Interferon-alpha resistance in renal carcinoma cells is associated with defective induction of signal transducer and activator of transcription 1 which can be restored by a supernatant of phorbol 12-myristate 13-acetate stimulated peripheral blood mononuclear cells.

Therapy of selected human malignancies with interferon-alpha is widely accepted but often complicated by the emergence of interferon-alpha resistance. Interferon is a pleiotropic cytokine with antiproliferative, antitumour, antiviral and immunmodulatory effect; it signals through the Jak-STAT signal transduction pathway where signal transducer and activator of transcription 1 plays an important role. Here we report both, a lack of signal transducer and activator of transcription induction in interferon-alpha resistant renal cell carcinoma cells and signal transducer and activator of transcription 1 reinduction of phorbol 12-myristate 13-acetate-stimulated peripheral blood mononuclear cells supernatant. Preliminary experiments on the identification of the molecules that reinducing signal transducers and activators of transcription 1 indicate that interferon-gamma may be the responsible candidate cytokine, but several others may be involved as well. This work provides the basis for therapeutic strategies directed at the molecular modulation of interferon-alpha resistance in human neoplasms.

Intracellular staining of Mx proteins in cells from peripheral blood, bone marrow and skin.

AIM/BACKGROUND: The Mx proteins are known to be specifically and dose dependently induced in mononuclear cells (MNC) by type I interferons (IFN). The aim of this study was to establish a staining method for the human intracellular Mx proteins, MxA and MxB, in leucocytes and bone marrow and skin cells. METHODS: Several monoclonal antibodies directed against the MxA and MxB proteins were generated. These antibodies were used to stain Mx proteins in both frozen and paraffin wax sections using the standard alkaline phosphatase anti-alkaline phosphatase (APAAP) method. RESULTS: Granulocytes, monocytes and lymphocytes extracted from freshly collected blood from 21 healthy subjects did not stain. After incubating MNC from these subjects with IFN alpha 2b for 48 hours, Mx proteins were detected in monocytes and lymphocytes. Within two days of starting treatment with subcutaneous IFN alpha 2b, granulocytes, monocytes and lymphocytes of 16 patients with cancer stained strongly for Mx proteins. The intensity of staining was correlated with the Mx content of whole blood measured using a specific ELISA. Prior to IFN treatment, cells from bone marrow and skin tissue specimens were negative for Mx proteins with the exception of endothelial cells. During treatment with IFN alpha 2b, nearly all cells from bone marrow and skin stained intensely. CONCLUSIONS: These new monoclonal antibodies facilitate the detection of Mx positive cells in peripheral blood and in frozen or paraffin wax specimens. The advantage of this staining method is that individual cells which have responded to viruses or biologically active IFN alpha, beta or omega can be identified.

Strong transient expression of the type I interferon-induced MxA protein in hepatitis A but not in acute hepatitis B and C.

The human MxA protein is a new specific marker for type I interferon activity both in vitro and in vivo. In the study presented here, this interferon-induced marker, as well as the 2',5'-oligoadenylate synthetases, was measured in circulating mononuclear cells from 21 patients with acute hepatitis A, 20 patients with acute hepatitis B and 14 patients with acute hepatitis C for determination of the activation of the interferon system in these viral diseases. In acute hepatitis A a strong expression (10 of 10 patients) of the MxA protein and the 2',5'-oligoadenylate synthetase activity in peripheral-blood mononuclear cells was observed during the first 2 wk after onset of clinical symptoms. In this period the MxA protein concentrations reached levels similar to those measured in patients treated with up to 5 x 10(6) IU interferon-alpha three times a week. Beyond wk 3, in eight of eight patients with hepatitis A no increased MxA protein levels were found. In contrast, peripheral-blood mononuclear cells from patients with acute hepatitis B contained either no measurable MxA protein or only slightly higher levels of the MxA protein, as did those of most patients (12 of 14) with acute hepatitis C. The MxA protein levels of both hepatitis B and C patients were significantly lower (p < 0.05) than those found in hepatitis A patients. Furthermore, sera from 6 of 10 patients with hepatitis A, but none of 10 patients with acute hepatitis B and C, contained measurable MxA protein. This serum MxA protein may originate from interferon-exposed and subsequently damaged liver cells.(ABSTRACT TRUNCATED AT 250 WORDS)

A whole blood immunoassay for the interferon-inducible human Mx protein.

Mx protein, an intracellular protein induced by type I interferons (IFNs), is useful as a marker for the IFN-induced state. It is detectable, for example, in leukocytes of patients undergoing IFN-alpha treatment as well as in patients suffering from viral or autoimmune diseases. For immunizations and standardizations, recombinant human MxA protein was expressed in Escherichia coli and purified from inclusion bodies by several steps of chromatography. Two monoclonal antibodies against nonoverlapping epitopes and specific for human Mx protein were selected to establish a simple two-site immunometric enzyme assay. In addition, a monoclonal antibody also reacting with Mx proteins of other species was identified. Prior to assay, whole blood samples were lysed with a nonionic detergent. The sample was incubated on wells coated with a first monoclonal antibody (1304.5.32) together with a second biotinylated monoclonal (1302.34.16), which, after washing, was revealed by an avidin-alkaline phosphatase system. Limit of detection was 5 ng/ml. In two-thirds of normal blood samples (n = 87), Mx protein levels were below 5 ng/ml; 25 samples (29%) had Mx levels between 5 and 50 ng/ml; and 4 samples (5%) were above 50 ng/ml. No Mx was found in plasma, and the mononuclear cell fraction accounted for the bulk of Mx in blood. In vitro, as determined by flow cytometry, monocytes and lymphocytes accumulated Mx protein for 24 h with similar kinetics and remained at plateau levels for more than 70 h. Monocytes contained around eight times more Mx than lymphocytes. The immunoassay was also suitable for detecting Mx after IFN induction in heparinized blood.

Effective natural interferon-alpha therapy in recombinant interferon-alpha-resistant patients with hairy cell leukemia.

To explore the relationship between anti-interferon-alpha (anti-IFN-alpha) antibodies and loss of clinical responsiveness to IFN-alpha treatment, we examined sera from 59 patients with hairy cell leukemia who responded to therapy with recombinant IFN-alpha-2a (rIFN-alpha-2a). During the first 2 years of therapy, 10 patients developed rIFN-alpha-2a-neutralizing and 15 rIFN-alpha-2a-binding antibodies. Nine of the 59 initially responding patients became resistant to rIFN-alpha-2a and suffered a relapse of the disease at 7 to 24 months of treatment. All nine relapsing patients tested positive for both neutralizing and binding antibodies with titers above 400 INU/mL, while none of the antibody-negative patients relapsed. Six patients with detectable binding antibody titers below 400 INU/mL continued to respond to treatment. By measuring the IFN kinetics and the levels of the IFN-induced Mx-homologous protein in mononuclear cells after a single injection each of rIFN-alpha-2a and nIFN-alpha the IFN antibodies of eight of the nine resistant rIFN-alpha patients were found to be highly specific for rIFN-alpha-2a. Therefore, these eight patients were switched to natural IFN-alpha (nIFN-alpha) therapy at doses of 3 million IU, three times a week. All eight patients responded to treatment with nIFN-alpha, achieving durable objective responses similar to those obtained previously with rIFN-alpha-2a. These data clearly demonstrate that rIFN-alpha antibody-positive patients can effectively be treated with nIFN-alpha.

Treatment of anti-recombinant interferon-alpha 2 antibody positive CML patients with natural interferon-alpha.

Of 38 patients with a Philadelphia-chromosome positive chronic myeloid leukaemia treated with recombinant interferon alpha (rIFN-alpha) 2a or 2b and monitored for emergence of IFN-antibodies in their sera 11 patients developed rIFN-alpha 2 binding and 10 rIFN-alpha 2 neutralizing antibodies. rIFN-alpha neutralizing antibody positive patients experienced significantly (P less than 0.025) more clinical relapses (6/10) than IFN-antibody negative patients (6/28) during continuous IFN-therapy. Furthermore, IFN-antibody-positive patients with titre above 400 INU/ml were more likely to relapse under rIFN-alpha-therapy than IFN-antibody-negative patients with titre below 400 INU/ml (P less than 0.05). Seven rIFN-antibody-positive patients experiencing a clinical relapse or a primary non-responsiveness were treated with two- to three-fold increased doses of rIFN-alpha 2. Only one of these seven patients developed a partial haematological remission upon intensification of the rIFN-alpha 2 therapy. Consecutively, the six patients failing high dose rIFN-alpha treatment were switched to a natural IFN-alpha preparation (3 x 9 x 10(6) I.U. weekly s.c.). Under such treatment two of the six patients achieved a long-lasting complete, one a partial haematological remission. In high-titred IFN-antibody positive patients significantly altered serum-IFN-titre and minimal IFN-inducible Mx-homologue concentrations were measured; in contrast, nIFN-alpha induced normal IFN-titre and dose-equivalent Mx-homologue amounts in these patients. The data prove that high-titred rIFN-alpha neutralizing antibodies abrogate the biological action of rIFN-alpha, but not of nIFN-alpha in vivo and explains why nIFN-alpha can be effective in the anti rIFN-alpha 2 positive patients.

Correlation of the antiproliferative effect and the Mx-homologous protein induction by IFN in patients with malignant melanoma.

The human interferon-induced intracellular protein homologous to the murine Mx-protein has recently been identified by means of a specific monoclonal antibody. Three of six melanoma cell lines elicited this intracellular human Mx-homolog upon incubation with IFN-alpha or IFN-gamma, yet all six melanoma cell lines tested were susceptible to the antiproliferative effect of IFN-alpha and IFN-gamma. Compared per antiviral unit, IFN-gamma had weaker Mx-inducing but stronger antiproliferative activity than IFN-alpha. These data suggest that the IFN-induced Mx-homologous protein is not involved in the antiproliferative action of IFN on malignant melanoma cell lines. Furthermore, 51 patients with advanced malignant melanoma were treated thrice weekly with 10 x 10(6) IU rIFN-alpha-2b and 6 x 10(6) nIFN-alpha, respectively. Nine of the 51 patients experienced systemic objective tumor responses (3 complete response, 6 partial response), but had Mx concentrations in their mononuclear cells equal to the Mx levels of non-responders during IFN-alpha therapy. Therefore, the level of Mx-homologous protein induced during IFN therapy is not a predictive marker for an antitumor response in malignant melanoma.

The human intracellular Mx-homologous protein is specifically induced by type I interferons.

The murine Mx-1 protein is one of the best biochemically and functionally characterized interferon (IFN)-induced proteins that is necessary, and sufficient, for providing resistance to murine cells against viral influenza infection. Recently an intracellular human protein homologous to the murine Mx-1 protein has been identified by means of a specific monoclonal antibody. The restricted induction of this intracellular protein in human mononuclear cells (MNC) by various cytokines was investigated. MNC from 26 of 28 healthy people and 35 of 36 cancer patients before IFN-alpha therapy had no detectable Mx-homologous protein. Incubation of human MNC with IFN-alpha and IFN-beta for 24 h at different concentrations led to a dose-dependent induction of the Mx-homologous protein. All IFN-alpha or IFN-beta preparations tested were equally effective in eliciting this intracellular protein. IFN-gamma induced only 1% of the Mx amount elicited by type-1 IFN compared on a weight basis. Neither interleukin (IL) 1 nor IL3, IL4, IL5, IL6, tumor necrosis factor-alpha/beta, granulocyte colony-stimulating factor (CSF) or granulocyte macrophage-CSF at any of the concentrations tested were capable of eliciting any detectable amount of the Mx homolog, while IL2 was a poor Mx-homologous protein inducer. In the presence of high-titered IFN-alpha antisera both IL2 and IFN-gamma were unable to stimulate this protein, proving that IFN-gamma and IL2 indirectly induce the Mx homolog via IFN-alpha. Therefore, the human Mx-homologous protein is a strictly by type I IFN-regulated protein in human peripheral blood lymphocytes.

Emergence and decay of the human Mx homolog in cancer patients during and after interferon-alpha therapy.

The human Mx, an interferon (IFN)-alpha- and IFN-beta-induced 76-kd protein, is a homolog (Mx-homolog) to the murine Mx protein, which is necessary and sufficient to provide adequate resistance against influenza virus in murine cells and in mice. Leukocytes from 36 patients with tumors (chronic myelogenic leukemia, hairy cell leukemia, and malignant melanoma) were monitored for their Mx-homolog content before, during, and after rIFN-alpha-2b therapy. Before therapy, only one patient was slightly positive for Mx-homolog. All 36 patients showed a significant increase of Mx-homolog in their mononuclear cells within the first day of IFN therapy. During therapy, the Mx-homolog levels remained elevated. After cessation of treatment, the Mx-homolog content in the mononuclear cells decreased slowly; within 2 weeks, it was about 20-30% of its value during therapy. However, even after 3 weeks, the Mx-homolog was still detectable. The maximally induced Mx-homolog concentration showed a significant correlation to the IFN dose given in vivo. These data indicate that the Mx-homolog is an excellent marker for monitoring the activity of IFN during IFN therapy. In addition, the in vivo endogenous activation of the IFN system might be detectable by the determination of the Mx-homolog despite the lack of circulating IFN.

The interferon-induced Mx-homologous protein in people with symptomatic HIV-1 infection.

Twenty-six people with symptomatic HIV-1 infection were screened for the presence of interferon (IFN) alpha and IFN alpha antibodies in their sera and the presence of the IFN-induced intracellular Mx-homologous protein in their peripheral blood leukocytes. Eleven people had measurable IFN alpha levels ranging from 1 to 40 IU/ml. None of the sera tested was positive for IFN alpha binding or IFN alpha neutralizing antibodies in the assays employed. Twenty-five of the 26 people had significant levels of the Mx-homologous protein in their peripheral mononuclear cells. The Mx concentrations varied from 0.3 to 6 U/ml in the people studied. IFN alpha-positive people had significantly higher levels of the Mx homolog than IFN alpha-negative people (P less than 0.03). Furthermore, the Mx homolog content in Walter-Reed class 2 people was significantly lower than in Walter-Reed class 5/6 people (P less than 0.01). Our results suggest that the IFN system is activated in more than 90% of the people with lymphadenopathy-associated syndrome, AIDS-related complex and AIDS. Since acid-labile IFN alpha can induce the Mx homolog in vitro endogenously produced IFN alpha seems likely to be responsible for the high Mx homolog levels detected.

Humoral response to recombinant interferon-alpha 2b in patients receiving recombinant interferon-alpha 2b therapy.

Twenty-seven patients in the chronic phase of a Philadelphia chromosome-positive chronic myelogenic leukemia (CML) underwent a monotherapy with recombinant interferon-alpha 2b (rIFN-alpha 2b), receiving a mean dose of 3 x 5 Mio. IU per week. Eight of the 27 patients developed both rIFN-alpha 2b-binding and -neutralizing antibodies during therapy. Sera of these patients abrogated both antiviral and antiproliferative activities of rIFN-alpha 2b, but not of nIFN-beta or rIFN gamma. Only one serum neutralized nIFN-alpha. The IFN binding and neutralizing titers rose during therapy, while a decrease occurred over months after cessation of therapy. The IgG fraction obtained by DEAE chromatography contained more than 90% of the IFN-neutralizing capacity of the purified sera. This result strongly suggested that IgG antibodies are responsible for the IFN-alpha-neutralizing activity.

MX homologous protein in mononuclear cells from patients with systemic lupus erythematosus.

The 76-kd human interferon (IFN)-induced MX protein is the homolog to the murine protein, which is necessary and sufficient to provide adequate resistance to influenza virus infection in murine cells and in mice. Fifty-one patients with systemic lupus erythematosus (SLE) were screened for the presence of the MX homolog in mononuclear cells and for IFN and anti-IFN antibodies in serum. In 47 of 51 patients, significant levels of the MX homolog were found, while only 15 of 51 patients had measurable alpha-IFN in their serum. The IFN activity found in these sera was characterized as a partially acid-labile alpha-IFN, by means of acid-stability cross-reactivity on heterologous cells, trypsin sensitivity, and neutralization by homologous or heterologous antisera. Four of the patients had no detectable MX homolog in their leukocytes; 3 of these 4 possessed an anti-alpha-IFN antibody that was able to neutralize both a natural alpha-IFN preparation and the acid-labile IFN in SLE sera. Also, acid-labile alpha-IFN-containing SLE sera induced the MX homolog in vitro in mononuclear cells from healthy donors. These observations suggest that endogenously produced alpha-IFN is responsible for the observed induction of the MX homolog in SLE and that the IFN system is activated in more than 90% of SLE patients.

Presence of interferon and anti-interferon in patients with systemic lupus erythematosus.

Sera from 61 patients with systemic lupus erythematosus (SLE) were serially screened over a period of at least 2 years for IFN and anti-IFN antibodies. IFN concentrations were measured both with a cytopathic effect assay and a more sensitive radioimmunoassay. Of the patients 15% (9/61) had IFN in their serum at one or more occasions as measured in the bioassay (greater than or equal to 6 IU/ml); employing a RIA (greater than or equal to 1 IU/ml) 28% (17/61) of the patients studied were positive for IFN-alpha. Fifteen patients had a measurable interferonemia over 2-16 months; only two patients had detectable IFN in their serum at only one occasion. In five patients, hourly and daily variations of the IFN titer as measured by RIA were found to amount to less than 80%. The IFN activity found in these sera was characterized as IFN-alpha by means of acid stability, cross-reactivity on heterologous cells, trypsin sensitivity, and neutralization by homologous and heterologous antisera. IFN antibodies were quantified with a neutralization bioassay, an ELISA, and a radioimmunoassay. Of the 61 patients 5% (3) possessed high titers of anti-IFN antibodies which persisted over 2 years. The IFN-alpha antibody positive patients had an inactive form of the disease over years without visceral involvement but decreased serum complement levels (C4, C3, CH50) and repeated episodes of Quincke-like edema.