Serum interferon (IFN)-neutralizing antibodies and bioactivities of IFNs in patients with severe type II essential mixed cryoglobulinemia

Abstract

The efficacy of alpha interferon (IFN-alpha) in the treatment of severe type II essential mixed cryoglobulinemia (EMC) has been reported previously. In some patients, the development of neutralizing antibodies to recombinant IFN-alpha (rIFN-alpha) can affect the clinical response achieved with rIFN-alpha; a second treatment with natural IFN-alpha preparations may reinduce the clinical response. In the present study the ability of leukocyte IFN (LeIFN) to restore the response was investigated from a pharmacodynamic viewpoint. Specifically, the pharmacodynamic profiles of different IFN-alpha preparations were studied by measuring the serum neopterin levels and the levels of expression of protein MxA mRNA in in vivo peripheral blood mononuclear cells in two patients with EMC whose resistance to rIFN-alpha2a treatment increased concomitantly with the development of neutralizing antibodies. These markers were measured before injection and at 24 and 48 h after a single injection of rIFN-alpha2a, consensus IFN [(C)IFN], or LeIFN. No increase or only a slight increase in MxA mRNA levels was detectable after administration of rIFN-alpha2a or (C)IFN, whereas a significant increase (>/=10-fold) in MxA mRNA expression was recorded following administration of LeIFN. The neutralizing antibodies to rIFN-alpha2a cross-react with (C)IFN. Sera from these patients neutralized most but not all of the subtypes present in the natural IFN-alpha (LeIFN) mixture, and no significant increase in neopterin levels was observed after these patients were switched to LeIFN treatment. In summary, the data demonstrate that the problem of neutralizing antibodies still exists and that LeIFN may induce an increase in the level of MxA mRNA expression but not an increase in neopterin levels in patients who are resistant to treatment with rIFN-alpha2a or (C)IFN.

FIG. 1.

Treatment and blood sampling sequences during the study.

FIG. 2.

QC PCR analysis of MxA mRNA in PBMCs from patient 1. PBMCs were collected before and at 24 and 48 h after a single injection of rIFN-a2a, (C)IFN, and LeIFN. Serial logarithmic dilutions of extracted RNA, normalized by using b-actin mRNA as a control, were mixed with 2 3 106 copies of the internal competitor and subjected to RT-PCR (for details, see Materials and Methods). The arrows indicate the equivalence points.

FIG. 3.

QC PCR analysis of MxA mRNA in PBMCs from patient 2. PBMCs were collected before and at 24 and 48 h after a single injection of rIFN-α2a, (C)IFN, and LeIFN. Serial logarithmic dilutions of extracted RNA, normalized by using β-actin mRNA as a control, were mixed with 2 × 10⁶ copies of the internal competitor and subjected to RT-PCR (for details, see Materials and Methods). The arrows indicate the equivalence points.

FIG. 4.

Neopterin protein levels in the sera of patients 1 (A) and 2 (B). Serum neopterin concentrations were measured by enzyme-linked immunosorbent assay before and at 24 and 48 h after a single injection of rIFN-α2a, (C)IFN, and LeIFN. Bars represent means ± standard deviations for three separate determinations.

FIG. 5.

Increases in serum neopterin levels at 24 and 48 h compared with that at the baseline in NAB-negative patients with EMC after a single injection of recombinant IFN-α2a. Each histogram represents the mean ± standard deviations for five patients. ∗, P < 0.05 compared with the level at time zero.