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Biomedical subjects

S E Grossberg

Publications and source records attributed to S E Grossberg.

At least 19 recordsLinked to original sources
PubMed2001

The neutralization of interferons by antibody. I. Quantitative and theoretical analyses of the neutralization reaction in different bioassay systems.

The highly specific ability of antibodies to inhibit the biologic activity of cytokines or other therapeutic proteins is widely used in research and a subject of increasing clinical importance. The need exists for a standardized approach to the reporting of neutralizing antibody potency soundly based on theoretical and practical considerations and tested by experimental data. Pursuant to the original studies of Kawade on the theoretical and functional aspects of neutralization of interferons (IFN), experimental data were obtained by different laboratories employing varied methodology to address two hypotheses concerning the nature of IFN neutralization reactions, based on a derived formula that allows expression of neutralizing power as the reduction of 10 laboratory units (LU)/ml to 1 LU/ml, the end point of most bioassays. Two hypotheses are posed: (1) antibody acts to neutralize a fixed amount of biologically active IFN molecules, or (2) antibody reduces IFN activity in a set ratio of added/residual biologically active IFN. The first, or fixed amount, hypothesis relates to the reactivity of high-affinity antibodies neutralizing equimolar amounts of antigen, whereas the second, or constant proportion, hypothesis postulates a reduction in the ratio of total added IFN to residual active IFN molecules, such as a low-affinity antibody might exhibit. Analyses of data of the neutralization of IFN-alpha and IFN-beta are presented, employing human polyclonal antibodies and murine monoclonal antibodies (mAb). The theoretical constructs of Kawade are extended in the Appendix and correlated with new experimental data in the text. The data clearly indicate that the low-antibody affinity, constant proportion hypothesis, rather than the high-antibody affinity, fixed amount hypothesis, is applicable, if the bioassay is sensitive to IFN. The findings presented here and in the following paper (pp. 743-755, this issue) taken together provide the basis for a standardized method of expression of neutralizing potency and substantiate the earlier operational 10/1 LU/ml approach recommended by the World Health Organization. The accompanying paper relates neutralization results to the sensitivity of the bioassay to IFN and describes the rationale for a recommended unit of antibody neutralization.

Adenocarcinoma, Bronchiolo-Alveolar↗
PubMed2001

The neutralization of interferons by antibody. II. Neutralizing antibody unitage and its relationship to bioassay sensitivity: the tenfold reduction unit.

The importance of establishing a common method of reporting neutralizing antibody levels is emphasized by the fact that patients injected repeatedly with a human interferon (HuIFN) may develop such antibodies that can abrogate the beneficial effects of the treatment. The earlier experimental and theoretical constructs of Kawade led to certain recommendations by the World Health Organization (WHO) concerning the methodology of neutralization tests and how to report the resultant data. A WHO international collaborative study on two human sera with antibodies against HuIFN-alpha and HuIFN-beta provided the opportunity not only to test the theoretical concepts concerning the neutralization reaction with data obtained in different bioassay systems in different laboratories but also to obtain enough data points for statistical evaluation with bioassays having a great range of sensitivity to IFN. The analyses substantiate and extend the original conclusions of Kawade that the neutralization follows the reaction mode of low-affinity antibody, in accord with the constant proportion hypothesis by which antibody reduces IFN activity in a set ratio of added/residual biologically active IFN, a consequence of the low molar concentration of free IFN at the neutralization end point. The present results support the recommendation that the preferred way to state the index of neutralization of antibodies is a titer (t), calculated by the formula t = f(n - 1)/9, where f is the reciprocal of the antibody dilution achieving the end point, and n is the IFN concentration measured in that day's titration. The tenfold reduction unit (TRU) of neutralization is proposed for use in expressing the quantity, or unitage, of IFN neutralizing antibody. The utility of its application is explained. The use of the index of neutralization described and the proposed derivative term of antibody unitage, TRU, should help make the results from different laboratories employing different bioassay systems more readily comparable and interpretable, provided the bioassays are sufficiently sensitive to IFN.

Antibodies↗
PubMed2001

Mapping of IFN-beta epitopes important for receptor binding and biologic activation: comparison of results achieved using antibody-based methods and alanine substitution mutagenesis.

The epitopes important for receptor binding and activation of human interferon-beta1a (IFN-beta1a) were mapped with monoclonal antibodies (mAb), grouped on the basis of their specificity and ability to neutralize biologic activity, and alanine scanning mutagenesis (ASM). The binding properties of nine mAb were defined, using ASM-IFN-beta mutants having alanine substituted at targeted, surface-exposed residues. The results were correlated with the mAb neutralizing potency. Of six mAb that bound either at or adjacent to the IFNAR-2 receptor chain binding site defined by the ASM epitopes, only three had measurable neutralizing activity. Two of these inhibited IFN-beta/IFNAR-2 complex formation, suggesting that steric hindrance of receptor binding constitutes their mechanism of neutralization. However, two mAb that bound to sites remote from the IFNAR-2 binding site on IFN-beta also inhibited IFN-beta/IFNAR-2 complex formation and demonstrated potent neutralizing activity. Thus, neutralizing mAb may employ mechanisms other than steric blockade to inhibit directly the binding of receptor by cytokine, limiting their usefulness as tools to define precise receptor-ligand interaction sites.

Alanine↗
PubMed2000

Characterization of genetically engineered mengoviruses in mice.

We have shown that genetically engineered mengoviruses containing artificially shortened 5' noncoding poly(C) tracts (e.g., C0 or C13UC10) are dramatically attenuated in adult Swiss/ICR mice when compared to wild-type virus or to a genetically engineered virus containing a wild-type length poly(C) tract (C44UC10). To explore further the relationship between poly(C) tracts and virulence, we have conducted more extensive characterizations of several engineered viruses in the murine model. Both short and long poly(C) tract viruses were highly virulent in newborn mice, underscoring the importance of age in poly(C)-mediated attenuation. Virus vMC24, with a tract sequence of C13UC10, was as attenuated in 4-week-old BALB/c, C.C3-H2k/LiMcdJ, and DBA/2 mice as in Swiss/ICR mice. But it was more pathogenic for C57BL/6 mice, and highly virulent for C3H/Hej and C3H/Hen mice, demonstrating the importance of murine genotype. As expected from its virulence in all mouse strains, vMwt, with a poly(C) of C44UC10, induced higher levels of viremia than vMC24. The vMwt also induced higher levels of circulating interferon and had reduced pathogenicity in chemically immunosuppressed Swiss/ICR mice. Similar immunosuppression did not increase the virulence of vMC24. Collectively, the data suggest that endogenous immune components and the immune competence of the host play significant roles in determining the susceptibility of mice to mengovirus infection.

Animals↗
PubMed1999

Quantitative liquid-phase chemiluminescence ELISA: detection of subtle epitope differences in HuIFN-beta.

We have developed a new liquid-phase, chemiluminescence-enhanced, inhibition ELISA (LP-CEI-ELISA) to explore the binding sites recognized by two neutralizing monoclonal antibodies (mAb) against recombinant human IFN-(beta)ser (rHuIFN-(beta)ser). In this assay, the initial antigen-antibody reaction occurs in solution under more physiologic conditions than in a standard solid-phase ELISA. Subsequently, the reaction mixture is applied to a membrane that is exposed to a second, peroxidase-labeled mAb, chemiluminescent reagents are added, and the membrane is photographically recorded. Competitive inhibition of binding of a second, labeled mAb by the first mAb decreases the signal detected. Two well-characterized mAb A1 and A7, have been shown to recognize distinct epitopes on rHuIFN-(beta)ser and to neutralize its antiviral and antiproliferative activity (Proc. Natl. Acad. Sci. USA 88, 4040-4044, 1991). In conventional solid-phase ELISA, mAb A1 does not inhibit the binding of A7 to rHuIFN-(beta)ser, but we observed partial inhibition in the new liquid-phase assay. In contrast, A7 did not inhibit the binding of A1, consistent with the solid-phase ELISA results. This observation suggests that in the LP-CEI-ELISA, A1 and A7 may recognize epitopes differently than in solid-phase assays. Thus, the LP-CEI-ELISA, which is simple, sensitive, and quantifiable, appears also to be able to detect subtle, conformational differences of epitopes not evident in a standard solid-phase ELISA.

Binding, Competitive↗
PubMed1998

Constitutive production of a murine retrovirus in the human B-lymphoblastoid cell line, DG-75.

During the screening of human lymphoblastoid cells as suitable hosts for retrovirus transmission studies, the Epstein-Barr virus (EBV)-negative, B-lymphoblastoid cell line DG-75 was found to be chronically infected with a heretofore unrecognized retrovirus. Two DG-75 sublines obtained from different sources (designated UW and KAR) were found to produce constitutively particles identified as retroviral by electron microscopy and reverse transcriptase activity. The ultrastructure, morphogenesis, and density in sucrose of the particles were typical of C-type retroviruses. Immunoblot analysis of the DG-75(UW) retrovirus proteins showed antigenic similarity to Moloney murine leukemia virus. A third DG-75 subline in early passage, designated HAD, was free of retrovirus. The DG-75(UW) retrovirus was infectious and produced progeny virions that could be passaged to uninfected cells. We have thus demonstrated that DG-75 cells, which have been used extensively in studies of the biological effects of EBV-encoded genes and their promoters, may be chronically infected with a murine retrovirus and that an early passage subline is retrovirus free and available for such studies.

Animals↗
PubMed1998

A novel bioassay for the determination of neutralizing antibodies to IFN-beta1b.

We have adapted the new MxA gene-induction bioassay to measure neutralizing antibodies to interferon-beta1b (IFN-beta1b, the active ingredient in Betaseron) in sera from patients treated with Betaseron. This antibody assay has been validated to quantify neutralizing titers of 1:20 and above, with a precision of +/- 0.20 in log10. We have used this MxA gene-induction antibody assay to reinvestigate serum samples from multiple sclerosis (MS) patients treated with Betaseron. The titers measured were closely comparable to those obtained in antiviral assays. Data obtained by both methods show that neutralizing antibodies may appear and subsequently disappear over time in the sera of some patients treated with Betaseron. Sera from some patients contain binding antibodies to IFN-beta1b. It was shown that binding antibody titers do not correlate quantitatively or qualitatively with neutralizing antibody titers, and indeed, a number of patients develop high levels of binding antibodies but never form measurable levels of neutralizing antibodies.

Antigen-Antibody Reactions↗
PubMed1998

Ruthenium red preserves glycoprotein peplomers of C-type retroviruses for transmission electron microscopy.

Peplomers, the glycoprotein projections of the outer viral envelope, are distinctive for many viruses. Peplomers of retroviral C-type particles are fragile and are not preserved in standard preparations for transmission electron microscopy of thin sections, whereas the peplomers of B- and D- type retroviruses are usually preserved. Ruthenium red, extensively used in transmission electron microscopy to enhance the preservation of glycosylated proteins, was used in the preparation of three retrovirus-producing lymphoblastoid cell lines: murine SC-1 cells producing the C-type murine leukemia retrovirus LP-BM5 that causes immunodeficiency, human DG-75 cells producing a murine leukemia retrovirus, and human C5/MJ cells producing human T-cell lymphotropic virus type I (HTLV-I). Fixation of cells was carried out with ruthenium red present in the glutaraldehyde, osmium tetroxide, and the ethanol dehydration through the 70% ethanol step. The detailed structure of peplomers of these three different viruses was well preserved.

Animals↗
PubMed1998

The effects of interferon-alpha on the production and action of other cytokines.

Various interferon-alpha (IFN-alpha) preparations, either as individual subtypes or natural mixtures, induce or inhibit expression of several other cytokines, as well as cytokine receptors and chemokines. The cytokines and receptors reportedly affected by IFN-alpha include interleukin-1 (IL-1), IL-2, IL-6, IL-8, IL-1 receptor, IL-1 receptor antagonist, tumor necrosis factor, tumor necrosis factor receptor, and IFN-gamma, all of which may amplify the effects of IFN-alpha treatment. The mechanism by which IFN-alpha induces expression of these cytokines is not clear. Some of the therapeutic and toxic effects associated with IFN-alpha therapy may be caused by the induction or inhibition of other cytokines and their respective cellular effects. Side effects including fever, anorexia, and fatigue can be caused by one or more of the cytokines induced by IFN-alpha. The response of different cell types, normal or malignant, to cytokines can vary. Such variation in cell type-specific responses may contribute to the diverse array of physiologic effects associated with IFN-alpha therapy. Further research is required to systematically uncover how other cytokines, receptors, or cellular factors contribute to the therapeutic and toxic effects of IFN-alpha.

Cytokines↗
PubMed1997

The expression of potency of neutralizing antibodies for interferons and other cytokines.

The occurrence of antibody formation in patients administered biologically active human proteins as biotherapy for different diseases emphasizes the importance of establishing a common method of reporting neutralizing antibody levels for such cytokines. For quantitative neutralization bioassays, the preferred expression of the neutralizing potency of an antiserum is a titer, that is, the dilution of serum that reduces 10 Laboratory Units (LU)/ml of the cytokine to 1 Laboratory Unit/ml, the endpoint of most bioassays. This 10-to-1 LU/ml expression, which has been recommended by the World Health Organization for recording the results of interferon neutralization by the constant interferon method (with varying dilutions of serum) can also be used with the constant antibody method (with varying concentrations of interferon). For various reasons, interferon doses in International Units (IU)/ml should not be used for the neutralization test. Should the interferon concentration vary, intentionally or otherwise, from the intended dose of 10 LU/ml, a simple calculation allows expression of the neutralizing potency as the recommended reduction of 10-to-1 LU/ml as follows: the titer to be reported is the reciprocal of the antibody dilution (achieving the endpoint), multiplied by the interferon concentration (measured in that day's titration) minus one, divided by 9. This index of neutralization is the preferred method to represent the neutralizing potency of polyclonal and monoclonal antibodies and should make the results from different laboratories more readily interpretable and enable comparison.

Antibodies↗
PubMed1997

Molecular and biologic characterization of recombinant interferon-alpha2b.

Recombinant IFN-alpha2b (Intron A, Schering-Plough Corp, Kenilworth, NJ), derived from Escherichia coli, is currently approved worldwide for the therapy of various cancers and chronic hepatitis B and C. We describe here the purity and identity tests for both physicochemical properties and bioactivity that have been developed for the characterization of highly purified IFN-alpha2b with a specific activity of 2.5 x 10(8) IU/mg protein. The data indicate that a product of high purity can be consistently produced without DNA contamination. The antiviral bioassay chosen to measure potency is based on the ability of IFN-alpha2b to protect human foreskin fibroblast FS-71 cells from the cytopathic effects of encephalomyocarditis virus. Various immunoassays are described that have been used to quantitate the presence of binding and neutralizing antibodies in patients treated with IFN-alpha2b. Overall, the available data indicate a very low incidence of neutralizing antibody formation. We also summarize the current state of knowledge with respect to IFN reference standards for the biologic assay as well as recommendations for the calculation of antibody neutralization titers.

Antiviral Agents↗
PubMed1996

Synergy, activity and tolerability of zidovudine and interferon-alpha in patients with symptomatic HIV-1 infection: AIDS Clincal Trial Group 068.

Thirty-four subjects with symptomatic HIV-1 infection, p24 antigenaemia, and CD4 cell counts > 200/mm3 were randomly assigned to receive treatment with either zidovudine (ZDV) orally, interferon-alpha (IFN-alpha) subcutaneously, or both at respective low (200 mg ZDV/ 2 million international units IFN-alpha (MIU)), middle (400 mg/4 MIU) or high (600 mg/6 MIU) daily dose levels for 12 weeks. Thereafter, all patients received combination therapy at the initially assigned dose level to a total of 96 weeks. This design permitted analysis by the combination index (CI) method, which demonstrated antiretroviral synergy between ZDV and IFN-alpha with respect to p24 antigen suppression. Over the first 12 weeks, combination therapy was acceptably tolerated, more so than IFN-alpha monotherapy, and it was significantly more active in suppressing antigenaemia than either of the monotherapies. Similarly, the high-dose combination was the most active dose level over weeks 12 to 96. Combination ZDV/IFN-alpha at the optimal dose level defined by this trial merits further study. In addition, the CI design strategy employed here may be useful for the investigation of new antiretroviral combinations.

Administration, Cutaneous↗
PubMed1995

Transmissible retrovirus in Epstein-Barr virus-producer B95-8 cells.

Epstein-Barr virus (EBV) released from the B95-8 marmoset cell line has served as a prototype for biologic and biochemical studies of EBV. Here we identify and characterize a retrovirus carried by many cultures of B95-8 cells. The experiments were stimulated by the isolation of a cDNA clone from B95-8 cells in which sequences from the EBV large internal repeat were linked to gag sequences similar to those of squirrel monkey retrovirus, human isolate, SMRV-H. However, among 413 amino acids predicted from the nucleotide sequence of the gag region of the B95-8 SMRV isolate there were 48 amino acid changes that distinguished this virus from SMRV-H originally isolated from a human lymphoid cell line by Oda et al. (1988, Virology 167, 468-476). Nucleic acid and antibody probes were developed for the B95-8 isolate of SMRV. Using such probes, we found that SMRV-B95-8 was readily transmissible, independent of EBV, as an infectious virus to human B and T cell lines. SMRV-B95-8 was highly fusogenic in the presence or absence of EBV. The ultrastructural appearance of the B95-8 retrovirus was characteristic of a type D retrovirus. Cells dually infected with EBV and SMRV-B95-8 did not demonstrate increased levels of lytic EB viral replication. SMRV-B95-8 did not by itself cause lymphocyte immortalization or enhance immortalization by EBV. Thus SMRV-B95-8 does not contribute to the major biologic properties of the B95-8 strain of EBV.

Amino Acid Sequence↗
PubMed1994

Clostridium difficile toxin A therapy for HCT 116 human colon cancer in nude mice.

Clostridium difficile toxin A was evaluated for an antitumor effect in vivo on HCT 116 human colon carcinoma cells growing subcutaneously in nude mice. A mean reduction in tumor volume of at least 65%, by measurement in three dimensions, was observed in mice who received two 9- to 13-day courses of daily intraperitoneal injections of toxin A as compared to mice receiving diluent alone. Reversible adverse effects of toxin A were noted in some animals, consisting primarily of liver toxicity and skin rash. HCT 116 cells in toxin A-treated mice grew as flattened tumors with ulcerated centers compared to rounded tumors without ulceration in controls. Histologic examination of tumors from representative mice revealed that two thirds of the tumor in a treated mouse was necrotic compared to only one third in a control, suggesting greater antitumor efficacy of toxin A than estimated by tumor measurements alone.

Animals↗
PubMed1993

Phase I trial of an oral immunomodulator and interferon inducer in cancer patients.

Imiquimod [1-(2-methylpropyl)-1H-imidazo[4,5c]quinolin-4-amine] is a compound of low molecular weight that, when administered p.o., induces interferon-alpha in several animal species and inhibits tumor growth in mice. To determine maximum tolerated dose, toxicity, and biological response in humans, a phase I clinical trial was conducted with 14 eligible cancer patients who received 100-500 mg imiquimod p.o. either once or twice weekly. Imiquimod induced interferon-alpha in serum in 10 of 19 doses of 200-300 mg. Interferon serum levels peaked 8-24 h after treatment and reached a maximum of 23,000 IU/ml in one patient. Significant mean increases (P < 0.01) in serum beta 2-microglobulin (1.5-fold), serum neopterin (3.5-fold), and 2-5A synthetase activity in peripheral blood mononuclear cells (7.9-fold) indicated that 200-300 mg imiquimod had biological and immunological activity in all evaluable patients. Increases in serum interferon, beta 2-microglobulin, and neopterin correlated significantly with dose (P < 0.001). No patient developed measurable antibody to interferon-alpha. Dose-limiting side effects included fatigue, malaise, fever, headache, and lymphocytopenia; no hepatic or renal toxicity or other hematological changes exceeded the normal range. Patients tolerated weekly doses of up to 500 mg, with the longest treatment lasting 4 weeks at 200 mg weekly. Twice-weekly doses up to to 300 mg were tolerated, with the longest twice-weekly treatments being 200 mg for 9 weeks and 100 mg for 25 weeks. No clinical responses were observed. Imiquimod, as an oral inducer of interferon, may have therapeutic usefulness in human cancers, viral infections, and other diseases. However, before initiation of phase II trials, additional work will be required to establish a tolerated dose and schedule for continued administration.

2',5'-Oligoadenylate Synthetase↗