Biomedical subjects

W J Grimes

Publications and source records attributed to W J Grimes.

At least 19 recordsLinked to original sources

PubMed1998

Baculoviral expressed HLA class I heavy chains used to screen a synthetic peptide library for allele-specific peptide binding motifs.

Recombinant baculoviruses encoding truncated HLA-A*0101 and HLA-A*0201 class I heavy chains have been isolated and used to infect lepidopteran cells. Proteins overexpressed in this system were glycosylated, and consisted of 282 amino acid residues after signal sequence cleavage. These class I heavy chains could fold into their native conformation in the presence of recombinant human beta2-microglobulin expressed in Escherichia coli and a synthetic peptide library of nonamers bound to resin-support beads. Reconstitution into native ternary complexes was detected using a conformation specific monoclonal antibody followed by isolation and sequencing of the bound peptides. The motifs obtained for HLA-A1.1 and HLA-A2.1 peptides are similar although more extensive than those derived from sequencing endogenous peptides. This approach selects peptides which form very stable complexes regardless of whether these peptides are generated under physiological conditions, thereby providing unique supplementary data for predicting and designing CTL epitopes. This method is based solely on peptide binding to the class I molecule and is therefore independent of any constraints imposed by endogenous intracellular processing or transport systems. A comparison of the two motifs provides an opportunity to distinguish between the requirements of binding from those arising as a function of intracellular processing or transport. Our findings are not consistent with a recent report suggesting that constraints on the COOH termini of these peptides can be attributed to the effects of either intracellular processing or transport. We find that the carboxy termini in the class I peptides analyzed to date mimic the endogenous data, suggesting that residues in this position contribute to binding affinity.

Alleles↗

PubMed1995

A human monoclonal antimelanoma single-chain Fv antibody derived from tumor-infiltrating lymphocytes.

With the development of recombinant DNA technology, it has become feasible to clone, construct, and express fully human immunoglobulin molecules. Here we report a novel methodology to make human antitumor single-chain Fv (scFv) antibodies from tumor-infiltrating B lymphocytes. We isolated and expanded tumor-infiltrating B lymphocytes from melanomas in the presence of Epstein-Barr virus. The transformed B cells secreting tumor-specific antibodies were identified and cloned by limiting dilution. From one B cell clone with specific melanoma reactivity, we captured the immunoglobulin variable region genes VH and Vk by PCR, sequenced the genes, and linked them together by PCR assembly with the use of a (Gly4Ser)3 linker. The scFv gene was then cloned into the pET21d vector and expressed. The obtained scFv protein with a M(r), of 29,000 was purified and biotinylated for further characterization. The scFv demonstrated specific tumor reactivity to 21 of 24 different melanoma cell lines and not to 14 nonmelanoma tumor cell lines, such as breast, ovarian, and colon cancer cells lines; normal human melanocytes as well as normal human leukocytes. These results were obtained in (a) a tumor cell ELISA, (b) fixed cell immunofluorescence, and (c) live cell flow cytometry. The immunoprecipitation results indicated that a protein antigen of M(r) 45,000 was recognized by the scFv. Since we reported previously that about 70% of human tumors of different histological types contain tumor-infiltrating B lymphocytes producing specific antitumor antibodies, this approach offers a rapid, effective method by combining in vitro B-cell expansion and PCR gene cloning to elucidate the repertoire of the human antitumor immune response and to make human monoclonal antitumor antibody molecules.

Amino Acid Sequence↗

PubMed1995

Antibodies to tumor necrosis factor: a component of B cell immune responses with a role in tumor/host interaction.

Infiltrating B lymphocytes are found within tumors, where their role and the antigens they recognize are poorly defined. After in vitro expansion of these cells, we were able to detect the production of antibodies to tumor necrosis factor alpha (TNF) in 13 of 17 human tumors studied. These antibodies were detected by both enzyme-linked immunosorbent assay and by neutralization. Anti-TNF antibodies were not produced by resting peripheral blood B cells of normal subjects. However, anti-TNF antibodies were produced by B cells obtained from healthy individuals, after either in vivo or in vitro antigenic stimulation. This suggests that anti-TNF antibody production may constitute part of the overall B cell response to antigens. The intratumoral production of anti-TNF antibody may play a role in tumor/host interactions.

Antibodies↗

PubMed1994

Anti-tumor antibody produced by human tumor-infiltrating and peripheral blood B lymphocytes.

Cell suspensions from 69 human tumor biopsies and malignant effusions depleted of infiltrating T cells were incubated for 10-14 days with mitomycin-C-treated cells of the transformed T cell line MOT as feeder cells. B lymphocytes proliferated and differentiated as indicated by immunoglobulin (Ig) secretion in the culture supernatants (B cell expansion). Ig was present in culture supernatants of tumor cell suspensions incubated without MOT feeder cells (non-expanded cells), but the addition of MOT feeder cells to these cultures invariably resulted in a significant increase in Ig concentration. While IgG, IgA, and IgM isotypes were all detected in supernatants of both expanded- and non-expanded tumor cell suspensions, the increase in total Ig induced by MOT feeder cells was mainly due to an increase in IgG. Peripheral blood B lymphocytes (PBBL) from 15 cancer patients and 4 healthy individuals were also successfully expanded by the same method. In these it was shown that IgA was the predominant Ig isotype. Using a modified enzyme-linked immunosorbent assay, IgG of 25/36 expansions from tumor cell suspensions showed reactivity with autologous tumor targets, and that from 10/13 expansions reacted with allogeneic tumor targets of the same histological diagnosis. No reactivity was found against tumor targets of different histology. IgG of 4/10 expansions of PBBL from cancer patients showed reactivity with allogeneic tumor targets of the same histology, while no reactivity was demonstrated against tumor targets of different histology. IgG of expanded PBBL from healthy individuals showed no reactivity against tumor targets. This method allows detailed study of the specific humoral antitumor immune response of intratumoral and peripheral blood B lymphocytes in cancer.

Antibodies, Neoplasm↗

PubMed1994

Peptide sequences binding to MHC class I proteins.

Motifs for peptides which bind specifically to the human class I major histocompatibility complex molecules HLA-A2 and B7 were determined by sequence analysis of class I-bound peptides selected from a random synthetic library of nonamers. Thirteen individual peptides were sequenced for HLA-A2, twelve individual and nine pooled peptides were sequenced for HLA-B7. Analysis of sequence alignment implicated four peptide positions in potential contact with the class I HLA-A2 molecule and three positions for the HLA-B7 molecule. The results demonstrate that a synthetic peptide library can be used to identify allele-specific motifs for class I molecules, providing information comparable to the results obtained from sequencing endogenous peptides. This method utilizes denatured class I heavy chains, and similar results were obtained using a class I protein purified from mammalian cells or by expression in Escherichia coli. This method has the potential to detect peptides which may not be generated physiologically, but due to their binding properties, may be valuable to predict or engineer immunomodulatory T cell epitopes.

Amino Acid Sequence↗

PubMed1991

Ability of cell-sized beads bearing tumor cell membrane proteins to stimulate LAK cells to secrete interferon-gamma and tumor necrosis factor-alpha.

We recently reported that lymphokine activated killer (LAK) cells were stimulated to release both interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) when stimulated by a variety of tumor cells. We proposed then that the released cytokines may play a role in mediating tumor cell regression in vivo. In this paper, we provide further information on the nature of the signals, provided by the tumor cells (K562 erythroleukemia), that stimulate LAK cells to secrete IFN-gamma and TNF-alpha. Using a previously published protocol for coating tumor-membrane molecules onto cell-sized hydrophobic beads (also called pseudocytes), we demonstrate that the signal provided by the tumor cell is membrane associated. Beads coated with K562 membranes stimulated LAK cells to release IFN-gamma and TNF-alpha. The pretreatment of these beads with trypsin and sodium periodate eliminated the ability of these pseudocytes to stimulate cytokine release in LAK cells. The glycoproteins that stimulate LAK cells to secrete IFN-gamma and TNF-alpha were further enriched by their ability to bind concanavalin A (Con A, Jack Bean). To determine if the tumor-associated molecules that stimulate LAK cells to release IFN-gamma and TNF-alpha are also the molecules involved in mediating tumor cell lysis, we tested the ability of the Con A binding and nonbinding proteins to inhibit the LAK cell-mediated lysis of K562 cells. Our results demonstrate that molecules that inhibited LAK cell-mediated cytotoxicity were not enriched by Con A. These results are therefore consistent with the conclusion that different sets of tumor-associated molecules are involved in the stimulation of LAK cells to secrete cytokine and in the induction of LAK cells to mediate tumor cell cytolysis.

Antigens, Neoplasm↗

PubMed1991

The effect of steroid therapy on recovery from tonsillectomy in children.

A prospective, randomized, double-blind study to determine the postoperative effects of steroids in tonsillectomy was performed on 25 children from 4 to 12 years of age. A single intravenous dose of dexamethasone or a placebo was administered at onset of surgery. Other preoperative and postoperative medications, including antibiotics, anesthesia, and surgical techniques were standardized as noted in this article. The ability to return to a full or semifull diet occurred on the third and fourth postoperative days, significantly sooner in the steroid-treated patients than in the control patients. By the fifth and sixth days, the control group were eating as well as those children who received steroids. No significant differences were observed in postoperative pain, nausea, emesis, fever, or in the need for postoperative pain medications. This preliminary article concludes that a single preoperative dose of steroid results in an earlier return to a normal (full) diet in children who had undergone tonsillectomy.

Adenoidectomy↗

PubMed1991

Gamma-irradiated peripheral blood mononuclear cells can express LAK activity.

Peripheral blood mononuclear cells (PBMC) irradiated with high dose gamma-radiation (1000-5000 rad) are commonly used as feeder cells during the cloning of T lymphocytes, natural killer (NK) and lymphokine activated killer (LAK) cells. We report here that such gamma-irradiated PBMC can be stimulated with interleukin 2 (IL-2) to express the ability to lyse a variety of tumor cell targets. The non-major histocompatibility complex (MHC) restricted cytotoxicity demonstrated by irradiated PBMC is, however, lower than that expressed by their non-irradiated counterparts. The numbers of viable, gamma-irradiated LAK cells are significantly increased by the addition of the mitogen, phytohemagglutinin (PHA). Purification of the gamma-irradiated cells expressing cytotoxic activity by flow cytometry determined that the effector cells were predominantly CD3- cells, although some CD3+ cells also expressed moderate LAK activity. The ability of gamma-irradiated cells to proliferate in the presence of PHA alone, or with IL-2 + PHA, was maximal at day 4-5; but proliferation, as detected by 3H-thymidine uptake, was not detectable beyond 12-15 days of in vitro culture. Because many of the LAK, T cell and NK cell cloning procedures require the presence of feeder layers, growth factors (usually IL-2) and mitogens, the presence of residual feeder cells expressing cytotoxic activity may affect the specificity of such clones. Thus, efforts should be made to ensure that such gamma-radiation-resistant cells capable of expressing cytotoxic activity are completely eliminated before the cloned cells are used for further experiments.

Cell Division↗

PubMed1990

Phenotypic analyses of lymphokine-activated killer cells that release interferon gamma and tumor necrosis factor alpha.

We recently reported that interleukin-2(IL-2)-activated peripheral blood lymphocytes and CD3+, lymphokine-activated killer (LAK) cell clones release tumor necrosis factor alpha(TNF alpha) and interferon gamma (IFN gamma) when stimulated with K562 erythroleukemia cells. We examined the phenotype of IL-2-activated peripheral blood leukocytes that secrete TNF alpha and IFN gamma when stimulated with K562 cells and demonstrated that TNF alpha secretion is not due to the presence of contaminating mononuclear phagocytes. Further, we demonstrate that IL-2-activated natural killer (NK) cells release only IFN gamma when stimulated with K562 cells while T lymphocytes exposed to monoclonal anti-CD3 and K562 cells secrete both TNF alpha and IFN gamma. However, T cells stimulated only with K562 cells did not release IFN gamma or TNF alpha while the admixture of these T cells with NK cells, when stimulated with K562 cells, released levels of TNF alpha comparable to those produced by the unseparated cells. At present it is unclear whether only one or both effector cell types respond to K562 by releasing TNF alpha or why the presence both cell types is needed.

Antigens, Differentiation↗

PubMed1989

Tumor targets stimulate IL-2 activated killer cells to produce interferon-gamma and tumor necrosis factor.

Lymphokine-activated killer (LAK) cells are cytotoxic for a variety of autologous and allogeneic tumor cells as well as modified autologous cells. It is assumed that LAK cells lyse their targets solely by direct cell to cell contact, possibly involving the degranulation and exocytosis of pore-forming elements, similar to that observed with cytotoxic T lymphocytes and NK cells. Reported here are studies demonstrating that LAK cells release factor(s) that are cytotoxic for a human breast carcinoma cell line, MCF-7, when stimulated with tumor cells. The factor(s) are slow acting and maximum cytotoxicity is observed only in a 72-h cytotoxic assay. The ability of LAK cells to secrete cytotoxic factor(s) is dependent on both the ratio of LAK cells to stimulating tumor cells as well as the length of their coincubation. A number of similarly slow acting cytokines that are cytostatic and/or cytotoxic for tumor cells have been described. We tested the ability of specific polyclonal antibodies directed against TNF, IFN-alpha, IFN-beta, and IFN-gamma to neutralize the cytotoxic supernatant activity. Only antibodies specific for IFN-gamma and TNF were neutralizing. We measured the amounts of IFN-gamma and TNF in the cytotoxic supernatants and determined that increased amounts of IFN-gamma and TNF were released after LAK cell-tumor cell interactions compared to supernatants of LAK cells alone or tumor cell alone. Comparable concentrations of human rIFN-gamma and rTNF resulted in similar levels (50 to 90%) of MCF-7 cell cytotoxicity as those observed with the stimulated LAK cell supernatants. We thus concluded that the majority of the cytotoxic activity released by LAK cells when stimulated with tumor cells was attributed to the synergistic activities of IFN-gamma and TNF. The significance of these observations in relation to the possible mechanisms by which LAK cells mediate cytolysis is discussed.

Cell Line↗

PubMed1989

Phenotypic and functional analysis of lymphokine-activated killer (LAK) cell clones. Ability of CD3+, LAK cell clones to produce interferon-gamma and tumor necrosis factor upon stimulation with tumor targets.

Lymphokine-activated killer (LAK) cells are generated by the culture of peripheral blood lymphocytes with interleukin-2 (IL-2). A variety of cells, including T-lymphocytes and natural killer (NK) cells, can be activated by IL-2 to exhibit the ability to kill multiple tumor and "modified-self" targets. Recent reports indicate that culture conditions can determine the phenotype of cells expressing LAK activity. Using limiting dilution techniques, we first generated cloned LAK cells with three culture conditions: autologous human serum (AHS) + IL-2; AHS + IL-2 + 0.1 micrograms/ml phytohemagglutinin and fetal bovine serum and IL-2. We determined that all but one of the 47 LAK cell clones generated with the three culture conditions were CD3+ and T-cell like; one NK-like clone was observed. Clones that were cytotoxic for one target could generally kill multiple targets, and the absence of phytohemagglutinin did not significantly affect the ability of the LAK cell clones to kill multiple targets. The presence of phytohemagglutinin was, however, necessary for the long-term maintenance of proliferation and cytotoxic activity of the LAK cell clones. The mechanism by which LAK cells kill tumor targets is not known. We here demonstrate that LAK cells and LAK cell clones can produce interferon-gamma and tumor necrosis factor (TNF) when stimulated with an erythroleukemia cell, K562. Five of the six CD3+, LAK cell clones tested could be stimulated by K562 cells to produce both interferon-gamma and TNF. However, the ability of the cloned LAK cells to kill K562 cells, as measured in a 4-h 51Cr-release assay, did not correlate with their ability to produce these cytokines. Furthermore, specific antibodies that neutralize the cytotoxic activity of interferon-gamma and TNF did not inhibit killing of K562 cells by LAK cells as measured with a 4-h cytotoxic assay. The cytostatic and cytotoxic activities of interferon-gamma and TNF for tumor cells are well documented, but these cytolytic activities are slower acting and exhibit their maximum effect after 48-96 h. We here propose that LAK cells kill tumor targets by a combination of cell-to-cell-mediated killing and by the release of slower acting cytostatic/cytotoxic cytokines that can inhibit the growth of tumors some distance from the effector cells.

Antibodies, Monoclonal↗

PubMed1989

Cytostatic and cytotoxic activity of lymphokine-activated killer cell supernatants.

Incubation of peripheral blood mononuclear cells with interleukin-2 (IL-2) results in the release of a factor which is cytostatic and cytotoxic both to tumor cell lines (A375M, A375P, C480, MCF-7, Hey) and fresh tumor cells (in the human tumor cloning assay), including breast cancer, colon cancer, melanoma, myeloma and ovarian cancer. The factor cannot be detected in a 4-h chromium-release assay, but is best demonstrated after tumor cells have been to it for exposed 3 days. The factor is not cytotoxic to normal peripheral blood leukocytes or normal fibroblasts, and is not toxic to certain targets sensitive to lymphokine-activated killer (LAK) cells, such as K562 and Daudi cells. The factor is diffusible, non-dialyzable, relatively stable to heat and acid and does not contain appreciable amounts of targets resistant to interferon-alpha and beta, tumor necrosis factor beta and interleukin-1. The data suggest that there are several mechanisms of LAK cell activity against tumor cells including one which requires direct interaction of LAK and tumor cells and one which is mediated by LAK cell supernatant. The former is detected by 4-h chromium release while the latter is not.

Cytotoxicity, Immunologic↗

PubMed1988

Blocking of lymphokine activated killer (LAK) cell mediated cytotoxicity by cell-sized beads bearing tumor cell proteins.

Lymphokine activated killer cells (LAK) have been demonstrated to be cytotoxic for a variety of tumor-derived cells. Little is known of the nature of the cell surface molecules that mediate LAK cell-target cell interactions. Reported here are studies designed to develop the methodology that can lead to the identification and characterization of tumor cell surface molecules recognized by LAK cells. Results from experiments involving the pre-treatment of LAK cells and target cells (51Cr-labeled target cells or cold-blocking cells) with trypsin, neuraminidase, or sodium periodate suggest that proteins on the surface of LAK cells specifically recognized trypsin-sensitive molecules on the tumor cell surface. We extracted tumor cell membranes with detergents, and incorporated membrane proteins together with phospholipids and cholesterol onto the surfaces of cell-sized hydrophobic beads. The resulting "pseudocytes" block LAK mediated killing of 51Cr-labeled targets. Trypsin pretreatment of these pseudocytes significantly reduced their blocking activity. These observations suggested that we have incorporated onto the surface of pseudocytes tumor-membrane derived molecules that are specifically recognized by LAK cells. When membrane proteins from LAK resistant PBMC were incorporated onto beads, the resulting pseudocytes did not block LAK mediated cytotoxicity. It is of interest that beads coated with membrane proteins from one tumor were able to reduce LAK cell lysis of a different tumor target. Our results are consistent with the possibility that each LAK cell is polyspecific or that the LAK cell recognizes a common marker on many tumors. The methodology using pseudocytes should allow the purification and characterization of target acceptor molecule(s) and permit us to distinguish between these possibilities.

Binding, Competitive↗

PubMed1985

Arylphorin from Manduca sexta: carbohydrate structure and immunological studies.

The major hemolymph protein in the last larval stage of Manduca sexta is a hexameric glycoprotein, arylphorin (Mr = 450,000). Sodium dodecyl sulfate polyacrylamide gel electrophoresis of purified arylphorin reveals the presence of two subunits, A1 and A2. Both subunits are glycosylated and have apparent Mr = 77,000 and 72,000, respectively. Pronase digestion of arylphorin yielded a single major glycopeptide. 250 MHz NMR spectroscopy of arylphorin glycopeptide revealed a Man9GlcNAc2 oligosaccharide structure similar to that observed in mammalian glycoproteins. Endoglycosidase-H treatment of arylphorin was employed to remove covalently linked carbohydrate residues. The carbohydrate removal lowered the apparent Mr of subunits A1 and A2 to 72,000 and 69,000, respectively, indicating that the difference in arylphorin subunit size is not due to levels of glycosylation. Immunoblotting experiments with anti-arylphorin antiserum and Bombyx mori storage proteins indicated cross reactivity with the corresponding arylphorin of this insect. Preparation of subunit A2 monospecific antibodies, followed by immunoblotting of arylphorin showed a close immunological relationship between subunits A1 and A2.

Animals↗

PubMed1985

Application of microcomputers to the interpretation of high-resolution nuclear magnetic resonance spectra of asparagine-linked oligosaccharides: evaluation of high-mannose structures.

A microcomputer program for determining primary oligosaccharide structures by high-resolution nuclear magnetic resonance is reported. The program uses the data base obtained from spectra of model compounds reported in the literature. A number of high-mannose-type asparagine-linked glycopeptide spectra containing tabulated chemical shifts measured at room temperature were tested to determine the ability of the computer to correctly assign carbohydrate units into a complete structure. High-mannose structures ranging from five to nine oligomannosyl units were completely and unambiguously analyzed to give complete structural assignments. The computer program will also resolve glycopeptide mixtures. The method provides investigators with a useful tool for accurately evaluating nuclear magnetic resonance spectra and should greatly facilitate the complicated and time-consuming evaluation of microheterogeneity in samples commonly isolated from biological systems.

Asparagine↗

PubMed1985

Major carbohydrate structures at five glycosylation sites on murine IgM determined by high resolution 1H-NMR spectroscopy.

Mouse myeloma immunoglobulin IgM heavy chains were cleaved with cyanogen bromide into nine peptide fragments, four of which contain asparagine-linked glycosylation. Three glycopeptides contain a single site, including Asn 171, 402, and 563 in the intact heavy chain. Another glycopeptide contains two sites at Asn 332 and 364. The carbohydrate containing fragments were treated with Pronase and fractionated by elution through Bio-Gel P-6. The major glycopeptides from each site were analyzed by 500 MHz 1H-NMR and the carbohydrate compositions determined by gas-liquid chromatography. The oligosaccharide located at Asn 171 is a biantennary complex and is highly sialylated. The amount of sialic acid varies, and some oligosaccharides contain alpha 1,3-galactose linked to the terminal beta 1,4-galactose. The oligosaccharides at Asn 332, Asn 364, an Asn 402 are all triantennary and are nearly completely sialylated on two branches and partially sialylated on the triantennary branch linked beta 1,4 to the core mannose. The latter is sialylated about 40% of the time for all three glycosylation sites. The major oligosaccharide located at Asn 563 is of the high mannose type. The 1H-NMR determination of structures at Asn 563 suggests that the high mannose oligosaccharide contains only three mannose residues.

Amino Acids↗

PubMed1983

Incomplete glycosylation of Asn 563 in mouse immunoglobulin M.

Mouse immunoglobulin IgM was prepared from MOPC 104E ascites fluid and [3H]-mannose labeled tumor cells. The purified protein was used to prepare Fc fragments which were cleaved by cyanogen bromide. Gel filtration allows complete separation of the C-terminal glycosylation site. Amino acid and carbohydrate analyses show that Asn 563 of murine IgM is glycosylated only about 44% of the time.

Amino Acids↗

PubMed1983

Heterogeneity of sialoglycoproteins purified from normal and malignant cells.

We have identified previously a group of sialoglycoproteins with an apparent molecular weight of 110,000, which appear homogeneous by sodium dodecyl sulfate:polyacrylamide gel electrophoresis but exhibit isoelectric point heterogeneity by two-dimensional polyacrylamide gel electrophoresis. This technique demonstrated that there are multiple glycoproteins of similar molecular weight which differ between normal cells and transformants. We have now purified glycoproteins by concanavalin A:Sepharose chromatography and preparative polyacrylamide gel electrophoresis from both a nontumorigenic normal mouse fibroblast line, A31, and a highly malignant nonimmunogenic transformant, 3T12T. Differences in the isoelectric point distribution of the sialoglycoproteins which were observed between the normal and transformed two-dimensional gel patterns using crude membranes could also be demonstrated with the purified glycoproteins. Treatment of the isolated sialoglycoproteins with neuraminidase to remove sialic acid resulted in significant isoelectric point shifts but did not eliminate all of the heterogeneity. Even following neuraminidase treatment, the purified glycoprotein fraction upon isoelectric focusing showed differences in patterns between normal and transformed cells. Preliminary characterization of the alterations seen in the two cell lines are presented and show decreased fucose and increased sialic acid in transformed cell glycoprotein.

Animals↗