Carlos Alberto von Mühlen

Induction of Cytoplasmic Rods and Rings Structures by Inhibition of the CTP and GTP Synthetic Pathway in Mammalian Cells

Background Cytoplasmic filamentous rods and rings (RR) structures were identified using human autoantibodies as probes. In the present study, the formation of these conserved structures in mammalian cells and functions linked to these structures were examined. Methodology/Principal Findings Distinct cytoplasmic rods (∼3–10 µm in length) and rings (∼2–5 µm in diameter) in HEp-2 cells were initially observed in immunofluorescence using human autoantibodies. Co-localization studies revealed that, although RR had filament-like features, they were not enriched in actin, tubulin, or vimentin, and not associated with centrosomes or other known cytoplasmic structures. Further independent studies revealed that two key enzymes in the nucleotide synthetic pathway cytidine triphosphate synthase 1 (CTPS1) and inosine monophosphate dehydrogenase 2 (IMPDH2) were highly enriched in RR. CTPS1 enzyme inhibitors 6-diazo-5-oxo-L-norleucine and Acivicin as well as the IMPDH2 inhibitor Ribavirin exhibited dose-dependent induction of RR in >95% of cells in all cancer cell lines tested as well as mouse primary cells. RR formation by lower concentration of Ribavirin was enhanced in IMPDH2-knockdown HeLa cells whereas it was inhibited in GFP-IMPDH2 overexpressed HeLa cells. Interestingly, RR were detected readily in untreated mouse embryonic stem cells (>95%); upon retinoic acid differentiation, RR disassembled in these cells but reformed when treated with Acivicin. Conclusions/Significance RR formation represented response to disturbances in the CTP or GTP synthetic pathways in cancer cell lines and mouse primary cells and RR are the convergence physical structures in these pathways. The availability of specific markers for these conserved structures and the ability to induce formation in vitro will allow further investigations in structure and function of RR in many biological systems in health and diseases.

Evaluation of Multiplexed Fluorescent Microsphere Immunoassay for Detection of Autoantibodies to Nuclear Antigens

ABSTRACT Antibodies to extractable nuclear antigens (ENA) are found in a variety of collagen vascular diseases. Determining the individual specificities of these antibodies is extremely useful in establishing the disease diagnosis and in some cases the prognosis. With a multiplexed fluorescent microsphere immunoassay, reactivity to five of the most diagnostically useful ENA was measured in 249 serum samples, including samples from 56 patients previously documented to have systemic lupus erythematosus (SLE). Results of the multiplexed assay were compared to results from established ENA enzyme-linked immunosorbent assays (ELISAs), and the agreement, sensitivity, and specificity, respectively, for the five ENA evaluated were as follows: SSA, 99.1, 100.0, and 98.8%; SSB, 98.6, 88.9, and 99.5%; Sm, 97.6, 95.8, and 97.9%; RNP, 97.2, 92.7, and 98.8%; Scl-70, 93.6, 50.0, and 99.0%. In the 56 confirmed SLE patients, the frequency of significant concentrations of autoantibodies with the multiplexed assay was 21.4% for SSA, 7.1% for SSB, 10.7% for Sm, 32.1% for RNP, and 0% for Scl-70. The new flow cytometric bead-based multiplexed assay showed excellent correlation with the well-established single-analyte ELISA methods for four of five the ENA markers investigated in this study. The most notable discrepancies between the two assays were for the Scl-70 antigen, which was most often resolved in favor of the multiplexed assay. Our studies show that the multiplexed microsphere-based immunoassay is a sensitive and specific method for the detection and semiquantitation of ENA antibodies in human sera.

International multicenter evaluation of autoantibodies to ribosomal P proteins.

ABSTRACT Autoantibodies to the ribosomal phosphoproteins (Rib-P) are a serological feature of patients with systemic lupus erythematosus (SLE). The reported prevalence of anti-Rib-P antibodies in SLE ranges from 10 to 40%, being higher in Asian patients. The variation in the observed frequency may be related to a number of factors but is dependent in large part on the test system used to detect the autoantibodies. An association of anti-Rib-P with central nervous system involvement and neuropsychiatric manifestations of SLE has been controversial. In the present international multicenter study, we evaluated the clinical accuracy of a new sensitive Rib-P-specific enzyme-linked immunosorbent assay based on recombinant Rib-P polypeptides. The results showed that 21.3% of 947 SLE patients, but only 0.7% of 1,113 control patients, had a positive test result (P < 0.0001). The sensitivity, specificity, positive and negative predictive values, and diagnostic efficiency were determined to be 21.3%, 99.3%, 95.6%, 62.2%, and 65.3%, respectively. When evaluated in the context of participating centers, the prevalence of anti-Rib-P antibodies was found in descending frequency, as follows: China (35%) > Poland (34%) > Japan (28%) > United States (26%) > Germany (Freiburg; 23.3%) > Denmark (20.5%) > Germany (Berlin; 19%) > Mexico (15.7%) > Israel (11.7%) > Brazil (10%) > Canada (8%). The substantial data from this study indicate that the prevalence of anti-Rib-P antibodies may not be restricted to the genetic background of the patients or to the detection system but may depend on regional practice differences and patient selection. We confirm previously reported associations of antiribosomal antibodies with clinical symptoms and serological findings. Remarkably, we found a lower occurrence of serositis in Rib-P-positive lupus patients.

Report of the First International Consensus on Standardized Nomenclature of Antinuclear Antibody HEp-2 Cell Patterns 2014–2015

During the 12th International Workshop on Autoantibodies and Autoimmunity held in Sao Paulo, Brazil, on August 28, 2014, a full day session was devoted to establishing a consensus on the nomenclature of staining patterns observed in the antinuclear antibody (ANA) indirect immunofluorescence test on HEp-2 cells. The current report summarizes the collective agreements with input from the host Brazilian and international communities that represented research, clinical, and diagnostic service laboratories. Patterns are categorized in three major groups (nuclear, cytoplasmic, and mitotic patterns) and each pattern has been defined and described in detail. The consensus nomenclature and representative patterns are made available online at the international consensus on antinuclear antibody pattern (ICAP) website (www.ANApatterns.org). To facilitate continuous improvement and input, specific comments on ICAP are encouraged and these will be discussed in subsequent ICAP meetings. The ultimate goal with the establishment of the ICAP is to promote harmonization and understanding of autoantibody test nomenclature, as well as interpretation guidelines for ANA testing, thereby optimizing usage in patient care.

Postmenopausal Estrogen and Increased Risk of Clinical Osteoarthritis at the Hip, Hand, and Knee in Older Women

OBJECTIVE We examined postmenopausal estrogen (PME) use and prevalence of clinical osteoarthritis (OA) at the hand, knee, and hip in 1001 community-dwelling postmenopausal women aged 43-97 years (mean age 72). METHODS OA at the hip, hand, and knee was defined by validated and standardized criteria based on pain history plus a clinical examination performed by a specially trained nurse. RESULTS PME, validated by examination of pills and prescriptions, had been used for at least 1 year by 638 women (63.4%) for an average duration of 14.6 (+/-10.6) years. OA prevalence was 34.5% among women who had used PME for at least 1 year and 30.9% among women who did not use PME (age adjusted p = 0.02). Knee OA prevalence did not differ by PME use (p > 0.05). A significantly larger proportion of women who used PME for at least 1 year had hip and hand OA compared with women not using PME (4.1% vs. 1.1%, age-adjusted p = 0.002, and 15.8% vs. 13.5%, age-adjusted p = 0.02, respectively). In analyses adjusted for the potential confounding effects of age, body mass index (BMI), smoking, exercise, and type of menopause, women who used PME still were more likely to have hip OA (odds ratio [OR] = 5.03, confidence interval [CI] = 1.70-14.84, p = 0.003) and hand OA ([OR] = 1.57, CI = 1.05-2.33, p = 0.03). Among estrogen users, duration of PME use was longer for women with OA than for women without OA (16 vs. 11 median years, p = 0.01). CONCLUSIONS PME is associated with a higher prevalence of clinical OA.

EVALUATION OF IMMUNE PARAMETERS IN DEPRESSED PATIENTS

The association between depression and altered immunological activities has repeatedly been suggested, but experimental data show contradictory results. In this work, cellular and humoral immunological activities were evaluated in patients presenting major depression, unipolar subtype. Natural killer cell activity (NKCA) was significantly reduced in patients as compared to healthy controls (p < 0.001). However, lymphocyte mitogenic responses and immunoglobulin titers (IgG, IgM, and IgA) were similar for all samples. Hematological, hormonal, and nutritional variables presented normal values in patients and in controls. A familial history of depression was related to lower NKCA and higher phytohemagglutinin responses (p < 0.01). These data suggest possible differential inhibition of cellular immune responses in depressed patients.

Cytoplasmic rods and rings autoantibodies developed during pegylated interferon and ribavirin therapy in patients with chronic hepatitis C.

BACKGROUND Serum autoantibodies are frequently detected in patients with chronic HCV infection, reflecting the wide spectrum of immune reactions related to this virus. In the present study, a novel autoantibody to cytoplasmic rods and rings (RR) in chronic HCV patients was characterized. METHODS Sera from 75 previously untreated HCV patients were investigated by indirect immunofluorescence using HEp-2 cell substrate before and during pegylated interferon (PEG-IFN)/ribavirin (RBV) therapy. HEp-2 cells were cultured and fixed either following standard protocols or with the addition of RBV in culture medium. RESULTS In 15 out of 75 (20%) patients, analysis revealed the presence of antibodies to rod-like cytoplasmic structures ranging approximately 3-10 μm in length and rings approximately 2-5 μm in diameter. These RR structures became detectable in >95% of cells after addition of RBV in culture medium, whereas they were absent in untreated cells. Anti-RR antibodies were found in sera collected during PEG-IFN/RBV treatment only, but never detected before antiviral therapy nor in control groups. More importantly, these anti-RR antibodies were more often detected in non-responder/relapsers than in responder patients (33% versus 11%; P-value =0.037). CONCLUSIONS An RBV-induced autoantibody was identified to a new cytoplasmic autoantigenic structure developed in HCV patients after PEG-IFN/RBV and this same structure can be induced by RBV in in vitro culture. Owing to the onset of anti-RR antibodies in PEG-IFN/RBV-treated patients and their association with a treatment failure, studies are deemed necessary to clarify whether anti-RR plays a role in the response to PEG-IFN/RBV therapy.

Antibodies to the atherosclerotic plaque components beta2-glycoprotein I and heat-shock proteins as risk factors for acute cerebral ischemia

One third of cases of cerebral ischemia have no clear etiology. A humoral response to the atherosclerotic plaques components beta2-glycoprotein l (beta2-gpl) and heat-shock proteins (Hsp) might be involved in the pathogenesis of stroke. This case-control study includes a complete profile of anti-beta2-gpl antibodies and testing of IgG antibodies to the 60/65 kilodaltons (kDa) Hsp in stroke patients. Ninety-three patients with acute ischemic stroke and 93 controls were evaluated for age, sex, race, hypertension, smoking, previous cardiopathy, diabetes mellitus, hypercholesterolemia and previous history of cerebral ischemia. lgG/lgM/lgA anticardiolipin (aCL) and anti-beta2-gpl antibodies, as well as lgG antibodies to human 60 kDa Hsp and to Mycobacterium bovis 65 kDa Hsp, were detected by immunoassay. Adjusted odds ratios (OR) were calculated by logistic regression. The adjusted OR for IgA anti-beta2-gpl antibodies was 4.6 (90%Cl 1.5 to 14.3; p = 0.025). The non-adjusted OR for IgG antibodies to Hsp 60 was 26.1. The adjusted OR for IgG antibodies to Hsp 65 was 3.2 (90%Cl 1.2 to 8.3; p = 0.044). The adjusted OR for lgG to any Hsp (60 or 65) was 4.8 (90%Cl 1.9 to 12.1; p = 0.006). This study demonstrates that elevated IgA anti-beta2-gpl and lgG anti-Hsp 60/65 antibodies are associated with increased risk of ischemic stroke. The association occurred independently of other risk factors. This humoral response might link autoimmunity, thrombophilia and atherosclerosis in stroke patients.

Anticorpos contra beta2-glicoproteína I como fatores de risco para infarto agudo do miocárdio

OBJECTIVE: To determine whether high levels of antibodies against the phospholipid beta2-glycoprotein I (beta2-gpI) cofactor are associated with an increase in the risk of acute myocardial infarction. METHODS: The study comprised 82 patients with acute myocardial infarction and 82 controls, who were assessed in regard to age, sex, race, hypertension, smoking, previous heart disease, history of diabetes mellitus, and hypercholesterolemia. The following antibodies were detected using immunoassay: anticardiolipin and anti-beta2-gpI IgA, IgG, and IgM. Adjusted odds ratios (OR) for risk factors were obtained through logistic regression. RESULTS: The mean ages of the cases and controls were, respectively, 57.7 and 51.1 years (P=0.003). Men (P=0.005) and the white race predominated in both groups (P=0.798). Of the risk factors, a history of diabetes (OR=5.3; 95% CI: 1.9 to 14.9; P=0.001) and previous heart disease (OR=4.7; 95% CI: 2.0 to 10.7; P<0.001) were the most consistent associations with myocardial infarction. The frequency of anticardiolipin IgG, IgM, and IgA antibodies did not differ between cases and controls (P=1.000). Anti-beta2-gpI IgA antibodies were more frequent in cases than in controls (P=0.054). The adjusted OR for anti-beta2-gpI IgA antibodies was 3.4 (95% CI: 1.3 to 9.1; P=0.015). CONCLUSION: Anti-beta2-gpI IgA antibodies, but not anticardiolipin antibodies, seemed to behave as independent risk factors for myocardial infarction, which may represent a link between autoimmunity and atherosclerosis in patients with acute myocardial infarction.

Giantin is the major Golgi autoantigen in human anti-Golgi complex sera

Anti-Golgi complex antibodies (AGAs) are primarily associated with systemic lupus erythematosus and Sjögrens syndrome. Here we report on the immunoreactivity of AGAs against five Golgi autoantigens (giantin, golgin-245, golgin-160, golgin-95/GM130, and golgin-97) and provide data from epitope mapping on the most common Golgi autoantigen, namely giantin. A total of 80 human sera containing AGAs, as defined by indirect immunofluorescence on HEp-2 cells, were analyzed by ELISA using recombinant autoantigens and immunoprecipitation. The proportion of AGA sera that reacted with the five Golgi autoantigens was correlated with the molecular mass of the Golgi antigens. Autoantibodies to giantin, the largest Golgi autoantigen, were the predominant AGAs, being found in 50% of the AGA sera. Epitope mapping of giantin was performed using six recombinant fragments spanning the entire protein. Antigiantin-positive sera with low titer autoantibodies recognized epitopes in the carboxyl-terminal fragments that are proximal to the Golgi membrane, whereas higher titer sera exhibited strong reactivity to amino-terminal and central domains that are likely to extend from the Golgi membrane into the cytoplasm. Our working hypothesis is that aberrantly expressed Golgi complex autoantigens may be released into the immune system when cells undergo lysis. By virtue of a carboxyl-terminal transmembrane domain, giantin is likely to be more stably associated with the cytoplasmic face of the Golgi complex than are other golgins, which are peripheral proteins. The stable association of giantin with the putative released Golgi complex may contribute to its preferential autoantigenicity.