Blueprinting extendable nanomaterials with standardized protein blocks.

A wooden house frame consists of many different lumber pieces, but because of the regularity of these building blocks, the structure can be designed using straightforward geometrical principles. The design of multicomponent protein assemblies, in comparison, has been much more complex, largely owing to the irregular shapes of protein structures1. Here we describe extendable linear, curved and angled protein building blocks, as well as inter-block interactions, that conform to specified geometric standards; assemblies designed using these blocks inherit their extendability and regular interaction surfaces, enabling them to be expanded or contracted by varying the number of modules, and reinforced with secondary struts. Using X-ray crystallography and electron microscopy, we validate nanomaterial designs ranging from simple polygonal and circular oligomers that can be concentrically nested, up to large polyhedral nanocages and unbounded straight 'train track' assemblies with reconfigurable sizes and geometries that can be readily blueprinted. Because of the complexity of protein structures and sequence-structure relationships, it has not previously been possible to build up large protein assemblies by deliberate placement of protein backbones onto a blank three-dimensional canvas; the simplicity and geometric regularity of our design platform now enables construction of protein nanomaterials according to 'back of an envelope' architectural blueprints.

Light-sensitive phosphorylation regulates retinal IMPDH1 activity and filament assembly.

Inosine monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in guanosine triphosphate (GTP) synthesis and assembles into filaments in cells, which desensitizes the enzyme to feedback inhibition and boosts nucleotide production. The vertebrate retina expresses two splice variants IMPDH1(546) and IMPDH1(595). In bovine retinas, residue S477 is preferentially phosphorylated in the dark, but the effects on IMPDH1 activity and regulation are unclear. Here, we generated phosphomimetic mutants to investigate structural and functional consequences of S477 phosphorylation. The S477D mutation resensitized both variants to GTP inhibition but only blocked assembly of IMPDH1(595) filaments. Cryo-EM structures of both variants showed that S477D specifically blocks assembly of a high-activity assembly interface, still allowing assembly of low-activity IMPDH1(546) filaments. Finally, we discovered that S477D exerts a dominant-negative effect in cells, preventing endogenous IMPDH filament assembly. By modulating the structure and higher-order assembly of IMPDH, S477 phosphorylation acts as a mechanism for downregulating retinal GTP synthesis in the dark when nucleotide turnover is decreased.

Light-sensitive phosphorylation regulates enzyme activity and filament assembly of human IMPDH1 retinal splice variants.

Inosine monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in de novo guanosine triphosphate (GTP) synthesis and is controlled by feedback inhibition and allosteric regulation. IMPDH assembles into micron-scale filaments in cells, which desensitizes the enzyme to feedback inhibition by GTP and boosts nucleotide production. The vertebrate retina expresses two tissue-specific splice variants IMPDH1(546) and IMPDH1(595). IMPDH1(546) filaments adopt high and low activity conformations, while IMPDH1(595) filaments maintain high activity. In bovine retinas, residue S477 is preferentially phosphorylated in the dark, but the effects on IMPDH1 activity and regulation are unclear. Here, we generated phosphomimetic mutants to investigate structural and functional consequences of phosphorylation in IMPDH1 variants. The S477D mutation re-sensitized both variants to GTP inhibition, but only blocked assembly of IMPDH1(595) filaments and not IMPDH1(546) filaments. Cryo-EM structures of both variants showed that S477D specifically blocks assembly of the high activity assembly interface, still allowing assembly of low activity IMPDH1(546) filaments. Finally, we discovered that S477D exerts a dominant-negative effect in cells, preventing endogenous IMPDH filament assembly. By modulating the structure and higher-order assembly of IMPDH, phosphorylation at S477 acts as a mechanism for downregulating retinal GTP synthesis in the dark, when nucleotide turnover is decreased. Like IMPDH1, many other metabolic enzymes dynamically assemble filamentous polymers that allosterically regulate activity. Our work suggests that posttranslational modifications may be yet another layer of regulatory control to finely tune activity by modulating filament assembly in response to changing metabolic demands.

Blueprinting expandable nanomaterials with standardized protein building blocks.

A wooden house frame consists of many different lumber pieces, but because of the regularity of these building blocks, the structure can be designed using straightforward geometrical principles. The design of multicomponent protein assemblies in comparison has been much more complex, largely due to the irregular shapes of protein structures 1 . Here we describe extendable linear, curved, and angled protein building blocks, as well as inter-block interactions that conform to specified geometric standards; assemblies designed using these blocks inherit their extendability and regular interaction surfaces, enabling them to be expanded or contracted by varying the number of modules, and reinforced with secondary struts. Using X-ray crystallography and electron microscopy, we validate nanomaterial designs ranging from simple polygonal and circular oligomers that can be concentrically nested, up to large polyhedral nanocages and unbounded straight "train track" assemblies with reconfigurable sizes and geometries that can be readily blueprinted. Because of the complexity of protein structures and sequence-structure relationships, it has not been previously possible to build up large protein assemblies by deliberate placement of protein backbones onto a blank 3D canvas; the simplicity and geometric regularity of our design platform now enables construction of protein nanomaterials according to "back of an envelope" architectural blueprints.

Anti-rods/rings autoantibody and IMPDH filaments: an update after fifteen years of discovery.

Autoantibodies to unknown subcellular rod and ring-shaped structures were first discovered in sera from hepatitis C patients in 2005. Early studies showed a strong association between these anti-rods/rings antibodies (anti-RR) and the standard of care interferon-α plus ribavirin combination therapy (IFN/RBV), suggesting that anti-RR are drug-induced autoantibodies. In the context of hepatitis C, anti-RR have been linked with relapse from or lack of response to IFN/RBV in some patient cohorts. However, examples of anti-RR in other diseases and healthy individuals have also been reported over the years, although anti-RR remains a rare autoantibody response in general. The advent of new direct-acting antiviral drugs for chronic hepatitis C and studies of anti-RR from different parts of the world are also beginning to change the perception of anti-RR. The nucleotide biosynthetic enzyme inosine monophosphate dehydrogenase (IMPDH) has been identified as the major autoantigen recognized by anti-RR. Coincidentally, the assembly of IMPDH into micron-scale rod and ring-shaped structures was discovered around the same time as anti-RR. Knowledge of the fundamental biological properties and cellular functions of these structures, referred to as "IMPDH filaments" by cell biologists, has advanced in parallel to anti-RR antibodies. Recent studies have revealed that IMPDH filament assembly is a mechanism to prevent feedback inhibition of IMPDH and is therefore important for the increased nucleotide production required in hyperproliferating cells, like activated T cells. Fifteen years later, we review the history and current knowledge in both the anti-RR autoantibody and IMPDH filament fields. TAKE-HOME MESSAGE: Anti-rods/rings are recognized as an example of a drug-induced autoantibody in hepatitis C patients treated with interferon and ribavirin, although new studies suggest anti-rods/rings may be detected in other contexts and may depend on unknown environmental or genetic factors in different populations. Recent data suggest that the assembly of IMPDH into rod and ring structures, the targets of anti-rods/rings autoantibody, is a mechanism for hyperproliferating cells, like activated T cells, to maintain increased guanine nucleotide levels to support rapid cell division.

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.

Immune Response-Dependent Assembly of IMP Dehydrogenase Filaments.

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the conversion of IMP to xanthosine monophosphate, the rate-limiting step in de novo guanosine monophosphate (GMP) synthesis. In cultured cells, IMPDH polymerizes into micron-scale filamentous structures when GMP synthesis is inhibited by depletion of purine precursors or by various drugs, including mycophenolic acid, ribavirin, and methotrexate. IMPDH filaments also spontaneously form in undifferentiated mouse embryonic stem cells and induced pluripotent stem cells, hinting they might function in various highly proliferative cell types. Therefore, we investigated IMPDH filament formation in human and murine T cells, which rely heavily on de novo guanine nucleotide synthesis to rapidly proliferate in response to antigenic challenge. We discovered extensive in vivo IMPDH filament formation in mature T cells, B cells, and other proliferating splenocytes of normal, adult B6 mice. Both cortical and medullary thymocytes in young and old mice also showed considerable assembly of IMPDH filaments. We then stimulated primary human peripheral blood mononuclear cells ex vivo with T cell mitogens phytohemagglutinin (PHA), concanavalin A (ConA), or antibodies to CD3 and CD28 for 72 h. We detected IMPDH filaments in 40-60% of T cells after activation compared to 0-10% of unstimulated T cells. Staining of activated T cells for the proliferation marker Ki-67 also showed an association between IMPDH filament formation and proliferation. Additionally, we transferred ovalbumin-specific CD4+ T cells from B6.OT-II mice into B6.Ly5a recipient mice, challenged these mice with ovalbumin, and harvested spleens 6 days later. In these spleens, we identified abundant IMPDH filaments in transferred T cells by immunofluorescence, indicating that IMPDH also polymerizes during in vivo antigen-specific T cell activation. Overall, our data indicate that IMPDH filament formation is a novel aspect of T cell activation and proliferation, and that filaments might be useful morphological markers for T cell activation. The data also suggest that in vivo IMPDH filament formation could be occurring in a variety of proliferating cell types throughout the body. We propose that T cell activation will be a valuable model for future experiments probing the molecular mechanisms that drive IMPDH polymerization, as well as how IMPDH filament formation affects cell function.

A Comprehensive Overview on Myositis-Specific Antibodies: New and Old Biomarkers in Idiopathic Inflammatory Myopathy.

Autoantibodies specific for idiopathic inflammatory myopathy (myositis-specific autoantibodies (MSAs)) are clinically useful biomarkers to help the diagnosis of polymyositis/dermatomyositis (PM/DM). Many of these are also associated with a unique clinical subset of PM/DM, making them useful in predicting and monitoring certain clinical manifestations. Classic MSAs known for over 30 years include antibodies to Jo-1 (histidyl transfer RNA (tRNA) synthetase) and other aminoacyl tRNA synthetases (ARS), anti-Mi-2, and anti-signal recognition particle (SRP). Anti-Jo-1 is the first autoantibodies to ARS detected in 15-25 % of patients. In addition to anti-Jo-1, antibodies to seven other aminoacyl tRNA synthetases (ARS) have been reported with prevalence, usually 1-5 % or lower. Patients with any anti-ARS antibodies are associated with anti-synthetase syndrome characterized by myositis, interstitial lung disease (ILD), arthritis, Raynaud's phenomenon, and others. Several recent studies suggested heterogeneity in clinical features among different anti-ARS antibody-positive patients and anti-ARS may also be found in idiopathic ILD without myositis. Anti-Mi-2 is a classic marker for DM and associated with good response to steroid treatment and good prognosis. Anti-SRP is specific for PM and associated with treatment-resistant myopathy histologically characterized as necrotizing myopathy. In addition to classic MSAs, several new autoantibodies with strong clinical significance have been described in DM. Antibodies to transcription intermediary factor 1γ/α (TIF1γ/α, p155/140) are frequently found in DM associated with malignancy while anti-melanoma differentiation-associated gene 5 (MDA5; CADM140) are associated with clinically amyopathic DM (CADM) complicated by rapidly progressive ILD. Also, anti-MJ/nuclear matrix protein 2 (NXP-2) and anti-small ubiquitin-like modifier-1 (SUMO-1) activating enzyme (SAE) are recognized as new DM-specific autoantibodies. Addition of these new antibodies to clinical practice in the future will help in making earlier and more accurate diagnoses and better management for patients.

Anti-rods/rings autoantibody seropositivity does not affect response to telaprevir treatment for chronic hepatitis C infection.

PURPOSE: Autoantibodies to intracellular 'rods and rings' structures (anti-rods/rings or anti-RR) are strongly associated with hepatitis C (HCV) patients treated with interferon-α/ribavirin (IFN/RBV) and are linked with non-responsiveness to IFN/RBV or relapse, especially in Italian patients. This is the first study to determine whether there is any correlation of anti-RR with non-responsiveness to IFN/RBV treatment in patients also treated with telaprevir (TPV), one of several new therapies for chronic HCV recently implemented. METHODS: From 2013 to 2014, 52 HCV-infected patients were treated with IFN/RBV and TPV at five Italian clinics. Patient sera were collected and analyzed by indirect immunofluorescence for the presence of anti-RR antibodies. Patients were classified as anti-RR positive or anti-RR negative, and then various biological and clinical variables were analyzed to compare the two groups, including gender, age, HCV genotype, previous IFN/RBV treatment, and IFN/RBV/TPV treatment outcome. RESULTS: Of these 52 HCV patients treated with IFN/RBV/TPV, 10/32 (31%) who previously received IFN/RBV were anti-RR positive, compared to 0 of 20 treatment-naïve patients. Anti-RR-positive patients relapsed more than anti-RR-negative patients (3/10, 30% vs. 2/42, 5%; p < 0.05). However, zero anti-RR-positive patients were non-responsive, and frequencies of sustained virological response were similar (anti-RR positive: 7/10, 70% vs. anti-RR negative: 33/42, 79%). CONCLUSIONS: Overall, the data suggest that anti-RR seropositivity is not associated with resistance to TPV treatment in this patient cohort, but monitoring anti-RR-positive patients for relapse within the first 6 months after treatment may be useful.

Periodontal bacterial colonization in synovial tissues exacerbates collagen-induced arthritis in B10.RIII mice.

BACKGROUND: It has been previously hypothesized that oral microbes may be an etiological link between rheumatoid arthritis (RA) and periodontal disease. However, the mechanistic basis of this association is incompletely understood. Here, we investigated the role of periodontal bacteria in induction of joint inflammation in collagen-induced arthritis (CIA) in B10.RIII mice. METHODS: CIA-prone B10.RIII mice were infected orally with a polybacterial mixture of Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia for 24 weeks before induction of CIA. The ability of polybacterial mixture to colonize the periodontium and induce systemic response, horizontal alveolar bone resorption in infected B10.RIII mice was investigated. Arthritis incidence, severity of joint inflammation, pannus formation, skeletal damage, hematogenous dissemination of the infection, matrix metalloproteinase 3 (MMP3) levels, and interleukin-17 expression levels were evaluated. RESULTS: B10.RIII mice had gingival colonization with all three bacteria, higher levels of anti-bacterial immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies, significant alveolar bone resorption, and hematogenous dissemination of P. gingivalis to synovial joints. Infected B10.RIII mice had more severe arthritis, and higher serum matrix metalloproteinase 3 levels and activity. Histopathological analysis showed increased inflammatory cell infiltration, destruction of articular cartilage, erosions, and pannus formation. Additionally, involved joints showed had expression levels of interleukin-17. CONCLUSION: These findings demonstrate that physical presence of periodontal bacteria in synovial joints of B10.RIII mice with collagen-induced arthritis is associated with arthritis exacerbation, and support the hypothesis that oral bacteria, specifically P. gingivalis, play a significant role in augmenting autoimmune arthritis due to their intravascular dissemination to the joints.

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

'Rods and rings' (RRs) are conserved, non-membrane-bound intracellular polymeric structures composed, in part, of inosine monophosphate dehydrogenase (IMPDH), a key enzyme leading to GMP and GTP biosynthesis. RR formation is induced by IMPDH inhibitors as well as glutamine deprivation. They also form upon treatment of cells with glutamine synthetase inhibitors. We now report that depriving cells of serine and glycine promotes RR formation, and we have traced these effects to dihydrofolate reductase (DHFR) and serine hydroxymethyltransferase-2 (SHMT2), pivotal enzymes in one-carbon metabolism and nucleotide biosynthesis. RR assembly is likewise induced upon DHFR inhibition by methotrexate or aminopterin as well as siRNA-mediated knockdown of DHFR or SHMT2. Because RR assembly occurs when guanine nucleotide biosynthesis is inhibited, and because RRs rapidly disassemble after the addition of guanine nucleotide precursors, RR formation might be an adaptive homeostatic mechanism, allowing IMPDH to sense changes in the one-carbon folate pathway.

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.

Anti-rods/rings: a human model of drug-induced autoantibody generation.

In recent years, autoantibodies targeting subcellular structures described as the rods and rings pattern in HEp-2 ANA have been presented as a unique case of autoantibody generation. These rod and ring structures (RR) are at least partially composed of inosine monophosphate dehydrogenase type 2 (IMPDH2), and their formation can be induced in vitro by several small-molecule inhibitors, including some IMPDH2 inhibitors. Autoantibodies targeting these relatively unknown structures have been almost exclusively observed in hepatitis C virus (HCV) patients who have undergone treatment with pegylated interferon-α/ribavirin (IFN/RBV) combination therapy. To date, anti-RR antibodies have not been found in treatment-naïve HCV patients or in patients from any other disease groups, with few reported exceptions. Here, we describe recent advances in characterizing the RR structure and the strong association between anti-RR antibody response and HCV patients treated with IFN/RBV, detailing why anti-RR can be considered a human model of drug-induced autoantibody generation.

Molecular cell biology and immunobiology of mammalian rod/ring structures.

Nucleotide biosynthesis is a highly regulated process necessary for cell growth and replication. Cytoplasmic structures in mammalian cells, provisionally described as rods and rings (RR), were identified by human autoantibodies and recently shown to include two key enzymes of the CTP/GTP biosynthetic pathways, cytidine triphosphate synthetase (CTPS) and inosine monophosphate dehydrogenase (IMPDH). Several studies have described CTPS filaments in mammalian cells, Drosophila, yeast, and bacteria. Other studies have identified IMPDH filaments in mammalian cells. Similarities among these studies point to a common evolutionarily conserved cytoplasmic structure composed of a subset of nucleotide biosynthetic enzymes. These structures appear to be a conserved metabolic response to decreased intracellular GTP and/or CTP pools. Antibodies to RR were found to develop in some hepatitis C patients treated with interferon-α and ribavirin. Additionally, the presence of anti-RR antibodies was correlated with poor treatment outcome.

Glutamine deprivation initiates reversible assembly of mammalian rods and rings.

Rods and rings (RR) are protein assemblies composed of cytidine triphosphate synthetase type 1 (CTPS1) and inosine monophosphate dehydrogenase type 2 (IMPDH2), key enzymes in CTP and GTP biosynthesis. Small-molecule inhibitors of CTPS1 or IMPDH2 induce RR assembly in various cancer cell lines within 15 min to hours. Since glutamine is an essential amide nitrogen donor in these nucleotide biosynthetic pathways, glutamine deprivation was examined to determine whether it leads to RR formation. HeLa cells cultured in normal conditions did not show RR, but after culturing in media lacking glutamine, short rods (<2 µm) assembled after 24 h, and longer rods (>5 µm) formed after 48 h. Upon supplementation with glutamine or guanosine, these RR underwent almost complete disassembly within 15 min. Inhibition of glutamine synthetase with methionine sulfoximine also increased RR assembly in cells deprived of glutamine. Taken together, our data support the hypothesis that CTP/GTP biosynthetic enzymes polymerize to form RR in response to a decreased intracellular level of glutamine. We speculate that rod and ring formation is an adaptive metabolic response linked to disruption of glutamine homeostasis.

Differential reactivity to IMPDH2 by anti-rods/rings autoantibodies and unresponsiveness to pegylated interferon-alpha/ribavirin therapy in US and Italian HCV patients.

PURPOSE: Autoantibodies to cytoplasmic structures called rods and rings (RR) are primarily specific to patients with hepatitis C virus (HCV) infection treated with pegylated interferon-alpha/ribavirin (IFN/R). Our aim is to examine anti-RR antibodies specificity and correlation with the response to IFN/R therapy in two independent cohorts (US and Italy) of HCV patients. METHODS: Sera from the US cohort (n = 47) and the Italian cohort (n = 46) pre-selected for anti-RR antibodies were analyzed by immunofluorescence and radioimmunoprecipitation. The prevalence and titers of anti-RR were analyzed for correlation with the response to IFN/R therapy. RESULTS: In the US cohort, anti-RR antibodies were more frequently non-responders to IFN/R (71 % vs 29 % responders). Titers in responder patients (n = 11) were ≤1:3200, whereas titers in non-responder patients (n = 27) reached 1:819,200 (p = 0.0016). In the Italian cohort, anti-RR titers ranged from 1:200 to >1:819,200 and only relapsers had the highest anti-RR titers. Radioimmunoprecipitation demonstrated that anti-RR autoantibodies were mainly anti-inosine monophosphate dehydrogenase 2 (IMPDH2) - 96 % in the Italian cohort vs. 53 % in the US cohort. CONCLUSIONS: In the two cohorts analyzed, the anti-IMPDH2 response as a component of the anti-RR response is much more prominent in the Italian cohort. The reason for the difference between the US and Italian cohorts is unclear but it possibly illustrates the heterogeneity in response and the overall negative correlation between the production of these autoantibodies and response to IFN/R therapy. Patients with high titer anti-RR antibodies are either relapsers (Italian) or non-responders/relapsers (US).