Fraction 1 of the glycoprotein agglutinated sheep blood cells (1%), whereas Fraction 2 of the glycoprotein did not. Hemagglutinating activity of the fraction was compared to that of phytohemagglutinin P; maximal agglutination by Fraction 1 of glycoprotein 1.25mg/ml, whereas phytohemagglutinin-P showed maximal agglutination at a concentration of 0.04mg/ml. The glycoprotein fraction was also shown to stimulate deoxyribonucleic acid synthesis in baby hamster kidney (BHK 21) cells. Heat-treatment of the fraction abolished the stimulation of DNA synthesis, which suggests that the active component is the native protein moiety of glycoprotein. The therapeutic effect of aloe on burns was attributed to the induction of blastmitogenesis by the stimulation of DNA synthesis by the glycoprotein.
ATF1011, A LECTIN AUGMENTING TUMOR-SPECIFIC IMMUNITY
A lectin that did not have hemagglutinating activity or mitogenic activity but which did have agglutinating activity against tumor cells was separated from whole leaves of A. arborescens (Yoshimoto etal, 1987). The lectin differed from aloctins which had already been separated from A. arborescens and augmented tumor-specific immunity through activation of T cells specific for the lectin.
The supernatant solution of homogenates of whole leaves of A. arborescens was fractionated with 40% saturation ammonium sulfate as the final concentration. After dialysis of the precipitated fraction, non-dialyzates were dissolved with 50 mM phosphate buffer, pH 7.5 and applied to a series of a DEAE-cellulose column chromatography three times; first elution with 0.4M NaCl/50mM phosphate buffer: second elution with a linear gradient of 0M-0.4M NaCl/50mM phosphate buffer; and, third elution with 0 M—0.3 M NaCl/50 mM phosphate buffer. The active fraction was lyophilized.
ATF 1011 is a glycoprotein containing 0.4—0.7% neutral sugars as mannose and less than 0.3% amino sugars as glucosamine, has isoelectric point 4.3—5.2 and has a molecular mass of about 64 kDa.
Affects on tumor cells. i No direct cytotoxicity of ATF 1011 to the tumor cells
Since Aloctin A was shown to have antitumor effects, antitumor effects of ATF 1011 were also examined using tumor cells such as MM46 and MM102 (mammary tumor cell lines originating from C3H/He, inbred mouse strain), MH134 (hepatoma cell line originating from C3H/He), Meth-A (fibrosarcoma cell line originating from BALB/C, inbred mouse strain), P815 (mastocytoma cell line from DBA/2, inbred mouse substrain) and EL4 (thymoma cell line from C57/BL6, inbred mouse strain).
Effects of ATF 1011 on the growth of NN46, MM102, MH134 and EL4 were examined and all the tumor cell lines tested showed almost the same growth in the presence of ATF 1011 as that of the control culture, indicating that there was no direct cytotoxicity to the tumor cells. However, MM46, MH134 and EL4 did not grow in the presence of Con A (low concentration). Ricin and abrin were also cytotoxic to these tumor cell lines (Fodstad and Pihl, 1978). MM102 was resistant to low concentrations of Con A but growth was suppressed at high concentration.
The following experiments were attempted to determine the relationship to the immune systems of ATF 1011; activation of Thymus helper (Th) cells in antibody production by ATF 1011; augmentation of cytotoxic Thymus(T) cell response by ATF 1011; and, induction of antitumor cytotoxic T cells in syngeneic tumor-bearing mice by intralesionally administered ATF 1011.
These results demonstrated the potential of ATF 1011 as a carrier protein in the immune system. That is, intralesionally administered ATF 1011 binds to the tumor cell membrane and activates T cells specific for this carrier lectin in situ, resulting in the augmented induction of systemic antitumor immunity.
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