Natural antibodies have been considered to be important in the primary defense against invading pathogens , the clearance of damaged structures, dying cells and oxidized epitopes , and the modulation of cell functions . But also, naturally occurring antibodies could play a role in the protection against neoplastic transformation [25-29]. In this study, we describe the presence of antibodies against NeuGcGM3 ganglioside, circulating in the sera of healthy adult individuals. NeuGcGM3 ganglioside is not only overexpressed on tumor cell membranes, but are also important for tumor development due to its suppressive effect on immune system function . Sixty-five healthy donors’ sera out of 100 tested bound to NeuGcGM3 by ELISA, and did not recognize the acetylated form of this ganglioside. This result is in concordance with a previous result about reactivity against different N-glycolylated compounds of 16 healthy donors, reported by Padler-Karavani et al. . Previous reports have shown the existence of a naturally occurring immunity against glycolipidic antigens, specifically gangliosides. Some of these reactivities have been associated with the induction of pathological alterations, as is the case for the antibodies against ganglioside complexes, such as GD1a and GD1b, or GM1 and GD1a in Guillian–Barre syndrome . However, other studies suggest that naturally occurring antiganglioside antibodies may play an important role in immune surveillance against tumors in humans [25, 27]. Different studies about the antibody response against Neu5Gc containing molecules have shown opposite findings regarding its impact on tumor growth. In a mouse model of human-like Neu5Gc deficiency, transferred polyclonal syngeneic mouse anti-Neu5Gc antibodies interacted with Neu5Gc-positive tumors generating chronic inflammation and facilitating tumor progression . On the other hand, the same group later reported a reduction in tumor growth in mice passively treated with higher amounts of human anti-Neu5Gc antibodies, arguing that the effect on tumor progression or suppression depends on the dose of the anti-Neu5Gc antibodies . Another explanation for the contrasting results could reside in the fact that Neu5Gc-containing glycans are diverse and presented on many different glycoconjugates, with further structural diversity due to different possible Neu5Gc modifications and linkage differences . Thus, in a polyclonal anti-Neu5Gc pool there can be antibodies with different fine specificities and properties. In fact, the anti-Neu5Gc antibodies purified in the previous reports  had minimal reaction with NeuGcGM3 ganglioside, the Neu5Gc-containing antigen recognized by the healthy donors’ sera evaluated in our study.
The anti-NeuGcGM3 antibodies present in the healthy donors’ sera were not only able to recognize NeuGcGM3 coated on ELISA plates, but also when NeuGcGM3 was expressed on tumor cell membranes. We confirmed that the binding to L1210 cells was dependent on the presence of NeuGcGM3. First, we demonstrated that the sera detected an N-glycolylated molecule, by showing that the antibodies in the sera did not recognize L1210-cmah-kd cells. Next, we demonstrated that the detected glycolylated molecule was not a glycoprotein, since the binding was not affected by trypsin treatment. Finally, we blocked cell line recognition by preincubation of the sera with NeuGcGM3. This binding was not inhibited by NeuAcGM3, a ganglioside that differs only in the presence of a hydroxyl group in the N-glycolylated variant. Furthermore, we demonstrated that these antibodies were able not only to recognize but also to induce the death of NeuGcGM3-expressing tumor cells by complement cascade activation, and also by a complement-independent mechanism. This cell death mechanism is different from apoptosis, since it was temperature independent, did not induce caspase activation, and chromatin condensation or apoptotic body formation were not detectable. The incubation of the cells with sera increased the size of the cells and disrupted cell membranes. These characteristics resemble the oncotic cell death reported for anti-NeuGcGM3 mAb 14F7, and for anti-NeuGcGM3 antibodies induced in NSCLC patients treated with 1E10 anti-idiotypic vaccine [18, 20]. This cell death depended on the expression of antigen on the cell membrane, since the cytotoxicity was completely abrogated on tumor cells that do not express NeuGcGM3, or by preincubation of the donors’ sera with saturating amounts of this ganglioside. Nguyen et al. reported the capacity of healthy donors’ sera to bind and kill human leukemic cells and activated T cells that were exogenously fed with Neu5Gc, but in these studies the detected cell death was mediated only by a complement-mediated mechanism . The antibodies that recognized NeuGcGM3-expressing cells were of the IgM isotype. The IgM fraction isolated from one of the healthy donor's sera retained the capacity to induce complement-independent death of the tumor cells. To our knowledge, this is the first report of anti-NeuGcGM3 antibodies that are able to induce the oncotic cell death of antigen-expressing tumor cells without the necessity of any other immune component. These results suggest the existence of antibodies with antitumor potential, which could contribute to tumor immune surveillance. It is interesting to observe that the levels of anti-NeuGcGM3 antibodies decreased as the age of the donors increased. Not only is the level of anti-NeuGcGM3 antibodies lower in elderly donors, but also the percentage of responding donors decreases with age. An age-associated decrease in antibody levels against foreign antigens was first reported more than 70 years ago , supporting the idea of an immune deficiency state in the elderly. However, this seems to be a phenomenon dependent on the nature of the antigen and the cells involved in the different responses, since other studies have shown that the concentration of serum antibodies against a variety of self-antigens such as thyroglobulin, DNA, and IgG, increases with age . In fact our results demonstrate that the total amount of IgG and IgM did not decrease with age, suggesting that it is not the amount of antibodies but the antibody repertoire that changes with age. One possible explanation for the decrease in antibody levels with increasing age involves an impaired capacity of T cells to facilitate the maturation of B cells in the periphery and the generation of a diverse B-cell repertoire from precursors within the bone marrow . According to this theory, the response against T-independent antigens should not be affected by age . However, the antibody response against not only NeuGcGM3 but also against other tumor related gangliosides (T-independent antigens), significantly decrease with increasing donor age . Another possibility could be a reduction in the B-cell population responsible for the production of naturally occurring antibodies. Recently, Griffin et al. described a human B-cell population equivalent to mouse B1 cells , the main source of murine natural antibodies . These researchers showed that human B1 cells decline with age. The reduction of B cells secreting antibodies with immune surveillance properties could explain, at least in part, the increased susceptibility of aged individuals to neoplastic transformation. In fact, it is interesting to observe that in NSCLC patients, who had not been exposed to any antitumor treatment (including radio or chemotherapy), we could not detect cytotoxic anti-NeuGcGM3 antibodies in the conditions used for our study. This behavior was observed even in those patients less than 60 years of age. Only six of the 53 NSCLC patients studied had a low response against NeuGcGM3, and their sera were not able to bind to tumor cells expressing the antigen. The levels of IgG and IgM antibodies did not decrease with the age of the cancer patients, however, we did detect a significantly lower total IgM concentration in the cancer patients’ sera when compared with healthy donors’. In contrast, the IgG concentrations were similar, suggesting that the IgM reduction is not due to a general state of immunosuppression in these patients. The reduced level of anti-NeuGcGM3 antibodies detected in these patients could be a consequence of the low total IgM levels, the isotype of the antibodies that recognize NeuGcGM3. But this specificity could be particularly affected, resembling what we observed for elderly healthy donors. In the case of these cancer patients, the observed behavior could be due to the anti-NeuGcGM3 antibody-secreting B-cell population being affected, or to the capacity of this B-cell population to secrete antibodies with this specificity being inhibited. By idiotypic vaccination, however, we have been able to boost this kind of immune response in cancer patients, which suggests that these cells are not completely deleted . Another possibility is that, in NSCLC patients, anti-NeuGcGM3 antibodies form immune complexes with gangliosides released from the tumor cells, which might affect their detection. This phenomenon could also result from the recruitment of such antibodies to the tumors since the presence of NeuGcGM3 in NSCLC tumor samples has been reported [41-43]. To our knowledge this is the first report showing that the levels of anti-NeuGcGM3 antibodies are lower in cancer patients in comparison with healthy donors. Previous work reported that, depending on the ganglioside and the kind of tumor, higher or lower concentrations of antibodies against gangliosides in the sera of cancer patients with respect to healthy donors, could have a prognostic value [25, 44]. Further studies are needed to evaluate whether this is also the case for the antibody response against NeuGcGM3.