Longitudinal Study of a Human Drug-Induced Model of Autoantibody to Cytoplasmic Rods/Rings following HCV Therapy with Ribavirin and Interferon-α

Abstract

BackgroundA novel pattern in the indirect immunofluorescence antinuclear antibody assay on HEp-2 cells (IIF-HEp-2) characterized by cytoplasmic rods and rings (RR) was reported in HCV patients, but stringent disease specificity studies and longitudinal analysis are lacking. We investigated the clinical significance of anti-RR in an HCV cohort with up to a 12-month treatment follow up.Methodology/Results597 patients (342 HCV, 55 HCV/HIV, 200 non-HCV) were screened and titered for anti-RR. Serial samples were available from 78 of 176 treated and 27 of 166 untreated patients. Anti-RR was detected in 14.1% of 342 HCV patients, 9.1% of 55 HCV/HIV, 3.4% of 29 Hepatitis B, and none of 171 non-HCV (p<0.0001; HCV versus non-HCV). Anti-RR was present in 38% of 108 patients receiving interferon-α/ribavirin, but none in 26 receiving either interferon-α or ribavirin, or 166 untreated patients (p<0.0001). Other IIF-HEp-2 patterns were more frequently associated with interferon-α treatment alone (52.2%) as compared to interferon-α/ribavirin (25%), ribavirin alone (33.3%), and no therapy (26.5%). Anti-RR frequency was not associated with sex, age, ethnicity, HCV genotype or viral load. Anti-RR occurred only after initiation of treatment, beginning as early as 1 month (6%), but by the sixth month >47% tested positive for anti-RR. The anti-RR titer generally increased with sustained treatment and remained high in 53% of patients. After treatment, anti-RR titer was negative in 41%. Non-responders to HCV therapy were 77% in anti-RR-positive versus 64% in anti-RR-negative patients. Response to treatment was not associated with anti-RR titer or the dynamics of anti-RR reactivity during and after treatment.ConclusionsThe exquisite association of anti-RR reactivity with combined interferon-α/ribavirin therapy in HCV patients represents a unique model for drug-induced autoantibody generation in humans as demonstrated by the fact that a significant fraction of patients who have anti-RR during therapy becomes anti-RR-negative after completion of therapy.

Ultrastructure of Cytoplasmic and Nuclear Inosine-5’-Monophosphate Dehydrogenase 2 “Rods and Rings” Inclusions

“…All presented experiments were performed on Hep2 cells because these cells have been widely used in IMPDH field, and because the R&R pattern was originally found using commercial Hep2 cell slides used for autoantibody screening (Carcamo et al 2011;Keppeke et al 2012). Importantly, human U-2 OS, Hep G2 and mouse MH22A and NIH 3T3 cell lines were also tested using fluorescence microscopy, with similar results identified to those obtained with Hep2 cells (Supplemental Fig.…”

“…Coverslips were washed several times in PBS and once in water, then mounted onto cells using home-made mowiol containing DAPI (4',6-diamidino-2-phenylindole, Sigma-Aldrich) to counterstain the DNA. As a primary antibody, rabbit anti-IMPDH2 (12948-1-AP, Proteintech; Manchester, United Kingdom), or human autoimmune sera with reactivity to R&R (a gift from the laboratory of Prof. Luis Eduardo C. Andrade, Universidade Federal de São Paulo, Brazil; Keppeke et al 2012) were used. Secondary antibodies used were goat anti-rabbit and goat anti-human antibodies conjugated with FITC or Cy3 (Jackson ImmunoResearch Laboratories; West Grove, USA).…”

Cellular Serpents and Dreaming Spires: New Frontiers in Arginine and Pyrimidine Biology

“…Gerson Keppeke, a PhD student from Luis E.C. Andrade's lab (Federal University of Sao Paulo) described the relationship between CTPsyn and IMPDH using novel patient derived autoantibodies (Keppeke et al, 2012). Barbara Zimmerman (Universidad de los Andes) gave an introduction into the regulation of CTPsyn in the parasitic protozoan Toxoplasma gondii and described the changes occurring in CTPsyncontaining structures throughout its novel life-cycle.…”

‘Rod and ring’ formation from IMP dehydrogenase is regulated through the one-carbon metabolic pathway

“…the Methotrexate Leucovorin Rescue protocol) and during immunosuppression for rheumatoid arthritis. We and others have demonstrated an autoantibody response against RR structures (IMPDH as a major autoantigen) in a subset of hepatitis C patients that have undergone interferon-α and ribavirin therapy (Carcamo et al, 2013;Covini et al, 2012;Keppeke et al, 2012). Prolonged exposure to interferon-α and ribavirin increases the prevalence of this autoantibody response (Keppeke et al, 2014), and in rare cases anti-RR antibody can be detected in non-hepatitis patients (Calise et al, 2014a.…”