Immunological delineation in normal and malignant cells of a membrane protein involved in glucose transport. I. Preparation and properties of the antibody

1981 ◽  
Vol 52 (1) ◽  
pp. 99-120
Author(s):  
R.D. Gingrich ◽  
M. Wouters ◽  
M.E. Bramwell ◽  
H. Harris
Keyword(s):  
Monoclonal Antibodies ◽  
Limit Of Detection ◽  
Rat Kidney ◽  
Immunoglobulin M ◽  
Malignant Cells ◽  
Malignant Tumours ◽  
Embryonic Mouse ◽  
Normal Tissues ◽  
Mouse Tissues

Partially purified preparations of the 200000 Mr membrane glycoprotein described by Bramwell & Harris were used as antigens to generate monoclonal antibodies. Whether the protein was prepared from human or mouse tumours, the mice immunized with it yielded monoclonal antibodies of broadly similar specificities. All the antibodies obtained were of the immunoglobulin M class, despite a prolonged immunization schedule, and recognized an antigen that was present on the surface of mouse, rat and human cells. One such antibody, M/27, was studied in detail. With the exception of 2 rat kidney cell lines, the antigen detected by M/27 was found on the surface of all cells grown in culture, whether malignant or not. However, in the mouse, the species in which the antibody was generated, M/27 discriminated sharply between malignant tumours and normal tissues excised directly from the animal. In normal adult or embryonic mouse tissues, whether they were composed of multiplying cells or not, the antigen detected by M/27 was present on the cell surface only at the lower limit of detection, if at all. Malignant mouse tumours grown in the animal produced large amounts of the antigen. Explantation of embryonic mouse fibroblasts induced the expression of the antigen, which was not detected on these same cells in their normal habitat in the growing embryo. Conversely, non-malignant cells that express the antigen in vitro cease to do so when they are re-introduced into the animal. The antigenic determinant appeared to be polypeptide in nature rather than polysaccharide.

scholarly journals Modulation of Polymorphonuclear Cell Interleukin-8 Secretion by Human Monoclonal Antibodies to Type 8 Pneumococcal Capsular Polysaccharide

2003 ◽  
Vol 71 (12) ◽  
pp. 6775-6783 ◽  
Author(s):  
Tamika Burns ◽  
Zhaojing Zhong ◽  
Michael Steinitz ◽  
Liise-anne Pirofski
Keyword(s):  
Monoclonal Antibodies ◽  
Capsular Polysaccharide ◽  
Immunoglobulin M ◽  
Interleukin 8 ◽  
Blue Staining ◽  
Enzyme Linked Immunosorbent Assay ◽  
Polymorphonuclear Cells ◽  
Human Monoclonal Antibodies ◽  
Classical Complement Pathway

ABSTRACT Pneumococcal capsular polysaccharide (PS) vaccines induce type-specific immunoglobulin M (IgM), IgG, and IgA. Type-specific IgG to the PS is sufficient to confer protection against the homologous serotype of the pneumococcus, but the efficacies of type-specific IgM and IgA are less well understood. We examined the in vitro activities and efficacies in mice of two human monoclonal antibodies (MAbs) to type 8 PS, NAD (IgA) and D11 (IgM). MAb-mediated opsonophagocytic killing was evaluated after coculture of type 8 pneumococci with human polymorphonuclear cells (PMNs), type-specific or control MAbs, and human complement sources. The effects of the MAbs on PMN interleukin-8 (IL-8) and IL-6 secretion were determined in supernatants from cocultures containing pneumococci and PMNs by enzyme-linked immunosorbent assay. MAb efficacy was determined in an intratracheal model of type 8 infection in mice with classical complement pathway deficiency. Both MAbs were protective in 100% of infected mice. Neither MAb promoted a significant amount of killing of type 8 pneumococci compared to its isotype control MAb. Both type-specific MAbs mediated complement-dependent modulation of PMN IL-8 secretion, with increased secretion at effector/target (E:T) ratios of 500:1 and 50:1 and reduced secretion at 1:5. Trypan blue staining revealed that PMNs cocultured with D11 were less viable at an E:T ratio of 1:5 than PMNs cocultured with the control MAb. PMN IL-6 secretion was increased by both type-specific and control MAbs. These results suggest that certain type-specific IgM and IgAs might contribute to host defense by modulation of the inflammatory response to pneumococci.

scholarly journals In Vitro and In Vivo Neutralization of the Relapsing Fever Agent Borrelia hermsii with Serotype-Specific Immunoglobulin M Antibodies

2001 ◽  
Vol 69 (2) ◽  
pp. 1009-1015 ◽  
Author(s):  
Alan G. Barbour ◽  
Virgilio Bundoc
Keyword(s):  
Monoclonal Antibodies ◽  
Antibody Response ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Relapsing Fever ◽  
Borrelia Hermsii ◽  
Intact Cells ◽  
Whole Cells

ABSTRACT The antigenic variation of the relapsing fever agent Borrelia hermsii is associated with changes in the expression of the Vlp and Vsp outer membrane lipoproteins. To investigate whether these serotype-defining proteins are the target of a neutralizing and protective antibody response, monoclonal antibodies were produced from spleens of infected mice just after clearance of serotype 7 cells from the blood. Two immunoglobulin M monoclonal antibodies, H7-7 and H7-12, were studied in detail. Both antibodies specifically agglutinated serotype 7 cells and inhibited their growth in vitro. Administered to mice before or after infection, both antibodies provided protection against infection or substantially reduced the number of spirochetes in the blood of mice after infection. Whereas antibody H7-12 bound to Vlp7 in Western blotting, enzyme-linked immunosorbent assay, and immunoprecipitation assays, as well as to whole cells in other immunoassays, antibody H7-7 only bound to wet, intact cells of serotype 7. Antibody H7-7 selected against cells expressing Vlp7 in vitro and in vivo, an indication that Vlp7 was a conformation-sensitive antigen for the antibody. Vaccination of mice with recombinant Vlp7 with adjuvant elicited antibodies that bound to fixed whole cells of serotype 7 and to Vlp7 in Western blots, but these antibodies did not inhibit the growth of serotype 7 in vitro and did not provide protection against an infectious challenge with serotype 7. The study established that a Vlp protein was the target of a neutralizing antibody response, and it also indicated that the conformation and/or the native topology of Vlp were important for eliciting that immunity.

scholarly journals EA-1, a novel adhesion molecule involved in the homing of progenitor T lymphocytes to the thymus.

1991 ◽  
Vol 114 (5) ◽  
pp. 1069-1078 ◽  
Author(s):  
B A Imhof ◽  
P Ruiz ◽  
B Hesse ◽  
R Palacios ◽  
D Dunon
Keyword(s):  
Endothelial Cell ◽  
T Cell ◽  
T Lymphocytes ◽  
Cell Line ◽  
Adhesion Molecule ◽  
Newborn Mice ◽  
Embryonic Mouse ◽  
Endothelial Cell Line ◽  
Mouse Tissues

The mouse progenitor T lymphocyte (pro-T) cell line FTF1 binds in vitro to thymus blood vessels, the thymic capsule, and liver from newborn mice. A mAb, EA-1, raised against an embryonic mouse endothelial cell line, blocked adhesion. The antibody also interfered with pro-T cell adhesion to a thymus-derived mouse endothelial cell line; it had no effect on the adhesion of mature T lymphocytes and myeloid cells. The antigen recognized by EA-1 is located on the vascular endothelium of various mouse tissues and absent on pro-T cells. EA-1 antibody precipitates molecules with apparent molecular weights of 110,000, 140,000, 160,000, and 200,000. Immunoclearing and binding-inhibition studies with antibodies against known adhesion molecules suggest that the EA-1 antigen is a novel adhesion molecule involved in colonization of the embryonic thymus by T cell progenitors.

scholarly journals Role of Bordetella bronchisepticaFimbriae in Tracheal Colonization and Development of a Humoral Immune Response

2000 ◽  
Vol 68 (4) ◽  
pp. 2024-2033 ◽  
Author(s):  
Seema Mattoo ◽  
Jeff F. Miller ◽  
Peggy A. Cotter
Keyword(s):  
Immune Response ◽  
Humoral Immune Response ◽  
Limit Of Detection ◽  
Immunoglobulin M ◽  
Tissue Culture Cell ◽  
Wild Type ◽  
Humoral Immune ◽  
Tract Infections

ABSTRACT Fimbriae are filamentous, cell surface structures which have been proposed to mediate attachment of Bordetella species to respiratory epithelium. Bordetella bronchiseptica has four known fimbrial genes: fim2, fim3,fimX, and fimA. While these genes are unlinked on the chromosome, their protein products are assembled and secreted by a single apparatus encoded by the fimBCD locus. ThefimBCD locus is embedded within the fha operon, whose genes encode another putative adhesin, filamentous hemagglutinin (FHA). We have constructed a Fim− B. bronchiseptica strain, RB63, by introducing an in-frame deletion extending from fimB through fimD. Western blot analysis showed that RB63 is unable to synthesize fimbriae but is unaffected for FHA expression. Using this mutant, we assessed the role of fimbriae in pathogenesis in vitro and in vivo in natural animal hosts. Although RB63 was not significantly defective in its ability to adhere to various tissue culture cell lines, including human laryngeal HEp-2 cells, it was considerably altered in its ability to cause respiratory tract infections in rats. The number of ΔfimBCD bacteria recovered from the rat trachea at 10 days postinoculation was significantly decreased compared to that of wild-type B. bronchiseptica and was below the limit of detection at 30 and 60 days postinoculation. The number of bacteria recovered from the nasal cavity and larynx was not significantly different between RB63 and the wild-type strain at any time point. The ability of fimbriae to mediate initial attachment to tracheal tissue was tested in an intratracheal inoculation assay. Significantly fewer RB63 than wild-type bacteria were recovered from the tracheas at 24 h after intratracheal inoculation. These results demonstrate that fimbriae are involved in enhancing the ability of B. bronchiseptica to establish tracheal colonization and are essential for persistent colonization at this site. Interestingly, anti-Bordetella serum immunoglobulin M (IgM) levels were significantly lower in animals infected with RB63 than in animals infected with wild-type B. bronchiseptica at 10 days postinoculation. Even at 30 days postinoculation, RB63-infected animals had lower serum anti-Bordetella antibody titers in general. This disparity in antibody profiles suggests that fimbriae are also important for the induction of a humoral immune response.

scholarly journals Sites of methylation of purified transfer ribonucleic acid preparations by enzymes from normal tissues and from tumours induced by dimethylnitrosamine and 1,2-dimethylhydrazine

1974 ◽  
Vol 137 (2) ◽  
pp. 239-248 ◽  
Author(s):  
Anthony E. Pegg
Keyword(s):  
Rat Kidney ◽  
Trna Sequence ◽  
Normal Tissues ◽  
Mouse Colon ◽  
Mammalian Tissues ◽  
Trna Species ◽  
Micro Organisms ◽  
Methylase Activity

1. The sites within the tRNA sequence of nucleosides methylated by the action of enzymes from mouse colon, rat kidney and tumours of these tissues acting on tRNAAsp from yeast and on tRNAGlu2, tRNAfMet and tRNAVal1 from Escherichia coli were determined. 2. The same sites in a particular tRNA were methylated by all of these extracts. Thus tRNAGlu2 was methylated at the cytidine residue at position 48 and the adenosine residue at position 58 from the 5′-end of the molecule; tRNAAsp was methylated at the guanosine residue at position 26 from the 5′-end of the molecule; tRNAfMet was methylated at the guanosine residues 9 and 27, the cytidine residue 49 and the adenosine residue 59 from the 5′-end; tRNAVal1 was methylated at the guanosine residue 10, the cytidine residue 48 and the adenosine residue 58 from the 5′-end. 3. All of these sites within the clover leaf structure of the tRNA sequence are occupied by a methylated nucleoside in some tRNA species of known sequence. It is concluded that methylation of tRNA from micro-organisms by enzymes from mammalian tissues in vitro probably does accurately represent the specificity of these enzymes in vivo. However, there was no evidence that the tumour extracts, which had considerably greater tRNA methylase activity than the normal tissues, had methylases with altered specificity capable of methylating sites not methylated in the normal tissues.

scholarly journals Two Monoclonal Antibodies That Specifically Recognize Aspergillus Cell Wall Antigens and Can Detect Circulating Antigens in Infected Mice

2021 ◽  
Vol 23 (1) ◽  
pp. 252
Author(s):  
Xihua Lian ◽  
Stephen Chambers ◽  
John G. Lewis ◽  
Amy Scott-Thomas ◽  
Madhav Bhatia
Keyword(s):  
Cell Wall ◽  
Monoclonal Antibodies ◽  
Invasive Aspergillosis ◽  
Candida Species ◽  
Sandwich Elisa ◽  
Diagnostic Techniques ◽  
Patient Treatment ◽  
Diagnostic Tools ◽  
Mouse Tissues

Invasive aspergillosis (IA) is a life-threatening disease mainly caused by Aspergillus fumigatus and Aspergillus flavus. Early diagnosis of this condition is crucial for patient treatment and survival. As current diagnostic techniques for IA lack sufficient accuracy, we have raised two monoclonal antibodies (1D2 and 4E4) against A. fumigatus cell wall fragments that may provide a platform for a new diagnostic approach. The immunoreactivity of these antibodies was tested by immunofluorescence and ELISA against various Aspergillus and Candida species in vitro and by immunohistochemistry in A. fumigatus infected mouse tissues. Both monoclonal antibodies (mAbs) showed intensive fluorescence with the hyphae wall of A. fumigatus and A. flavus, but there was no staining with other Aspergillus species or Candida species. Both mAbs also showed strong immunoreactivity to the cell wall of A. fumigatus hyphae in the infected liver, spleen and kidney of mice with IA. The antigens identified by 1D2 and 4E4 might be glycoproteins and the epitopes are most likely a protein or peptide rather than a carbohydrate. An antibody-based antigen capture ELISA detected the extracellular antigens released by A. fumigatus, A. flavus, A. niger and A. terreus, but not in Candida species. The antigen could be detected in the plasma of mice after 48 h of infection by double-sandwich ELISA. In conclusion, both 1D2 and 4E4 mAbs are potentially promising diagnostic tools to investigate invasive aspergillosis.

In Vitro and In Vivo Tumor Models for Studies of Distribution of Radiolabelled Monoclonal Antibodies and Fragments

1986 ◽  
Vol 25 (06) ◽  
pp. 207-209 ◽  
Author(s):  
S. E. Halpern ◽  
R. M. Sutherland ◽  
M. Schreyer ◽  
J.-P. Mach ◽  
F. Buchegger
Keyword(s):  
Monoclonal Antibodies ◽  
Urinary Excretion ◽  
Multicellular Spheroids ◽  
Normal Tissues ◽  
Fab Fragments ◽  
Tumor Accumulation ◽  
Radiolabelled Monoclonal Antibodies ◽  
Antibody Localization

Colon carcinoma multicellular spheroids were incubated in vitro with radiolabeled MAbs. The more rapid penetration of fragments as compared to intact MAbs was clearly demonstrated. For the study of antibody localization in tumors in vivo, the model of nude mice with ligated kidneys was used. Although very artificial, this model allowed to demonstrate that, without urinary excretion, Fab fragments accumulated more rapidly into the tumor than intact MAbs and disappeared faster from the blood. This difference was less striking for F(ab’)2 fragments. In the liver a decreased accumulation of both types of fragments as compared to intact MAbs was observed. Concerning radioimmunotherapy we think that Fab fragments are not useful because of their too short half-life in the circulation and in tumor and because they will probably be too toxic for the kidneys. Intact MAbs and F(ab’)2 fragments have each their advantages. Intact MAbs show highest tumor accumulation in mice without ligated kidney, however, they remain mostly on the periphery of tumor nodules, as shown by autoradiography. F(ab’)2 fragments have been found to penetrate deeper into the tumor and to accumulate less in the liver. It might be therefore an advantage to combine intact MAbs with F(ab’)2 fragments, so that in the tumor two different regions could be attacked whereas in normal tissues toxicity could be distributed to different organs such as to the liver with intact MAbs and to the kidney with F(ab’)2 fragments.

Isoenzyme transitions of creatine phosphokinase, aldolase and phosphoglycerate mutase in differentiating mouse cells

Development ◽  
1976 ◽  
Vol 35 (2) ◽  
pp. 355-367
Author(s):  
Eileen D. Adamson
Keyword(s):  
Creatine Phosphokinase ◽  
Phosphoglycerate Mutase ◽  
Embryonic Mouse ◽  
Embryonic Tissues ◽  
Differentiated Cells ◽  
Teratocarcinoma Cells ◽  
Mouse Tissues ◽  
Mouse Cells

Extracts of embryonic mouse tissues (skeletal, cardiac and smooth muscle, and brain) were analysed by Cellogel electrophoresis, for their isoenzymic distributions of three enzymes, creatine phosphokinase, aldolase and phosphoglycerate mutase. Embryonic tissues from the 12th day to the end of gestation were examined for isoenzyme transitions, and it was found that the adult forms of these enzymes appeared during gestation. Extracts from cloned teratocarcinoma cells were similarly examined in order to determine their degree of biochemical differentiation. Undifferentiated embryonal carcinoma cells contained only the early embryonic forms of all three enzymes, while differentiated cells formed in vivo, and in some cases in vitro, started to express the adult types of creatine phosphokinase and aldolase. Thus, biochemical parallels have been demonstrated between developing embryonic tissues and teratocarcinoma cells differentiating in vitro.

scholarly journals Remyelination-Promoting DNA Aptamer Conjugate Myaptavin-3064 Binds to Adult Oligodendrocytes In Vitro

2020 ◽  
Vol 13 (11) ◽  
pp. 403
Author(s):  
Mahboubeh Fereidan-Esfahani ◽  
Wei Ying Yue ◽  
Brandon Wilbanks ◽  
Aaron J. Johnson ◽  
Arthur E. Warrington ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Immunoglobulin M ◽  
Dna Aptamer ◽  
Live Cells ◽  
Embryonic Mouse ◽  
Adult Rat ◽  
Cortical Cultures ◽  
Mixed Cortical Cultures

We previously applied Systematic Evolution of Ligands by EXponential enrichment (SELEX) technology to identify myelin-specific DNA aptamers, using crude mouse central nervous system myelin as bait. This selection identified a 40-nucleotide aptamer (LJM-3064). Multiple biotinylated LJM-3064 molecules were conjugated to a streptavidin core to mimic a multimeric immunoglobulin M (IgM) antibody, generating 3064-BS-streptavidin (Myaptavin-3064). We previously showed that Myaptavin-3064 induces remyelination in the Theiler’s murine encephalomyelitis virus (TMEV) model of chronic spinal cord demyelination. While details of target binding and the mechanism of action remain unclear, we hypothesized that Myaptavin-3064 induces remyelination by binding to oligodendrocytes (OLs). We now report the results of binding assays using the human oligodendroglioma (HOG) cell line, applying both flow cytometry and immunocytochemistry (IC) to assay aptamer conjugate binding to cells. IC assays were applied to compare aptamer conjugate binding to primary embryonic mouse mixed cortical cultures and primary adult rat mixed glial cultures. We show that Myaptavin-3064 binds to HOG cells, with increased binding upon differentiation. In contrast, a negative control aptamer conjugate, 3060-BS, which did not promote central nervous system (CNS) remyelination, does not bind to HOG cells. Myaptavin-3064 did not bind to lung (L2) or kidney (BHK) cell lines. Total internal reflection fluorescence (TIRF) imaging indicates that Myaptavin-3064 binds at the cell membrane of live cells. In addition to HOG cells, Myaptavin-3064 binds to adult rat OLs, but not to embryonic mouse mixed cortical cultures. These data support the hypothesis that Myaptavin-3064 binds to a surface molecule on both rodent and human OLs in a manner that triggers a remyelination signal pathway.

Electron microscopic study of the membrane glycoproteins in the rat kidney after cisplatin treatment

1989 ◽  
Vol 47 ◽  
pp. 912-913
Author(s):  
S.K. Aggarwal
Keyword(s):  
Alkaline Phosphatase ◽  
Rat Kidney ◽  
Light And Electron Microscopy ◽  
Kidney Tissue ◽  
Membrane Glycoproteins ◽  
Electron Microscopic ◽  
Before And After ◽  
Interstrand Cross Links ◽  
Cross Links

The proposed primary mechanism of action of the anticancer drug cisplatin (Cis-DDP) is through its interaction with DNA, mostly through DNA intrastrand cross-links or DNA interstrand cross-links. DNA repair mechanisms can circumvent this arrest thus permitting replication and transcription to proceed. Various membrane transport enzymes have also been demonstrated to be effected by cisplatin. Glycoprotein alkaline phosphatase was looked at in the proximal tubule cells before and after cisplatin both in vivo and in vitro for its inactivation or its removal from the membrane using light and electron microscopy.Outbred male Swiss Webster (Crl: (WI) BR) rats weighing 150-250g were given ip injections of cisplatin (7mg/kg). Animals were killed on day 3 and day 5. Thick slices (20-50.um) of kidney tissue from treated and untreated animals were fixed in 1% buffered glutaraldehyde and 1% formaldehyde (0.05 M cacodylate buffer, pH 7.3) for 30 min at 4°C. Alkaline phosphatase activity and carbohydrates were demonstrated according to methods described earlier.

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