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Biomedical subjects

R W Knowles

Publications and source records attributed to R W Knowles.

At least 37 records · Page 2Linked to original sources
PubMed1985

The origin of ABH antigens on human platelets.

ABH antigens are present on platelets from individuals of the corresponding red cell phenotype, but the extent to which these antigens are intrinsic or adsorbed remains undefined. To evaluate platelets for intrinsic H substance, an IgM mouse monoclonal antibody against type 2H chain (the intrinsic H structure found on erythrocytes) was labeled with 125I and incubated with platelets from donors of different ABO type. The antibody showed dose-response saturation curves, and binding to platelets paralleled that of the red cell ABO type, with O greater than B greater than A1 greater than A1B greater Oh cells, giving a single factor variance F of 190 (P less than .0005). Passive adsorption of A antigens by platelets has been previously reported. To verify this phenomenon for A and B antigens and to quantitate the elution of A and B antigens from platelets, the following assay system was used. Platelets from group A1 and B donors were incubated in plasma from group O donors, and platelets from group O donors were incubated in plasma from different ABO, Lewis, and presumed secretor-type donors. Human IgG anti-A or anti-B was added to the platelets. The amount of antibody bound was determined with a 125I-labeled mouse monoclonal anti-human IgG. When incubated for 96 hours in group O plasma, group A1 platelets showed a 45% to 50% decrease in binding of anti-A. There was no significant change in the level of type 2H antigen on these platelets during the same incubation period. Group O platelets incubated in A or B plasmas rapidly acquired the antigens, but if returned to their original plasma, 95% of this passively adsorbed antigen eluted off within 18 hours. The maximum uptake of A and B substances was influenced by the Lewis and secretor type of donor plasma. Our present study demonstrates that ABH antigens on platelets consist of type 2H chains, which are presumably intrinsic as when found on red cells, and of passively adsorbed ABH structures, which are presumably type 1H chains.

ABO Blood-Group System↗
PubMed1985

Ultrastructural documentation of M241 glycoprotein on dendritic and endothelial cells in normal human skin.

M241 is a glycoprotein that recently has been demonstrated to be present in human thymus in a distribution similar to T6. Studies in skin, however, suggest that M241, in contrast to T6, is detected only on a subset of Langerhans cells and on a population of dendritic cells in the superficial dermis. We compared the reactivities of monoclonal antibodies to M241 and T6 with dendritic cells in normal human skin using immunoelectron microscopy. Our findings indicate that M241 is present on a minority of Langerhans cells and on a substantial number of other predominantly dermal dendritic cells with morphologic features of indeterminate cells. Anti-M241 reactivity was generally restricted to the cell membrane, although cytoplasmic reactivity was detected in some Langerhans cells. Dermal endothelial cells, which like dendritic cells are capable of antigen presentation, were also reactive with anti-M241 antibody. M241 is a glycoprotein, different from T6, with a tissue distribution potentially relevant to the understanding of antigen-presenting cells in the skin.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1984

Human cutaneous dendritic cells express two glycoproteins T6 and M241 which are biochemically identical to those found on cortical thymocytes.

Monoclonal antibodies anti-T6 and anti-M241 define unique cell populations within different lineages: cortical thymocytes and dendritic cells in the skin. T6 positive cutaneous dendritic cells are located predominantly in the epidermis and belong to the Langerhans/indeterminate lineage, whereas, most of the M241 positive cells are located in the perivascular regions of the dermis. Biochemical analysis of thymocytes and cutaneous dendritic cells was performed in order to determine whether the reactivity of these antibodies with these cell types is due to sharing of antigenic determinants by two unrelated proteins, or whether similar proteins are present on cells of different lineages. Our results indicate that T6 antigens are borne by the same glycoprotein (49K) on cortical thymocytes and Langerhans/indeterminate cells. Similarly, M241 antigens isolated from thymocytes and cutaneous dendritic cells are found on the same glycoprotein (43K).

Antibodies, Monoclonal↗
PubMed1984

Biochemical and functional evidence that an MT3 supertypic determinant defined by a monoclonal antibody is carried on the DR molecule on HLA-DR7 cell lines.

A cytotoxic monoclonal antibody, PL3, was produced by immunizing mice with a cell line homozygous for the HLA class II antigenic specificity DR7. The serologic specificity of PL3 was completely concordant with the MT3 supertypic specificity, which is tightly associated with HLA-DR4, -DR7, and -DRw9. This was confirmed by the finding that F(ab')2 fragments of PL3 blocked the cytotoxicity of anti-MT3 alloantisera. Although PL3 bound to each of the MT3-positive cell lines, it showed significantly weaker binding to HLA-DR4 and -DRw9 cells relative to -DR7 cells, both in titration and in quantitative absorption assays. This differential pattern of binding was not found for the polyclonal MT3 alloantisera, suggesting that the PL3 determinant may be one of several closely related determinants that comprise the MT3 allospecificity. To identify which of the subpopulations of class II molecules carry the PL3 determinant, several approaches have been used. F(ab')2 fragments of PL3 which block the anti-MT3 alloantisera were also tested with anti-MB2 and anti-DR7 sera. Binding of the PL3 F(ab')2 fragments to DR7 homozygous target cells had no effect on the anti-MB2 sera, but significantly enhanced the cytotoxic reactivity of some anti-DR7 sera. This finding suggested that the PL3 determinant is distinct from the DR7 determinant, but is carried on the same molecule. PL3 was also used in blocking studies with allocytotoxic T cell clones which only recognize DR7-positive cell lines. Binding of PL3 to the DR7-positive target cells was found to completely inhibit these T cell clones. Complete blocking was also found with a monoclonal antibody, PL8, which recognizes a monomorphic determinant found on the DR subpopulation of class II molecules. This finding suggested that the PL3 determinant is carried on the same molecule that carries these T cell-defined DR7 allodeterminants. In biochemical studies with DR7-positive cell lines, PL3 and PL8 were found to immunoprecipitate the same subpopulation of class II molecules recognized by other DR-specific antibodies, SG157 and TAL-1B5. Two-dimensional gel analysis demonstrated that the pattern of alpha- and beta-chains immunoprecipitated by PL3, PL8, and TAL-1B5 were identical. In sequential immunoprecipitation studies, both PL3 and TAL-1B5 were capable of removing the same DR subpopulation of molecules recognized by PL3, PL8, TAL-1B5, or SG157 while leaving the additional class II molecules (DS) recognized by SG171 on DR7 cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Specific inhibition of in vitro lymphocyte transformation by an anti-pan T cell (gp67) ricin A chain immunotoxin.

The toxin A chain of ricin has been conjugated by a disulfide bond to a murine monoclonal antibody that recognizes the gp67kD antigen present on 95% of peripheral T lymphocytes. The immunotoxin retains both functions of its component parts: it binds to human peripheral blood lymphocytes, and it inhibits protein synthesis in a cellfree reticulocyte system. The immunotoxin has been evaluated for its ability to inhibit in vitro T lymphocyte transformation. In the presence of 20 mM NH4Cl, the immunotoxin decreases lymphocyte proliferation in response to phytohemagglutinin to less than 8% of untreated controls. The proliferative response in mixed lymphocyte culture and the development of allocytotoxic T cells is also dramatically inhibited by this immunotoxin. Monoclonal antibody alone does not inhibit these responses. Specificity of the immunotoxin has been established: the effect of the immunotoxin can be blocked by unconjugated monoclonal antibody, but not by a control monoclonal antibody that recognizes another T lymphocyte differentiation antigen or by a control monoclonal antibody that does not recognize human peripheral blood leukocytes. Treatment of human bone marrow cells with the immunotoxin preserves hematopoietic progenitor cells, as measured by granulocyte-macrophage, erythroid, and multipotential hematopoietic progenitor cell assays. These results indicate that an anti-pan T lymphocyte-ricin A chain immunotoxin is an effective agent against immunocompetent T lymphocytes in vitro, and may be an effective agent for use in clinical bone marrow transplantation.

Animals↗
PubMed1983

Two monoclonal antibodies identifying a subset of human peripheral mononuclear cells with natural killer cell activity.

Two monoclonal antibodies (H25 and H366) raised against the cultured T lymphoid cell line HSB-2 were shown to define a subset of peripheral mononuclear cells with natural killer and killer cell activity. The two antibodies show similar tissue distributions. Radioimmune trace binding assay shows that H25 and H366 react with all T cell lines tested and with the monocyte line U937, but weakly or not at all with cell lines of B, myeloid or erythroid origin. Both antibodies react with about 10% of E rosette-forming cells and a proportion of nonrosetting lymphocytes and monocytes. They do not react with B lymphocytes, granulocytes, red cells or platelets. A proportion of thymocytes and low numbers of tonsil and spleen mononuclear cells are positive. H25+ and H366+ lymphocytes are medium-sized cells with abundant granular cytoplasm, and most carry Fc receptors for IgG. H25 and H366 immunoprecipitate two polypeptide chains of 96 and 53kDa from surface-labeled HSB-2 cells.

Animals↗
PubMed1983

Allocytotoxic T cell clones: both Leu 2+3- and Leu 2-3+ T cells recognize class I histocompatibility antigens.

T cell clones were selected which were cytotoxic for human class I major histocompatibility target antigens. Specificity was based on target cell panel studies and inhibition by monoclonal antibodies to class I determinants. Eight clones were Leu 2+3-. The cytotoxicity of these clones was inhibited by antibody to the Leu 2 antigen. Two clones expressed the Leu 2-3+ phenotype and were not inhibited by anti-Leu 2a or anti-Leu 3a antibodies. These studies indicate that class I-specific cytotoxic T cells are distributed in both T cell subsets, though predominantly in the Leu 2+3- group. In addition, these studies suggest that the Leu 3 molecule may not function in identical fashion in Leu 3+ cytotoxic T cells, which recognize class I target antigens, as in those which recognize class II targets.

Antibodies, Monoclonal↗
PubMed1983

The thymic differentiation markers T6 and M241 are two unusual MHC class I antigens.

The human beta 2-microglobulin (beta-2m)-associated human thymocyte differentiation antigens T6 and M241 were compared using biochemical techniques. T6 and M241 antigens reside on different molecules with apparent m.w. of 49,000 and 43,000, respectively. Here we show that both proteins have a protein backbone m.w. of 33,000. In addition, T6 and M241 have a large portion of their peptides in common. When we compared the protein backbone m.w. of T6 and M241 with the murine beta-2m-associated thymus leukemia (TL) antigens, we found a considerable difference in size, suggesting that T6 and M241 may not be human homologues of TL antigens and constitute a novel type of major histocompatibility (MHC) class I antigens.

Animals↗
PubMed1983

Analysis of two new leukemia-associated antigens detected on human T-cell acute lymphoblastic leukemia using monoclonal antibodies.

Two monoclonal antibodies (anti-3-3 and anti-3-40) were produced, which identify two new leukemia-associated antigens. Both antibodies reacted with most cell lines derived from patients with T lymphoblastic leukemia (T-ALL), but were not detected on suspensions of normal hematopoietic cells (including thymocytes) by cytotoxicity, absorption, or indirect immunofluorescence assays. Analysis of fresh leukemic cells indicated that anti-3-3 only reacted with T-ALL cells, while anti-3-40 also reacted with some non-T, non-B ALL cells and a few acute myelocytic leukemia (AML) cells. The 3-40 antigen was also found histopathologically in frozen sections of several normal tissues, including the epithelial cells and a few lymphoid cells of the thymus, and some malignant tissues. The 3-3 antigen was not found in any tissue studied. A "double absorption"assay provided additional serologic evidence that the two antibodies identify different antigenic determinants. Biochemical analysis indicated that the molecules immunoprecipitated by anti-3-3 and anti-3-40 have molecular weights of 35,000-40,000 daltons. This study demonstrated that the 3-3 and 3-40 antigens are markers for human T-ALL and can be used along with the normal T-lymphocyte antigen, 3A1, to discriminate T-ALL from cutaneous T-cell lymphoma (CTCL), adult T-cell leukemia (ATL), and T-cell chronic lymphocytic leukemia (T-CLL).

Adult↗
PubMed1982

A monoclonal antibody recognizing a human thymus leukemia-like antigen associated with beta 2-microglobulin.

A monoclonal antibody, M241, was produced which binds to a human cell surface molecule with properties similar to the murine thymus leukemia (TL) antigen. This human TL-like antigen was found on thymocytes and some T cell lines derived from patients with acute lymphocytic leukemia, but was not found on peripheral blood lymphocytes or B cell lines. The monoclonal antibody M241 was used to immunoprecipitate a molecule from lysates of 125I surface-labeled MOLT 4 cells which had two subunits, a 43-kDa chain and a 12-kDa chain. The small subunit was shown to be beta 2-microglobulin (beta 2m) by immunoprecipitation with a monoclonal antibody, BBM.1, which recognizes human beta 2 m. The TL-like molecule recognized by M241 was shown to be serologically distinct from the HLA-A,B,C molecules recognized by three monoclonal antibodies W6/32, PA2.6 and BB7.8, and distinct from another human thymocyte antigen, the 49 kDa HTA 1 molecule, recognized by the monoclonal antibody NA1/34. Following removal of the HLA-A,B,C molecules, the HTA 1 molecules, and the M241-defined TL-like molecules from MOLT 4 lysates, additional beta 2m-associated molecules were immunoprecipitated with BBM.1. These molecules contained a 45-kDa subunit attached to beta 2m.

Animals↗
PubMed1982

Detection of human cancer in an animal model using radio-labelled tumour-associated monoclonal antibodies.

Monoclonal antibodies to epithelial-cell antigenic determinants, labelled with 123I and 125I, were administered parenterally to immunodeficient mice bearing human tumours derived from a human cancer cell line. Anterior, posterior and lateral radioscans of the body were taken with a gamma scintillation camera at various times from immediately to 65 days after injection. Visual displays of the images were processed by standard computer techniques. The model used a human colon-cancer cell line, HT29, and the monoclonal antibody, AUA1, which is specific to an epithelial proliferating antigen. Tumour detection was achieved in all the mice. The smallest tumour detectable appeared to be about 1 mm in diameter. The degree of antibody uptake in a tumour depended on its size and the blood supply of its surrounding tissues We believe that the technology and skills are now available for accurate radioimmunodetection of cancer in man.

Animals↗
PubMed1982

Monoclonal anti-type 2 H: an antibody detecting a precursor of the A and B blood group antigens.

A monoclonal antibody was produced which binds specifically to Type 2 H antigen of the ABO blood group system. The antibody, H11, is an IgM molecule which reacts by direct agglutination of red cells with the same pattern as other anti H reagents such as Ulex europaeus lectin. The specificity was determined by inhibition and adsorption with chemically defined oligosaccharides. H11 also reacted with poly (glycosyl) ceramide purified from group 0 red cells and glycoprotein H purified from human stomach mucosa and meconium. H11 differs from Ulex lectin, however, in that it was not inhibited by saliva from ABH secretor individuals or by glycoprotein H purified from human ovarian cysts or submaxillary glands.

ABO Blood-Group System↗