Anti-Citrullinated Protein Antibodies With Multiple Specificities Ameliorate Collagen Antibody-Induced Arthritis in a Time-Dependent Manner.

OBJECTIVE: Anti-citrullinated protein antibodies (ACPAs) are highly specific for rheumatoid arthritis (RA) and have long been regarded as pathogenic. Despite substantial in vitro evidence supporting this claim, reports investigating the proinflammatory effects of ACPAs in animal models of arthritis are rare and include mixed results. Here, we sequenced the plasmablast antibody repertoire of a patient with RA and functionally characterized the encoded ACPAs. METHODS: We expressed ACPAs from the antibody repertoire of a patient with RA and characterized their autoantigen specificities on antigen arrays and enzyme-linked immunosorbent assays. Binding affinities were estimated by bio-layer interferometry. Select ACPAs (n = 9) were tested in the collagen antibody-induced arthritis (CAIA) mouse model to evaluate their effects on joint inflammation. RESULTS: Recombinant ACPAs bound preferentially and with high affinity (nanomolar range) to citrullinated (cit) autoantigens (primarily histones and fibrinogen) and to auto-cit peptidylarginine deiminase 4 (PAD4). ACPAs were grouped for in vivo testing based on their predominant cit-antigen specificities. Unexpectedly, injections of recombinant ACPAs significantly reduced paw thickness and arthritis severity in CAIA mice as compared with isotype-matched control antibodies (P ≤ 0.001). Bone erosion, synovitis, and cartilage damage were also significantly reduced (P ≤ 0.01). This amelioration of CAIA was observed for all the ACPAs tested and was independent of cit-PAD4 and cit-fibrinogen specificities. Furthermore, disease amelioration was more prominent when ACPAs were injected at earlier stages of CAIA than at later phases of the model. CONCLUSION: Recombinant patient-derived ACPAs ameliorated CAIA. Their antiinflammatory effects were more preventive than therapeutic. This study highlights a potential protective role for ACPAs in arthritis.

Oral mucosal breaks trigger anti-citrullinated bacterial and human protein antibody responses in rheumatoid arthritis.

Periodontal disease is more common in individuals with rheumatoid arthritis (RA) who have detectable anti-citrullinated protein antibodies (ACPAs), implicating oral mucosal inflammation in RA pathogenesis. Here, we performed paired analysis of human and bacterial transcriptomics in longitudinal blood samples from RA patients. We found that patients with RA and periodontal disease experienced repeated oral bacteremias associated with transcriptional signatures of ISG15+HLADRhi and CD48highS100A2pos monocytes, recently identified in inflamed RA synovia and blood of those with RA flares. The oral bacteria observed transiently in blood were broadly citrullinated in the mouth, and their in situ citrullinated epitopes were targeted by extensively somatically hypermutated ACPAs encoded by RA blood plasmablasts. Together, these results suggest that (i) periodontal disease results in repeated breaches of the oral mucosa that release citrullinated oral bacteria into circulation, which (ii) activate inflammatory monocyte subsets that are observed in inflamed RA synovia and blood of RA patients with flares and (iii) activate ACPA B cells, thereby promoting affinity maturation and epitope spreading to citrullinated human antigens.

A novel intradermal tattoo-based injection device enhances the immunogenicity of plasmid DNA vaccines.

In recent years, tattooing technology has shown promising results toward evaluating vaccines in both animal models and humans. However, this technology has some limitations due to variability of experimental evaluations or operator procedures. The current study evaluated a device (intradermal oscillating needle array injection device: IONAID) capable of microinjecting a controlled dose of any aqueous vaccine into the intradermal space. IONAID-mediated administration of a DNA-based vaccine encoding the glycoprotein (GP) from the Ebola virus resulted in superior T- and B-cell responses with IONAID when compared to single intramuscular (IM) or intradermal (ID) injection in mice. Moreover, humoral immune responses, induced after IONAID vaccination, were significantly higher to those obtained with traditional passive DNA tattooing in guinea pigs and rabbits. This device was well tolerated and safe during HIV vaccine delivery in non-human primates (NHPs), while inducing robust immune responses. In summary, this study shows that the IONAID device improves vaccine performance, which could be beneficial to the animal and human health, and importantly, provide a dose-sparing approach (e.g., monkeypox vaccine).

Augmented Reality for Retrosigmoid Craniotomy Planning.

While medical imaging data have traditionally been viewed on two-dimensional (2D) displays, augmented reality (AR) allows physicians to project the medical imaging data on patient's bodies to locate important anatomy. We present a surgical AR application to plan the retrosigmoid craniotomy, a standard approach to access the posterior fossa and the internal auditory canal. As a simple and accurate alternative to surface landmarks and conventional surgical navigation systems, our AR application augments the surgeon's vision to guide the optimal location of cortical bone removal. In this work, two surgeons performed a retrosigmoid approach 14 times on eight cadaver heads. In each case, the surgeon manually aligned a computed tomography (CT)-derived virtual rendering of the sigmoid sinus on the real cadaveric heads using a see-through AR display, allowing the surgeon to plan and perform the craniotomy accordingly. Postprocedure CT scans were acquired to assess the accuracy of the retrosigmoid craniotomies with respect to their intended location relative to the dural sinuses. The two surgeons had a mean margin of d avg = 0.6 ± 4.7 mm and d avg = 3.7 ± 2.3 mm between the osteotomy border and the dural sinuses over all their cases, respectively, and only positive margins for 12 of the 14 cases. The intended surgical approach to the internal auditory canal was successfully achieved in all cases using the proposed method, and the relatively small and consistent margins suggest that our system has the potential to be a valuable tool to facilitate planning a variety of similar skull-base procedures.

Clonally expanded B cells in multiple sclerosis bind EBV EBNA1 and GlialCAM.

Multiple sclerosis (MS) is a heterogenous autoimmune disease in which autoreactive lymphocytes attack the myelin sheath of the central nervous system. B lymphocytes in the cerebrospinal fluid (CSF) of patients with MS contribute to inflammation and secrete oligoclonal immunoglobulins1,2. Epstein-Barr virus (EBV) infection has been epidemiologically linked to MS, but its pathological role remains unclear3. Here we demonstrate high-affinity molecular mimicry between the EBV transcription factor EBV nuclear antigen 1 (EBNA1) and the central nervous system protein glial cell adhesion molecule (GlialCAM) and provide structural and in vivo functional evidence for its relevance. A cross-reactive CSF-derived antibody was initially identified by single-cell sequencing of the paired-chain B cell repertoire of MS blood and CSF, followed by protein microarray-based testing of recombinantly expressed CSF-derived antibodies against MS-associated viruses. Sequence analysis, affinity measurements and the crystal structure of the EBNA1-peptide epitope in complex with the autoreactive Fab fragment enabled tracking of the development of the naive EBNA1-restricted antibody to a mature EBNA1-GlialCAM cross-reactive antibody. Molecular mimicry is facilitated by a post-translational modification of GlialCAM. EBNA1 immunization exacerbates disease in a mouse model of MS, and anti-EBNA1 and anti-GlialCAM antibodies are prevalent in patients with MS. Our results provide a mechanistic link for the association between MS and EBV and could guide the development of new MS therapies.

Silencing of Dok-7 in Adult Rat Muscle Increases Susceptibility to Passive Transfer Myasthenia Gravis.

Myasthenia gravis (MG) is an autoimmune disease mediated by autoantibodies that target proteins at the neuromuscular junction, primarily the acetylcholine receptor (AChR) and the muscle-specific kinase. Because downstream of kinase 7 (Dok-7) is essential for the full activation of muscle-specific kinase and consequently for dense clustering of AChRs, we hypothesized that reduced levels of Dok-7 increase the susceptibility to passive transfer MG. To test this hypothesis, Dok-7 expression was reduced by transfecting shRNA-coding plasmids into the tibialis anterior muscle of adult rats by in vivo electroporation. Subclinical MG was subsequently induced with a low dose of anti-AChR monoclonal antibody 35. Neuromuscular transmission was significantly impaired in Dok-7-siRNA-electroporated legs compared with the contralateral control legs, which correlated with a reduction of AChR protein levels at the neuromuscular junction (approximately 25%) in Dok-7-siRNA-electroporated muscles, compared with contralateral control muscles. These results suggest that a reduced expression of Dok-7 may play a role in the susceptibility to passive transfer MG, by rendering AChR clusters less resistant to the autoantibody attack.

HIV-1-Mediated BAFF Secretion in Macrophages Does Not Require Endosomal TLRs, Type-I IFN, and Nef, but Depends on the Cellular Phenotype Status.

HIV-1 infection is characterized by persistent viral replication, chronic immune activation, and CD4(+) T cell depletion. Moreover, several immune dysfunctions are observed in cells that are not targeted by the virus, such as B cells. Some B cell abnormalities include hypergammaglobulinemia, nonspecific B cell activation, class switching, increased cell turnover, breakage of tolerance, and a loss of the capacity to generate and maintain memory. Several cytokines and growth factors that are increased in the serum of HIV-1-infected individuals have been suggested to directly or indirectly trigger B cell activation, and one of these is BAFF. In this study, we investigate the ability of fully competent (R5-tropic) HIV-1 to induce BAFF production by monocyte-derived macrophages (MDMs). We demonstrate here that HIV-1 drives BAFF production in MDMs in a type-I IFN- and TLR-independent manner. Moreover, we determine that HIV-1 Nef accessory protein is dispensable in BAFF upregulation as a nef-deleted HIV-1 strain is still able to increase BAFF at levels similar to the wild type strain. Finally, we show that the macrophage phenotype status affects HIV-1 replication and BAFF induction, as both were abrogated in MDMs displaying a M1 phenotype. This study provides new useful information about the increased levels of BAFF observed during HIV-1 infection and highlights the importance of macrophages as a source of BAFF, a phenomenon that might contribute to B cell dysfunctions at inflammatory tissue sites in infected individuals.

HIV-1-triggered release of type I IFN by plasmacytoid dendritic cells induces BAFF production in monocytes.

HIV-1 infection leads to numerous B cell abnormalities, including hypergammaglobulinemia, nonspecific B cell activation, nonspecific class switching, increased cell turnover, breakage of tolerance, increased immature/transitional B cells, B cell malignancies, as well as a loss of capacity to generate and maintain memory, all of which contribute to a global impairment of the immune humoral compartment. Several cytokines and soluble factors, which are increased in sera of HIV-1-infected individuals, have been suggested to directly or indirectly contribute to these B cell dysfunctions, and one of these is the B cell-activating factor (BAFF). We report in this study that HIV-1 (X4- and R5-tropic) upregulates BAFF expression and secretion by human monocytes. Moreover, we show that the virus-mediated production of BAFF by monocytes relies on a type I IFN response by a small percentage of plasmacytoid dendritic cells (pDCs) present in the monocyte cultures. HIV-1-induced type I IFN by pDCs triggers BAFF production in both classical and intermediate monocytes, but not in nonclassical monocytes, which nonetheless display a very strong basal BAFF production. We report also that basal BAFF secretion was higher in monocytes obtained from females compared with those from male donors. This study provides a novel mechanistic explanation for the increased BAFF levels observed during HIV-1 infection and highlights the importance of pDC/monocyte crosstalk to drive BAFF secretion.

Proteasome inhibition with bortezomib depletes plasma cells and specific autoantibody production in primary thymic cell cultures from early-onset myasthenia gravis patients.

Bortezomib is a potent inhibitor of proteasomes currently used to eliminate malignant plasma cells in multiple myeloma patients. It is also effective in depleting both alloreactive plasma cells in acute Ab-mediated transplant rejection and their autoreactive counterparts in animal models of lupus and myasthenia gravis (MG). In this study, we demonstrate that bortezomib at 10 nM or higher concentrations killed long-lived plasma cells in cultured thymus cells from nine early-onset MG patients and consistently halted their spontaneous production not only of autoantibodies against the acetylcholine receptor but also of total IgG. Surprisingly, lenalidomide and dexamethasone had little effect on plasma cells. After bortezomib treatment, they showed ultrastructural changes characteristic of endoplasmic reticulum stress after 8 h and were no longer detectable at 24 h. Bortezomib therefore appears promising for treating MG and possibly other Ab-mediated autoimmune or allergic disorders, especially when given in short courses at modest doses before the standard immunosuppressive drugs have taken effect.

Proteomic analysis of rat tibialis anterior muscles at different stages of experimental autoimmune myasthenia gravis.

Myasthenia gravis (MG) is an autoimmune disease in which autoantibodies, most commonly directed against the acetylcholine receptor (AChR), impair neuromuscular transmission and cause muscle weakness. In this study, we utilized two-dimensional difference in-gel electrophoresis (2D-DIGE) to analyze the muscle's proteomic profile at different stages of experimental autoimmune myasthenia gravis (EAMG). We identified twenty-two differentially expressed proteins, mainly related to metabolic and stress-response pathways. Interestingly, these identified proteins have also been associated with other contraction-impairing muscle pathologies (e.g. inclusion body myositis), suggesting a similar response of the muscle to such conditions.

Pathophysiology of myasthenia gravis with antibodies to the acetylcholine receptor, muscle-specific kinase and low-density lipoprotein receptor-related protein 4.

Myasthenia gravis is caused by antibodies to the acetylcholine receptor, muscle-specific kinase, low-density lipoprotein receptor-related protein 4, or possibly yet unidentified antibodies. The mechanisms by which these antibodies interfere with the function of postsynaptic proteins include complement activation, antigenic modulation by crosslinking of the target proteins, competition with ligand binding sites, or steric hindrance which inhibits conformational changes or binding to associated proteins. Screening for auto-antibodies to different postsynaptic targets, and also for low-affinity antibodies, is contributing to a more accurate diagnosis of MG patients. Further studies into the specific pathophysiological pathways of the several MG subforms might help to develop new, more antigen specific, therapies.

Targeting plasma cells with proteasome inhibitors: possible roles in treating myasthenia gravis?

Myasthenia gravis (MG) is treated primarily with broad-spectrum immuno-suppressants such as prednisone or azathioprine, which normally require several months to reduce autoantibody titers significantly. This delay may be caused by the resistance of the main antibody-producing cells, the plasma cells, to these drugs. In particular, long-lived plasma cells are resistant to immunosuppressive treatments and can produce (auto-) antibodies for months. Bortezomib is a proteasome inhibitor approved for treating patients with multiple myeloma, a plasma cell malignancy. Recent preclinical studies in cell cultures and animal models, and clinical studies in organ-transplant recipients, have demonstrated that bortezomib can kill non-neoplastic plasma cells within hours. This suggests that proteasome inhibitors could also be used for rapidly reducing autoantibody production in autoimmune diseases. We have begun to assess their potential in MG.

Proteasome inhibition with bortezomib depletes plasma cells and autoantibodies in experimental autoimmune myasthenia gravis.

Bortezomib, an inhibitor of proteasomes, has been reported to reduce autoantibody titers and to improve clinical condition in mice suffering from lupus-like disease. Bortezomib depletes both short- and long-lived plasma cells; the latter normally survive the standard immunosuppressant treatments targeting T and B cells. These findings encouraged us to test whether bortezomib is effective for alleviating the symptoms in the experimental autoimmune myasthenia gravis (EAMG) model for myasthenia gravis, a disease that is characterized by autoantibodies against the acetylcholine receptor (AChR) of skeletal muscle. Lewis rats were immunized with saline (control, n = 36) or Torpedo AChR (EAMG, n = 54) in CFA in the first week of an experimental period of 8 wk. After immunization, rats received twice a week s.c. injections of bortezomib (0.2 mg/kg in saline) or saline injections. Bortezomib induced apoptosis in bone marrow cells and reduced the amount of plasma cells in the bone marrow by up to 81%. In the EAMG animals, bortezomib efficiently reduced the rise of anti-AChR autoantibody titers, prevented ultrastructural damage of the postsynaptic membrane, improved neuromuscular transmission, and decreased myasthenic symptoms. This study thus underscores the potential of the therapeutic use of proteasome inhibitors to target plasma cells in Ab-mediated autoimmune diseases.

Antibody effector mechanisms in myasthenia gravis-pathogenesis at the neuromuscular junction.

Myasthenia gravis (MG) is an autoimmune disorder caused by autoantibodies that are either directed to the muscle nicotinic acetylcholine receptor (AChR) or to the muscle-specific tyrosine kinase (MuSK). These autoantibodies define two distinct subforms of the disease-AChR-MG and MuSK-MG. Both AChR and MuSK are expressed on the postsynaptic membrane of the neuromuscular junction (NMJ), which is a highly specialized region of the muscle dedicated to receive and process signals from the motor nerve. Autoantibody binding to proteins of the postsynaptic membrane leads to impaired neuromuscular transmission and muscle weakness. Pro-inflammatory antibodies of the human IgG1 and IgG3 subclass modulate the AChR, cause complement activation, and attract lymphocytes; together acting to decrease levels of the AChR and AChR-associated proteins and to reduce postsynaptic folding. In patients with anti-MuSK antibodies, there is no evidence of loss of junctional folds and no apparent loss of AChR density. Anti-MuSK antibodies are predominantly of the IgG4 isotype, which functionally differs from other IgG subclasses in its anti-inflammatory activity. Moreover, IgG4 undergoes a posttranslational modification termed Fab arm exchange that prevents cross-linking of antigens. These findings suggest that MuSK-MG may be different in etiological and pathological mechanisms from AChR-MG. The effector functions of IgG subclasses on synapse structure and function are discussed in this review.

HIV type 1 genetic diversity surveillance among newly diagnosed individuals from 2003 to 2005 in Buenos Aires, Argentina.

To perform a diversity surveillance study we characterized viral subtypes among newly diagnosed individuals in Buenos Aires city. Plasma samples were collected from 322 drug-naive newly diagnosed HIV-1 individuals attending two voluntary counseling and testing centers. Sequences of pol and vpu genes were obtained from 283 samples and viral subtype was characterized by Neighbor-joining trees and Bootscanning analysis. BF recombinants were found in 56.9% followed by subtype B strains (39.2%). CRF12_BF structure was found in 27% of BF while another 27% had that structure only in one of both genes analyzed. Unusual non-B-non-BF strains were found in 3.9% (11/283). They were further analyzed by database searching and maximum likelihood trees in order to track their origin. Two subtype C sequences were found to be related to South American isolates while another two subtype C sequences and the subtype C segment of a BC recombinant were found to be related to isolates from Senegal. We also identified the CRF16_A2D previously found in Argentina and the CRF06_cpx commonly prevalent in Africa. The B segment of a BD recombinant was also found to be related to the Argentinean Bs suggesting a recombination between an African and a local strain. We also found a BK and two BA recombinants. In conclusion, CRF16_A2D and a new line of subtype C (of Senegalese origin) seem to be successfully established and are now spreading in Buenos Aires. BF recombinants keep recombining with local strains losing the CRF12_BF structure. Altogether they are changing the diversity of HIV in Argentina.

Drug-resistance surveillance among newly HIV-1 diagnosed individuals in Buenos Aires, Argentina.

OBJECTIVE: Our objective was to estimate primary resistance in an urban setting in a developing country with a long history of antiretroviral delivery and high coverage levels. DESIGN: We carried out a resistance surveillance study according to WHO HIV-Resistance Guidelines. METHODS: Blood samples were collected from 323 drug-naive HIV-1 infected individuals diagnosed at two HIV voluntary counselling and testing centers in Buenos Aires. Viral-load, CD4 cell counts and detuned assays were performed on all samples. The pol gene was sequenced and the resistance profile determined. Phylogenetic analysis was performed by neighbor-joining trees and bootscanning analysis. RESULTS: We found that 12 (4.2%) of the 284 samples sequenced harbored primary resistance mutations, of which K103N, M41L and V108I were most prevalent. Phylogenetic analysis revealed evidence for the transmission of the K103N mutation among the drug-naive population. The proportion of recent infections identified by the detuned assay was 10.1%. CONCLUSIONS: Levels of primary resistance in Buenos Aires are still low, despite a long history of ARV delivery and high coverage levels.