminase from jumbo squid (Dosidicus gigas) mantle muscle from the Gulf of California, Mexico.

Adenosine monophosphate (AMP) deaminase was purified from jumbo squid mantle muscle by chromatography in cellulose phosphate, Q-Fast and 5'-AMP sepharose. Specific activity of 2.5U/mg protein, 4.5% recovery and 133.68 purification fold were obtained at the end of the experiment. SDS-PAGE showed a single band with 87kDa molecular mass, native PAGE proved a band of 178kDa, whereas gel filtration detected a 180kDa protein, suggesting the homodimeric nature of this enzyme, in which subunits are not linked by covalent forces. Isoelectric focusing of this enzyme showed a pI of 5.76, which agrees with pI values of AMP deaminase from other invertebrate organisms. AMP deaminase presented a kinetic sigmoidal plot with Vmax of 1.16µM/min/mg, Km of 13mM, Kcat of 3.48µM.s(-1) and a Kcat/Km of 267 (mol/L)(-1).s(-1). The apparent relative low catalytic activity of jumbo squid muscle AMP deaminase in the absence of positive effectors is similar to that reported for homologous enzymes in other invertebrate organisms.