Biomedical subjects

M F Wolf

Publications and source records attributed to M F Wolf.

12 recordsLinked to original sources

PubMed1992

Biostability and blood-contacting properties of sulfonate grafted polyurethane and Biomer.

Sulfonate-containing polyurethanes were evaluated for in vivo biodegradation using subcutaneously implanted tensile bars. In addition, these anionically charged polyurethanes were evaluated for in vivo activation of human complement C3a and ex vivo platelet deposition in arteriovenously-shunted canines. The sulfonate derivatized polymers included laboratory synthesized polyurethane and Biomer. Other polymers used for references included Intramedic polyethylene, Silastic and a poly(ethylene oxide) based polyurethane. The biodegradation results indicated that Biomer and the laboratory sulfonated Biomer (both manufactured with stabilizers), remained mechanically stable, retaining both tensile strength and elasticity after 4 weeks of subcutaneous implantation. The unstabilized polyurethanes (with or without sulfonation), however, showed marked cracking and a loss of mechanical properties after the same period of subcutaneous implantation. Sulfonated polyurethanes depressed human complement C3a activation in plasma, as indicated by decreased levels of anaphylatoxin production. The results of canine ex vivo blood contacting experiments were conducted in both an acute and chronic model and demonstrated decreased platelet deposition and activation for the sulfonated polyurethanes.

Absorption↗

PubMed1989

Decrease in estradiol-stimulated progesterone receptor production in MCF-7 cells by epidermal growth factor and possible clinical implication for paracrine-regulated breast cancer growth.

These studies have evaluated the modulation by epidermal growth factor (EGF) of estrogen receptor (ER) levels and estradiol-stimulated progesterone receptor (PgR) synthesis. Short-term culture of MCF-7 cells in an "estrogen (phenol red indicator)-free" environment caused a rise in ER concentration that is inhibited by EGF at 10(-8) M and 10(-7) M. Estradiol at 10(-10) M induced a 5-fold increase of PgR over a 5-day assay period. However, the rise in PgR was diminished or prevented by increasing concentrations of EGF (10(-9) M to 10(-7) M). Similarly, the concentration-related rise in PgR caused by estradiol (10(-13) M to 10(-9) M) was abolished after a 7-day pretreatment with EGF (10(-7) M). For both the ER and PgR receptor, EGF treatment caused a decrease in receptor number without an apparent change in receptor affinity. Thus, EGF appears to down regulate the ER by approximately 50% and to diminish the ability of estradiol to induce PgR. In addition, a survey of ER+PgR+ and ER+PgR- values of primary breast tumors from women between the ages of 55 and 70 demonstrated significantly less (50%) (85 to 39 fmol/mg of cytosol protein) ER in ER+PgR- tumors (P = 0.0005). The median PgR values for the PgR-positive tumors were 139 fmol/mg of cytosol protein. We propose that ER+ breast cancer that has changed to a paracrine growth factor-driven system (from stromal cells or ER- breast cancer cells) is less responsive to gonadal steroids. The loss of PgR in these ER+ carcinomas may be an indicator of this type of hormone independence.

Breast Neoplasms↗

PubMed1989

Expression of Thomsen-Friedenreich (TF) antigens on lymphocytes. I. Distribution of cryptic and exposed TF antigens on murine lymphocytes from different lymphoid organs: detection with an anti-TF monoclonal antibody and peanut agglutinin.

Cryptic Thomsen-Friedenreich (TF) antigens were detected by the lectin peanut agglutinin (PNA) on the surface of murine lymphocytes after treatment of cells with neuraminidase. Thereby, a particular TF antigen could be distinguished using a monoclonal anti-TF antibody 49H8. In contrast to the known general galactoside specificity of PNA, the mAb was restricted to Gal beta(1-3)GalNAc/GlcNAc. Preincubation of cells with PNA abolished mAb 49H8 binding completely. However, only the intensity of staining with PNA was reduced by prior incubation of cells with the mAb. Cryptic TF antigens detected by the mAb were expressed on 39% of murine bone marrow cells, 88% of thymocytes, 62% of lymph node cells, and 65% of spleen cells. On the other hand, over 80% of the lymphatic cells carried cryptic PNA binding sites independent of the lymphoid organ they derived. In the thymus, a subpopulation of cells (76%) could be detected by PNA without neuraminidase treatment. Twenty-eight percent of thymocytes carried exposed mAb binding sites, too. All of them were shown to express further binding sites for PNA constantly. Therefore, a subpopulation of PNA-reactive, immature thymocytes can be distinguished by the mAb 49H8. During activation of splenic lymphocytes with PHA, the lymphoblasts completely lost their cryptic mAb binding sites while PNA reactivity was not affected. We conclude that the anti-TF mAb recognizes a particular TF antigen exposed on thymocytes and present in a cryptic form on other lymphocytes. The number of cells carrying mAb 49H8 binding sites varied, dependent on the organ from which the lymphocytes derived. PNA-reactive lymphocytes are distributed homogeneously in the lymphoid organs.

Animals↗

PubMed1989

Expression of Thomsen-Friedenreich (TF) antigens on lymphocytes. II. Loss of cryptic TF antigens during mitogenic activation of human T and B lymphocytes.

Human peripheral blood lymphocytes (HPBL) were examined for the presence of cryptic Thomsen-Friedenreich (TF) antigens as detected by PNA or an anti-TF mAb (49H8) after neuraminidase treatment of the cell surface. Neither PNA nor the mAb bound to the cells before treatment with neuraminidase. After removal of surface sialic acid, all lymphocytes were PNA-reactive, and 85% of HPBL reacted with the mAb 49H8. Seventy-seven percent of nylon wool (NW)-eluted T cells (96% Leu 1+), 80% of enriched helper T cells (83% Leu 3a+), and 78% of suppressor/cytotoxic T cells (63% Leu 2a+) carried the cryptic TF determinant recognized by the mAb 49H8. Ninety-one percent of NW-adherent cells (68% Leu 10+, 5% Leu 1+) were also TF positive. In contrast to NW-eluted T cells which showed low to moderate mAb 49H8 binding, 48% of NW-adherent cells revealed strong binding of anti-TF mAb. With progressive activation of T cells by PHA, binding of mAb to the cryptic TF antigen completely disappeared on blast cells. The presence of TF antigens on small cells in the culture was only poorly affected. The same was observed for activation of B cells with PWM. On the other hand, binding sites for PNA did not change during blastogenesis. The disappearance of the particular, mAb 49H8-reactive TF antigen on T blast cells is not due to the loss of antigen in a distinct T cell subset, but occurs to an equal extent in the helper and suppressor/cytotoxic T cell subpopulations. Thus, the majority of peripheral T and B lymphocytes carries cryptic TF antigens. Activated T or B cell blasts, on the other hand, are deficient for the particular TF antigen detected by the mAb 49H8. We interpret these data as a modulation of certain TF antigens on effector cells in the course of lymphocyte activation.

Antibodies, Monoclonal↗

PubMed1988

Estrogenic activity of phenol red.

It has recently been reported that phenol red, a pH indicator present in most tissue culture media, is a weak estrogen that can stimulate some estrogen-sensitive cells. However, the relative impact of phenol red on various cell lines is controversial. We examined the effect of phenol red on several estrogen-responsive cell systems that we use to study estrogen action. These included estrogenic stimulation of progesterone receptor and growth in human breast cancer-derived MCF-7 cells, stimulation of growth in human breast cancer-derived T47D cells, stimulation of prolactin synthesis in primary cultures of immature rat pituitary cells, and stimulation of progesterone receptor in primary cultures of immature rat uterine cells. Estrogenic responses in MCF-7 cells were the most sensitive to the presence of phenol red, while the other three cell cultures showed lesser effects of the indicator. In addition to intrinsic differences in cell responses, there were several other factors involved. These included differences in the estrogenic activity of phenol red-containing media and phenol red itself from different commercial suppliers, and differences in the concentration of free phenol red in final media due to binding of the indicator by serum. Higher concentrations of serum reduced the impact of phenol red on estrogenic responses in primary pituitary cells. Phenol red added to rat uterine cytosol competed with estradiol for binding to the estrogen receptor (relative binding affinity (RBA) approx. 0.001), and the acidic and basic forms of the indicator showed similar activity. Some commercial phenol red samples inhibited cell growth at levels of 100 mg/l; these effects were toxic rather than antiestrogenic, because growth inhibition could not be competitively reversed by an excess of estradiol. The amount of the indicator bound to serum in the final media, the source of the phenol red and the sensitivity of different cell types to the indicator ultimately determine its influence to the response of cells in tissue culture.

Animals↗

PubMed1988

Increased expression of Thomsen-Friedenreich antigens during tumor progression in breast cancer patients.

Paraffin-embedded tissue sections of primary tumors and lymph node metastases of 80 breast cancer patients were tested for the expression of Thomsen-Friedenreich (TF) antigens with the aid of a monoclonal IgM antibody (49H8) highly specific for phenyl-beta-galactoside. TF antigens were not expressed in 16 different normal tissues with the exception of some structures in the kidney. In tumor cells, two types of antigen expression were found; namely, cryptic and exposed. From stage T1/No to stages T2-4/N1,2 the number of cases expressing high amounts of TF antigens increased from 9% (2/22) to 22% (4/18) while the percentage of patients with low intensity of antibody binding was reduced from 59% (13/22) to 39% (7/18). The total amount of TF-positive primary tumors at stages T2-4/No increased from 42% (8/19) to 69% (18/26) when lymph nodes were infiltrated (T2-4/N1,2). At this stage 80% (21/26) of the patients with lymph node infiltration carried TF antigens in the nodes. The distribution of antigens was heterogeneous among the tumor cells and was expressed mainly in an apical or luminal position. The increased expression of antigens was attributed to exposed TF antigens, while cryptic antigens remained constant. When primary tumors expressed exposed TF antigens, the corresponding lymph nodes also contained exposed antigen. The same was true for the cryptic antigen. The data demonstrate an increase in the intensity of TF antigen expression during tumor progression and a spread of TF-positive tumor cells into the axillary lymph nodes with an increasing number of breast cancer patients being TF-positive at this stage.

Antigens, Tumor-Associated, Carbohydrate↗

PubMed1988

Estrogen receptor distribution in enucleated breast cancer cell lines.

The intracellular location of estrogen receptors in hormone-responsive cells has been studied with a number of techniques which indicate that the unoccupied receptors are nuclear and not cytoplasmic proteins. We used cell enucleation of two human breast cancer-derived cell lines, MCF-7 and T47D, to determine whether the unoccupied receptors were also nuclear in these cells and to determine whether the weak estrogen phenol red, present in nearly all tissue culture media, affected the distribution of the receptors seen with this technique. Nucleoplasts prepared from the breast cancer cells contained most of the estrogen receptors that were present in whole cells. The cytoplast fraction, which contained some contaminating whole cells, also contained some receptors. However, incubating cells with estradiol before enucleation did not translocate any receptors out of the cytoplast fraction (to the nucleoplasts). The unoccupied receptors appeared to be almost exclusively nuclear in these cells. The same results were obtained with either radioligand binding or enzyme-linked immunoassay used to measure estrogen receptor, and the distribution of receptors was unaffected by the presence of the pH indicator phenol red. In addition, we observed changes in the estrogen receptor content of incubated cytoplasts that were consistent with receptor synthesis, and this may prove to be a useful model system to characterize receptor synthesis and degradation.

Breast Neoplasms↗

PubMed1988

Hormone receptor assays: clinical usefulness in the management of carcinoma of the breast.

The revision of the subcellular model of hormone action is described, with an incorporation of potential autocrine mechanisms. A general overview of available assay methodologies considers the major disadvantages of earlier methods and describes in detail the current methodologies (sucrose gradient analysis, dextran-coated charcoal assays, ER-EIA, ERICA). A major concern with clinical correlations of response to hormone receptor levels is the quality assurance of the multicentric programs. Results from national and international programs are considered. The clinical correlations are divided into four major categories: (1) the response to hormone deprivation (oophorectomy or adrenalectomy), (2) the development of specific agents which exploit receptor mechanisms (antiestrogens) or inhibit steroid biosynthesis (aminoglutehimide), (3) the rates of recurrence of tumors following mastectomy, and (4) the correlation of hormone receptors with current adjuvant therapies.

Breast Neoplasms↗

PubMed1987

Inhibition of homotypic aggregation of a human Burkitt-lymphoma cell line.

The molecules involved in homotypic aggregation of the human Burkitt-lymphoma cell line Raji were investigated by inhibition of reaggregation with carbohydrates and glycoconjugates, by inhibition of glycosylation, and enzyme treatment of the cell surface. Complete inhibition of reaggregation was achieved with bovine submaxillary mucin. Asialomucin, on the other hand, was not effective in this assay. Another potent inhibitor of reaggregation was the ganglioside GMI. The common carbohydrate structure of these molecules is NeuNAc-(gal)-galNAc. Lactosamine, fucosyllactosamine, sialyllactosamine, complex mannose type, or Thomsen-Friedenreich antigen sequences are not involved in aggregation. Neuraminidase and chloroquine also abolished agglutination of cells. The finding that mucin, but not asialomucin, inhibits the reaction, demonstrates the importance of sialic acid in this process. Homotypic aggregation was shown to be resistant to trypsin. Using the glycosylation inhibitor tunicamycin we show that N-glycosidically linked carbohydrate chains are involved in aggregation. Swainsonine or castanospermine, which inhibit processing of terminal sialyllactosamines to the mannose core, did not interfere with the reaction supporting the results of the inhibition assay. The data presented suggest the involvement of 2 molecules in homotypic aggregation of human Burkitt-lymphoma cells. One component is a lectin-like molecule containing N-linked carbohydrate chains. The other component carries the neuraminidase-sensitive and trypsin-resistant determinant NeuNAc-(gal)-galNAc and, therefore, appears to be a glycolipid. This proposed lectin-carbohydrate interaction in homotypic aggregation is further supported by the frequently observed dependence of lectins on divalent cations as indicated by inhibition of aggregation with EDTA and EGTA.

Burkitt Lymphoma↗

PubMed1987

Characterization of Thomsen-Friedenreich antibody subpopulations from normal human serum.

Thomsen-Friedenreich (TF) antibodies were prepared from human serum by different enrichment procedures. This resulted in three antibody preparations all of which agglutinated neuraminidase-treated erythrocytes. On the other hand, each of the three antibody populations showed a distinct specificity pattern. Anti-TF1 antibodies could be inhibited in the hemagglutination inhibition assay by asialofetuin, asialotransferrin, asialoglycophorin and asialomucin. The sialylated form of these glycoproteins showed no inhibition. No significant inhibition could be achieved with several mono- or disaccharides. This suggests that anti-TF1 recognizes common structures on glycoproteins normally hidden by sialic acid. Anti-TF2 antibodies showed specificity for asialofetuin, bovine submaxillary mucin, asialomucin, asialoglycophorin, the disaccharide gal-beta (1-3)N-acetyl-galactosamine (galNAc) and nitrophenyl-beta-galactoside. Because asialotransferrin or unbound lactosamine were not inhibitory, we suppose that the residual common structure of the inhibitors is (gal)-galNAc-O-Ser/Thr, which is present in high amounts in submaxillary mucin. Anti-TF3 antibodies were inhibited by asialoglycophorin but not by asialomucin or asialofetuin. Strong saccharide inhibitors were gal-beta (1-3)galNAc, nitrophenyl-beta-galactoside, as well as galactose. Therefore, both of the antibody preparations, anti-TF2 and anti-TF3, could be inhibited by gal-beta-(1-3)galNAc, but showed preference to one or the other sugar component of the disaccharide resulting in a differential recognition of glycoconjugate inhibitors. Anti-TF2 and anti-TF3 seem to recognize the carbohydrates in the context of a protein backbone, because gal-beta-(1-3)galNAc in connection with a ceramide backbone (GM1) was not inhibitory. When tested on three human breast cancer cell lines, only anti-TF2 recognized epitopes exposed on the cell surface. We, therefore, conclude that human serum contains at least three subpopulations of TF antibodies with distinct specificities. Only anti-TF2 can detect cryptic erythrocyte epitopes which are also exposed on human breast cancer cell lines.

Antibodies↗

PubMed1986

Specificity of reagents directed to the Thomsen-Friedenreich antigen and their capacity to bind to the surface of human carcinoma cell lines.

The Thomsen-Friedenreich antigen (T-antigen) is a cryptic disaccharide structure on human erythrocytes and is supposed to be expressed in an unhidden form on carcinoma cells. We tested the ability of four anti-T reagents (i.e., peanut agglutinin, human and rabbit anti-T antisera, and monoclonal anti-T antibodies) to agglutinate neuraminidase treated human erythrocytes and compared their capacity to bind to the surface of human carcinoma cell lines or neuraminidase treated lymphocytes. We found that all of the reagents strongly agglutinated neuraminidase treated erythrocytes. In contrast, only peanut agglutinin and the monoclonal antibody bound to the surface of carcinoma cell lines and to neuraminidase treated lymphocytes. Peanut agglutinin was inhibited by D-galactose and is known to be specific for the T-disaccharide. The determinants on erythrocytes, lymphocytes, and carcinoma cells, recognized by peanut agglutinin, are resistant to trypsin treatment. The monoclonal antibody was specifically inhibited by phenyl-beta-D-galactoside. The binding sites on erythrocytes and lymphocytes for the monoclonal antibody can be removed by treatment with trypsin or Pronase. On the other hand, the binding sites on carcinoma cells are resistant to trypsin but can be removed with Pronase. In contrast to the peanut agglutinin binding sites on carcinoma cells the structures recognized by the monoclonal antibody cannot be further increased by neuraminidase treatment. Human and rabbit anti-T antisera did not bind to tumor cell surface or to neuraminidase treated lymphocytes. Hemagglutination of human anti-T could be inhibited by asialofetuin; the specificity of rabbit anti-T could not be established in this study. Hemagglutination with both antisera is resistant to trypsin but partially sensitive to Pronase treatment. These results indicate that each of the reagents has a distinct specificity and recognizes different antigenic determinants on different molecules. Only peanut agglutinin and monoclonal anti-T antibodies are able to detect common structures on the surface of neuraminidase treated erythrocytes and carcinoma cell lines.

Animals↗

PubMed1985

Rat uterine growth and induction of progesterone receptor without estrogen receptor translocation.

The rat uterus responds to estradiol (E2) and E2 benzoate stimulation with an increase in progesterone receptor production and with growth. These responses were also elicited to varying degrees by a series of estrogenic (ICI 77,949 and ICI 47,699) and antiestrogenic triphenylethylene derivatives [tamoxifen (TAM), 4-hydroxy-TAM (4-OH-TAM), and 4-CH3-TAM]. These compounds have a range of affinities for the estrogen receptor (ER) and are able to compete with [3H]E2 binding in the uterus in vivo. Within 1-2 h of a sc injection of high affinity ligands (E2, E2B, and 4-OH-TAM), there was decrease in cytosol ER. This decrease was also observed with TAM, which is metabolized to 4-OH-TAM in vivo. In contrast, there was no decrease in cytosolic ER in animals treated with low affinity compounds (ICI 77,949, ICI 47,699, and 4-CH3-TAM) at any time before the onset of an estrogenic response. Furthermore, the nuclear ER increased after administration of a high affinity ligand (E2), as measured by exchange assay, but no increase in nuclear ER was observed after administration of low affinity ligands (ICI 77,949 and 4-CH3-TAM), although estrogenic responses were produced. From these data we have suggested a functional model to explain ER-mediated events in the rat uterus that supports the recent proposal that unoccupied ER is located in the nuclear compartment. In this model, the majority of unoccupied ER may reside in the nucleus in vivo; however, when the cells are disrupted in vitro, the unoccupied receptor or dissociation of low affinity ligand-ER complex causes unoccupied receptor to fall out of the nucleus and be incorporated into the cytosolic fraction. The high affinity ligand-ER complexes are retained in the nucleus. This would suggest that the apparent translocation of the ER from the cytoplasm to the nucleus may be an artifact. The data may reflect differential extraction of unoccupied receptors from the nucleus rather than transfer of receptor complexes to the nucleus.

Animals↗