Clinical and Experimental Immunology

The term ‘neuromyelitis optica’ (‘Devic's syndrome’, NMO) refers to a syndrome characterized by optic neuritis and myelitis. In recent years, the condition has raised enormous interest among scientists and clinical neurologists, fuelled by the detection of a specific serum immunoglobulin (Ig)G reactivity (NMO-IgG) in up to 80% of patients with NMO. These autoantibodies were later shown to target aquaporin-4 (AQP4), the most abundant water channel in the central nervous system (CNS). Here we give an up-to-date overview of the clinical and paraclinical features, immunopathogenesis and treatment of NMO. We discuss the widening clinical spectrum of AQP4-related autoimmunity, the role of magnetic resonance imaging (MRI) and new diagnostic means such as optical coherence tomography in the diagnosis of NMO, the role of NMO-IgG, T cells and granulocytes in the pathophysiology of NMO, and outline prospects for new and emerging therapies for this rare, but often devastating condition.

In addition to disturbed apoptosis and insufficient clearance of apoptotic cells, there is recent evidence for a role of neutrophils in the aetiopathogenesis of systemic lupus erythematosus (SLE). In response to various stimuli, neutrophils can rapidly release DNA fibres decorated with citrullinated histones and anti-microbial peptides. These structures are referred to as neutrophil extracellular traps (NETs). In addition to apoptotic cell-derived microparticles, these NETs may comprise a further source of autoantigens, able to drive the autoimmune response in SLE. Our group recently identified specific histone modifications occurring during apoptosis that play an important role in the autoimmune response in SLE. In the current study, we evaluated the presence and immunostimulatory potential of these previously identified histone modifications in NETs. Compared to NETs from healthy donors, the histones present in NETs formed by SLE-derived neutrophils contain increased amounts of acetylated and methylated residues, which we previously observed to be associated with apoptosis and SLE. Treatment of neutrophils with histone deacetylase (HDAC) inhibitor Trichostatin A (TSA), prior to induction of NETosis, induced NETs containing hyperacetylated histones, endowed with an increased capacity to activate macrophages. This implies that specific histone modifications, in particular acetylation, might enhance the immunostimulatory potential of NETs in SLE.

Dermatomyositis (DM) and polymyosits (PM) are systemic autoimmune diseases whose pathogeneses remain unclear. Neutrophil extracellular traps (NETs) are reputed to play an important role in the pathogenesis of autoimmune diseases. This study tests the hypothesis that NETs may be pathogenic in DM/PM. Plasma samples from 97 DM/PM patients (72 DM, 25 PM) and 54 healthy controls were tested for the capacities to induce and degrade NETs. Plasma DNase I activity was tested to further explore possible reasons for the incomplete degradation of NETs. Results from 35 DM patients and seven PM patients with interstitial lung disease (ILD) were compared with results from DM/PM patients without ILD. Compared with control subjects, DM/PM patients exhibited a significantly enhanced capacity for inducing NETs, which was supported by elevated levels of plasma LL-37 and circulating cell-free DNA (cfDNA) in DM/PM. NETs degradation and DNase I activity were also decreased significantly in DM/PM patients and were correlated positively. Moreover, DM/PM patients with ILD exhibited the lowest NETs degradation in vitro due to the decrease in DNase I activity. DNase I activity in patients with anti-Jo-1 antibodies was significantly lower than in patients without. Glucocorticoid therapy seems to improve DNase I activity. Our findings demonstrate that excessively formed NETs cannot be degraded completely because of decreased DNase I activity in DM/PM patients, especially in patients with ILD, suggesting that abnormal regulation of NETs may be involved in the pathogenesis of DM/PM and could be one of the factors that initiate and aggravate ILD.

Increasing evidence indicates that aberrant neutrophil extracellular trap (NET) formation could contribute to the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Recent research has provided evidence that a novel type of ANCA autoantibody, anti-lysosomal membrane protein-2 (LAMP-2) antibody, may have a pathogenic role in AAV. We have shown previously that anti-LAMP-2 antibody-stimulated NET formation contains autoantigens and anti-microbial peptides. The current study sought to determine whether LAMP-2, as a novel antigen of ANCA, was present on NETs in AAV patients, the influence of the anti-LAMP-2 antibody on the neutrophil apoptosis rate and the role of autophagy in anti-LAMP-2 antibody-induced NET formation. NET formation was assessed using immunofluorescence microscopy, scanning electron microscopy or live cell imaging. The neutrophil apoptosis rate was analysed using fluorescence activated cell sorting (FACS). Autophagy was detected using LC3B accumulation and transmission electron microscopy. The results showed that enhanced NET formation, which contains LAMP-2, was observed in kidney biopsies and neutrophils from AAV patients. The apoptosis rate decreased significantly in human neutrophils stimulated with anti-LAMP-2 antibody, and this effect was attenuated by the inhibitors of autophagy 3-methyladenine (3MA) and 2-morpholin-4-yl-8-phenylchromen-4-one (LY294002). The anti-LAMP-2 antibody-stimulated NET formation was unaffected by benzyloxycarbonyl-Val- Ala-Asp (OMe)-fluoromethylketone (zVAD-fmk) and necrostatin-1 (Nec-1), which are inhibitors of apoptosis and necrosis, respectively, but was inhibited by 3MA and LY294002. Moreover, the proportion of LC3BI that was converted to LC3BII increased significantly (P = 0·0057), and massive vacuolizations that exhibited characteristics typical of autophagy were detected in neutrophils stimulated with anti-LAMP-2 antibody. Our results provide further evidence that autophagy is involved in ANCA-induced NET formation in human neutrophils.

The interaction between neutrophils and activation of alternative complement pathway plays a pivotal role in the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). ANCAs activate primed neutrophils to release neutrophil extracellular traps (NETs), which have recently gathered increasing attention in the development of AAV. The relationship between NETs and alternative complement pathway has not been elucidated. The current study aimed to investigate the relationship between NETs and alternative complement pathway. Detection of components of alternative complement pathway on NETs in vitro was assessed by immunostain and confocal microscopy. Complement deposition on NETs were detected after incubation with magnesium salt ethyleneglycol tetraacetic acid (Mg-EGTA)-treated human serum. After incubation of serum with supernatants enriched in ANCA-induced NETs, levels of complement components in supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Complement factor B (Bb) and properdin deposited on NETs in vitro. The deposition of C3b and C5b-9 on NETs incubated with heat-inactivated normal human serum (Hi-NHS) or EGTA-treated Hi-NHS (Mg-EGTA-Hi-NHS) were significantly less than that on NETs incubated with NHS or EGTA-treated NHS (Mg-EGTA-NHS). NETs induced by ANCA could activate the alternative complement cascade in the serum. In the presence of EGTA, C3a, C5a and SC5b-9 concentration decreased from 800·42 ± 244·81 ng/ml, 7·68 ± 1·50 ng/ml, 382·15 ± 159·75 ng/ml in the supernatants enriched in ANCA induced NETs to 479·07 ± 156·2 ng/ml, 4·86 ± 1·26 ng/ml, 212·65 ± 44·40 ng/ml in the supernatants of DNase I-degraded NETs (P < 0·001, P = 0·008, P < 0·001, respectively). NETs could activate the alternative complement pathway, and might thus participate in the pathogenesis of AAV.

The aim of this study was to assess the synovial fluid (SF) neurotransmitter excitatory amino acid (EAA) levels, including glutamate (Glu) and aspartate (Asp), in the context of SF levels of other amino acids, TNF-α and chemokines from patients with active arthropathies. The SF was collected from patients with active rheumatoid arthritis (RA), gout, or osteoarthritis (OA). The SF samples were analysed for levels of neurotransmitters glutamate and aspartate, tumour necrosis factor-alpha (TNF-α), Regulated upon Activation Normally T-cell Expressed and Secreted (RANTES), macrophage inhibitory factor-1 alpha (MIP-1α) and interleukin 8 (IL-8). SF WBC counts were also determined. Correlations between SF EAA, TNF-α and chemokines were determined by the Pearson product-moment correlation. Primary cultures derived from SF from active RA and gout patients were incubated with added l-glutamate, to assess if exposure to Glu could increase TNF-α levels. There were significant elevations in SF EAA, SF TNF-α and SF RANTES in RA patients compared to gout or OA patients. Significant correlations between SF EAA and SF RANTES, MIP-1α and IL-8 levels were seen, and SF EAA and SF TNF-α or SF WBC levels approached significance. Addition of exogenous neurotransmitter glutamate significantly increased TNF-α levels in primary cell cultures derived from RA and gout patients. The SF neurotransmitter EAA levels significantly correlated to selected SF chemokine levels, in clinically active RA, gout and OA patients, independent of disease. Added Glu resulted in significantly increased TNF-α levels in primary synovial cell cultures. These data expand the relationship of SF neurotransmitter EAA levels to SF cytokines and chemokines in patients with clinically active arthritis, and suggest that neurotransmitters Glu and Asp contribute to peripheral inflammatory processes.

In the last decade, an unprecedented genetic diversity has been disclosed among Mycobacterium tuberculosis strains found worldwide. However, well-conserved genotypes seem to prevail in areas with high incidence of tuberculosis. As this may be related to selective advantages, such as advanced mechanisms to circumvent [M. bovis Bacille Calmette–Guerin (BCG)-induced] host defence mechanisms, we investigated the influence of strain diversity on the course of experimental disease. Twelve M. tuberculosis strains, representing four major genotype families found worldwide today, and the laboratory strain H37Rv were each used to infect BALB/c mice by direct intratracheal injection. Compared with H37Rv, infections with Beijng strains were characterized by extensive pneumonia, early but ephemeral tumour necrosis factor-alpha (TNF-α) and inducible isoform of nitric oxide synthetase (iNOS) expression, and significantly higher earlier mortality. Conversely, Canetti strains induced limited pneumonia, sustained TNF-α and iNOS expression in lungs, and almost 100% survival. Strains of the Somali and the Haarlem genotype families displayed less homogeneous, intermediate rates of survival. Previous BCG vaccination protected less effectively against infection with Beijing strains than against the H37Rv strain. In conclusion, genetically different M. tuberculosis strains evoked markedly different immunopathological events. Bacteria with the Beijing genotype, highly prevalent in Asia and the former USSR, elicited a non-protective immune response in mice and were the most virulent. Future immunological research, particularly on candidate vaccines, should include a broad spectrum of M. tuberculosis genotypes rather than a few laboratory strains.

Các kháng thể chống tế bào nội mô (AECA) đã được tìm thấy đóng vai trò quan trọng trong nhiều rối loạn mạch máu. Để xác định sự hiện diện của AECA ở trẻ em mắc hội chứng Henoch-Schönlein (HSP), và để làm rõ giá trị bệnh lý và lâm sàng của việc đo lường chúng trong bệnh này, AECA đã được phát hiện qua phương pháp nhuộm miễn dịch huỳnh quang và xét nghiệm miễn dịch enzyme liên kết hấp thụ (ELISA) dựa trên tế bào nội mô tĩnh mạch rốn người (HUVEC) ở 20 trẻ em mắc HSP, 10 trẻ em bị viêm khớp dạng thấp ở trẻ vị thành niên (JRA) không có viêm mạch và 10 trẻ em khỏe mạnh bình thường. Các kháng thể chống lại tế bào nội mô khác, các tế bào nội mô vi mạch da người (HMVEC-d) cũng đã được phát hiện thông qua ELISA dựa trên tế bào. Trong một số thí nghiệm, chúng tôi đã so sánh hoạt động gắn kết của kháng thể đối với HUVEC với và không có điều trị trước bằng yếu tố hoại tử khối u-α (TNF-α) hoặc interleukin-1 (IL-1). Các bệnh nhân có khởi phát cấp tính của HSP có mức độ IgA kháng thể huyết thanh cao hơn, cả đối với HUVEC và HMVEC-d, so với nhóm kiểm soát khỏe mạnh (P = 0,001, P = 0,008, tương ứng). Bốn mươi lăm phần trăm bệnh nhân có AECA IgA dương tính đối với HUVEC, và 35% có AECA IgA dương tính đối với HMVEC-d. Nồng độ kháng thể IgA đối với HUVEC phản ánh hoạt động của bệnh. Sau điều trị TNF-α, các giá trị AECA IgA đối với HUVEC ở bệnh nhân HSP đã tăng đáng kể (P = 0,02). Đối với AECA IgG và IgM, không có sự khác biệt giữa bệnh nhân HSP và nhóm đối chứng (P = 0,51, P = 0,91). Mười trẻ em JRA không bị viêm mạch không có hoạt động AECA IgG, IgM hoặc IgA nào được phát hiện. Kết quả của nghiên cứu này cho thấy trẻ em mắc HSP có AECA IgA, điều này được tăng cường bởi điều trị TNF-α. Mặc dù vai trò của các kháng thể này chưa rõ ràng, nhưng AECA IgA cung cấp thêm một gợi ý miễn dịch cho việc hiểu biết về HSP.

Việc xác định mức độ kháng thể trong huyết thanh chống lại kháng nguyên bề mặt virus viêm gan B (anti-HBs) sau khi tiêm vắc-xin viêm gan B hiện nay là phương pháp đơn giản duy nhất có sẵn để dự đoán sự suy giảm sự bảo vệ và lập kế hoạch tiêm liều nhắc lại. Tổng cộng 3085 người nhận vắc-xin từ huyết tương và vắc-xin tái tổ hợp đã được theo dõi trong 10 năm để xác định động học sản xuất anti-HBs và xây dựng một mô hình toán học có thể dự đoán hiệu quả sự suy giảm mức độ anti-HBs. Mức đỉnh anti-HBs đạt được 68 ngày sau liều vắc-xin tái tổ hợp cuối cùng và 138 ngày sau liều vắc-xin từ huyết tương cuối cùng. Tuổi của người được tiêm vắc-xin ảnh hưởng tiêu cực đến mức độ anti-HBs và cũng ảnh hưởng đến thời gian cần thiết để đạt đến đỉnh anti-HBs. Mô hình toán học hai chiều (mức độ log10, thời gian log10) của sự suy giảm anti-HBs đã được xây dựng trên mẫu những người nhận vắc-xin tái tổ hợp và sau đó được kiểm chứng trên các mẫu khác của người nhận vắc-xin tái tổ hợp hoặc từ huyết tương. Tuổi tác, giới tính, loại vắc-xin (tái tổ hợp hoặc từ huyết tương), số liều vắc-xin (ba hoặc bốn) không ảnh hưởng đến mô hình toán học của sự suy giảm kháng thể. Chương trình có thể được tải xuống tại trang web: http://www2.stat.unibo.it/palareti/vaccine.htm. Việc đưa vào một xác định anti-HBs được thu thập sau khi đạt đỉnh, chương trình tính toán dự đoán sự suy giảm anti-HBs cá nhân và cho phép lập kế hoạch chính sách tiêm nhắc lại hiệu quả.