No abstract

A new hemocyanin was isolated from the hemolymph of garden snails Helix vulgaris, composed of two isoforms, HvH1 and HvH2 separated on an ion exchange column DEAE-Sepharose 6CL. Structural and immunological properties of Helix vulgaris hemocyanin were studied in comparison with molluscan Hcs Rapana venosa and Megathura crenulata. The possibility of using HvH and RvH as carriers of small molecules (haptens) in immunizing protocols was studied in comparison with KLH, which is a widely used, highly immunogenic carrier protein. By using HvH as a carrier of the well-known hapten TNBS (2,4,6-trinitrobenzene sulfonic acid), an increasing with time production of hapten-specific TFN-gamma was detected in splenocyte cultures of mice, which lasted longer than in case of KLH and RvH carriers. Also, use of HvH or RvH as a carrier of the hapten ProT alpha[101-109] (i.e., the synthetic C-terminal fragment of the poorly immunogenic protein prothymosin alpha) showed that antisera of higher titres than that of the control conjugate (ProT alpha[101-109]-KLH) were obtained immediately after the second bleeding. HvH and RvH may prove to be useful for the development of new antiviral, antibacterial and antitumor vaccines, since they seem to launch strong and specific immune response against the conjugated antigens.

Prothymosin alpha (proTalpha) is a 109 amino acid long polypeptide presenting distinct immunoenhancing activity in vitro and in vivo. Recent reports suggest that in apoptotic cells, proTalpha is cleaved by caspases at its carboxy(C)-terminus generating potentially bioactive fragments. In this study, we identified the peptide segment of proTalpha presenting maximum immunomodulatory activity. Calf thymus proTalpha was trypsinised, and the five fragments produced (spanning residues 1-14, 21-30, 31-87, 89-102 and 103-109) were tested for their ability to stimulate healthy donor- and cancer patient-derived peripheral blood mononuclear cell (PBMC) proliferation in autologous mixed lymphocyte reaction (AMLR), natural killer and lymphokine-activated killer cell activity, intracellular production of perforin, upregulation of adhesion molecules and CD25 expression. ProTalpha(89-102) and proTalpha(103-109) significantly fortified healthy donor-lymphocytes' immune responses to levels comparable to those induced by intact proTalpha. These effects were more pronounced in cancer patients, where peptides proTalpha(89-102) and proTalpha(103-109) partly, however significantly, restored the depressed AMLR and cytolytic ability of PBMC, by simulating the biological activity exerted by intact proTalpha. ProTalpha(1-14), proTalpha(21-30) and proTalpha(31-87) marginally upregulated lymphocyte activation. This is the first report showing that proTalpha's immunomodulating activity can be substituted by its C-terminal peptide(s). Whether generation and externalization of such immunoactive proTalpha fragments occurs in vivo, needs further investigation. However, if these peptides can trigger immune responses, they may eventually be used therapeutically to improve some PBMC functions of cancer patients.

No abstract

Prothymosin α (ProTα) is a nuclear polypeptide of great biological and, possibly clinical, importance, because its expression levels have been associated with early diagnosis/prognosis of human cancer. It is therefore interesting to raise easily available and cost-effective antibodies that would be applied to develop reliable ProTα immunodiagnostics. In this study, New Zealand white rabbits and laying hens were parallel immunized against intact ProTα or the synthetic fragments ProTα[1-28], ProTα[87-109], and ProTα[101-109], all conjugated to keyhole limpet hemocyanin (KLH). The corresponding antibodies G and Y were immunochemically evaluated in parallel with ELISA and Western blot systems and applied to fluorescence immunocytology experiments using various cancer cell lines and normal cells. The antibody G raised against ProTα[101-109]/KLH had excellent functional characteristics in the Western blot and immunocytology experiments, where the fluorescent signal was almost exclusively shown in the cell nucleus independently of the cells assayed. The above antibody has been applied to preliminary IHC staining of human cancer prostate tissues, leading to a high percentage of clearly and intensively stained nuclei in the adenocarcinoma tissue; this antibody can be further used in cancer tissue immunostaining and in research concerning the role of ProTa in tumorigenesis.