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Biomedical subjects

R F Irie

Publications and source records attributed to R F Irie.

At least 37 records · Page 2Linked to original sources
PubMed1994

Attachment of monophosphoryl lipid A (MPL) to cells and liposomes augments antibody response to membrane-bound gangliosides.

Antibodies to gangliosides are found in low levels in normal individuals, and attempts to augment their production have had limited success. Murine studies suggest that the antibody response to membrane-bound cryptic antigens, such as phospholipids and gangliosides, can be induced and augmented by attaching lipid A to membranes. Therefore, we assessed the ability of monophosphoryl lipid A, a non-toxic derivative of lipid A, to augment antibody response against membrane-associated gangliosides. Anti-ganglioside antibodies were IgM after the first and second immunizations; in contrast, anti-phospholipid antibodies were IgM after the first immunization and IgG after the second immunization. Mice (BALB/c) immunized with MPL-attached human cells as well as mice (C57BL/6J) immunized with MPL-attached syngeneic tumor cells (B16 melanoma) produced a significant IgM response. Mice (C57BL/6J) immunized with MPL-attached liposomes containing GM3 developed significantly higher IgM responses than those immunized with purified gangliosides, MPL or MPL-free B16 cells. However, the antibody response after immunization with MPL-GM3-liposomes is similar to that after immunization with MPL-attached tumor cells, even though the MPL-liposomes contained a 27-fold higher level of gangliosides than the tumor cells. Our results emphasize that co-expression of MPL with membrane-bound gangliosides is necessary to augment the anti-ganglioside antibody response. These findings may shed light on the elevated titers of anti-ganglioside IgM antibodies found in patients with motor neuron diseases, various neuropathies and classical ALS, and are relevant to clearance of circulating immunosuppressive gangliosides in cancer patients.

Adjuvants, Immunologic↗
PubMed1994

An improved method for the measurement of total lipid-bound sialic acids after cleavage of alpha 2,8 sialic acid linkage with Vibrio cholerae sialidase in the presence of cholic acid, SDS and Ca2+.

In the measurement of total lipid-bound sialic acids involving periodic acid oxidation, as in the periodate-resorcinol assay, the inner sialic acids of disialoglycolipids (such as GD3 and GD2) are not involved because their alpha 2,8 ketosidic linkages are resistant to periodic acid oxidation, even after acid/enzyme hydrolysis or alkali pretreatment. However, the sialic acids from these glycolipids can be recovered completely after cleavage of alpha 2,8 linkages by V. cholerae sialidase in the presence of cholic acid, sodium dodecyl sulphate and calcium. Interestingly, removal of calcium or detergent(s) or both significantly minimizes the sialidase action on the disialyl residues of these gangliosides. Therefore, we recommend sialidase (Vibrio cholerae) pretreatment of the glycolipids in the presence of cholic acid, SDS and Ca2+ for complete recovery of sialic acids from di- and polysialogangliosides and for accurate measurement of total lipid-bound sialic acids by periodate-resorcinol assay.

Calcium↗
PubMed1994

Both VH and VL regions contribute to the antigenicity of anti-idiotypic antibody that mimics melanoma associated ganglioside GM3.

Previously we developed a murine monoclonal anti-idiotype (anti-id) antibody (4C10) that mimics the melanoma-associated ganglioside antigen GM3, that is, it carries the internal image of GM3. 4C10 was made against the human monoclonal antibody (HuMAb) L612, which reacts with several types of human cancer cells, including melanoma and breast cancer. To reduce mouse components of 4C10, the constant region was replaced by a human constant domain to form the murine/human chimeric anti-id antibody TVE-1. In the present study, we sought to determine which chain (VH or VL) of the anti-id is responsible for the antigenicity of GM3. The TVE-1 VH and VL expression vectors were simultaneously transfected with either the VH or VL expression vector of a murine-human chimeric IgG antidansyl haptenic antibody, resulting in the construction of three different combinations of VH and VL chimeric antibodies. These IgG molecules were produced from the transfectomas, and their reactivity to HuMAb L612 was tested. Neither of the IgG proteins that had cross-combined the VH-VL pair showed positive results, suggesting that both heavy and light chains are required to express the antigenicity. The in vivo antigenicity of this chimeric anti-id was confirmed by skin tests in melanoma patients receiving active specific immunotherapy.

Animals↗
PubMed1994

Serum preparation and methods for the large-scale production of IgG monoclonal antibody.

In this study we demonstrated that fetal calf serum (FCS) depleted of IgG by protein G affinity chromatography (G-FCS) is superior to whole FCS or serum-free culture media as a culture supplement for the production of purified IgG monoclonal antibodies (MAb). One hundred ml FCS was applied to a 25 ml protein G Sepharose 4 Fast Flow Column, which was shaken gently for 2 days at 4 degrees C. The procedure was repeated using a protein G column for an additional day. G-FCS was used at a concentration of 5% in RPMI 1640 medium to grow the mouse myeloma cell line P3X63.Ag8.653, which secretes an IgG-1 mouse-human chimeric monoclonal antibody (TVE-1). Cell density, viability, doubling time, and antibody production were used as indices to compare the efficacy of this medium with that of whole FCS medium, AIM-V (Gibco, USA) and other serum-free media. The results demonstrate that cell growth and antibody production in -GFCS medium did not differ significantly from that in FCS medium, but were significantly better than in the serum-free media (p < 0.001). TVE-1 antibody in the spent tissue culture media was purified by 50% ammonium sulfate precipitation and Protein A affinity chromatography. An antibody that was more than 99% pure was obtained. Endotoxin analysis revealed that the IgG depletion process does not generate a significant level of endotoxin in FCS (< 0.06 EU/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Molecular cloning of a human monoclonal antibody reactive to ganglioside GM3 antigen on human cancers.

In this study we report the characterization of a human monoclonal antibody (HuMab), L612, that reacts with ganglioside GM3 and has therapeutic application for the treatment of human neoplasms, particularly melanoma. A permanent IgM-secreting Epstein-Barr virus-transformed B-cell line L612 was established. L612 HuMab bound specifically to neoplastic cell lines in culture and in tissue biopsy specimens such as melanoma, colon, breast, and lung cancer. The antibody did not bind to normal cells or biopsy tissue. HuMab L612 showed the highest reactivity to melanoma cells, particularly to those with high concentrations of GM3. Immunostaining on high-performance thin-layer chromatography plates demonstrated that L612 HuMab bound to GM3 purified from melanoma cells. Removal of the sialic acid from GM3 abolished antibody binding. HuMab L612 also reacted to GM4 purified from egg yolk, indicating that it recognizes an NeuAc alpha 2-3 galactose antigen determinant. HuMab L612 heavy and light chains were sequenced and determined to belong to the mu heavy chain variable subgroup III and kappa chain variable subgroup IV families, respectively. The studies indicate that the L612 HuMab has significant therapeutic potential for a wide variety of human cancers.

Adenocarcinoma↗
PubMed1993

A protein fraction from aged garlic extract enhances cytotoxicity and proliferation of human lymphocytes mediated by interleukin-2 and concanavalin A.

Fraction 4 (F4), a protein fraction isolated from aged garlic extract, enhanced cytotoxicity of human peripheral blood lymphocytes (PBL) against both natural-killer (NK)-sensitive K562 and NK-resistant M14 cell lines. Although F4 treatment alone increased cytotoxicity, the effect was more remarkable when F4 was administered together with suboptimal doses of interleukin-2 (IL-2); combination treatment of 5 micrograms/ml F4 plus 10 U/ml IL-2 for 72 h generated lymphokine-activated killer activity equivalent to that produced by 100 U/ml IL-2 alone against M14. F4 enhanced IL-2-induced proliferation and IL-2 receptor (Tac) expression of PBL without significant increase of IL-2 production. The enhancement of cytotoxicity both by F4 alone and by F4 plus IL-2 was abolished by anti-IL-2 antibody. F4 also enhanced concanavalin-A(ConA)-induced proliferation of PBL. Radiolabeled-ConA binding assays revealed that F4 treatment greatly augmented the affinity and slightly increased the number of ConA binding sites in PBL. F4 also enhanced ConA-induced IL-2 receptor (Tac) expression and IL-2 production of PBL. Anti- IL-2 antibody inhibited the effect of F4 on ConA-induced proliferation. These data suggest that IL-2 is involved in augmentative effects of F4. Our results indicate that F4 is a very efficient immunopotentiator and may be used for immunotherapy.

Concanavalin A↗
PubMed1993

Aberrant expression of gangliosides in human renal cell carcinomas.

Aberrant and elevated ganglioside expression has been observed in neoplasms, and has been shown to be an important marker of tumor progression. We therefore studied the gangliosides of renal cell carcinoma (RCC) by analyzing gangliosides from 18 RCC biopsies, 10 RCC lines and 5 normal kidney biopsies. A comparison of tumor with normal tissue revealed a significant difference in individual ganglioside expression in which the former consistently expressed eight major gangliosides, GM3, GM2, GM1, GD3, GD1A, GD2, GD1B and GT1B, according to the nomenclature of Svennerholm. There was a notable significant mean increase in the expression of GM2, GM1 and GD1A and a significant decrease in the expression of GD3 in tumor tissue compared with normal kidney tissue. Compared with tumor biopsy tissue, RCC cell lines showed a significant decrease in the expression of GM3, but a significant increase in GM2, GM1 and GD2. There was a marked increase in a pathway gangliosides (GM2, GM1, and GD1a) in RCC biopsies and cell lines compared with normal kidney. These studies indicating that RCC have markedly aberrant ganglioside expression similar to neural origin tumors may relate to the activation of a ganglioside pathway enzymes. Gangliosides expressed on RCC tumors may be important markers of tumor progression and target antigens for immunotherapy.

Biomarkers, Tumor↗
PubMed1993

Immunotherapy of melanoma: current status and prospects for the future.

As one of the most immunogenic human malignancies, melanoma has served as a prototype for the development of immunotherapy against human cancer in a number of treatment modalities over the past two decades. These studies have included augmentation of host immunity with lymphokines and other biological response modifiers, lymphokine-activated killer cells, and cytotoxic T-cells; active immunotherapy using purified antigens, tumor cell vaccines, and anti-id antibodies; and passive immunotherapy with monoclonal antibodies. The results of these investigations are discussed as well as the prospect for future treatments of melanoma incorporating immunotherapeutic techniques. A report on a recent trial of gene therapy of human melanoma performed by Dr. Steven Rosenberg is also presented.

Humans↗
PubMed1993

Growth inhibition and modulation of cell markers of melanoma by S-allyl cysteine.

A sulfur-containing amino acid compound, S-allyl cysteine (SAC), derived from garlic extract inhibited proliferation of nine human and murine melanoma cell line in a dose-dependent manner (1.2-10 mM) assessed by a [3H]thymidine incorporation assay. Three control human lymphoblastoid cell lines were not inhibited by SAC concentrations < 5 mM. Four human melanoma cell lines in a soft-agar assay also showed dose-dependent inhibition of colony formation by SAC. Melanin content was increased up to 95% compared to the same untreated cell lines in these four human melanoma and two B16 murine melanoma sublines. Expression of cell surface gangliosides, cellular-differentiation and transformation markers, decreased after SAC treatment. Significant morphological changes including 'flattening and/or dendritic-like elongations' were also observed. Thus SAC inhibited cellular growth and proliferation and modulated major cell differentiation markers of melanoma.

Animals↗
PubMed1992

Variations in the ganglioside profile of uveal melanoma correlate with cytologic heterogeneity.

Gangliosides may play an important role in the proliferation and spread of human malignant melanoma. Because the frequency of metastases in uveal and cutaneous melanoma differs, it is possible that they may express different gangliosides. We analyzed the ganglioside profiles of primary uveal melanoma in 14 cases and of cutaneous melanoma metastasis in 19 cases. In cutaneous melanoma, GM3 ranged from 4.2% to 74.6% and GD3 from 22.1% to 91.8% of total lipid-bound sialic acid. GM2 (found in 13 of 19 cases, ranging from 0.5% to 11.7%), GD2 (11/19, 0.5%-22.0%) and 9-O-acetyl-GD3 (13/19, 0.5%-12.6%) were also frequently observed. By contrast, in 11 cases of uveal melanoma, GM3 was > 90%, GD3 was < 10%, GM2 was < 1.1%; neither GD2 nor 9-O-acetyl-GD3 were detected. The ganglioside profiles of these uveal melanomas were virtually identical to those of normal melanocytes obtained from foreskins. Histological examination of these 11 biopsies showed a monomorphous cell composition, but neither infiltration of lymphocytes or melanophages nor cell necrosis was observed. In 3 other cases, GD3 was increased to 19.5%-46.0%. Histological examination of these 3 biopsy specimens showed at least 2 populations of tumor cells that were separable based on morphological grounds, and mononuclear inflammatory cells interspersed among the tumor cells. An increase in GD3 appears to be related to tumor polyclonality and infiltration of the tumor by lymphocytes and macrophages. These results suggest that ganglioside expression of uveal melanoma is associated with host immune responses to the tumor. Furthermore, the low metastatic capacity of uveal melanoma, in contrast to the high metastatic rate of cutaneous melanoma, may be a result of its differentiated ganglioside expression, which is strikingly similar to that of normal melanocytes.

Gangliosides↗
PubMed1992

Production and characterization of a murine/human chimeric anti-idiotype antibody that mimics ganglioside.

The VL and VH from a murine anti-idiotypic antibody that mimics ganglioside have been cloned, sequenced, and expressed as a chimeric mouse/human IgG1 antibody. The chimeric antibody retained a binding specificity indistinguishable from the original murine antibody. The VH was a member of Vgam 3.8 family. The sequences are discussed in terms of ways in which proteins may mimic ganglioside epitopes.

Amino Acid Sequence↗
PubMed1992

Modulation of ganglioside expression in human melanoma cell lines; increased resistance to chemo- and radiation treatment.

Cell surface gangliosides in human melanoma cell lines were modulated by pretreatment and adaptation to 6-thioguanine and 5-bromo-deoxyuridine. Chemo- and radiation sensitivities were compared in original cell lines and modulated cells by the human tumor colony-forming assay. Modulated cells showed decreased expression of cell surface GM2 and GD2 gangliosides. This reduction was correlated with increased resistance to bleomycin, vincristine, cisplatin and radiation treatment. These results suggest that cell surface GM2 and GD2 ganglioside expression in human melanoma cells is intimately associated with several cellular biological properties, such as drug or radiation sensitivity and cellular differentiation.

Antineoplastic Agents↗
PubMed1992

Ganglioside GM2 levels in human melanoma cells: inverse correlation with lysosomal beta-hexosaminidase A activity.

GM2 is usually significantly elevated in human melanoma cells. The lysosomal hydrolase beta-hexosaminidase A (Hex A), is an isoenzyme required for terminal GalNAc hydrolysis from intact GM2 to GM3. The objective of the present studies is to determine if the elevated levels of gangliosides, particularly GM2, correlate with the activity of Hex A in cultured melanoma cells. The Hex A activity in 13 melanoma cell lines ranged from 26%-88.3% There was a inverse correlation between GM2 nmole/g and Hex A activity (r = -0.48; p less than 0.003). An inverse correlation (p less than 0.03) occurred between GD2 nmole/g and Hex A activity. There was an inverse correlation (r = -0.53; p less than 0.05 and r = -0.51; p less than 0.05, respectively) between the ratio of substrate/product (GM2/GM3) and (GD2/GD3) and Hex A activity. These results indicate that the level of GM2 in melanoma is inversely correlated with the level of activity of Hex A.

Analysis of Variance↗
PubMed1991

Interleukin 4 alone and with gamma-interferon or alpha-tumor necrosis factor inhibits cell growth and modulates cell surface antigens on human renal cell carcinomas.

Immune cytokines have been shown to play important roles in regulating immune cell functions as well as neoplastic cells. Interleukin-4 (IL4), primarily known as a B-cell growth factor, can also activate and differentiate other immune cells. This cytokine has recently been shown to have immunotherapeutic benefit in tumor-bearing hosts. The present study assessed the effect on human renal cell carcinoma cell lines of recombinant IL4 alone and in combination with recombinant gamma-interferon (IFN) or recombinant alpha-tumor necrosis factor (TNF). IL4 inhibited cell growth of all lines at 250-500 units/ml in a differential manner. Expression of IL4 receptors was demonstrated on renal cell carcinomas. Overall, IFN (500 units/ml) alone inhibited cell growth; however, TNF (500 units/ml) was not as strong an inhibitor. When IL4 was combined with IFN or TNF there was a significant augmentation of cell growth inhibition and modulation of cell morphology of the cell lines. Tumor-associated ganglioside antigens (NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer, NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer, GalNAc beta 1-4 (NeuAc alpha 2-3)Gal beta 1-4Glc beta 1-1'Cer, and GalNAc beta 1-4(NeuAc alpha 2-8NeuAc alpha 2-3)Gal beta 1-4Glc beta 1-1'Cer) HLA class I, HLA-DR, and beta 2-microglobulin on the cell surface of renal cancer lines were assessed by flow cytometry and radiometric binding assay. IL4 alone or in combination with other cytokines modulated HLA class I and HLA-DR expression. IL4 and IFN consistently enhanced NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer and GalNAc beta 1-4(NeuAc alpha 2-8NeuAc alpha 2-3)Gal beta 1-4Glc beta 1-1'Cer expression on individual cell lines. The study demonstrated that IL4 alone or in combination with other cytokines can significantly inhibit growth, and modulate the expression of surface major histocompatibility and tumor-associated antigens of renal cell carcinomas.

Carcinoma, Renal Cell↗
PubMed1991

Ganglioside GM3:GD3 ratio as an index for the management of melanoma.

This report evaluates the relevance of the ratio of the melanoma-associated ganglioside, GD3, and its precursor, GM3, to prognosis and management of Stage II disease. Tumor biopsy specimens from 42 melanoma patients were examined for the ratio and found that GD3 and GM3 constitute 80% of the total lipid bound sialic acids (LBSA) of melanoma. Although the ratio of GM3:GD3 in melanocytes, the progenitors of melanoma, is 19:1 (based on %LBSA), it ranged from 15:1 to 1:5 in tumor biopsy specimens. Patients are categorized into three groups based on their GM3:GD3 ratio (%LBSA) of tumor tissues as Group I (ratio ranged from 15:1 to 1.5:1) (10 of 42), Group II (1.4:1 to 1:1.4) (13 of 42), and Group III (1:1.5 to 1:5) (19 of 42). When the overall survival of patients from the onset of Stage II disease was evaluated among different groups, patients belonging to Group I survived significantly longer than patients of Group II (P = 0.02) and Group III (P = 0.01). Interestingly, Group III expressed immunogenic gangliosides (GD2, GM2, and O-AcGD3) better than the other groups and may benefit more from therapies targeted against these gangliosides antigens. The results of this study indicate that GM3:GD3 ratio is an unusual but well-defined biochemical criteria that may be useful for prognosis and therapeutic management of the disease. Therefore, we propose a routine analysis of the GM3:GD3 ratio of tumors excised after surgery. Screening the ratio is feasible since melanoma expresses a simple profile of gangliosides unlike other forms of cancer.

Adolescent↗