Biomedical subjects

S E Grossberg

Publications and source records attributed to S E Grossberg.

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↗

PubMed1992

Endogenous interferon and triglyceride concentrations to assess response to zidovudine in AIDS and advanced AIDS-related complex.

To improve evaluation of new antiretroviral drugs in the acquired immunodeficiency syndrome (AIDS), sensitive biological markers that accurately predict response to treatment are needed. Two possible markers are endogenous interferon (E-IFN), which is a cytokine involved in the pathophysiology of AIDS, and serum triglycerides (TG), which are raised in patients with AIDS, possibly reflecting enhanced cytokine activity. E-IFN, TG, body-mass index, CD4 count, and HIV p24 were measured in 19 patients (15 with AIDS, 4 with AIDS-related complex), who were part of the phase II licensing trial of zidovudine (ZDV). 10 received ZDV and 9 received placebo. Rapid, significant, and sustained declines from initial values in E-IFN and TG concentrations were observed in ZDV patients but not in placebo patients. Baseline values of E-IFN and TG concentrations after 4 months on ZDV treatment were both important contributors to long-term survival. The findings suggest that these indicators of abnormal cytokine expression may be useful measures of not only disease severity but also efficacy of antiretroviral therapy in AIDS.

AIDS-Related Complex↗

PubMed1992

Absence of biological effects of orally administered interferon-beta ser.

To assess biological effectiveness of interferon (IFN) administered orally, we measured serum IFN and several proteins and metabolites induced by IFN after oral administration of 2.5 mg or 7.5 mg of recombinant IFN-beta ser to 6 healthy volunteers. These IFN-induced metabolites, beta 2-microglobulin, and neopterin in serum, and 2',5'-oligoadenylate (2-5A) synthetase in peripheral blood mononuclear cells, are more sensitive to the presence of IFN than bioassay of IFN in serum. Up to 48 h after oral IFN was administered, serum IFN, beta 2-microglobulin, neopterin, or 2-5A synthetase were not generally increased compared to pretreatment levels, indicating that oral IFN had no significant biological effects.

Adenine Nucleotides↗

PubMed1991

Antibodies that neutralize human beta interferon biologic activity recognize a linear epitope: analysis by synthetic peptide mapping.

The location of biologically relevant epitopes on recombinant human beta interferon in which Ser-17 replaces Cys-17 (rh[Ser17]IFN-beta) was evaluated by testing the immunoreactivity of antibodies against 159 sequential, overlapping octamer peptides. Three monoclonal antibodies (mAbs) that neutralize rh[Ser17]IFN-beta biologic activity, designated A1, A5, and A7, bound to peptides spanning only residues 39-48, whereas nonneutralizing mAb bound less specifically at multiple sites near the amino terminus. The immunoreactivity of peptides spanning residues 40-47 that contained a series of single amino acid substitutions suggested that residues 41-43 (Pro-Glu-Glu) and 46 (Gln) are important for the binding of neutralizing mAbs. The reactivity of mAbs to larger synthetic peptides containing rh[Ser17]IFN-beta sequences from residue 32 through residue 56 was evaluated. All mAbs except A7 reacted with synthetic peptides representing rh[Ser17]IFN-beta residues 32-47, 40-56, and 32-56, but only mAbs A1 and A5 bound to the core peptide composed of residues 40-47. Peptide 32-56 effectively blocked the binding of mAbs A1 and A5 to rh[Ser17]IFN-beta and markedly inhibited their neutralizing activity. Biologic activity of the peptides was undetectable. Rabbit antisera raised against peptides 32-47 and 40-56 recognized rh[Ser17]IFN-beta but did not neutralize its antiviral activity. Thus, structure-function analysis by peptide mapping has permitted the identification of a linear epitope recognized by neutralizing antibody on a biologically active cytokine. We conclude that the region spanning residues 32-56 is of major importance in the expression of the biologic activity of human IFN-beta.

Amino Acid Sequence↗

PubMed1990

Immunochemical characterization of antigenic domains on human interferon-beta: spatially distinct epitopes are associated with both antiviral and antiproliferative activities.

The use of a panel of monoclonal antibodies (mAb) raised against recombinant (serine-17) human interferon-beta (rHuIFN-beta ser) has permitted the identification of three epitopes on HuIFN-beta, designated as sites I, II and III, based solely on functional differences, i.e., the neutralization of antiviral and antiproliferative activities of natural and recombinant HuIFN-beta (Redlich, P.N. and Grossberg, S. E., J. Immunol. 1989. 143: 1887). Site I- and II-directed mAb possessed neutralizing activity whereas none was noted by mAb recognizing site III. To characterize further these epitopes by immunochemical means, we studied their (a) spatial relationship by competitive binding assays, (b) antigenic structure by Western blotting, and (c) sensitivity to chemical modification by the measurement of mAb reactivity after radioiodination. Competitive antibody binding studies revealed site II to be spatially distinct from sites I and III. Furthermore, site I- and II-directed mAb could easily recognize rHuIFN-beta ser on a Western blot, suggesting that both these epitopes are primarily sequential in structure or denaturation resistant. Chemical modification by radioiodination, which did not alter the biologic activity of rHuIFN-beta ser, had likewise little effect on mAb reactivity to site I; however, reactivity to site II was diminished and reactivity to site III was minimal following the radioiodination process. Both site I- and II-directed mAb inhibited the binding of 125I-rHuIFN-beta ser to intact Daudi cells, suggesting that inhibition of receptor binding is their mechanism of neutralization. Thus, we conclude that epitopes I and II, which are associated with both antiviral and antiproliferative activities of rHuIFN-beta, are spatially and immunochemically distinct.

Antibodies, Monoclonal↗

PubMed1990

Recent progress in interferon research: molecular mechanisms of regulation, action, and virus circumvention.

A complex system of cis regulatory elements exists by which induction of IFN gene expression is initiated in response to a variety of inducers; cis elements also appear to be involved in the down-regulation of IFN production. IFN gene activation or inhibition of expression may be tightly regulated by the specific binding of newly synthesized or modified proteins to be regulatory regions of the IFN genes. IFN itself acts as a potent modulator of multiple cellular activities. By binding to specific cell surface receptors and probable internalization via receptor-mediated endocytosis and transport into the dense chromatin, IFN treatment leads to activation of numerous genes, some of which possess known antiviral or immunoregulatory functions, whereas the function of others remains to be identified. As with the IFN genes themselves, many of the IFN-inducible genes appear to possess complex regulatory mechanisms, including domains for binding of specific trans-acting proteins. To add to this molecular complexity some viruses have successfully developed methods to circumvent, among other mechanisms, the 2',5'-A-mediated system and the P1 protein kinase system.

Animals↗

PubMed1990

Biological and clinical effects of the oral immunomodulator 3,6-bis(2-piperidinoethoxy)acridine trihydrochloride in patients with malignancy.

CL 246,738 is a synthetic heterocyclic of the acridine class that has immunomodulating, interferon (IFN)-inducing, and antitumor activity by the oral route in mice. We have completed a phase I ascending dose trial to determine the maximum tolerated oral dose and biologic modifying effects. Twenty-three patients received CL 246,738 orally at five dose levels, escalating from 5 to 50 mg/kg. The major side effects were gastrointestinal disturbances such as nausea, vomiting, and diarrhea. No hematologic, hepatic, or symptomatic dose-limiting toxicities were encountered. The mean half-life of CL 246,738 in whole blood was at least 300 h and remained relatively constant over the dose range studied. Higher doses resulted in increased whole blood levels. Biologic response modification included stimulation of the IFN-induced proteins, 2',5'-oligoadenylate synthetase and beta 2-microglobulin, at higher doses of CL 246,738, and enhanced T-cell proliferation to alloantigens at lower doses. Increases in lymphocytes bearing the Leu-7 phenotypic marker were observed and some patients had enhanced natural killer (NK) cell cytotoxicity. Enhanced NK cell cytotoxicity was demonstrated in vitro. Thus, CL 246,738 was orally relatively well tolerated and has immunomodulating properties in humans.

2',5'-Oligoadenylate Synthetase↗

PubMed1989

Analysis of antigenic domains on natural and recombinant human IFN-beta by the inhibition of biologic activities with monoclonal antibodies.

Human IFN-beta (HuIFN-beta) is a biologically potent protein with both antiviral and antiproliferative activities. To understand better its mode of action, a number of murine mAb were developed against a recombinant (serine-17) HuIFN-beta (rHuIFN-beta ser) and screened by ELISA and neutralization of antiviral activity. The panel of antibodies, composed of both IgA and IgG immunoglobulins, were specific for HuIFN-beta and did not crossreact with HuIFN-alpha or gamma. Furthermore, three functionally distinct epitopes (designated as sites I, II, and III) were identified based on mAb neutralization of antiviral and antiproliferative activities of recombinant and natural HuIFN-beta. Antibodies directed to sites I and II neutralized the antiviral and antiproliferative activities of rHuIFN-beta ser, though the antiviral neutralization potency of the mAb to site II was approximately 10-fold less than mAb to site I. Antibodies directed to site I neutralized both recombinant and natural HuIFN-beta, although the antiviral neutralization potency was approximately 10-fold higher against rHuIFN-beta ser than the native protein. The mAb directed to site II did not demonstrate any significant neutralization of the antiviral or antiproliferative activity of natural HuIFN-beta but neutralized a recombinant HuIFN-beta containing the native sequence. Antibodies recognizing site III did not neutralize the biologic activity of either recombinant or natural HuIFN-beta. Thus, three epitopes on HuIFN-beta have been identified, two of which are associated with both antiviral and antiproliferative activities.

Animals↗

PubMed1989

Interferon receptors and their role in interferon action.

Interferon (IFN) proteins interact with cells through specific cell surface receptors, some of which have been purified and cloned. The alpha-IFNs and beta-IFN bind to a common receptor (type I), whereas gamma-IFN binds to a separate receptor (type II). Both types of high-affinity receptors have been demonstrated on a variety of receptors and the ways in which IFNs may affect cellular physiology and gene expression is discussed.

Animals↗

PubMed1989

Inhibitory effects of mitochondrial metabolic inhibitors on interferon action.

The expression of the antiviral action of the human interferons (IFNs) HuIFN-alpha and HuIFN-gamma was inhibited in human cells treated with antimetabolites affecting different mitochondrial functions. We confirmed earlier observations that cycloheximide, a specific inhibitor of cytoplasmic protein synthesis, failed to inhibit IFN action completely. Chloramphenicol, which inhibits mitochondrial protein synthesis, suppressed IFN effect when present in high concentration; in human foreskin cells, the inhibitory effect on HuIFN-alpha activity of 500 micrograms/ml chloramphenicol (which caused only 20% inhibition of overall cellular protein synthesis) was greater than that observed with 25 micrograms/ml cycloheximide (which caused 98% inhibition of overall cellular protein synthesis). Cycloheximide combined with chloramphenicol further inhibited the antiviral effect of IFN than that observed by either drug alone. Similar observations were made with mouse IFN-alpha/beta in mouse L cells. Treatment with cycloheximide, in combination with oligomycin, an inhibitor of oxidative phosphorylation, also produced an inhibition of the antiviral effect. Oligomycin, dinitrophenol, and 1799, a fluorinated uncoupler of oxidative phosphorylation, all produced IFN-suppressive effects in heteroploid human cells. These data indicate that intact mitochondrial functions are required for the full expression of the antiviral actions of IFN-alpha and IFN-gamma.

Antimetabolites↗

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

Antiviral and antidifferentiative activities of interferon beta and gamma in relation to their induction of double-stranded RNA-dependent protein kinase activity in 3T3-L1 cells.

Mouse interferons beta (IFN-beta) and gamma (IFN-gamma) inhibit the differentiation of 3T3-L1 fibroblasts into adipocytes when added to cultures at the time of induction of differentiation. Differentiation, as measured by incorporation of radiolabeled leucine into lipids, was inhibited 50% by approximately 1-3 units/ml of either IFN-beta or IFN-gamma, with maximum inhibition of differentiation achieved with 100 units/ml of either IFN. The magnitude of antiviral activity induced by IFN-beta and IFN-gamma was similar in differentiated and undifferentiated 3T3-L1 cells, although the slopes of the dose-response curves were different; IFN-gamma induced an antiviral state with greater efficiency than IFN-beta in differentiated and undifferentiated 3T3-L1 cells. By contrast, IFN-beta induced the double-stranded RNA-dependent P1 protein kinase more efficiently than did IFN-gamma in both differentiated and undifferentiated cells. However, IFN-beta and IFN-gamma both induced greater phosphorylation of protein P1 in cell-free extracts prepared from differentiated adipocytes than in extracts from undifferentiated fibroblasts. Cultures treated with either beta or gamma IFN throughout 8 days of differentiation continued to produce double-stranded RNA-dependent protein kinase in a manner dependent on IFN dose. These results suggest that the antiviral and antidifferentiative activities of IFN-beta and IFN-gamma in 3T3-L1 cells involve different molecular mechanisms.

1-Methyl-3-isobutylxanthine↗

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↗

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

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↗