14F7 murine monoclonal antibody (MAb) is an IgG1 immunoglobulin that is generated by immunizing Balb/c mice with GM3(NeuGc) ganglioside hydrophobically conjugated with human very-low-density lipoproteins and in the presence of Freund's adjuvants. 14F7 MAb binds specifically to GM3(NeuGc), whereas neither N-glycolyl or N-acetyl gangliosides, nor a sulfated glycolipid, are recognized as assessed by enzyme-linked immunosorbent assay or immunostaining on thin layer chromatograms. Immunohistochemical studies in fresh tumor tissues showed that 14F7 MAb strongly recognized in antigen expressed in human breast and melanoma tumors.

We generated the 1E10 γ-type anti-idiotype mAb (Ab2) specific to an Ab1 mAb able to react specifically with N-glycolyl-containing gangliosides and with Ags expressed on human melanoma and breast carcinoma cells. This Ab2 mAb induced an Ab response in animal models sharing immunochemically defined idiotopes with the Ab1. The treatment of tumor-bearing mice with 1E10 mAb induced a strong antitumor activity. A clinical trial was conducted in 20 patients with advanced malignant melanoma. Patients were treated with six intradermal injections of aluminum hydroxide-precipitated 1E10 anti-Id mAb given at 2-wk intervals. Sixteen of the 17 patients who received at least four doses of the anti-Id vaccine develop Ab3 Abs capable of inhibiting Ab2 binding to Ab1 (Ab3Id+). In contrast to the incapacity of 1E10 mAb to generate Ab3 Abs with the same antigenic specificity as the Ab1 mAb in mice, a very specific and strong Ab3 response against N-glycolyl-containing gangliosides was induced in 16 patients (Ab3Ag+). No evidence of serious or unexpected adverse effects has been observed in this clinical trial. 1E10 anti-Id vaccine was safe, well tolerated, and immunologically effective, with most patients being able to generate a specific immune response against 1E10 and Neu-glycolyl-GM3 ganglioside.

The P3 murine monoclonal antibody (MAb) was generated by immunizing BALB/c mice with NeuGcGM3 in¬ cluded into liposomes. The specificity of this MAb was defined by an enzyme-linked immunosorbent assay and immunostaining on thin-layer chromatograms. P3 MAb binds to NeuGc-containing gangliosides and was shown also to react with sulfated glycolipids. A preliminary immunohistochemical study showed that the P3 MAb was able to recognize antigens expressed in human breast tumors.

No abstract

An IgM monoclonal antibody (MAb), named P3, has the characteristic to react specifically with a broad battery of N-glycolyl containing-gangliosides and with antigens expressed on breast tumors. When this MAb was administered alone in syngeneic mice, an specific IgG anti-idiotypic antibody (Ab2) response was induced, this Ab2 response was increased when P3 MAb was injected coupled to a carrier protein and in the presence of Freund's adjuvant. Spleen cells from these mice were used in somatic-cell hybridization experiments, using the murine myeloma cell line P3-X63-Ag8.653 as fusion partner. Five Ab2 MAbs specific to P3 MAb were selected. These IgG1 Ab2 MAbs were able to block the binding of P3 MAb to GM3(NeuGc) ganglioside and to a human breast carcinoma cell line. Cross-blocking experiments demonstrated that these Ab2 MAbs are recognizing the same or very close sites on the Abl MAb. The five Ab2 MAbs were injected into syngeneic mice and four of them produced strong anti-anti-idiotypic antibody (Ab3) response. While these Ab2 MAbs were unable to generate Ab3 antibodies with the same antigenic specificity than P3 MAb, three of them induced antibodies bearing P3 MAb idiotopes (Ag-Id+ Ab3). These results demonstrated that these Ab2 MAbs are not "internal image" antibodies, but they could define "regulatory idiotopes."

The variable regions from P3, a murine monoclonal antibody (MAb) against NeuGc-containing gangliosides, and two anti-idiotype MAbs directed to P3 MAb were cloned and sequenced. Comparisons with previously reported sequences showed that P3 is a germline antibody encoded by genes from the V(H)Q52 and V(kappa)19 families. Analysis of nucleotides at the heavy chain CDR3 (H-CDR3) showed the presence of an extensive 3' N region that contains almost 50% of the nucleotides of this CDR. In addition, amino acid sequence analysis of the H-CDRs of this MAb revealed the presence of three arginines, two of which are present in the H-CDR3, that could be involved in the interaction of P3 MAb with its electronegative epitope on gangliosides. Anti-idiotype 1E10, which seems to define a "regulatory" idiotope on P3 MAb (it induces Id+ Ab3), represents a germline Ab2 that belongs to the V(H)J558 and V(kappa)10 gene families. By contrary, the anti-idiotype 3B11 is an extensively mutated antibody that belongs to the V(H)3660 and V(kappa)4/5 gene families, defining a "private" idiotope on P3 MAb. Even when different V genes contribute to the variable regions of 1E10 and 3B11 MAbs, they share an acidic motif E/D-D-Y/D-Y-D in H-CDR3, suggesting that both Ab2s recognize paratope positive residues on the Ab1. Therefore, complementary electrostatic interactions involving H-CDR3 from both Ab1 and Ab2, might provide a clue to understand the molecular basis for the generation of gamma-type anti-idiotype antibodies to V regions recognizing glycolylated ganglioside antigens.

The objective of this on-farm study was to determine the effect of corn planting density on the nutritional quality of whole-plant corn for silage. This study was performed in a commercial 1,900-cow dairy farm located in Piedritas (Buenos Aires, Argentina). Two commercial hybrids (A and B) were planted in experimental plots within a cornfield destined for corn silage. Hybrids were sown at a theoretical seeding rate of 60,000, 70,000, 80,000, and 90,000 seeds/ha in 4 replicates per hybrid. Plots were eight 50-m-long rows separated by 52cm. Corn was planted with a no-till seeder equipped with a pneumatic dosing machine. Ten plants within each plot were cut by hand at 15cm above ground. Whole plants were chopped, weighed, mixed thoroughly, and frozen until analysis. Nutritional composition was determined by near-infrared reflectance spectroscopy. Harvesting occurred at one-quarter milk-line [31.4% dry matter (DM)] and one-half milk-line (34.5% DM) stages of maturity for hybrids B and A, respectively. No interactions between hybrid and planting density were observed for any of the variables of interest. Planting density did not affect either plant DM weight or DM, crude protein, neutral detergent fiber, acid detergent fiber, or starch concentrations of whole-plant corn. Dry matter yield was significantly increased at higher planting densities. The similar per-plant biomass and nutritional quality among different densities can be explained by the abundant precipitation observed during this growing season (719mm since the beginning of fallow until harvest). In conclusion, greater yields of silage can be obtained by increasing corn planting density without affecting its nutritional composition, although the effect of planting density with limiting resources (e.g., precipitation) still needs to be elucidated.

Purified GM1 and GM2 gangliosides incorporated into liposomes were injected subcutaneously in BALB/c mice every 3-4 days after pretreatment of the animals with low-dose cyclophosphamide. Serum samples were collected at different intervals and tests by ELISA for the presence of anti-ganglioside antibodies. Four doses (50 micrograms each) were sufficient to raise a measurable primary type of response to GM1, while nine doses were required to obtain measurable IgM antibody titers to GM2. Three monoclonal antibodies (MAbs) wer generated by fusing splenocytes with mouse myeloma cells. The specificity of MAbs was determined by ELISA and HPTLC-immunostaining using a panel of purified glycolipids. The MAb designated E1 showed a high degree of specificity because it reacted only with N-acetyl GM2. Monoclonal antibody A3 reacted predominantly with GM2 and GM1, but also reacted moderately with the GM3 ganglioside. The epitope recognized by this MAb is suggested to be the trisaccharide sequence GalNAc beta 1-4(NeuAc alpha 2-3)Gal. The third MAb (F6) reacted strongly with GM1 but a weak reactivity was also observed with GD1b as well as with asialo-GM1, indicating that the terminal tetrasaccharide Gal beta 1-3GalNAc beta 1-4(NeuAc alpha 2-3)Gal- structure is probably involved in antigenic recognition. Formalin-fixed and paraffin-embedded tissue sections were stained with the E1 and A3 MAbs, using the avidin-biotin complex (ABC) technique. Strong immunoreactivity for E1 appeared in the tumor cells of five primary lung carcinomas and in five malignant melanomas. No immunoreactivity was demonstrated in the parenchyma of a lung without malignancy, or in a metastasis from a colon carcinoma.