anti-DNA Antibodies Research Articles

Abstract 1345: ATM kinase activity is dispensable in mitochondrial autophagy

Abstract Ataxia telangiectasia mutated (ATM), a critical DNA damage sensor with protein kinase activity, is frequently deleted or mutated in human cancers including mantle cell lymphoma (MCL). Loss of ATM protein is associated with accumulation of nonfunctional mitochondria and defective mitophagy, in both murine thymocytes and in non-cancer cells, however, the molecular mechanism of ATM kinase in cancer cell mitophagy is unknown. Here, we provide evidence that mitophagy in B-Cell Lymphoma including MCL (Granta-519, Jeko-1 and Mino), HeLa and other cancer cell lines is dependent on ATM but independent of its kinase function. Routine ATM kinase activity was performed by FACS analysis following either neocarzinostatin or IR-induced PE-ATMSer1981 and FITC-H2AXSer139 phosphorylations status and reconfirmed by immunoblot assay by probing with phospho-ATMSer1981and ATM targets Kap1Ser824 or Smc1Ser966 phosphorylations. Stable knockdown of ATM in ATM proficient MCL cell lines conferred resistance to mitophagy and was associated with reduced ATP production, oxygen consumption, and increased mitochondrial ROS. ATM protein interacts with the E3 ubiquitin ligase Parkin in a kinase-independent manner in multiple cancer cell lines including MCL and HeLa, while stable knockdown of ATM in HeLa cells (Kd HeLa) resulted in proteasomal degradation of GFP-Parkin which was rescued by the proteasome inhibitor, MG132 suggesting that ATM-Parkin interaction is important for Parkin stability. Confocal analysis revealed the presence of extra-nuclear ATM in both cytoplasm and mitochondrial compartments in HeLa cells and CCCP-induced mitophagy resulted in significantly higher mitochondrial ATM-Tom20 colocalization compared to DMSO control. Moreover both cell fractionation and confocal analysis revealed more GFP-Parkin translocation and loss of Tom20 expression in the mitochondrial fraction in WT cells following CCCP treatment. In contrast, mitochondrial GFP-Parkin expression was undetectable in Kd HeLa cells indicating a specific defect in Parkin translocation in the absence of ATM during CCCP-induced mitophagy. Further, confocal analysis with anti-DNA antibody indicated significantly higher mass of mitochondrial nucleoids in Kd HeLa cells compared to WT controls suggesting that loss of ATM promotes mtDNA accumulation. Neither loss of ATM kinase activity in primary B cell lymphomas nor inhibition of ATM kinase by KU60019 in MCL, non-tumor A-T fibroblasts and HeLa cell lines rescued FCCP or CCCP-induced mitophagy signifying that ATM catalytic activity is dispensable for mitophagy. Malignant primary B-cell lymphomas including MCL without detectable ATM, Parkin, Pink1, and Parkin-Ubser65 phosphorylation were resistant to mitophagy. This data provides the first molecular evidence of the role of ATM kinase function in mitophagy in cancer. Citation Format: Aloke Kumar Sarkar, Varsha Gandhi. ATM kinase activity is dispensable in mitochondrial autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1345.

Systemic lupus erythematosus, gender differences in Colombian patients.

The objective of this study was to compare the clinical and immunological presentation of SLE between males and females in a Colombian SLE population. A cross-sectional, retrospective study was performed that evaluated patients with SLE over 6years. The dependent variables were systemic complications, duration of hospitalization, readmission, and death. Descriptive, group comparison, bivariate, and multivariate analysis were performed using Stata 12.0 software 200 patients were included in this study, 84.5% were females and 15.5% males. Longer hospitalizations, readmissions, respiratory compromise, higher activity disease (ECLAM score), smoking, and use of cyclophosphamide in the past 3months were more prevalent in males. In the bivariate and multivariate analysis, we found an increased risk in males of respiratory symptoms (OR 3.35), anti-DNA antibody (OR 2.46), smoking (OR 4.2), cyclophosphamide use (OR 3.23), chronic pulmonary alterations (OR 2.51), readmission (OR 2.88), long hospitalization (OR 3.12), and death (OR: 3.31). This is the first study that shows the differences related to gender in Colombian SLE patients. Males with SLE have more disease activity compare with females. Also, we found that males have more risk of pulmonary impairment, longer hospitalizations, hospital readmissions, and deaths.

Childhood-Onset Systemic Lupus Erythematosus: A Cohort Study.

To determine the clinical and immunological characteristics and short-term outcome of children with systemic lupus erythematosus (SLE). A descriptive cohort study. Paediatric Rheumatology Clinic, The Aga Khan University Hospital, Karachi, from January 2011 to December 2015. Clinical and immunological profile and short-term outcome of children less than 16 years of age admitted in the paediatric ward, with the diagnosis of SLE was studied. Demographic data, clinical presentation, laboratory findings, immunological profile and treatment regimens of these children were evaluated. Thirty-two children, satisfying the criteria of American College of Rheumatology (ACR) for SLE, were enrolled during the study period of five consecutive years. A female predominance was observed with 28 (87.5%) patients being female (F:M 7:1). Mean age at symptom onset was 10.5 +2.7 years; and 8.8 +2.1 years in females and males, respectively. The mean age at diagnosis was 11.3 +2.8 years in females and 9.4 +1.9 years in males. Prolonged fever was the most common non-specific symptom found in 27 (84%), followed by pallor in 13 (41%) patients. Twenty-two (69%) children were found to be anemic and 18 patients (56%) having signs of arthritis at presentation. Renal involvement was observed in 15 (47%) patients. The most common laboratory finding was anemia, found in 22 (69%) of cases. The most common immunological markers were serum anti-neutrophil antibodies (ANA), positive in 28 (88%) patients, followed by anti double-stranded DNA antibodies, raised in 26 (81%) of cases. Out of 32, 12 patients were lost to follow-up. Of the remaining 20 children who were followed for four years, ten (50%) went into remission. Childhood-onset SLE encompasses a wide variety of manifestations with a female preponderance. Fever, arthralgia and pallor are the most frequent clinical manifestations among the children. Hemolytic anemia (HA) is the most common laboratory abnormality, with ANA and anti ds-DNA antibodies positivity in the majority of padiatric patients.

Bacterial amyloids from biofilms break tolerance in lupus by simultaneous BCR/TLR signaling

Abstract Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease, in which infections are considered to play a pathogenic role, but the cellular and molecular culprits remain unknown. Bacterial biofilms are multicellular bacterial communities important in the establishment of chronic infection by pathogens. We have previously shown that curli fibers, functional bacterial amyloids present in Salmonella and E. coli biofilms, can complex with DNA. Systemic administration of these curli/DNA complexes accelerated autoimmunity in lupus-prone mice and induced autoantibody production in wild-type mice, suggesting curli/DNA complexes as novel players in SLE pathogenesis. This affect was diminished in TLR2 or TLR9 deficient mice. We present here evidence that curli/DNA complexes polyclonally activate B cells in vivo in wildtype mice, lupus-prone mice and 3H9 mice, the latter expressing an anti-DNA Ig heavy chain and whose B cells are normally tolerized. Interestingly, curli/DNA complexes can activate B cells in the absence of T cell help. Indeed, curli/DNA complexes induce polyclonal proliferation and activation in B cells purified from wild-type and lupus prone mice in vitro. Interestingly, curli/DNA complexes also induce isotype switching and aicda, the master regulator of class switch recombination, in the absence of T cells help in vitro. The induction of non-canonical NFkB in the absence of T cells suggests that the fibrillar structure of curli/DNA complexes can cross-link BCR, some recognizing DNA. Our results suggest that curli/DNA complexes may induce anti-DNA antibody production by simultaneous BCR/TLR signaling, which leads to B cell antibody production in the absence of T cell help.

A comprehensive phenotyping program for the MRL-lpr mouse lupus model

Abstract MRL/MpJ-Faslpr/J (MRL-lpr) mice develop an autoimmune disease resembling systemic lupus erythematosus, a disease affecting > 1.5 million Americans. The causative mutation, Faslpr, promotes survival of self-reactive lymphocytes, leading to immune proliferation, lymphadenopathy, emergence of anti-DNA antibodies, and fatal immune complex glomerulonephrosis. Like all spontaneous autoimmunity models, MRL-lpr colonies are susceptible to inadvertent selective pressure when mice with absent or delayed phenotypes are bred. We will explain a program for promoting genetic and phenotypic integrity within MRL-lpr breeding colonies. In addition to confirmation of genetic background and Faslpr genotype, breeder phenotypes were assessed at the conclusion of the breeding period. White blood cell counts (WBC) were used to measure lymphoproliferation: at 17 weeks of age, median WBC (103 cells/μL) was 14.2 for females (range = 3.4 – 97.3; N=155) and 10.5 for males (range = 3.7 – 51.4; N=125). Urinalysis to characterize renal damage at 18 weeks of age showed a median albumin/creatinine ratio of 623 for females (range = 1 – 22806; N=163) and 95 for males (range = 1 – 6013; N=123). Median spleen weights (grams) at 18 weeks of age were 0.5464 in females (range = 0.1112 – 2.1348; N=177) and 0.4410 in males (range = 0.2078 – 1.5516; N=135). We will describe a scoring system for clinical signs present at gross necropsy, including appearance of fur/skin, lymph nodes, and organs. We will show that phenotypic integrity can be ensured over time by breeding mice from lineages showing optimal disease development criteria. This program has value beyond breeder assessment, as it can be applied to MRL-lpr mice on study and at conclusion.

Lupus nephritis: the roles of C1q and C3 in preventing antibody mediated injury

Abstract Lupus nephritis (LN) is a common and serious complication of systemic lupus erythematosus (SLE). While defective clearance of apoptotic cells (AC) and immune complexes (IC) is implicated in LN pathogenesis, the precise way in which different complement components impact kidney protection and injury is unknown. To address this, we created C57BL/6 mice with defective clearance of AC (Mfge8 deficiency) and anti-chromatin antibodies (sle1 interval) that were also deficient in either C1q [C1q Triple mutant (C1qTM)] or C3 (C3TM). Both C1q and C3 TM mice spontaneously developed anti-DNA antibodies and glomerulonephritis associated with glomerular IgG deposits after the age of 6 months. To dissociate the effects of complement on B cells versus effects on the kidney, we examined the effects of antibody mediated kidney injury (nephrotoxic nephritis, NTN) in mice deficient in the clearance of AC with complement deficiency [double knockout (DKO) (Mfge8 C1q or Mfge8 C3) mice]. In NTN, 50% of C1q DKO and 25% of C3 DKO, but no Mfge8 KO, died after NTN induction and the surviving C1q DKO mice developed severe proteinuria. Surprisingly, both spontaneous and NTN-induced nephritis in C1q TM or C1q DKO had strong glomerular C3 and C3d staining, while strong staining for the membrane attack complex (MAC) was observed in C3 TM or C3 DKO mice. These findings indicate that: 1) C1q and C3 independently protect against kidney injury separate from their effects on B cell function 2) In the presence of excess AC and low C1q, antibody mediated kidney injury is associated with C3 activation, presumably by the lectin or alternative pathway and 3) In the presence of excess AC and low C3, antibodies can cause MAC activation by pathways that remain to be determined.

Systemic Lupus Erythematosus: Definitions, Contexts, Conflicts, Enigmas.

Systemic lupus erythematosus (SLE) is an inadequately defined syndrome. Etiology and pathogenesis remain largely unknown. SLE is on the other hand a seminal syndrome that has challenged immunologists, biologists, genetics, and clinicians to solve its nature. The syndrome is characterized by multiple, etiologically unlinked manifestations. Unexpectedly, they seem to occur in different stochastically linked clusters, although single gene defects may promote a smaller spectrum of symptoms/criteria typical for SLE. There is no known inner coherence of parameters (criteria) making up the disease. These parameters are, nevertheless, implemented in The American College of Rheumatology (ACR) and The Systemic Lupus Collaborating Clinics (SLICC) criteria to classify SLE. Still, SLE is an abstraction since the ACR or SLICC criteria allow us to define hundreds of different clinical SLE phenotypes. This is a major point of the present discussion and uses “The anti-dsDNA antibody” as an example related to the problematic search for biomarkers for SLE. The following discussion will show how problematic this is: the disease is defined through non-coherent classification criteria, its complexity is recognized and accepted, its pathogenesis is plural and poorly understood. Therapy is focused on dominant symptoms or organ manifestations, and not on the syndrome itself. From basic scientific evidences, we can add substantial amount of data that are not sufficiently considered in clinical medicine, which may change the paradigms linked to what “The Anti-DNA antibody” is—and is not—in context of the imperfectly defined syndrome SLE.

Transitional changes in the incidence of osteonecrosis in systemic lupus erythematosus patients: focus on immunosuppressant agents and glucocorticoids

The purpose of this study was to investigate transitional changes in the incidence of glucocorticoid-associated osteonecrosis in SLE patients, with a focus on immunosuppressive agent and glucocorticoid consumption. We retrospectively registered 185 SLE patients with 740 joints, who were newly diagnosed and hospitalized for initial high-dose glucocorticoid therapy from 1986 to 2015. Immunosuppressive agent, glucocorticoid dose, age, sex, organ lesion at hospitalization, complement (C3, C4, CH50) and anti-DNA antibody before initial glucocorticoid therapy, the frequency of use of anticoagulant and antilipidemic drugs, and incidence of osteonecrosis were documented. Based on trends in immunosuppressive agent use, 116 patients treated from 1986 to 1999, before calcineurin inhibitors were introduced, comprised the past group, and 69 patients treated from 2000 to 2015 comprised the recent group. Patient characteristics (age, sex and organ lesion at hospitalization, complement, anti-DNA antibody, the frequency of use of anticoagulant and antilipidemic drugs) were similar between groups. Glucocorticoid doses were significantly lower in the recent group than in the past group (highest daily glucocorticoid dose, 45.7 vs 59.0 mg/day, respectively; dose per weight, 0.88 vs 1.16 mg/day/kg, respectively; and cumulative dose at 3 months, 3118 vs 3985 mg). The incidence of osteonecrosis was significantly lower in the recent group than in the past group (26.4 vs 41.0%, respectively), particularly in the knee (25.4 vs 46.6%, respectively). The incidence of glucocorticoid-associated osteonecrosis in SLE patients decreased in association with a decrease in glucocorticoid administration after introduction of immunosuppressant agents.

Abstract WMP70: Yield of Screening Tests for Systemic Vasculitis in Ischemic Stroke in Young Adults

Background and Purpose: The causes of ischemic stroke in young adults are more heterogeneous than in the elderly. Sometimes ischemic stroke in young adults is complicated by a systemic vasculitis, which can involve the brain. Therefore, a panel for systemic vasculitis could be considered as one of the etiological work-up of ischemic stroke in young adults. However, little is known about the yield of screening tests for systemic vasculitis in young ischemic stroke patients. Methods: Consecutive patients aged 18 to 45 years with ischemic stroke between January 2011 and June 2017 were included. They all underwent screening tests for systemic vasculitis including rheumatoid factor (RF), antinuclear antibody (ANA), antineutrophil cytoplasmic antibodies (ANCAs), anticardiolipin antibody, lupus anticoagulant, antiphospholipid antibody, anti-DNA antibody, and anti-Ro/SSA and La/SSB antibodies. Results: Among consecutive 2,938 acute ischemic stroke patients during study period, 177 patients (6.0%) were aged 18-45 years. The mean age was 39.0 years. Vasculitis panel screening results were positive in three patients (1.7%). They all had other symptoms or signs of systemic vasculitis. Two female patients with positive ANA and anti-DNA antibody were finally diagnosed as systemic lupus erythematosus (SLE). One patient was compatible with the diagnosis of anti-phospholipid antibody syndrome, and the other patient’s stroke was thought to be related to SLE vasculitis. They all had clinical symptoms of SLE including malar rash and Raynaud phenomenon. One male patient showed positive pANCA. A year previously, he was diagnosed with asthma. Inflammation markers in peripheral blood exams were elevated (CRP 8.21 mg/dl, leukocyte count 17.7/nl), the rate of eosinophilic cells increased (42%). He fulfilled the criteria for the diagnosis of Churg-Strauss syndrome. Conclusions: In the absence of systemic symptoms or signs of vasculitis, vasculitis panel screening in young ischemic stroke patients has a low yield. It’s better to consider individual clinical features and cost effectiveness before screening tests for systemic vasculitis in ischemic stroke in young adults.

Age-associatedBcells expanded in autoimmune mice are memory cells sharing H-CDR3-selected repertoires.

Age-associated Bcells (ABCs) represent a distinct cell population expressing low levels of CD21 (CD21-/low ). The Ig repertoire expressed by ABCs in aged mice is diverse and exhibits signs of somatic hypermutation (SHM). A CD21-/low B-cell population is expanded in autoimmune diseases, e.g. systemic lupus erythematosus, as well as in lupus-prone NZB/W mice and in mice lacking a pre-Bcell receptor (SLC-/- ). However, the nature of the CD21-/low Bcells (hereafter ABCs) in autoimmunity is not well understood. Here we show that in young SLC-/- mice, the vast majority of the ABCs express memory B-cell (MBC) markers in contrast to wild-type controls. A similar population is present in lupus-prone MRL mice before and at disease onset. In SLC-/- mice, a majority of the ABCs are IgM+ , their VH genes have undergone SHM, show clonal diversification and clonal restriction at the H-CDR3 level. ABC hybridomas, established from SLC-/- mice, secrete typical lupus autoantibodies, e.g. anti-Smith antigen, and some of those that bind to DNA comprise a H-CDR3 that is identical to previously described IgM anti-DNA antibodies from lupus-prone mice. Together, these results reveal that ABCs in autoimmune mice are comprised of autoreactive MBCs expressing highly restricted H-CDR3 repertoires.

Stellar Neuroretinitis Revealing Systemic Lupus Erythematosus without Antiphospholopid Syndrome

Introduction: Systemic Lupus Erythematosus (SLE) is an autoimmune systemic disease with multiple faces, secondary to auto-reacting antibodies targeting nuclear antigen. The optic nerve involvement is reported in less than 1% of SLE, dominated by optic neuritis and optic ischemic neuropathy. neuroretinitis is defined as an inflammation of optic nerve and neural retina. We report a rare case of neuroretinitis as revealing form of SLE in young man. Case report: 14 y old teenager boy, previously healthy, presented one month before his admission, rapidly progressive bilateral visual loss with no associated signs. Visual acuity evaluation revealed visual loss estimated to 2/10 right eye and 3/10 left eye with correction. Eye fundus objectified bilateral stellar macular with intermacularoptic disc exudates and moderate papillar pallor. The macular OCT found exudates in the plexiform layer of the retina. Other paraclinical test found bicytopenia with positive antinuclear and anti-DNA antibody; without antipospholipid antibody. The patient underwent corticotherapy with favourable evolution. Discussion: Neuroretinitis did not figure as usual cause of visual loss in SLE; moreover it has been very rarely reported as a revealing form of SLE. The exact pathogenesis behind neuroretinitis in SLE stays unknown. Sudden onset of unilateral painless loss of vision is the typical clinical presentation of neuroretinitis. Several etiologies may lead to neuroretinitis, dominated by infectious disease (bartonellosis, borreliosis, syphilis, herpes, hepatitis, HIV, CMV, Varicelle, EBV, Toxoplasmosis, Tuberculosis). Also neuroretinitis may be idiopathic. Concerning therapy, no clear guidelines are reported in neuroretinitis occurring in SLE. The visual prognosis is excellent with above 90% cases achieving a final visual acuity. Conclusion: We learn from this case, that neuroretinitis may be the revealing form of SLE. This suggest the need of revision of SLE criterion, especially that neuroretinitis is a part of severe form.

First-degree atrioventricular block: A rare manifestation in systemic lupus erythematosus

Cardiac involvement is very common in Systemic Lupus Erythematosus (SLE), and some 30%-50% of patients suffer from some form of heart disease. Conduction defects represent a rare manifestation of SLE cardiac involvement in adults. We reported a rare case of first-degree atrioventricular block associated with SLE in a 15-year-old female. The patient was diagnosed with SLE, fulfilling the 1982 American College of Rheumatology (ACR) criteria, with malar rash, pericarditis, lymphopenia, thrombocytopenia and the presence of anti-DNA antibodies with positive ANA. The first-degree atrioventricular block was attributed to SLE and completely resolved within 3 days of corticosteroid therapy.

Prognostic Aspects of Lupus Nephritis at Aristide Le Dantec University Hospital in Dakar

Introduction: Kidney injury is common in the course of lupus and affects the functional and vital prognosis. The risk of progression to end-stage renal failure can reach 40% to 60%. Thus we carried out this work for the purpose of an evaluation of the renal and vital prognosis and to deduce the factors of poor prognosis. Patients and method: This was a retrospective, descriptive and analytical study conducted over a period of 10 years from January 1, 2007 to December 31, 2016, performed in the Nephrology Department of Aristide Le Dantec Hospital in Dakar. Patients with lupus nephritis were included. The studied parameters were epidemiological, clinical, paraclinical and progression. We had done a crossover of the patients to look for the factors of poor renal and vital prognosis. Results: Out of 93 cases of lupus patients, 64 were included, a prevalence of 69%. The mean age of the patients was 31.97 ± 10.44 years old. There were 81% women and 19% men, a sex ratio of 0.23. Class III was found in 24 cases (37.5%), Class IV in 20 cases (31.25%), Class V in 15 cases (23.4%), Class II in 4 cases (6.25%) and Class I in 1 case (1.6%). The combination of corticosteroids and immunosuppressants was used in 56.25% of cases. After a follow-up of six months, 19 patients were in complete remission, 21 had resistance and 9 had partial remission. Of the 21 patients who had resistance, 8 were in chronic renal failure. Death was observed in 5 patients and the causes were in 3 patients: pulmonary embolism, bacterial meningitis and pulmonary tuberculosis. The cause of death was unknown in 2 patients. The factors of poor renal prognosis were lymphopenia, the presence of anti-native DNA antibodies, nephrotic syndrome, microscopic hematuria, tubular atrophy and interstitial fibrosis. Risk factors affecting renal survival were the presence of native anti-DNA antibodies, microscopic hematuria, leukocyturia and the presence of a proliferative class. The factors of poor prognosis were renal failure, lymphopenia, nephrotic syndrome, glomerular sclerosis, arteriosclerosis, interstitial infiltration and tubular atrophy. Conclusion: The risk conferred by nephropathy is greater for proliferative glomerulonephritis; it is also correlated with the presence of persistent nephrotic syndrome or severe renal failure.

Comparative evaluation of the presence of serum antibodies SMA, Anti DNA, ANA and RF in the oral lichen planus and oral lichenoid drug and contact reactions

Introduction: Oral lichenoid reactions (OLR) are referred to a group of diseases that clinically and histologically could not be differentiated from the oral lichen planus (OLP) diseases. In the recent years, the two groups of diseases have been considered as T-Cell mediated inflammatory diseases. The aim of this study was to gather more information to help better distinguish these two lesions. Method and Materials: In this descriptive-analytical study, blood samples were taken from 80 patients (59 females and 29 males) and sent to the laboratory for histopathological diagnosis based on WHO criteria. After separating the blood sera of the samples, the sera were evaluated by enzyme linked immunosorbent assay (ELISA) for the presence of ANA, anti-DNA, RF, and SMA auto-antibodies. Data statistically was analyzed with SPSS using the chi-squared test and t-test (a = 0.05). Results: The mean ages were 48.62 and 49.38 for OLP and OLR patients, respectively. There were no significant differences in ANA and RF blood levels (p value g 0.05). Anti-DNA and SMA antibodies were not detected in the blood samples. No significant differences were noted in the blood levels of these antibodies (p value g 0.05). Conclusion: According to the results of the present study, no significant differences were observed between the OLP and OLR patients in relation to the presence or absence of ANA, anti-DNA, RF, and SMA auto-antibodies in the blood samples.

Development of cell-penetrating bispecific antibodies targeting the N-terminal domain of androgen receptor for prostate cancer therapy.

Castration-resistant prostate cancer cells exhibit continued androgen receptor signaling in spite of low levels of ligand. Current therapies to block androgen receptor signaling act by inhibiting ligand production or binding. We developed bispecific antibodies capable of penetrating cells and binding androgen receptor outside of the ligand-binding domain. Half of the bispecific antibody molecule consists of a single-chain variable fragment of 3E10, an anti-DNA antibody that enters cells. The other half is a single-chain variable fragment version of AR441, an anti-AR antibody. The resulting 3E10-AR441 bispecific antibody enters human LNCaP prostate cells and accumulates in the nucleus. The antibody binds to wild-type, mutant and splice variant androgen receptor. Binding affinity of 3E10-AR441 to androgen receptor (284 nM) was lower than that of the parental AR441 mAb (4.6 nM), but could be improved (45 nM) through alternative placement of the affinity tags, and ordering of the VH and VK domains. The 3E10-AR441 bispecific antibody blocked genomic signaling by wild-type or splice variant androgen receptor in LNCaP cells. It also blocked non-genomic signaling by the wild-type receptor. Furthermore, bispecific antibody inhibited the growth of C4-2 prostate cancer cells under androgen-stimulated conditions. The 3E10-AR441 biAb can enter prostate cancer cells and inhibits androgen receptor function in a ligand-independent manner. It may be an attractive prototype agent for prostate cancer therapy.

Type I interferon-mediated autoinflammation due to DNase II deficiency

Microbial nucleic acid recognition serves as the major stimulus to an antiviral response, implying a requirement to limit the misrepresentation of self nucleic acids as non-self and the induction of autoinflammation. By systematic screening using a panel of interferon-stimulated genes we identify two siblings and a singleton variably demonstrating severe neonatal anemia, membranoproliferative glomerulonephritis, liver fibrosis, deforming arthropathy and increased anti-DNA antibodies. In both families we identify biallelic mutations in DNASE2, associated with a loss of DNase II endonuclease activity. We record increased interferon alpha protein levels using digital ELISA, enhanced interferon signaling by RNA-Seq analysis and constitutive upregulation of phosphorylated STAT1 and STAT3 in patient lymphocytes and monocytes. A hematological disease transcriptomic signature and increased numbers of erythroblasts are recorded in patient peripheral blood, suggesting that interferon might have a particular effect on hematopoiesis. These data define a type I interferonopathy due to DNase II deficiency in humans.

Extensively re-organized systemic lymph nodes provide a feasible environment for self-reactivity in lupus-prone NZB × NZW F1 mice.

Lymphadenopathy is a frequently observed symptom in systemic lupus erythematosus, although the immunological role of lymph nodes (LNs) in systemic autoimmunity remains largely unknown. Here, we performed comprehensive and systematic analyses of LNs in lupus-prone NZB × NZW F1 (BWF1) mice, demonstrating extensive tissue re-organization of the systemic LNs with follicular expansion, hyper germinal center (GC) formation, atrophy of the paracortical T-cell area and expansion of the medulla in aged BWF1 mice bearing glomerulonephritis. The proportion of B cells was significantly increased in these reactive LNs but not in the spleen, and lymphocyte subsets involved in antibody production, i.e. GC B cells, follicular helper T cells and plasma cells, were elevated. Draining LNs of the affected organs, such as the renal and cervical nodes, showed enhanced tissue re-organization and accumulation of effector lymphocytes, suggesting the presence of a positive feedback loop of regional responses. LN cells isolated from disease-bearing animals produced anti-DNA antibody, indicating activation of autoreactive lymphocytes in situ. The substantial development of disease and LN alterations in mice that received a splenectomy at a young age points to the importance of other secondary lymphoid organs, most likely LNs, for the progression of autoimmune responses independent of the spleen. Taken together, our findings highlight the value of taking LN alterations and activities into consideration for understanding the pathogenesis of systemic autoimmunity.

Clonal evolution and antigen recognition of anti-nuclear antibodies in acute systemic lupus erythematosus

The evolutional process of disease-associated autoantibodies in systemic lupus erythematosus (SLE) remains to be established. Here we show intraclonal diversification and affinity maturation of anti-nuclear antibody (ANA)-producing B cells in SLE. We identified a panel of monoclonal ANAs recognizing nuclear antigens, such as double-stranded DNA (dsDNA) and ribonucleoproteins (RNPs) from acute SLE subjects. These ANAs had relatively few, but nonetheless critical mutations. High-throughput immunoglobulin sequencing of blood lymphocytes disclosed the existence of sizable ANA lineages shearing critical mutations intraclonally. We further focused on anti-DNA antibodies, which are capable to bind to both single-stranded (ss) and dsDNA at high affinity. Crystal structure and biochemical analysis confirmed a direct role of the mutations in the acquisition of DNA reactivity and also revealed that these anti-DNA antibodies recognized an unpaired region within DNA duplex. Our study unveils the unique properties of high-affinity anti-DNA antibodies that are generated through antigen-driven affinity maturation in acute phase of SLE.

Traitement du lupus érythémateux par le bélimumab en pratique courante : étude rétrospective de 15 malades

Belimumab (an anti-BLyS monoclonal antibody) was recently approved for the treatment of systemic lupus erythematosus (SLE). The aim of the study was to describe efficacy and safety of the drug as well as its impact on serologic parameters and the role of long-term systemic sparing of treatment in clinical practice in LE. We conducted a retrospective study at Reims University Hospital between 2012 and 2016 including consecutive patients with LE treated with belimumab. Efficacy was evaluated in terms of clinical progression, and normalisation of laboratory factors (anti-DNA antibody and C3 serum levels) and sparing of associated long-term systemic therapies for LE. Among the 15 patients included, a therapeutic response was obtained in 9 patients (60%), with partial remission in 8 of 9 cases. The median titre of anti-DNA antibody was 50IU/mL (range: 4-50) and the median C3 level was 0.82g/L (range: 0.36-1.23) before initiation of belimumab, vs. 25.5IU/mL (range: 2-50) and 0.89g/L (range: 0.34-1.22) at the last evaluation, respectively, without significant modification (P=0.12 and P=0.45). The median dose of prednisone at the time of the first belimumab infusion was reduced from 9.5mg/day (range: 0-18) to 6mg/day (range: 0-20) at the last clinical evaluation. Eight patients (53%) experienced adverse events, and these were very slight or moderate in all cases. Belimumab appears to be an effective and well-tolerated treatment for moderately severe systemic LE, allowing sparing of maintenance corticosteroid therapy in order to decrease its frequent adverse events.

Heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) function as endocytic receptors for an internalizing anti-nucleic acid antibody

A subset of monoclonal anti-DNA autoantibodies enters a variety of living cells. Here, we aimed to identify the endocytic receptors recognized by an internalizing anti-nucleic acid autoantibody, the 3D8 single-chain variable fragment (scFv). We found that cell surface binding and internalization of 3D8 scFv were inhibited markedly in soluble heparan sulfate (HS)/chondroitin sulfate (CS)-deficient or -removed cells and in the presence of soluble HS and CS. 3D8 scFv colocalized intracellularly with either HS proteoglycans (HSPGs) or CSPGs in HeLa cells. 3D8 scFv was co-endocytosed and co-precipitated with representative individual HSPG and CSPG molecules: syndecan-2 (a transmembrane HSPG), glypican-3 (a glycosylphosphatidylinositol (GPI)-anchored HSPG); CD44 (a transmembrane CSPG); and brevican (a GPI-anchored CSPG). Collected data indicate that 3D8 scFv binds to the negatively charged sugar chains of both HSPGs and CSPGs and is then internalized along with these molecules, irrespective of how these proteoglycans are associated with the cell membrane. This is the first study to show that anti-DNA antibodies enter cells via both HSPGs and CSPGs simultaneously. The data may aid understanding of endocytic receptors that bind anti-DNA autoantibodies. The study also provides insight into potential cell membrane targets for macromolecular delivery.