"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.

Combined 13-valent pneumococcal conjugate and 23-valent pneumococcal polysaccharide vaccine regimens for adults with systemic lupus erythematosus: Does the sequence of pneumococcal vaccination affect immunogenicity responses? A single-center cohort study in Brazil.

OBJECTIVE: A combination of 13-valent pneumococcal conjugate vaccine (PCV13) and 23-valent pneumococcal polysaccharide vaccine (PPSV23) is currently recommended for adults with systemic lupus erythematosus (SLE). However, data on the immunogenicity elicited by sequential pneumococcal vaccination in this patient population are scarce. In this study, we compared short-term antibody responses to both PCV13/PPSV23 (≥8-week interval) and PPSV23/PCV13 (≥12-month interval) vaccination strategies in pneumococcal vaccine-naive adults with SLE. METHODS: This longitudinal, open-label, quasi-randomized study was performed in a single-center cohort of adults (18 years or older) with SLE. In both vaccination groups, blood samples were collected immediately before administering the first dose of the pneumococcal vaccine (timepoint T0), 4-6 weeks after the priming dose (T1), and 4-6 weeks after the booster dose (T2). We focused on the 12 shared serotypes between PCV13 and PPSV23, and compared the following immunogenicity outcomes between the groups at T2: anti-pneumococcal antibody geometric mean concentration (ApAb GMC), fold increase in ApAb levels (FI-ApAb), overall seroprotection rate, and overall seroconversion rate. The protective level for each pneumococcal serotype was set at 1.3 µg/mL. We used the multi-analyte immunodetection method to determine serum levels of ApAbs. RESULTS: Thirty-four patients with SLE were screened between April 2019 and January 2020, and 16 of them (mean age: 39.4 years, 87.5% female, and 100% on immunosuppressants) had evaluable immunogenicity results at T2. The median time elapsed between the pneumococcal vaccinations was 56 days in the PCV13/PPSV23 group (n = 11 patients) and 16 months in the PPSV23/PCV13 group (n = 5 patients). Priming with PCV13 (PCV13/PPSV23 group), as opposed to PPSV23 (PPSV23/PCV13 group), yielded significantly better results regarding FI-ApAb, overall seroconversion rate, and overall seroprotection rate 4-6 weeks after each pneumococcal vaccination. A trend toward augmented ApAb GMC in the patients who received the PCV13/PPSV23 sequence was also observed. No relevant safety issues were identified with sequential pneumococcal vaccination. CONCLUSION: The PCV13-priming PPSV23-boost strategy in adults with SLE induced greater antibody responses for most immunogenicity outcomes than those elicited by the PPSV23/PCV13 strategy.

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.

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.

Diagnostic power and clinical impact of exome sequencing in a cohort of 500 patients with rare diseases.

Rare diseases comprise a diverse group of conditions, most of which involve genetic causes. We describe the variable spectrum of findings and clinical impacts of exome sequencing (ES) in a cohort of 500 patients with rare diseases. In total, 164 primary findings were reported in 158 patients, representing an overall diagnostic yield of 31.6%. Most of the findings (61.6%) corresponded to autosomal dominant conditions, followed by autosomal recessive (25.6%) and X-linked (12.8%) conditions. These patients harbored 195 variants, among which 43.6% are novel in the literature. The rate of molecular diagnosis was considerably higher for prenatal samples (67%; 4/6), younger children (44%; 24/55), consanguinity (50%; 3/6), gastrointestinal/liver disease (44%; 16/36) and syndromic/malformative conditions (41%; 72/175). For 15.6% of the cohort patients, we observed a direct potential for the redirection of care with targeted therapy, tumor screening, medication adjustment and monitoring for disease-specific complications. Secondary findings were reported in 37 patients (7.4%). Based on cost-effectiveness studies in the literature, we speculate that the reports of secondary findings may influence an increase of 123.2 years in the life expectancy for our cohort, or 0.246 years/cohort patient. ES is a powerful method to identify the molecular bases of monogenic disorders and redirect clinical care.

HLA-B*51 and its main subtypes in Brazilian patients with Behçet's disease.

OBJECTIVES: This study aimed to evaluate the frequency of HLA-B*51 and its subtypes in Brazilian patients with Behçet's disease (BD) and healthy controls (HC) and to assess possible associations with disease manifestations. METHODS: A cross-sectional study with sequential BD patients and HC. HLAB*51 presence was determined by sequence-specific polymerase chain reaction (SSP-PCR) and HLA-B*51 subtypes by the Sanger sequencing method. RESULTS: Eighty-three BD patients and 258 HC were evaluated. HLA-B*51 was found in 30.1% of DB patients and in 15.5% of HC (p=0.003). The most prevalent subtypes in DB patients were HLA-B*51:01 (60.0%), HLA-B*51:08 (20.0%), HLA-B*51:22 (8.0%), HLAB* 51:29 (8.0%) and HLA-B*51:02 (4.0%), while HLA-B*51:01 (77.5%) and HLA-B*51:55 (7.5%) were the most prevalent in HC. HLA-B*51 was less frequently found in patients with neurologic involvement (8.0% vs. 29.3%; p=0.034) while HLAB*51:01 was more observed in patients with ocular involvement (93.3% vs. 60.3%; p=0.014). No BD patient with neurologic or vascular involvement presented HLA-B*51:01. HLAB*51:08 was more frequent in patients with vascular manifestations (60.0% vs. 15.4%; p=0.012). In multivariate analysis, HLA-B*51 was an independent risk factor for BD (OR=2.410; 95%CI: 1.332-4.361; p=0.004) and HLA-B*51:08 had an independent association with vascular manifestations of BD (OR = 14.843; 95%CI: 1.550 - 142.115; p=0.019). CONCLUSIONS: The prevalence of HLAB*51 is higher in Brazilian BD patients compared to HC, and it is a risk factor for BD. The HLA-B*51:08 subtype was independently associated with vascular manifestations of BD.

Changes in the result of antinuclear antibody immunofluorescence assay on HEp-2 cells reflect disease activity status in systemic lupus erythematosus.

Background The objective of the study was to determine whether the staining pattern and titer of indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA) are associated with systemic lupus erythematosus (SLE) disease activity. Methods A total of 269 consecutive patients meeting the ACR and SLICC criteria for SLE were classified into three groups according to the SLE Disease Activity Index 2000 (SLEDAI2K): Remission (SLEDAI2K = 0; n = 47); Intermediate (SLEDAI2K = 1-5; n = 111); Active (SLEDAI2K ≥ 6; n = 111). All subjects were assessed for HEp-2 IFA titer and staining pattern and nine traditional parameters of SLE disease activity. After a 6 to 12-month interval, 101 of the 269 patients were reassessed. Results HEp-2 IFA homogeneous nuclear pattern (AC-1) occurred more frequently in the Active Group compared to the Remission Group (p < 0.001). Fine speckled nuclear pattern (AC-4) tended to occur more frequently in the Remission Group compared to the Active Group (p = 0.054). Subjects with AC-1 pattern had higher SLEDAI (8.8 ± 7.6) than those with AC-4 (4.8 ± 5.2) (p < 0.001). HEp-2 IFA titer and anti-nuclear antibody by enzyme-linked immunosorbent assay (ANA-ELISA) values were lower in the Remission Group compared to the other two groups (p < 0.001). Multivariate analyses identified only ELISA anti-dsDNA as an independent variable associated with disease activity. In follow-up analysis, HEp-2 IFA titer decreased significantly in the 33 subjects with decreased disease activity (p = 0.002). Receiver operator characteristic (ROC) curve analysis for determination of disease activity showed equivalent areas under the curve (AUC) for HEp-2 IFA titer and traditional disease activity parameters. Conclusions HEp-2 IFA pattern and titer can reflect SLE disease activity and may be considered in conjunction with other laboratory and clinical parameters in the assessment of SLE disease activity.

Clinical relevance of HEp-2 indirect immunofluorescent patterns: the International Consensus on ANA patterns (ICAP) perspective.

The indirect immunofluorescence assay (IIFA) on HEp-2 cells is widely used for detection of antinuclear antibodies (ANA). The dichotomous outcome, negative or positive, is integrated in diagnostic and classification criteria for several systemic autoimmune diseases. However, the HEp-2 IIFA test has much more to offer: besides the titre or fluorescence intensity, it also provides fluorescence pattern(s). The latter include the nucleus and the cytoplasm of interphase cells as well as patterns associated with mitotic cells. The International Consensus on ANA Patterns (ICAP) initiative has previously reached consensus on the nomenclature and definitions of HEp-2 IIFA patterns. In the current paper, the ICAP consensus is presented on the clinical relevance of the 29 distinct HEp-2 IIFA patterns. This clinical relevance is primarily defined within the context of the suspected disease and includes recommendations for follow-up testing. The discussion includes how this information may benefit the clinicians in daily practice and how the knowledge can be used to further improve diagnostic and classification criteria.

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.

The effects of freeze/thawing on the function and phenotype of CD4(+) lymphocyte subsets in normal individuals and patients with systemic lupus erythematosus.

Several studies report on lymphocyte phenotypic and functional abnormalities in Systemic Lupus Erythematosus (SLE). Freezing and thawing may alter functional and phenotypic properties of cells. We assessed the effect of the freezing/thawing process (F/T) on Th1 (CD3(+)CD4(+)CCR4(-)CXCR3(+)CCR5(+)), Th2 (CD3(+)CD4(+)CCR5(-)CXCR3(-)CCR4(+)), Th17 (CD3(+)CD4(+)CCR6(+)CD161(+)), and Treg (CD3(+)CD4(+)CD25(high)CD127(-)) cell cultures in healthy controls and SLE patients. F/T was associated with decreased frequency of Th2 and Th17 cells in cultures from SLE patients but not from controls. F/T was also associated with increased frequency of apoptotic cells, as measured by annexin V labeling, in all T cell subtypes analyzed, as well as increased cell proliferation, as measured by Ki-67 labeling, in all cells except Th1 from SLE patients. Thus, F/T can have differentiated effects on T lymphocyte subtypes from SLE patients and controls, and can have significant effects on cell death and proliferation. These findings should be carefully considered when designing and interpreting studies on functional and phenotypic aspects of T lymphocytes in SLE.

International recommendations for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies.

Anti-nuclear antibodies (ANA) are fundamental for the diagnosis of autoimmune diseases, and have been determined by indirect immunofluorescence assay (IIFA) for decades. As the demand for ANA testing increased, alternative techniques were developed challenging the classic IIFA. These alternative platforms differ in their antigen profiles, sensitivity and specificity, raising uncertainties regarding standardisation and interpretation of incongruent results. Therefore, an international group of experts has created recommendations for ANA testing by different methods. Two groups of experts participated in this initiative. The European autoimmunity standardization initiative representing 15 European countries and the International Union of Immunologic Societies/World Health Organization/Arthritis Foundation/Centers for Disease Control and Prevention autoantibody standardising committee. A three-step process followed by a Delphi exercise with closed voting was applied. Twenty-five recommendations for determining ANA (1-13), anti-double stranded DNA antibodies (14-18), specific antibodies (19-23) and validation of methods (24-25) were created. Significant differences between experts were observed regarding recommendations 24-25 (p<0.03). Here, we formulated recommendations for the assessment and interpretation of ANA and associated antibodies. Notably, the roles of IIFA as a reference method, and the importance of defining nuclear and cytoplasmic staining, were emphasised, while the need to incorporate alternative automated methods was acknowledged. Various approaches to overcome discrepancies between methods were suggested of which an improved bench-to-bedside communication is of the utmost importance. These recommendations are based on current knowledge and can enable harmonisation of local algorithms for testing and evaluation of ANA and related autoantibodies. Last but not least, new more appropriate terminologies have been suggested.

Clinical profiles and treatment outcomes of outpatients with interstitial lung disease and mechanic's hands: A retrospective and observational cohort.

Idiopathic inflammatory myopathies, especially antisynthetase syndrome, often appear outside of the muscles as interstitial lung disease (ILD). Another typical finding is the presence of mechanic's hands. The aim of the present study was to describe the clinical, functional, tomographic, and serological data of patients with ILD and mechanic's hands and their response to treatment and survival rates. This is a retrospective study of ILD with concurrent myopathy. Among the 119 patients initially selected, 51 had mechanic's hands. All the patients were screened for anti-Jo-1 antibodies. An expanded panel of myopathy autoantibodies was also performed in 27 individuals. Of the 51 patients, 35 had 1 or more antibodies. The most common were anti-Jo-1, anti-PL-7, and anti-PL-12, while of the associated antibodies, anti-Ro52 was present in 70% of the 27 tested individuals. A significant response to treatment was characterized by an increase in predicted forced vital capacity (FVC) of at least 5% in the last evaluation done after 6 to 24 months of treatment. A decrease in predicted FVC of at least 5%, the need for oxygen therapy, or death were all considered treatment failures. All patients were treated with corticosteroids, and 71% with mycophenolate. After 24 months, 18 patients had an increase in FVC, 11 had a decrease, and 22 remained stable. After a median follow-up of 58 months, 48 patients remained alive and three died. Patients with honeycombing on high-resolution chest tomography (log-rank = 34.65; P < .001) and a decrease in FVC ≥5% (log-rank = 18.28, P < .001) had a poorer survival rate. Patients with ILD and mechanic's hands respond well to immunosuppressive treatment.

CD4 T lymphocyte subsets display heterogeneous susceptibility to apoptosis induced by serum from patients with systemic lupus erythematosus.

BACKGROUND: Serum from systemic lupus erythematosus (SLE) patients has been shown to induce T-lymphocyte (TL) apoptosis. Given that different cells of the immune system display different sensitivity to apoptosis, we set to evaluate the in vitro effect of SLE serum on regulatory T-cells (Treg), Th17, Th1 and Th2 from SLE patients and healthy controls. METHODS: Peripheral blood mononuclear cells from SLE patients or normal controls were exposed to a pool of sera from SLE patients or normal controls. Annexin V was used to label cells in apoptosis or necrosis. Annexin V-labeled Treg, Th17, Th1 and Th2 cells were determined using flow cytometry. RESULTS: Total CD3 + and CD4 + cells from SLE patients showed higher frequency of spontaneous apoptosis/necrosis, whereas Th1 cells from SLE patients presented reduced spontaneous apoptosis/necrosis rate as compared with cells from controls. Incubation with SLE serum induced increased frequency of apoptotic/necrotic CD3 + , CD4 + and Th2 cells from normal controls or from SLE patients as compared with cultures incubated with normal human serum (NHS) or without human serum at all. Incubation with SLE serum did not increase the apoptosis/necrosis rate in Th1 or Th17 cells. Treg cells from SLE patients were more prone to apoptosis/necrosis induced by SLE serum than Treg cells from normal individuals. Th1, Th2, and Th17 cells presented increased apoptosis rates in cultures without human serum. CONCLUSION: Our findings indicate that the serum of patients with active SLE stimulates apoptosis of CD4 + T cells in general and exhibit differentiated effects on CD4 + T-cell subsets.