Detection of Autoantibodies Against the Acetylcholine Receptor, Evaluation of Commercially Available Methodologies: Fixed Cell-Based Assay, Radioimmunoprecipitation Assay and Enzyme-Linked Immunosorbent Assay1.

Background/Objective: Myasthenia Gravis (MG) is an autoimmune disorder characterized by pathogenic autoantibodies (AAbs) targeting nicotinic acetylcholine receptors (AChR), disrupting neuromuscular communication. RadioImmunoPrecipitation Assay (RIPA) is recommended to detect AChR AAbs, but its complexity and radioactive requirements limit widespread use. We compare non-RIPA anti-AChR immunoassays, including Cell-Based Assay (CBA) and two ELISA kits, against the gold standard RIPA. Methods/Results: 145 samples were included with medical indication for anti-AChR testing. By the RIPA method, 63 were negative (RIPA-Neg < 0.02 nmol/L), 18 were classified as Borderline (≥0.02 -1 nmol/L), and 64 were positive (RIPA-Pos > 1 nmol/L). The competitive ELISA showed poor agreement with RIPA (Kappa = 0.216). The indirect ELISA demonstrated substantial agreement with RIPA (Kappa = 0.652), with ∼76% sensitivity and ∼94% specificity for MG diagnostic. The CBA, where fixed cells expressing clustered AChR were used as substrate, exhibited almost perfect agreement with RIPA (Kappa = 0.984), yielding ∼98% sensitivity and 96% specificity for MG. In addition, a semiquantitative analysis showed a strong correlation between CBA titration, indirect ELISA, and RIPA levels (r = 0.793 and r = 0.789, respectively). Conclusions: The CBA displayed excellent analytical performance for MG diagnostic when compared to RIPA, making it a potential replacement for RIPA in clinical laboratories. Some solid-phase assays (such as the indirect ELISA applied here), as well as CBA titration, offer reliable options to estimate anti-AChR AAb levels after confirming positivity by the CBA.∥.

Effect on cell survival and cytoophidium assembly of the adRP-10-related IMPDH1 missense mutation Asp226Asn.

Introduction: Inosine monophosphate dehydrogenase 1 (IMPDH1) is a critical enzyme in the retina, essential for the correct functioning of photoreceptor cells. Mutations in IMPDH1 have been linked to autosomal dominant retinitis pigmentosa subtype 10 (adRP-10), a genetic eye disorder. Some of these mutations such as the Asp226Asn (D226N) lead to the assembly of large filamentous structures termed cytoophidia. D226N also gives IMPDH1 resistance to feedback inhibition by GDP/GTP. This study aims to emulate the adRP-10 condition with a long-term expression of IMPDH1-D226N in vitro and explore cytoophidium assembly and cell survival. We also assessed whether the introduction of an additional mutation (Y12C) to disrupt the cytoophidium has an attenuating effect on the toxicity caused by the D226N mutation. Results: Expression of IMPDH1-D226N in HEp-2 cells resulted in cytoophidium assembly in ∼70% of the cells, but the presence of the Y12C mutation disrupted the filaments. Long-term cell survival was significantly affected by the presence of the D226N mutation, with a decrease of ∼40% in the cells expressing IMPDH1-D226N when compared to IMPDH1-WT; however, survival was significantly recovered in IMPDH1-Y12C/D226N, with only a ∼10% decrease when compared to IMPDH1-WT. On the other hand, the IMPDH1 expression level in the D226N-positive cells was <30% of that of the IMPDH1-WT-positive cells and only slightly higher in the Y12C/D226N, suggesting that although cell survival in Y12C/D226N was recovered, higher expression levels of the mutated IMPDH1 were not tolerated by the cells in the long term. Conclusion: The IMPDH1-D226N effect on photoreceptor cell survival may be the result of a sum of problems: nucleotide unbalance plus a toxic long-life cytoophidium, supported by the observation that by introducing Y12C in IMPDH1 the cytoophidium was disrupted and cell survival significantly recovered, but not the sensibility to GDP/GTP regulation since higher expression levels of IMPDH1-D226N were not tolerated.

"Untargeting" autoantibodies using genome editing, a proof-of-concept study.

Autoantibodies (AAbs) are useful biomarkers and many have direct pathogenic role. Current standard therapies for elimination of specific B/plasma-cell clones are not fully efficient. We apply CRISPR/Cas9 genome-editing to knockout V(D)J rearrangements that produce pathogenic AAbs in vitro. HEK293T cell-lines were established stably expressing a humanized anti-dsDNA Ab (clone 3H9) and a human-derived anti-nAChR-α1 Ab (clone B12L). For each clone, five CRISPR/Cas9 heavy-chain's CDR2/3-targeting guided-RNAs (T-gRNAs) were designed. Non-Target-gRNA (NT-gRNA) was control. After editing, levels of secreted Abs were evaluated, as well as 3H9 anti-dsDNA and B12L anti-AChR reactivities. T-gRNAs editing decreased expression of heavy-chain genes to ∼50-60%, compared to >90% in NT-gRNA, although secreted Abs levels and reactivity to their respective antigens in T-gRNAs decreased ∼90% and âˆ¼ 95% compared with NT-gRNA for 3H9 and B12L, respectively. Sequencing indicated indels at Cas9 cut-site, which could lead to codon jam, and consequently, knockout. Additionally, remaining secreted 3H9-Abs presented variable dsDNA reactivity among the five T-gRNA, suggesting the exact Cas9 cut-site and indels further interfere with antibody-antigen interaction. CRISPR/Cas9 genome-editing was very effective to knockout the Heavy-Chain-IgG genes, considerably affecting AAbs secretion and binding capacity, fostering application of this concept to in vivo models as a potential novel therapeutic approach for AAb-mediated diseases.

A cell-based assay for detection of anti-fibrillarin autoantibodies with performance equivalent to immunoprecipitation.

Anti-fibrillarin autoantibodies are useful for the diagnosis and prognosis of systemic sclerosis (SSc). Anti-fibrillarin produces a clumpy nucleolar pattern in indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA). Here we develop and validate a reliable cell-based anti-fibrillarin assay (Fibrillarin/CBA) for use in clinical diagnostic laboratories. A TransMembrane Signal was fused to the human fibrillarin gene (TMS-fibrillarin). HEp-2 cells overexpressing transgenic TMS-fibrillarin at the cytoplasmic membrane were used as IFA substrate in the Fibrillarin/CBA. Sixty-two serum samples with nucleolar pattern in the HEp-2 IFA (41 clumpy; 21 homogeneous/punctate) were tested for anti-fibrillarin using Fibrillarin/CBA, immunoprecipitation (IP), line-blot and ELISA. In addition, samples from 106 SSc-patients were evaluated with Fibrillarin/CBA and the results were correlated with disease phenotypes. Thirty-eight of 41 samples with the clumpy nucleolar pattern (92.7%) were positive in the Fibrillarin/CBA, while all 21 samples with other nucleolar patterns were negative. Fibrillarin/CBA results agreed 100% with IP results. Among the 38 Fibrillarin/CBA-positive samples, only 15 (39.5%) and 11 (29%) were positive for anti-fibrillarin in line-blot and ELISA, respectively. Higher frequency of diffuse cutaneous SSc (dcSSc) phenotype (72.7% vs 36.8%; p=0.022), cardiac involvement (36.4% vs 6.5%; p=0.001) and scleroderma renal crisis (18.2% vs 3.3% p = 0.028) was observed in SSc patients with positive compared to negative Fibrillarin/CBA result. Performance of Fibrillarin/CBA in the detection of anti-fibrillarin autoantibodies was comparable to the gold standard IP. Positive Fibrillarin/CBA results correlated with disease phenotypes known to be associated with anti-fibrillarin autoantibodies, underscoring the clinical validation of this novel assay.

Molecular crowding facilitates bundling of IMPDH polymers and cytoophidium formation.

The cytoophidium is a unique type of membraneless compartment comprising of filamentous protein polymers. Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step of de novo GTP biosynthesis and plays critical roles in active cell metabolism. However, the molecular regulation of cytoophidium formation is poorly understood. Here we show that human IMPDH2 polymers bundle up to form cytoophidium-like aggregates in vitro when macromolecular crowders are present. The self-association of IMPDH polymers is suggested to rely on electrostatic interactions. In cells, the increase of molecular crowding with hyperosmotic medium induces cytoophidia, while the decrease of that by the inhibition of RNA synthesis perturbs cytoophidium assembly. In addition to IMPDH, CTPS and PRPS cytoophidium could be also induced by hyperosmolality, suggesting a universal phenomenon of cytoophidium-forming proteins. Finally, our results indicate that the cytoophidium can prolong the half-life of IMPDH, which is proposed to be one of conserved functions of this subcellular compartment.

CTPS forms the cytoophidium in zebrafish.

Cytidine triphosphate synthase (CTPS) catalyzes the rate-limiting step of de novo CTP biosynthesis. An intracellular structure of CTPS, the cytoophidium, has been found in many organisms including prokaryotes and eukaryotes. Formation of the cytoophidium has been suggested to regulate the activity and stability of CTPS and may participate in certain physiological events. Herein, we demonstrate that both CTPS1a and CTPS1b in zebrafish are able to form the cytoophidium in cultured cells. A point mutation, H355A, abrogates cytoophidium assembly of zebrafish CTPS1a and CTPS1b. In addition, we show the presence of CTPS cytoophidia in multiple tissues of larval and adult fish under normal conditions, while treatment with a CTPS inhibitor 6-diazo-5-oxo-l-norleucine (DON) can induce more cytoophidia in some tissues. Our findings reveal that forming the CTPS cytoophidium is a natural phenomenon of zebrafish and provide valuable information for future research on the physiological importance of this intracellular structure in vertebrates.

IMPDH forms the cytoophidium in zebrafish.

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in de novo guanine nucleotide biosynthesis. Its activity is negatively regulated by the binding of GTP. IMPDH can form a membraneless subcellular structure termed the cytoophidium in response to certain changes in the metabolic status of the cell. The polymeric form of IMPDH, which is the subunit of the cytoophidium, has been shown to be more resistant to the inhibition by GTP at physiological concentrations, implying a functional correlation between cytoophidium formation and the upregulation of GTP biosynthesis. Herein we demonstrate that zebrafish IMPDH1b and IMPDH2 isoforms can assemble abundant cytoophidium in most of cultured cells under stimuli, while zebrafish IMPDH1a shows distinctive properties of forming the cytoophidium in different cell types. Point mutations that disrupt cytoophidium structure in mammalian models also prevent the aggregation of zebrafish IMPDHs. In addition, we discover the presence of the IMPDH cytoophidium in various tissues of larval and adult fish under normal growth conditions. Our results reveal that polymerization and cytoophidium assembly of IMPDH can be a regulatory machinery conserved among vertebrates, and with specific physiological purposes.

Ribavirin induces widespread accumulation of IMP dehydrogenase into rods/rings structures in multiple major mouse organs.

Ribavirin (RBV) is a guanosine analogue triazole most commonly used in the treatment of chronic hepatitis C (HCV) infection. Although its mechanism of action is a matter of debate, several possibilities have been proposed, including depletion of guanine nucleotides through inhibition of inosine monophosphate dehydrogenase (IMPDH). IMPDH has been shown to assemble into micron-scale rod- and ring-shaped structures (rods/rings or RR), also called "IMPDH filaments," both in vitro and in vivo. Formation of RR structures can occur naturally, potentially to influence IMPDH activity, or when de novo guanosine monophosphate biosynthesis or IMPDH itself are inhibited by nutrient deprivation or drugs like RBV. Numerous studies have also reported the occurrence of autoantibodies targeting RR structures (anti-RR) in HCV patients previously treated or under treatment with interferon-α and ribavirin (IFN/RBV) combination therapy. For this brief study, we considered the strong association between RR autoantibodies and IFN/RBV treatment, and the lack of data assessing how RBV affects RR formation in a variety of tissues in vivo. First, RR structures formed in the spleen and pancreas of normal mice without any treatment. Then, in RBV-treated mice, we detected RR structures in a number of tissues, including stomach, liver, spleen, kidney, brain, skin, and cardiac and skeletal muscle. We made several intriguing observations: predominance of RR structures in the mucosa and submucosa layers of the stomach wall; a high proportion of RR-positive cells in the cerebral cortex, suggesting that RBV actually crosses the blood-brain barrier; and a higher ratio of rings to rods in the epidermis compared to the dermis layer of the skin. Screening for RR structures appears to be a useful method to track tissue penetration of RBV and the many RR-inducing drugs previously identified.

Psychological characteristics of caregivers of pediatric patients with chronic rheumatic disease in relation to treatment adherence.

BACKGROUND: Adherence to treatment for chronic diseases is lower in children than in adults, less extensively studied in children and is associated with multiple related factors. The aim of this study is to perform a descriptive analysis of psycho-cognitive aspects of primary caregivers of pediatric patients with chronic rheumatic diseases, as well as socioeconomic and clinical factors, family functioning and treatment satisfaction. METHODS: Primary caregivers of 90 patients were included. Pairs (caregiver plus patient) were grouped as presenting good adherence (n = 50) or poor adherence (n = 40) according to the Morisky Adherence Test. Psycho-cognitive aspects were evaluated by Adult Self-Report and Wechsler Adult Intelligence Scale tests. For statistical comparisons, quantitative variables with normal distribution were analyzed by Student's t test, and those with non-Gaussian distribution with the Mann Whitney test. Categorical variables were analyzed by Chi square test. A multivariate logistic regression analysis was performed to estimate the contribution of the independent variables to adherence. RESULTS: Compared to caregivers in the good adherence group, caregivers in the poor adherence group were more likely to be classified as clinical on the scales for attention problems and externalizing problems, which include impulsiveness and aggressiveness. They also scored higher on the depressive problem scale. In addition, the average number of children per caregiver and the mean age of caregivers and patients were significantly higher in the poor adherence group, while the proportion of caregivers with higher education was lower. The poor adherence group also included a higher incidence of pediatric patients assuming sole responsibility for managing medications. Economic status, clinical factors, treatment satisfaction, family functioning and caregiver cognitive profile were not related to adherence, except for working memory index. CONCLUSION: Older patients, patients as the one solely responsible for medication management, and caregivers with externalizing problems, were observed to be the most strongly associated to poor adherence. Interventions aimed at adolescent patients are needed. Also, psychological programs and interventional studies to better determine caregivers' behavioral/emotional status, and parent-child relationships are recommended.

IMP/GTP balance modulates cytoophidium assembly and IMPDH activity.

BACKGROUND: Inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in de novo GTP biosynthesis, plays an important role in cell metabolism and proliferation. It has been demonstrated that IMPDH can aggregate into a macrostructure, termed the cytoophidium, in mammalian cells under a variety of conditions. However, the regulation and function of the cytoophidium are still elusive. RESULTS: In this study, we report that spontaneous filamentation of IMPDH is correlated with rapid cell proliferation. Intracellular IMP accumulation promoted cytoophidium assembly, whereas elevated GTP level triggered disassociation of aggregates. By using IMPDH2 CBS domain mutant cell models, which are unable to form the cytoophidium, we have determined that the cytoophidium is of the utmost importance for maintaining the GTP pool and normal cell proliferation in the condition that higher IMPDH activity is required. CONCLUSIONS: Together, our results suggest a novel mechanism whereby cytoophidium assembly upregulates IMPDH activity and mediates guanine nucleotide homeostasis.

CTP synthase forms the cytoophidium in human hepatocellular carcinoma.

CTP synthase (CTPS) can aggregate into an intracellular macrostructure, the cytoophidium, in various organisms including human cells. Previous studies have shown that assembly of human CTPS cytoophidia may be correlated with the cellular metabolic status, and is able to promote the activity of CTPS. A correlation between the cytoophidium and cancer metabolism has been proposed but not yet been revealed. In the current study we provide clear evidence of the presence of CTPS cytoophidia in various human cancers and some non-cancerous tissues. Moreover, among 203 tissue samples of hepatocellular carcinoma, 56 (28%) samples exhibited many cytoophidia, whereas no cytoophidia were detected in adjacent non-cancerous hepatocytes for all samples. Our findings suggest that the CTPS cytoophidium may participate in the adaptive metabolism of human hepatocellular carcinoma.

Differential capacity of therapeutic drugs to induce Rods/Rings structures in vitro and in vivo and generation of anti-Rods/Rings autoantibodies.

Some HCV patients using ribavirin and interferon alpha (IFN-α) develop anti-rods and rings (RR) autoantibodies, the main target of which is inosine monophosphate dehydrogenase (IMPDH), the rate-determining enzyme in de novo GTP biosynthesis. In vitro inhibition of IMPDH by ribavirin induces RR formation. Here we investigate whether other commonly used drugs that interfere with GTP biosynthesis can induce RR structures in vitro and vivo and elicit generation of autoantibodies. HEp-2 cells treated for 24h with ribavirin, mycophenolic acid (MPA), azathioprine, methotrexate or acyclovir were positive for RR structures. However, adefovir, entecavir, tenofovir and lamivudine did not induce RR structures in these cells. Structures induced by ribavirin in HEp-2 cells are stable after 24h drug-washout, while structures induced by other drugs are relatively labile, disappearing within 2h. Looking at patients treated with these drugs, HCV patients treated with ribavirin (n=17) showed higher average percentage of RR-positive peripheral mononuclear cells than autoimmune patients treated with RR-inducing immunosuppressant drugs (n=21). Serum from 173 autoimmune patients who had been treated with MPA, azathioprine or methotrexate was tested for presence of anti-RR autoantibodies, and only one sample was found to be positive. Conversely, of 48 anti-RR autoantibody positive samples identified at Fleury Laboratories over 30months, 94% were from HCV patients treated with ribavirin plus IFN-α. These data indicate that RR structures can be induced by a variety of drugs in vitro and in vivo, but anti-RR autoantibody production is mostly restricted to HCV patients under ribavirin+IFN-α treatment.

Anti-rods/rings autoantibody generation in hepatitis C patients during interferon-α/ribavirin therapy.

Chronic inflammation associated with hepatitis C virus (HCV) infection can lead to disabling liver diseases with progression to liver cirrhosis and hepatocellular carcinoma. Despite the recent availability of more effective and less toxic therapeutic options, in most parts of the world the standard treatment consists of a weekly injection of pegylated interferon α (IFN-α) together with a daily dose of ribavirin. HCV patients frequently present circulating non-organ-specific autoantibodies demonstrating a variety of staining patterns in the indirect immunofluorescence assay for antinuclear antibodies (ANA). Between 20% to 40% of HCV patients treated with IFN-α and ribavirin develop autoantibodies showing a peculiar ANA pattern characterized as rods and rings (RR) structures. The aim of this article is to review the recent reports regarding RR structures and anti-rods/rings (anti-RR) autoantibody production by HCV patients after IFN-α/ribavirin treatment. Anti-RR autoantibodies first appear around the sixth month of treatment and reach a plateau around the twelfth month. After treatment completion, anti-RR titers decrease/disappear in half the patients and remain steady in the other half. Some studies have observed a higher frequency of anti-RR antibodies in relapsers, i.e., patients in which circulating virus reappears after initially successful therapy. The main target of anti-RR autoantibodies in HCV patients is inosine-5'-monophosphate dehydrogenase 2 (IMPDH2), the rate-limiting enzyme involved in the guanosine triphosphate biosynthesis pathway. Ribavirin is a direct IMPDH2 inhibitor and is able to induce the formation of RR structures in vitro and in vivo. In conclusion, these observations led to the hypothesis that anti-RR autoantibody production is a human model of immunologic tolerance breakdown that allows us to explore the humoral autoimmune response from the beginning of the putative triggering event: exposure to ribavirin and interferon.

Assembly of IMPDH2-based, CTPS-based, and mixed rod/ring structures is dependent on cell type and conditions of induction.

Inhibition of guanosine triphosphate (GTP) and cytidine triphosphate (CTP) biosynthetic pathways induces cells to assemble rod/ring (RR) structures, also named cytoophidia, which consist of the enzymes cytidine triphosphate synthase (CTPS) and inosine-5'-monophosphate dehydrogenase 2 (IMPDH2). We aim to explore the interaction of CTPS and IMPDH2 in the generation of RR structures. HeLa and COS-7 cells were cultured in normal conditions or in the presence of 6-diazo-5-oxo-L-norleucine (DON), ribavirin, or mycophenolic acid (MPA). Over 90% of DON-treated cells presented RR structures. In HeLa cells, 35% of the RR structures were positive for IMPDH2 alone, 26% were CTPS alone, and 31% were IMPDH2/CTPS mixed, while in COS-7 cells, 42% of RR were IMPDH2 alone, 41% were CTPS alone, and 10% were IMPDH2/CTPS mixed. Ribavirin and MPA treatments induced only IMPDH2-based RR. Cells were also transfected with an N-terminal hemagglutinin (NHA)-tagged CTPS1 construct. Over 95% of NHA-CTPS1 transfected cells with DON treatment presented IMPDH2-based RR and almost 100% presented CTPS1-based RR; when treated with ribavirin, over 94% of transfected cells presented IMPDH2-based RR and 37% presented CTPS1-based RR, whereas 2% of untreated transfected cells presented IMPDH2-based RR and 28% presented CTPS1-based RR. These results may help in understanding the relationship between CTP and GTP biosynthetic pathways, especially concerning the formation of filamentous RR structures.

Microinjection of specific anti-IMPDH2 antibodies induces disassembly of cytoplasmic rods/rings that are primarily stationary and stable structures.

BACKGROUND: Our laboratory previously reported interesting rods 3-10 µm long and rings 2-5 µm diameter (RR) in the cytoplasm of mammalian cells. Experimental evidence show that both inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) and cytidine triphosphate synthetase (CTPS) are components of RR structures. Several cell types, including mouse embryonic stem cells, and cell lines, such as mouse 3 T3 and rat NRK, naturally present RR structures, while other cells can present RR when treated with compounds interfering with GTP/CTP biosynthetic pathways. In this study, we aimed to investigate the dynamic behavior of these RR in live cells. RESULTS: RR were detected in >90% of COS-7 and HeLa cells treated with 1 mM ribavirin or 6-Diazo-5-oxo-L-norleucine (DON) for 24 h, and in 75% of COS-7 cells treated with 1 mM mycophenolic acid (MPA) for the same period of time. Microinjection of affinity-purified anti-IMPDH2 antibodies in live COS-7 cells treated with ribavirin, DON, or MPA showed mature forms of RR presented as stable and stationary structures in 71% of cells. In the remaining 29% of cells, RR acquired erratic movement and progressively disassembled into fragments and disappeared within 10 min. The specific stationary state and antibody-dependent disassembling of RR structures was independently confirmed in COS-7 and HeLa cells transfected with GFP-tagged IMPDH2. CONCLUSIONS: This is the first demonstration of disassembly of RR structures upon microinjection of anti-IMPDH2 antibodies that led to the disappearance of the molecular aggregates. The disassembly of RR after microinjection of anti-IMPDH2 antibody further strengthens the notion that IMPDH2 are major building blocks of RR. Using two independent methods, this study demonstrated that the induced RR are primarily stationary structures in live cells and that IMPDH2 is a key component of RR.

Temporal evolution of human autoantibody response to cytoplasmic rods and rings structure during anti-HCV therapy with ribavirin and interferon-α.

Autoantibodies to inosine monophosphate dehydrogenase-2 (IMPDH2), an enzyme involved in de novo biosynthesis of guanine nucleotides, are observed in a subset of hepatitis C virus (HCV) patients receiving interferon alpha (IFN-α) plus ribavirin. Anti-IMPDH2 antibodies display a peculiar cytoplasmic "rod/ring" (RR) pattern in IIF-HEp-2. We examined the dynamics of anti-RR autoimmune response with respect to immunoglobulin isotypes, titer, avidity, and protein targets in 80 sequential samples from 15 HCV patients (plus 12 randomly selected anti-RR-positive, totalizing 92 samples) collected over an 18-month period, including samples collected before, during, and after IFN-α + ribavirin treatment. Immunoprecipitation showed reactivity with the 55 kDa IMPDH2 protein in 12/15 patients (80 %) and 11/15 (73 %) reacted with IMPDH2 in a sandwich ELISA. During treatment, anti-IMPDH2 autoantibodies hit their highest levels after 6-12 months of treatment and decreased post-treatment, while anti-HCV antibodies levels were stable over time. Anti-IMPDH2 IgM levels increased up until the sixth month of treatment and remained stable thereafter, while IgG levels increased steadily up to the twelfth month. Both IgG and IgM decreased during the post-treatment period. IgG avidity increased steadily up to the twelfth month of treatment. In conclusion, this study showed that the temporal kinetics of IFN-α + ribavirin-induced humoral autoimmune response to IMPDH2 exhibited a considerably delayed pace of increase in antibody levels and avidity as well as in isotype class switch in comparison with a conventional humoral response to infectious agents. These unique findings uncover intriguing differences between the autoimmune response and the immune response to exogenous agents in humans.

Longitudinal study of a human drug-induced model of autoantibody to cytoplasmic rods/rings following HCV therapy with ribavirin and interferon-α.

BACKGROUND: A 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/RESULTS: 597 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. CONCLUSIONS: The 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.