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Biomedical subjects

K C Zoon

Publications and source records attributed to K C Zoon.

At least 19 recordsLinked to original sources
PubMed1992

Purification and characterization of multiple components of human lymphoblastoid interferon-alpha.

Twenty-two components of human interferon-alpha (IFN-alpha) derived from Sendai virus-induced Namalwa cells were purified by sequential immunoadsorbent affinity chromatography using four monoclonal antibody affinity columns followed by ultrafiltration and reversed-phase high-performance liquid chromatography. The specific activity ranged from 0.2 to 2.6 x 10(8) IU/mg protein on Madin-Darby bovine kidney cells, 0.3 to 4.6 x 10(8) IU/mg protein on human WISH cells, and 10(4) to 7 x 10(5) units/mg protein on mouse L929 cells. The apparent molecular weights of the components ranged from 17,500 to 23,300 using nonreducing sodium dodecyl polyacrylamide-gel electrophoresis and 17,500 to 27,600 using reducing sodium dodecyl polyacrylamide-gel electrophoresis. The amino-terminal amino acid sequences were similar among the components as well as to those reported for the cloned human IFN-alpha genes (Pestka, S. (1986) Methods Enzymol. 119, 3-14). However, four components, f, i, l, and m, have amino-terminal amino acid sequences which appear to be unique when compared to those predicted from the cDNA clones. One component, pre-a, has a potential N-linked glycosylation site on the Asn of residues 2 through 4, Asn-Leu-Ser.

Amino Acid Sequence↗
PubMed1991

Regulation of interferon production by human monocytes: requirements for priming for lipopolysaccharide-induced production.

Macrophages are uniquely responsive to bacterial lipopolysaccharide (LPS) for activation of a number of host defense functions and production of bioactive mediators. One potentially important mediator produced by LPS-stimulated macrophages is interferon (IFN-alpha/beta). In contrast to murine observations, we have observed that freshly isolated human monocytes, purified by counter-current centrifugal elutriation, do not produce interferon in response to LPS. This is not due to a lack of response to LPS, as assessed by the induction of other monokines, or to an incapacity for IFN production, since IFN was inducible by poly-I,C treatment of monocytes in the absence of any other exogenous stimulus. However, human monocytes can be primed for the production of IFN in response to LPS if they are cultured in the presence of either granulocyte-macrophage colony stimulating factor (GM-CSF) or interferon-gamma (IFN-gamma). The IFN secreted is of the alpha subtype. Monocytes primed with GM-CSF or IFN-gamma also maintained LPS responses for production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1). M-CSF did not prime monocytes for LPS-induced IFN production, although it did enhance production of TNF-alpha and promoted monocyte survival. Northern analysis indicated that the induction of IFN-alpha by LPS was regulated primarily at the mRNA level. The highly regulated production of IFN-alpha by monocytes/macrophages has important implications for autocrine action of interferons in the activation and differentiation of these cells.

Animals↗
PubMed1990

Aurin tricarboxylic acid, the anti-AIDS compound, prevents the binding of interferon-alpha to its receptor.

Binding of HIV to its receptor, the CD4 molecule of lymphocytes, can be prevented by chemical agents. These agents could be considered as potential anti-AIDS drugs. We have shown that aurin tricarboxylic acid (ATA, 3 microM) specifically blocks the binding of gp120, the HIV coat protein, to the CD4 molecule. We have also found that ATA prevents the binding of interferon-alpha to its receptor in a dose-dependent manner (12-50 microM range). Membrane potential shift, associated with binding of interferon-alpha to its receptor, was also blocked by ATA in a dose-dependent fashion. Our results indicate that potential anti-AIDS drugs should be screened for such undesired side effects.

Antiviral Agents↗
PubMed1990

Effects of interferon-alpha in patients with AIDS-associated Kaposi's sarcoma are related to blood interferon levels and dose.

Definition of improved therapeutic regimens of interferon-alpha (IFN-alpha) for the treatment of Kaposi's sarcoma (KS) would be useful since currently recommended doses are sometimes associated with unacceptable toxicity. IFN concentrations were measured in serum samples from men with AIDS-associated KS who were enrolled in a trial of IFN-alpha alone (16 patients) or a trial of IFN-alpha combined with zidovudine (25 patients). Analyses were done to examine the relationship between the dose of IFN-alpha, blood level of IFN, and the patient's clinical response to treatment. There was no correlation between dose of zidovudine given and response. As expected, there was a high correlation between dose of IFN-alpha and blood level in both studies (p less than 0.001). Furthermore, we found relationships between clinical response and both dose of IFN-alpha and blood level achieved. In the two studies combined, among men with greater than 200 CD4+ cells/mm3 of blood at baseline on average daily doses of greater than or equal to 10 million international units (MIU) of IFN-alpha, 13/19 (68%) responded compared to 6/17 (35%) on less than MIU (p = 0.05). Similarly, of men with IFN blood levels greater than or equal to 100 IU/mL 12/16 (75%) responded compared to 7/20 (35%) of those with blood levels less than 100 IU/mL (p = 0.02). The dose and blood levels of IFN achieved and maintained may be important factors in determining responses of KS. Additional clinical trials of IFN-alpha treatment of KS at doses about 10 MIU/day appear warranted.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Combined zidovudine and interferon-alpha therapy in patients with Kaposi sarcoma and the acquired immunodeficiency syndrome (AIDS)

STUDY OBJECTIVE: To evaluate the toxicity and potential clinical efficacy of combined therapy with zidovudine and interferon-alpha for patients with Kaposi sarcoma and the acquired immunodeficiency syndrome (AIDS). DESIGN: Nonrandomized, open trial study. SETTING: Outpatient clinic of a government referral-based research hospital. PATIENTS: Volunteer sample of 39 patients with human immunodeficiency virus (HIV) infection and Kaposi sarcoma. INTERVENTIONS: Patients received zidovudine, 250, 100, or 50 mg orally every 4 hours; 6 weeks after interferon-alpha was begun at a dose of 5 million U/d, and the dose was increased every 2 weeks until a maximum tolerated dose was determined. Patients then received the maximum tolerated dose of the combination for a minimum of 12 weeks before formal efficacy evaluations. MEASUREMENTS AND MAIN RESULTS: In the dose-escalation phase, the ability to tolerate interferon-alpha was clearly related to the zidovudine dose. Of the 13 patients receiving 250 mg of zidovudine, only 1 patient was able to tolerate at least 10 million U/d of interferon-alpha. Of the 12 patients receiving 100 mg of zidovudine, 8 tolerated 10 million U/d, 5 tolerated 15 million U/d, and none tolerated higher doses. Of the 12 patients receiving 50 mg of zidovudine, 8 tolerated 10 million U/d, 7 tolerated 15 million U/d, and 6 tolerated 20 million U/d or more. Dose-limiting toxicities included neutropenia (57%), fatigue (16%), thrombocytopenia (14%), and hepatic dysfunction (10%). Of the 22 patients who received a stable dose of both drugs for 12 weeks, 11 patients had a complete or partial tumor response and 8 showed an anti-HIV effect. Peak serum levels of interferon-alpha (32 to 250 U/mL) and zidovudine (0.40 to 3.85 microM) were in the ranges previously shown to be synergistic against HIV. CONCLUSIONS: Combination therapy with zidovudine and interferon-alpha can be administered to patients with HIV infection and Kaposi sarcoma in doses that effect antiviral and antitumor responses; it appears to have a potential role in managing such patients.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Amino terminus is essential to the structural integrity of recombinant human interferon-gamma.

Species lacking either 8 or 10 residues at the amino terminus of recombinant human interferon-gamma (Hu-IFN-gamma) were generated by limited digestion with Staphylococcus aureus V8 protease. A crude digest, consisting predominantly of these species, were completely inactive in inducing antiviral activity and the expression of HLA-DR antigens on HL-60 cells. The NH2-terminal deletion fragments were separated from residual intact IFN-gamma and from smaller polypeptides by reverse phase high performance liquid chromatography (HPLC) at pH 2.2. Intact IFN-gamma, purified by HPLC and subsequently refolded by dilution in 0.1 M sodium phosphate buffer (pH 7.5, 0.1% bovine serum albumin) was similar to untreated IFN-gamma in terms of binding to its cell surface receptor and in inducing antiviral activity and the expression of HLA-DR molecules. Conversely, biological activity was not detected in purified fragments 8-139 and 10-139. Examination of fragments 8-139 and 10-139 by far-UV circular dichroism revealed that cleavage of 8-10 residues at the amino terminus accompanied a dramatic change in secondary structure (6% alpha-helical and 36% beta-sheet content) as compared to untreated or HPLC-purified IFN-gamma (66% alpha-helix and 0% beta-sheet content). In summary, these results indicate that the amino terminus contributes to the structural integrity of the IFN-gamma molecule.

Amino Acid Sequence↗
PubMed1989

Phorbol ester and interferon-gamma modulation of epidermal growth factor receptors on human amniotic (WISH) cells.

In this study we report that pretreatment of human amniotic (WISH) cells with interferon gamma (IFN-gamma) in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in the down-modulation of epidermal growth factor (EGF) receptors with respect to both receptor number and affinity. Scatchard analysis of EGF binding in the absence of both IFN-gamma and TPA indicated biphasic binding whereas addition of TPA resulted in the loss of the higher affinity class of sites. Pretreatment with IFN-gamma for 24 h enhanced the TPA-induced inhibition of EGF binding whereas IFN-gamma alone had no effect on binding. Protein kinase C (Ca2+/phospholipid-dependent enzyme) was examined as a possible mediator of IFN-induced EGF-receptor modulation; pretreatment of cells with IFN-gamma affected neither the binding of [3H]phorbol 12,13-dibutyrate to membrane or cytosolic fractions nor the protein kinase C activity, suggesting that IFN-gamma pretreatment did not result in translocation or activation of protein kinase C.

Cell Compartmentation↗
PubMed1989

Enhancement of monocyte-mediated tumoricidal activity by multiple interferon-alpha species.

Twenty-one interferon (IFN)-alpha species were evaluated for their ability to enhance monocyte-mediated lysis of the human melanoma cell line, A375. A wide variation in the potency of the different species in inducing monocyte tumoricidal action was observed. In addition, many IFN-alpha species were found to induce as much or more tumoricidal activity than recombinant IFN-gamma. The degree of monocyte activation induced by the various species generally correlated with their antiviral activity. Those which were better at inducing monocyte tumoricidal action also gave the highest antiviral specific activities. Studies were conducted to determine if the relative potency of the IFN-alpha species could be changed by altering certain parameters of the cytotoxicity assay. All IFN-alpha species tested required only 30 min in culture with the monocytes to induce activation. There were no changes in the relative potency of the species when cytotoxicity was measured at different times, nor when the effector:target ratio was altered. Competitive binding studies revealed that those IFN-alpha species which induced little activity in the bioassays were also generally poor in their ability to bind the IFN-alpha receptor on human monocytes, while the IFN-alpha species which induced relatively more activity in the bioassays were better able to bind to the IFN-alpha receptor. These data indicate that there are dramatic differences in activities among the IFN-alpha species which may, in part, be explained by different binding affinities. In addition, the differences observed among the IFN-alpha species demonstrate the need for further functional and structural characterization of the individual IFN-alpha species which could lead to a more effective clinical application of IFN-alpha.

Antiviral Agents↗
PubMed1989

Cell receptor assays.

There are four general assay methods used to quantify a drug/biologic in a preparation, including: (1) in vivo bioassays; (2) in vitro bioassays; (3) immunoassays; and (4) receptor assays. The cell receptor assay is used to evaluate the first step in the molecular action of the drug biologic, its interaction with a specific cellular receptor. Subsequently, the drug biologic must initiate other events, such as internalisation, signal transduction, and/or alterations of one or more cellular constituents in order to elicit its biological effect. Major factors to consider in cell receptor assay development include: (1) establishment of a reference standard preparation; (2) labelling; purifying and characterisation of the biologic drug; (3) cell receptor source; (4) methodology, e.g. separation of bound and free, and other factors affecting accuracy and reproducibility; (5) ligand specificity; and (6) correlation with bioactivity. It should be emphasised that cell receptor binding cannot be assumed to correlate with biological activity because of the requirement that subsequent steps must take place prior to achieving the final response. Chemically altered drugs biologics may bind to a specific cell receptor without eliciting a biological activity. Thus, utilisation of a cell receptor assay requires careful evaluation at both the chemical and biological levels prior to its acceptance as a measure of potency.

Animals↗
PubMed1989

Products of stimulated monocytes enhance the activity of interferon-gamma.

We have investigated the interaction of interferon-gamma (IFN-gamma) with monocytes or products of stimulated monocytes. We have shown that IFN-gamma does not stimulate IFN-alpha production in monocytes. Staphylococcus aureus (SAC) but not lipopolysaccharide (LPS) induced IFN-alpha secretion by monocytes. However, it was observed that supernatants of monocytes stimulated with IFN-gamma in combination with either LPS or SAC had higher levels of antiviral activity than supernatants of monocytes stimulated only with IFN-gamma. Moreover, the degree of enhancement of antiviral activity was dependent on the dose of either LPS or SAC used to stimulate the monocytes. Supernatants of monocytes stimulated with LPS or SAC enhanced the antiviral activity of IFN-gamma but not IFN-alpha. Thus, LPS- or SAC-stimulated monocytes produced a factor(s) that augmented the biological activity of IFN-gamma. To identify the factor within stimulated monocyte supernatants that was responsible for this enhancement, several monokines were added to IFN-gamma. Tumor necrosis factor (TNF) significantly increased the antiviral activity of IFN-gamma, although TNF by itself had no antiviral activity. Interleukin 1 (IL-1) or granulocyte-monocyte colony-stimulating factor (GM-CSF) did not enhance the activity of IFN-gamma. Our data indicate that the interaction between IFN-gamma and monocytes is bidirectional. Not only can IFN-gamma activate monocytes, but products of stimulated monocytes also enhance the biological activities of IFN-gamma.

Cell Line↗
PubMed1989

Specific binding of human alpha interferon to high-affinity cell-surface binding sites on peripheral blood mononuclear cells.

To characterize receptors for alpha interferon (IFN-alpha) on human cells, we studied the binding of radioiodinated recombinant DNA-derived human IFN-alpha to human peripheral blood mononuclear cells (PBMCs) from normal individuals and from patients with chronic type B hepatitis. At 1 degree C, binding reached equilibrium after 2 to 3 hours of incubation, and saturation of specific binding occurred at a concentration of approximately 4000 fmol/ml. Binding of labeled IFN-alpha was specific; it was inhibited by an excess of unlabeled IFN-alpha or IFN-beta but not by cholera toxin or IFN-gamma. Scatchard analysis of binding data yielded for normal PBMCs an apparent dissociation constant (Kd) of 1.54 +/- 0.49 x 10(-9) mol/L (mean +/- SD) and an apparent maximum binding capacity (Bmax) of 7.35 +/- 1.22 x 10(-11) mol/L. Corresponding values for patients with chronic type B hepatitis who had not received treatment were similar, suggesting that such patients should respond normally to endogenous interferon. Analysis of data on the binding of labeled IFN-alpha to normal PBMCs from experiments in which a high specific activity ligand or subpopulations of PBMCs had been used revealed that receptors for IFN-alpha on PBMCs are heterogenous. In patients with chronic type B hepatitis who were receiving IFN-alpha therapy, the apparent Kd was increased (3.02 +/- 0.91 x 10(-9) mol/L) without any appreciable change in the apparent Bmax or any appreciable changes in the proportions of subpopulations of PBMCs. This decreased affinity induced by IFN-alpha treatment does not necessarily reflect an effect on a single binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1989

Antibodies to interferon-alpha 2 in patients treated with interferon-alpha 2 for hairy cell leukemia.

Some patients treated with interferon (IFN) have developed antibodies (ABs) to that IFN. We examined the incidence of such ABs in hairy cell leukemia (HCL) patients treated with IFN-alpha 2a and IFN-alpha 2b. In the initial enzyme-linked immunosorbent assay (ELISA) assays, the serum samples were tested against their corresponding IFNs. Of 73 evaluable patients treated with IFN-alpha 2b, 6 patients, who tested negative prior to treatment, were positive by ELISA following treatment with IFN. Two of the samples tested positive in the neutralization assay. Regarding the samples from patients treated with IFN-alpha 2a, 2 of the 44 patients were ELISA positive following IFN treatment (while being negative prior to treatment) and 2 others became positive in the neutralization assay following IFN treatment. Five serum samples with neutralizing activity were tested for their ability to bind to each of eight natural IFN-alpha species derived from human lymphoblastoid cells induced with Sendai virus; a different cross-reactivity profile was seen for each sample. In conclusion, a low incidence of neutralizing and nonneutralizing ABs against IFN-alpha 2 was observed in patients with HCL treated with recombinant DNA-derived IFN-alpha 2a or IFN-alpha 2b. Finally, ABs against these IFNs cross-react by ELISA with several naturally occurring IFN-alpha s.

Antibodies↗
PubMed1988

Modulation of class I and class II histocompatibility antigens on human T cell lines by IFN-gamma.

IFN-gamma is an immunomodulatory agent which is known to induce or enhance the expression of class II histocompatibility Ag (Ia Ag) on many lymphoid cells and cell lines of diverse origin. However, we have observed that IFN-gamma did not induce the expression of Ia Ag on Ia- human T cell lines. Neither did IFN-gamma enhance the expression of Ia Ag on Ia+ T cells. However, IFN-gamma was able to enhance the expression of class I histocompatibility Ag (HLA-A,B,C Ag) on a number of the T cell lines tested. Experiments with 125I-labeled IFN-gamma showed a relatively small degree of specific binding to these T cell lines. More extensive studies on two of the T cell lines demonstrated 1000 and 2600 IFN-gamma binding receptor sites/cell and binding affinities of 4.0 X 10(-10) M and 7.3 X 10(-10) M. Thus, although IFN-gamma can bind to human T cell lines and enhance class I histocompatibility Ag on these cells, IFN-gamma alone does not appear to regulate expression of class II histocompatibility Ag on T cell lines.

Cell Line↗
PubMed1988

Biological characterization of human interleukin-2 mutant proteins. Structure-activity relationship studies.

Several human interleukin-2 (IL-2) mutant proteins have been produced previously by site-directed mutagenesis and found to have different capacities to induce T-cell proliferative activity. In this study, the abilities of these IL-2 mutant proteins to activate natural killer cells and to induce interferon-gamma production have been evaluated, and the binding of these proteins to IL-2 receptors analyzed. Natural killer cell activation and interferon-gamma induction assays showed that the relative activities of IL-2 mutant proteins were consistent with their relative activities in T-cell proliferation assay. Receptor-binding studies showed that the activities of most proteins correlated well with their respective affinities for high-affinity IL-2 receptors on CTLL-2 cells. Interestingly, although the mutant protein with deletion of cysteine 125 (des-Cys125) was biologically less active than the protein with substitution of alanine for cysteine 105 (Ala105), both proteins exhibited similar affinity. Des-Cys125, like IL-2 and Ala105, also caused down-regulation of high-affinity IL-2 receptors. Binding studies on MLA-144, a cell line expressing mainly intermediate-affinity IL-2 receptors (IL-2R beta), however, showed that des-Cys125 had much lower affinity than Ala105. These results suggest that binding of IL-2 and mutant proteins to the IL-2R beta component of the high-affinity receptor is essential for the induction of biological effects.

Animals↗
PubMed1987

Differential effects of interferon-alpha and interferon-gamma on interleukin 1 secretion by monocytes.

We examined the effect of interferon (IFN)-alpha and IFN-gamma on the ability of human monocytes to secrete interleukin 1 (IL 1). IFN-alpha directly induced IL 1 secretion by monocytes. IFN-gamma did not induce any IL 1. IFN-gamma-stimulated monocyte supernatants were also negative for pyrogenic activity. However, IFN-gamma greatly enhanced the amount of IL 1 secreted when monocytes were stimulated by lipopolysaccharide or Staphylococcus aureus, even at concentrations which by themselves did not induce IL 1. IFN-alpha did not enhance IL 1 secretion induced by other stimuli. IFN-gamma enhanced IL 1 secretion by priming monocytes to be more sensitive to an IL 1-inducing stimulus. However, IFN-gamma does not enhance IL 1 induced by all stimuli, because there was no enhancement of IL 1 induced by PMA. Thus, IFN-alpha and IFN-gamma have very distinct roles in the induction and enhancement of IL 1 by monocytes.

Humans↗
PubMed1987

Suppressor T cell clones from patients with acute Epstein-Barr virus-induced infectious mononucleosis.

Suppression and/or cytotoxicity are believed to play an important role in the defense against Epstein-Barr virus (EBV) infection. To analyze the role of suppressor T cells in relation to EBV, we sought to clone and study these T cells. Analysis of 152 T cell clones derived from the peripheral blood of two patients with acute EBV-induced infectious mononucleosis (IM) yielded 11 highly suppressive clones that had no cytotoxic activity for the natural killer sensitive K562 cell line, an autologous EBV-infected cell line, or an allogeneic EBV-infected B cell line. Four of six suppressor T cell clones also profoundly inhibited EBV-induced immunoglobulin production, and five of five clones delayed the outgrowth of immortalized cells. These results indicate that during acute IM, suppressor T cells capable of inhibiting B cell activation in the absence of cytotoxicity can be identified, and may play a key role in the control of EBV infection.

Antibody Formation↗