Biomedical subjects

J J Sedmak

Publications and source records attributed to J J Sedmak.

At least 19 recordsLinked to original sources

PubMed1992

Cytotoxicity of Clostridium difficile toxin A for human colonic and pancreatic carcinoma cell lines.

The use of bacterial exotoxins may constitute novel adjuncts to treatment of gastrointestinal tract malignancies. Clostridium difficile toxin A was evaluated for its cytotoxic effect in vitro on 24 human cell lines and strains including carcinomas of the colon, pancreas, prostate, lung, breast, and lymphoid malignancies, as well as nonmalignant tissues. All nine colon and five pancreas cell lines were extraordinarily sensitive to the cytotoxic effect of Clostridium difficile toxin A at very low concentrations. This effect, which occurred rapidly and was dose dependent, was observed in all cells of seven colon and two pancreas cell lines at concentrations as low as 1-5 ng/ml (10(-12) to 10(-11) M), whereas cells derived from other sites required 60 to greater than 500 ng/ml to achieve an equivalent effect. The data suggest that Clostridium difficile toxin A may have potential therapeutic value in the treatment of some gastrointestinal tract cancers.

Bacterial Toxins↗

PubMed1990

Actin and lamin comprised filaments in the nuclei of Chinese hamster ovary cells affected with Clostridium difficile enterotoxin A.

The major change in the ultrastructure of Chinese hamster ovary (CHO) cells treated with Clostridium difficile enterotoxin A was the formation of bundles of filaments in the nucleoplasm. The filaments appeared after 2.5 h and disappeared by the fourth hour of incubation with the toxin. To partially characterize these filaments, laser diffractometry and ultrastructural immunocytochemistry were employed. The bundles consist of coiled filaments of about 11 nm and 16 nm diameter and a main long axial periodicity of about 26 nm. Postembedding immunogold labelling with monoclonal antibodies demonstrated that these intranuclear filaments are comprised mainly of actin, but also contain lamin and vinculin. Between 2.5 and 3 h of incubation with enterotoxin A, the lamins were located close to the nuclear envelope, but they were also consistently found scattered in the cytoplasm and nucleoplasm. Such an unusual distribution of lamins might mediate the adverse effects of enterotoxin A on nuclear organization and function and thus cause the eventual death of CHO cells.

Actin Cytoskeleton↗

PubMed1989

Effect of Clostridium difficile enterotoxin A on ultrastructure of Chinese hamster ovary cells.

Electron microscopical immunocytochemistry and light microscopy were used to study the effect of Clostridium difficile enterotoxin A (EA) on cultured Chinese hamster ovary (CHO) cells. At 4 degrees C, immunocytochemically detected EA was randomly distributed along the plasma membrane; when cells were subsequently transferred to 37 degrees C, the EA moved into coated pits and coated vesicles within 2 min. Within 2 h of incubation at 37 degrees C with EA, the perinuclear cytoplasm of the CHO cells became highly refractile, and in 4 h, most of the cells were round; however, the majority of rounded cells excluded erythrosin B while remaining firmly attached to the culture dish plastic. When EA was removed from the cultured cells within 2 h, the cells returned to a normal morphology and formed a confluent monolayer. The nuclei of rounded cells were irregularly shaped; cytoplasmic intermediate filaments were collapsed toward the nucleus. Long bundles of parallel, 11-nm-diameter filaments appeared in the nuclei after 3 h of incubation with EA and disappeared by the fourth hour of incubation. Intoxicated cells did not undergo mitosis. Thus, EA was internalized, at least in part, by receptor-mediated endocytosis and subsequently affected the nuclei of CHO cells.

Animals↗

PubMed1989

High levels of circulating neutralizing antibody in normal animals to recombinant mouse interferon-beta produced in yeast.

Plasma from normal outbred Swiss mice not previously given interferon (IFN) neutralized a glycosylated (high-mannose) recombinant murine IFN-beta made in yeast (rMuIFN-beta y). The neutralization antibody titers were as high as 1:6,000 versus rMuIFN-beta y, whereas the titers obtained with native murine IFN-beta (MuIFN-beta) were 100-fold less; a recombinant murine IFN-beta make in Escherichia coli (rMuIFN-beta ec) was not neutralized at 1:10 dilution of plasma. An ELISA using rMuIFN-beta y demonstrated that it is gamma immunoglobulins in normal mouse plasma that bind rMuIFN-beta y. Normal rabbit serum also had very high endogenous neutralizing activity (1:5,300) against rMuIFN-beta y. Significant neutralizing activity also was detected in newborn bovine and normal human serum as well as pooled human immune serum globulin. Thus, endogenous neutralizing activity directed presumably at the carbohydrate residues of highly glycosylated recombinant proteins produced in yeast may limit their clinical utility.

Animals↗

PubMed1988

The cellular internalization of recombinant gamma interferon differs from that of natural interferon gamma.

Purified natural and recombinant murine gamma interferons (MuIFN-gamma) bind at 4 degrees C to cultured L929 mouse fibroblasts with comparable receptor-binding affinity (Kd = 9 x 10(-10) M). Both 125I-labeled MuIFNs are rapidly internalized by cells at 37 degrees C, although recombinant IFN is internalized somewhat more slowly than natural IFN (t1/2 = 90 sec and 45 sec, respectively). Immunoelectronmicroscopy showed that the majority of bound recombinant MuIFN-gamma was located on the plasma membrane outside of coated areas, whereas natural interferon was found mainly in coated pits. At 37 degrees C most of the recombinant molecules entered the cytoplasm in pinocytotic vesicles, while natural interferon was internalized by the specific mechanism of receptor-mediated endocytosis [1]. However, nearly equal amounts of immunocytochemically detectable molecules of both IFNs were found in the cell nucleus within 2-3 min incubation at 37 degrees C. Thus, the process of translocation of the recombinant IFN-gamma appears to differ from that of the natural product.

Animals↗

PubMed1988

Quantitative analysis of mouse interferon-beta receptor-mediated endocytosis and nuclear entry.

The dynamics of internalization and transport of murine interferon-beta (MuIFN-beta) to the cell nucleus was studied in cultured L929 mouse fibroblasts employing postembedding, ultrastructural quantitative immunocytochemistry. Cells preincubated with MuIFN-beta at 4 degrees C were transferred to 37 degrees C for various times, and then fixed and embedded in Lowicryl K4M. Internalized native IFN molecules were identified and quantified on the surface of ultrathin sections with monoclonal (MAb) or polyclonal (PAb) anti-IFN antibodies and a colloidal gold-protein A (CGPA) conjugate. Immunocytochemically recognizable MuIFN-beta molecules entered the cytoplasm by receptor-mediated endocytosis and were translocated via transport vesicles to the nuclear envelope. IFN molecules entered the nucleus through nuclear pores and accumulated maximally in the dense chromatin within 3 min of incubation of the cells at 37 degrees C. The data presented constitute the first direct observation of the dynamics of the extremely rapid transfer of ligand to its intracellular target, in this case, the cell nucleus.

Animals↗

PubMed1988

Quantification of low numbers of immunogold label on Lowicryl K4M sections.

Postembedding immunogold staining was used to quantify low numbers of intracytoplasmic cytokine interferon molecules in cells embedded in Lowicryl K4M by the rapid technique of Altman et al. (1984). When labelling with colloidal gold-protein A (CGPA) was performed as suggested by the manufacturers, the relatively high background precluded the statistically significant distinction of small amounts of specific label. The standard procedure of postembedding staining was modified by introducing: (1) UV-irradiation of the sections before immuno-staining; (2) 10- to 50-fold greater dilution of CGPA than recommended by the manufacturers, and (3) washing the sections at 60 degrees C between staining steps. The resulting decrease in nonspecific binding of CGPA facilitated the quantification of as few as 6-10 CGPA particles per 1,000 micron2 of the surface of the Lowicryl K4M sections at greater than a 95% confidence level.

Animals↗

PubMed1986

Transport of gamma-interferon into the cell nucleus may be mediated by nuclear membrane receptors.

Purified mouse interferon gamma (MuIFN-gamma), a lymphokine having potent antiviral, immunomodulatory, and growth inhibitory activities, is internalized (t1/2 less than 1.0 min) by mouse L929 fibroblasts via receptor-mediated endocytosis. Individual MuIFN-gamma molecules, identified by a postembedding immuno-gold technique, are then transported to the cell nucleus, perhaps through nuclear pores, into areas of dense chromatin. Purified, isolated nuclei of L929 cells bind radiolabeled MuIFN-gamma specifically and with high affinity (Kd = 2 X 10(-10) M). These nuclear membrane receptors, distinct from those for MuIFN-beta, number about 24,000/nucleus. Treatment of nuclei with trypsin prevents binding of MuIFN-gamma. The demonstration of rapid cellular uptake and transport of MuIFN-gamma into the dense chromatin, perhaps facilitated by nuclear receptors, suggests that IFN-gamma molecules, alone or bound to receptor, may directly affect genome regulation.

Animals↗

PubMed1986

Lanthanide ion enhancement of interferon binding to cells.

Pretreatment of purified [125I]-labeled human and mouse beta interferons (IFN) with lanthanum chloride (LaCl3) enhanced 20-30 fold the binding of the [125I]-IFNs to human A549 and mouse L cells at 0 degree C and also enhanced antiviral activity in homologous cells. Although lanthanides enhanced cross-species binding of both human and mouse [125I]-IFNs, there was no increase in cross-species antiviral activity. Unlabeled IFN not treated with LaCl3 did not compete with [125I]-IFN treated with LaCl3 for cellular receptors. However, unlabeled IFN treated with LaCl3 did compete with LaCl3-treated [125I]-IFN. These results suggest that lanthanide treated IFNs do not bind to the same receptors as native IFNs.

Animals↗

PubMed1986

Internalization and transport of mouse beta-interferon into the cell nucleus.

An electron microscopic technique employing postembedding immunolabeling was used to identify unmodified, intracellular MuIFN-beta molecules. The MuIFN-beta was rapidly internalized by receptor-dependent endocytosis and within three minutes a majority of internalized MuIFN-beta molecules was detected within the cell nucleus.

Animals↗

PubMed1986

Mouse beta-interferon reduces RNA efflux from isolated nuclei.

Treatments of isolated L929 cell nuclei with MuIFN-beta reduced the energy-dependent efflux of 3H-RNA from these nuclei in a dose-dependent manner. These observations provide evidence for a direct physiological effect of IFN on the cell nucleus.

Animals↗

PubMed1985

Murine interferon-beta receptor-mediated endocytosis and nuclear membrane binding.

Radioiodinated mouse interferon-beta (125I-MuIFN-beta) bound with high affinity (Kd = 9.8 X 10(-10) M) to plasma membrane of L929 murine fibroblasts (4-6 X 10(3) receptor sites per cell). The binding was saturable and inhibited by a 100-fold excess of unlabeled MuIFN-beta but not by excess mouse IFN-gamma (MuIFN-gamma). MuIFN-beta bound at 4 degrees C was very rapidly internalized upon warming of the cells to 37 degrees C (t 1/2 = 1.5 min). Indirect immunoferritin labeling indicated that MuIFN-beta was initially located in coated pits and subsequently internalized by receptor-mediated endocytosis. Isolated L929 cell nuclei bound 125I-MuIFN-beta with a 7-fold higher affinity (Kd = 1.4 X 10(-10) M) and higher receptor density (about 10(4) per nucleus) than that for the plasma membrane. Binding to the nuclear membrane was inhibited by a 100-fold excess of unlabeled MuIFN-beta but not by excess MuIFN-gamma. Trypsin treatment of nuclei decreased IFN binding by 80%, suggesting that the putative nuclear receptors are protein. Specific binding of MuIFN-beta to nuclei was also shown by fluorescence and electron microscopy. We propose that the very rapid internalization of MuIFN-beta by receptor-mediated endocytosis is important in the cellular processing of IFN and that its high-affinity binding to the nuclear membrane suggests the nucleus as an intracellular site of IFN action.

Animals↗

PubMed1985

HuIFN-gamma antiviral activity correlates with immunological reactivity in a double-monoclonal antibody radiometric assay.

Several lines of evidence have been obtained that correlate biological activity with reactivity in an immunoradiometric assay (IRMA) employing two different monoclonal antibodies. Antiviral activity and immunological reactivity decreased in parallel when freeze-dried human gamma-interferon (HuIFN-gamma) was heated for prolonged periods of time at elevated temperatures. Likewise, aqueous preparations of HuIFN-gamma inactivated by acid pH or heat responded similarly in the biological assay and IRMA. These results indicate that the monoclonal antibodies used in this commercially available IRMA are specific for the epitopes on biologically activity HuIFN-gamma.

Antibodies, Monoclonal↗

PubMed1985

Cytotoxicity of human beta-interferon for differentiating leukemic HL-60 cells.

We examined the effect of human interferon (HuIFN) -alpha and -beta on the proliferation and differentiation induced by dimethyl sulfoxide (DMSO) of HL-60 human promyelocytic leukemia cells into mature granulocytes. Neither HuIFN-alpha nor -beta, alone, from 1 to 1000 IU/ml, nor the homologous mock HuIFN preparations affected HL-60 cell differentiation or proliferation. Whereas the combination of HuIFN-alpha (10 to 1000 IU/ml) with DMSO also did not affect the proliferation or differentiation of HL-60 cells, the addition of HuIFN-beta (1000 IU/ml) and DMSO (1.25%) to growing cultures reduced cell viability as much as 14% of that observed for cells treated with DMSO alone or to 4% of that observed for either untreated cells or those treated with HuIFN-beta alone. The cytotoxic effect declined with decreasing concentrations of HuIFN-beta. The cytotoxic effect of DMSO and HuIFN-beta was exerted only as cells began to differentiate. Removal of HuIFN-beta at Day 2 did not reverse the cytotoxic effect, and addition of HuIFN-beta at Day 2 did not inhibit cell proliferation. Addition of HuIFN-beta to postmitotic cells on Day 4 after DMSO treatment did not affect proliferation but did slow differentiation. The cytotoxic and antidifferentiative effects of naturally produced HuIFN-beta were confirmed with highly purified recombinant HuIFN-beta. Undifferentiated HL-60 cells were resistant to the antiviral effects of HuIFN-beta, requiring 4 to 6 times the concentration to protect 50% of the cells against vesicular stomatitis virus as that needed to produce a cytotoxic or antidifferentiative effect. The profoundly cytotoxic effects of HuIFN-beta reported here may provide a model to study this interferon in combination with inducers of leukemic cell differentiation as a possible strategy in cancer therapy.

Antibodies↗

PubMed1984

Markedly enhanced production of gamma interferon in murine T lymphocytes treated with lentil lectin and the diterpene ester, mezerein.

Gamma interferon (IFN-gamma) was induced in murine splenocytes first stimulated to grow by concanavalin A (Con A) and subsequently treated for 3 h with the diterpene ester, mezerein (MZN) and then with lectin from Lens culinaris for 24 h. Yields as high as 60,000 u/ml were obtained in cells from either male or female, random-bred, white Swiss mice or inbred C67B1/6 mice. Antibody to Thy 1.2 surface antigen completely obliterated the mouse gamma interferon (MuIFN-gamma) response, whereas anti-Lyt 1.2 and anti-Lyt 2.2 each destroyed a portion of the lymphocyte population responsible for MuIFN-gamma production. Kinetic analysis of production and release showed that IFN was detectable in culture fluids within 4 h after treatment with very little IFN remaining cell-associated (less than 10%). A simple, rapid, and economical two-step purification procedure involving ammonium sulfate fractionation and yeast RNA affinity chromatography resulted in as much as 770-fold purification to achieve specific activities greater than 10(7) u/mg protein. The purified MuIFN-gamma was shown to be predominantly acid-labile, inactivated by sodium dodecyl sulfate (SDS), and neutralized by antiserum to MuIFN-gamma. Approximately 10% of the MuIFN-gamma was acid-stable and SDS-resistant, but was still neutralized by anti-MuIFN-gamma serum. Two molecular weight peaks of about 40 and 20 kD were demonstrated by SDS-polyacrylamide gel electrophoresis. Isoelectric focusing in polyacrylamide slab gels gave a relatively heterogeneous band of activity between pH 5.5 and 6.5. The mechanism by which the combination treatment described enhances MuIFN-gamma production so markedly remains unknown, but the degree of enhancement is greater than additive.

Animals↗

PubMed1984

Diphtheria toxin receptor sites on membranes of cultured cells and erythrocytes demonstrated by fluorescence and electron microscopy.

Diphtheria toxin (DT) receptor sites were identified on cell membranes by indirect immunofluorescence and immunoferritin labelling. The results obtained were correlated (1) with the binding of 125I-labelled DT (to the very same cell populations used in the ultrastructural studies), and (2) with toxin sensitivity as shown by inhibition of 14C-amino acid incorporation into protein. Binding and immunocytochemical studies were carried out at 0 degrees C to preclude internalization of DT. Receptor sites for DT were observed on functionally responsive, cultured human diploid (BUD-8) fibroblasts as well as on a small fraction of erythrocytes (RBC) of DT-susceptible species, i.e. human and sheep. On all BUD-8 cell sections DT receptor sites were located in annular patches and not on microvilli as had been observed in the KB epithelial cell line. Immunoferritin label was revealed on only 10% +/- 2.5 (SD) of human RBC in discreet loci along the cell membrane. Specificity of binding of DT to human fibroblast and RBC receptor sites was further demonstrated by competition between 125I-labeled and non-labelled DT. There was no specific binding to DT-treated cultured mouse L929 fibroblasts or to RBC of mouse of rat, species resistant to the toxin. Thus, direct ultrastructural immunocytochemical evidence is provided for the presence of specific cellular DT receptors on cultured cells and erythrocytes of species sensitive to DT, but not on those of species resistant to DT.

Animals↗

PubMed1983

A unique set of polypeptides is induced by gamma interferon in addition to those induced in common with alpha and beta interferons.

Human immune interferon (IFN-gamma) differs from leukocyte interferon (IFN-alpha) and fibroblast interferon (IFN-beta) in cell origin, inducing agents, physical and biological properties and amino acid sequence. These differences have led to interest in possible differences in the biological properties of IFN-gamma compared with IFN-alpha and IFN-beta. IFN-gamma has the same broad range of biochemical and biological actions as do IFN-alpha and IFN-beta, although relative potencies vary depending on the cell type and function investigated. There has so far been no direct evidence that IFN-gamma alters normal cell functions differently from other interferons. We report here striking qualitative and quantitative differences in the intracellular response of human fibroblasts to IFN-gamma compared with IFN-alpha and IFN-beta. Two-dimensional gel electrophoresis demonstrates, in addition to the induction of a common group of polypeptides, the existence of a set of polypeptides whose synthesis is uniquely induced by IFN-gamma.

Cell Line↗

PubMed1983

Interferon and antibody titrations using haemagglutinating Togaviridae and trypsinized human erythrocytes.

Several Togaviridae of the alphavirus and flavivirus genera agglutinate trypsinized human group O erythrocytes (THOE) (Shortridge and Hu, 1976). Haemagglutinin titers of Semliki Forest virus (SFV) and Japanese encephalitis virus (JEV) measured with THOE were equivalent to, if not higher than, those obtained with Embden gander erythrocytes, even with unextracted haemagglutinin. Results obtained with THOE in JEV haemagglutination-inhibition tests on sera taken from a previously infected individual over a 20-yr period were similar to those measured during the initial JEV infection. The inhibition of SFV haemagglutinin production as measured with THOE was a very sensitive bioassay for chicken interferon: interferon titers were 6- to 10-fold higher than those obtained with the vesicular stomatitis virus plaque-reduction method. The generally greater availability of human erythrocytes (including those stabilized with glutaraldehyde), the simplicity of the trypsin treatment, and the possibility of using unextracted haemagglutinin recommend this technique for use with haemagglutinating Togaviridae.

ABO Blood-Group System↗