Successful tumour necrosis factor (TNF) blocking therapy suppresses oxidative stress and hypoxia- induced mitochondrial mutagenesis in inflammatory arthritis

Monika Biniecka
Aisling Kennedy
Chin T Ng
Ting C Chang
Emese Balogh
Edward Fox
Douglas J Veale
Ursula Fearon
Jacintha N O'sullivan
Successful tumour necrosis factor (TNF) blocking therapy suppresses oxidative stress and hypoxia- induced mitochondrial mutagenesis in inflammatory arthritis
R E S E A R C H A R T I C L E Open Access
Introduction:To examine the effects of tumour necrosis factor (TNF) blocking therapy on the levels of early mitochondrial genome alterations and oxidative stress.Methods: Eighteen inflammatory arthritis patients underwent synovial tissue oxygen (tpO 2 ) measurements and clinical assessment of disease activity (DAS28-CRP) at baseline (T0) and three months (T3) after starting biologic therapy. Synovial tissue lipid peroxidation (4-HNE), T and B cell specific markers and synovial vascular endothelial growth factor (VEGF) were quantified by immunohistochemistry. Synovial levels of random mitochondrial DNA (mtDNA) mutations were assessed using Random Mutation Capture (RMC) assay.Results: 4-HNE levels pre/post anti TNF-α therapy were inversely correlated with in vivo tpO 2 (P < 0.008; r = -0.60). Biologic therapy responders showed a significantly reduced 4-HNE expression (P < 0.05). High 4-HNE expression correlated with high DAS28-CRP (P = 0.02; r = 0.53), tender joint count for 28 joints (TJC-28) (P = 0.03; r = 0.49), swollen joint count for 28 joints (SJC-28) (P = 0.03; r = 0.50) and visual analogue scale (VAS) (P = 0.04; r = 0.48). Strong positive association was found between the number of 4-HNE positive cells and CD4+ cells (P = 0.04; r = 0.60), CD8+ cells (P = 0.001; r = 0.70), CD20+ cells (P = 0.04; r = 0.68), CD68+ cells (P = 0.04; r = 0.47) and synovial VEGF expression (P = 0.01; r = 063). In patients whose in vivo tpO 2 levels improved post treatment, significant reduction in mtDNA mutations and DAS28-CRP was observed (P < 0.05). In contrast in those patients whose tpO 2 levels remained the same or reduced at T3, no significant changes for mtDNA mutations and DAS28-CRP were found. Conclusions: High levels of synovial oxidative stress and mitochondrial mutation burden are strongly associated with low in vivo oxygen tension and synovial inflammation. Furthermore these significant mitochondrial genome alterations are rescued following successful anti TNF-α treatment.

Introduction

Mitochondria produce ATP through oxidative metabolism to provide cells with energy under physiological conditions. The mitochondrial electron transport chain (ETC) is also a major cellular source of reactive oxygen species (ROS) as some of the electrons passing to molecular oxygen are prone to leakage from the chain and get trapped by oxygen, which converts to superoxide [1]. Hypoxia characterised by an inadequate supply of molecular oxygen, can trigger mitochondria dysfunction through ineffective functioning of respiratory complexes of ETC [2,3].

Free oxygen radicals are highly active molecules and increased mitochondrial ROS generation promotes cellular oxidative stress resulting in oxidative mitochondrial DNA (mtDNA) damage and lipid peroxidation.

Moreover, ROS mediate the stress signalling pathways involving nuclear factor-kappa B (NF-B) [4]. mtDNA is in the proximity of ROS generation site and has relatively limited repair capacity, which makes it vulnerable to high mutation rates [5]. Mutations and deletions of the mitochondrial genome in genes encoding proteins for subunits of mitochondrial respiratory chain complexes I-V, rRNA and tRNA have been linked to a variety of degenerative human diseases and high levels of mtDNA mutations have been also found in many tumours and cancer cells [5,6].

Oxidative stress, which arises from an imbalance between ROS production and antioxidant defences, results also in lipid peroxidation of cell membrane polyunsaturated fatty acids [7]. The primary products of freeradical attack of biological membranes are lipid hydroperoxides, which can decompose to highly reactive, cytotoxic secondary end products, such as 4-hydroxy-2nonenal (4-HNE) [8]. 4-HNE is an endogenously generated α,β unsaturated aldehyde, which is not only a marker of extensive oxidative stress but also can modulate cellular metabolism, inflammatory responses and apoptosis via its effects on transcriptional regulation and protein modification [9]. 4-HNE-induced mitochondrial protein modifications include those involved in the ETC, cellular respiration and Krebs cycle [10]. Moreover, 4-HNE can form adducts on DNA bases and modifies mtDNA thus measurement of such modifications may reflect the level of mitochondrial alterations [11].

Inflammatory arthritis (IA) is a chronic, progressive disorder associated with joint inflammation, synovial tissue hypertrophy, joint effusions and degradation of articular cartilage and bone. The normal synovial tissue is a relatively acellular structure with a lining layer (one to two cells thick) comprised of macrophages and fibroblasts. The morphology of IA synovium is strikingly different. There is a significant increase in the number of blood vessels that are associated with differential vascular morphology. Furthermore, the early vascular changes are accompanied by increased recruitment of macrophages and synovial fibroblast cells in the lining layer, along with infiltration of T, B and plasma cells. The precise mechanisms involved in regulation of persistent synovial infiltration and invasion are unclear, but high levels of TNF-α may be crucial in mediating the pathogenesis of IA. TNF-α is a proinflammatory cytokine, activating the NF-B pathway, leading to a downstream cascade of other proinflammatory cytokines [12,13]. Moreover, it is known to increase mitochondrial ROS production [14,15] and induce the formation of lipidderived aldehydes [16]; however TNF-α-induced mitochondrial mutagenesis has not yet been examined in patients with IA. Current targeted biologic therapies, including anti-TNF-α inhibitors result in greater disease improvement and prevention of joint erosion, although clinical studies on the efficacy of TNF-α blocking agents clearly show that about 40% of patients receiving this therapy are non-responders.

Recently, we demonstrated that successful biologic therapy significantly improves in vivo synovial hypoxia and it is strongly associated with improvement of joint inflammation [17]. In this study we investigate if successful anti-TNF-α treatment alters the levels of early mitochondrial genome alterations, which can play a crucial role in governing clinical response or resistance. Furthermore, we determine if TNF-α blocking therapy changes the levels of synovial 4-HNE, further confirming the relation between hypoxia, oxidative damage and mitochondrial mutagenesis.

Materials and methods

Patient recruitment

All research was carried out in accordance with the Declaration of Helsinki, and approval for this study was granted by the St. Vincent's University Hospital Medical Research and Ethics Committee. Eighteen patients with active IA (rheumatoid arthritis (RA) n = 14 and psoriatic arthritis (PsA) n = 4) were recruited from outpatient clinics at Department of Rheumatology, St. Vincent's University Hospital. All patients fulfilled the diagnostic criteria for RA and PsA [18,19]. All patients provided fully informed consent and underwent arthroscopy at baseline (T0) and three months after commencement of TNF blocking therapy (T3). At baseline, 50% of patients were naive for disease-modifying anti-rheumatic drugs (DMARDs) and corticosteroids; however, all patients including those on DMARDs (methotrexate (MTX) alone 35%, MTX + salazopyrine 10%, and plaquenil alone 5%) were biologic naive, had active disease, had at least one inflamed knee joint and were due to commence biologic therapy. Clinical and laboratory assessment was performed using standard measures of 28 tender and swollen joint count (DAS28), rheumatoid factor, anti-cyclic citrullinated peptide antibody, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and global health visual analogue scale (VAS). All measurements were obtained on the same day prior to baseline and three months after anti TNF-α treatment arthroscopy.

Arthroscopy, measurement of in vivo tpO 2

and sample collection

Under local anaesthetic, patients (n = 18) underwent arthroscopy at baseline and three months after commencement of TNF blocking therapy. Arthroscopy of the inflamed knee was performed using a Wolf 2.7 mm needle arthroscope. Macroscopic synovitis and vascularity were scored on a VAS (0-100 mm). A LICOX ® combined pO 2 and temperature probe (Integra Life Sciences Corporation, New Jersey, USA) was used to obtain synovial tissue oxygen partial pressure as previously described [20]. Synovial membrane biopsies were obtained from the site of the oxygen tension measurement and immediately embedded in mounting media for immunohistochemical analysis or snap frozen in liquid nitrogen for mitochondrial mutagenesis analysis.

Immunohistochemistry and scoring

Immunohistochemistry was performed using 7 μm cryostat synovial tissue sections and the DAKO ChemMate Envision Kit (DAKO, Glostrup, Denmark). Sections were defrosted at room temperature for 20 minutes, fixed in acetone for 10 minutes and washed in PBS for 5 minutes. Non-specific binding was blocked using 10% casein in PBS for 20 minutes. The sections were incubated with primary antibodies against human 4-HNE (Genox, Baltimore, MD, USA), CD4, CD8, CD20, CD68 (all from DAKO, Glostrup, Denmark) and vascular endothelial growth factor (VEGF) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). IgG control antibodies were used as negative controls. Following primary antibody incubation endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide for seven minutes at room temperature. Slides were incubated with secondary antibody/HRP (DAKO, Glostrup, Denmark). DAB (1:50) was used to visualise staining, and Mayer's haematoxylin (BDH Laboratories, Poole, UK) was incubated for 30 seconds as a counterstain prior to mounting in Pertex mounting media. Images were captured using Olympus DP50 light microscope and AnalySIS software (Soft Imaging System Corporation, Lakewood, CO, USA). Slides were scored separately for lining and sublining layers using well established and validated semi-quantitative scoring method, where the percentage of cells that were positive for a specific marker was compared with the percentage of cells that were negative [21]. Percentage positivity was graded using 0 to 4 scale, where 0 represented no stained cells, 1 was 1 to 25% stained cells, 2 was 25 to 50% stained cells, 3 was 50 to 75% stained cells, and 4 was 75 to 100% stained cells.

Mitochondrial random mutation capture assay

A sub-group of eight patients were selected from the initial cohort to quantify the levels of mitochondrial point mutations before and after treatment. Levels of mitochondrial point mutations in snap frozen synovial biopsies were analysed in a blinded fashion using Mitochondrial Random Mutation Capture assay as described previously [22]. Biopsies were homogenised (Precellys 24, Stretton Scientific Ltd., Stretton, Derbyshire, United Kingdom) in 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 20 mM EDTA, 0.5% SDS buffer and digested with Proteinase K (Sigma-Aldrich, Dublin, Ireland) at a final concentration of 0.2 mg/ml and incubated overnight at 56°C

. The mtDNA was extracted using phenol-chloroformisoamyl alcohol (25:24:1 by volume, Sigma-Aldrich, Dublin, Ireland) added in a 1:1 ratio with the lysed tissue, mixed thoroughly by shaking, and centrifuged at more than 12,000 × g for 10 minutes. The aqueous phase was gently removed from the top of the solution, without disturbing the interphase. The aqueous solution was again mixed with phenol-chloroform-isoamyl alcohol in a 1:1 ratio and re-extracted. One-tenth volume of 3 M sodium acetate was added, and the samples were precipitated with 2 to 2.5 volumes of ethanol. The DNA samples were resuspended in 50 μl 10 mM TrisCl. Ten micrograms of mtDNA were digested with 100 units of TaqαI restriction enzyme (New England BioLabs, Herts, United Kingdom), 1 × BSA and a TaqαI-specific digestion buffer (10 mM Tris-HCl, 10 mM MgCl 2 , 100 mM NaCl, pH 8.4) for 10 hours; 100 units of TaqαI being added to the reaction mixture every hour.

PCR amplification was performed in 25 μl reactions, containing 12.5 μl 2 × SYBR Green Brilliant Mastermix (Stratagene, Agilent Technologies, Inc., Santa Clara, CA, USA), 0.1 μl UDG (New England Biosciences, Herts, United Kingdom), 0.7 μl of 10 pM/μl forward and reverse primers (Integrated DNA Technologies, Inc., San Diego, CA, USA), and 6.7 μl water. The samples were amplified using a Roche Lightcycler 480 using the following protocol: 37°C for 10 minutes and 95°C for 10 minutes followed by 45 cycles of 95°C for 15 seconds, 60°C for 1 minute. Samples were held at 72°C for 7 minutes and, following melt curve analysis, immediately stored at -80°C. The primer sequences used were as follows: for mtDNA copy number: 5'ACAGTTTATGTAGCTTACCTCC-3' (forward) and 5'-TTGCTGCGTGCTTGATGCTTGT-3' (reverse); for random mutations: 5'-CCTCAACAGTTAAATCAA-CAAAACTGC-3' (forward) and 5'-GCGCTTACTTT GTAGCCTTCA-3' (reverse).

Statistical analysis

Data are presented as medians and interquartile ranges. Data were assessed using Wilcoxon's signed-rank test or Spearman's rank correlation coefficient as appropriate using the Statistical Package for the Social Sciences (SPSS, Chicago, IL, USA). All P values are two-sided and P values less than 0.05 were considered statistically significant.

Results

In vivo changes of oxidative stress pre/post anti TNF-a therapy Eighteen IA patients underwent synovial tissue oxygen tension (tpO 2 ) measurements and clinical assessment of disease activity (28-joint count disease activity score using C-reactive protein (DAS28-CRP)) at baseline and three months after starting biologic therapy. At T3 patients were categorised according to remission criteria using the DAS28 cut-off less than or more than 2.6. Patients with DAS28-CRP less than 2.6 were defined as responders (n = 7) and patients with DAS28-CRP more than 2.6 were defined as non-responders (n = 11). In responders, the median baseline pO 2 in the synovial tissue was 18.07 mmHg (range 4.3 to 42.2 mmHg), and was lower than in those patients at T3 (median tpO 2 39.25 mmHg (range 24.7 to 68.2 mmHg)). Of clinical responders, 86% had a corresponding increase in their synovial tpO 2 measurements. In non-responders the median baseline pO 2 was 23.75 mmHg (range 6.8 to 46.4 mmHg), and their median pO 2 level after biologic therapy was 19.78 mmHg (range 10.5 to 39.6 mmHg).

In clinical non-responders, 64% patients showed decrease in their synovial tpO 2 levels at T3. Furthermore, tpO 2 levels did not differ significantly between baseline patients with RA and those with PsA (n = 14 RA, n = 4 PsA). The median oxygen tension for RA was 23.5 mmHg and for PsA was 14.5 mmHg (P = 0.3).

To determine whether biologic treatment changes the levels of synovial oxidative damage, the number of 4-HNE positive cells was assessed in both lining and sublining layers of synovial tissue. Figures 1a and 1b show representative images of 4-HNE expression levels in responders at T0 and T3, respectively. Figure 1c graphically illustrates significantly reduced cytoplasmic 4-HNE expression in sublining layer in patients who successfully responded to anti-TNF-α therapy (P < 0.05). No significant differences in the levels of cytoplasmic 4-HNE expression pre/post therapy were found in non-responders (Figures 1d to 1f). In addition, the levels of 4-HNE did not differ significantly between baseline patients with RA and those with PsA (P = 0.6).

Previously, we demonstrated significant baseline inverse correlation between tpO 2 measurements and 4-HNE expression [20]. In this study we extend these findings and demonstrate that change in tpO 2 is also significantly and inversely correlated with changes in 4-HNE levels pre/post biologic therapy (P < 0.008; r = -0.60; Table 1). It suggests that as synovial tissue becomes less hypoxic oxidative stress is decreased. Furthermore, when patients were categorised according to their changes in tpO 2 before and after therapy, a significant reduction in the number of 4-HNE positive cells was observed only in patients who had higher oxygen levels at T3 compared with T0 (data not shown).

Synovial oxidative stress and clinical markers

The relation of oxidative stress marker and clinical markers pre/post anti-TNF-α therapy is shown in Table 1.  Table 2). We have also performed the colocalisation staining between synovial 4-HNE and all cellular specific markers and observed 4-HNE expression in T-cells, B-cells, macrophages and cells of blood vessels.

As higher levels of 4-HNE are strongly associated with high VEGF expression and the number of inflammatory cells pre/post therapy, it may suggest a key role of oxidative stress in driving inflammation and angiogenesis, two crucial processes involved in progression of IA.

Effect of biologic therapy on mitochondrial mutagenesis

To determine whether biologic therapy alters mitochondrial genome instability, random mutation capture assay was performed at baseline and three months after treatment in a sub-group of eight patients. Patients were categorised into two groups, those whose tpO 2 levels improved after treatment (n = 4) and those whose in vivo oxygen level remained the same or reduced after three months therapy (n = 4). Figure 2a shows pre/post tpO 2 changes in patients who had a significant increase in in vivo oxygen measurements after treatment in comparison with their baseline levels (P < 0.05). This was associated with significantly reduced frequency of mitochondrial point mutations in comparison with baseline levels (P < 0.05; Figure 2b) and with significantly lower DAS28-CRP scores at T3 than before treatment (P < 0.05; Figure 2c). In contrast, no significant changes in the pre/post levels of mtDNA mutations (Figure 2e) and DAS28-CRP (Figure 2f) were observed in patients who showed no improvement in in vivo tpO 2 levels post treatment (P < 0.05; Figure 2d). This data may suggest mitochondrial genome alterations as a consequence of elevated synovial hypoxia. In addition, we found that hypoxia-induced mitochondrial mutagenesis was positively correlated with clinical markers of IA. As shown in Table 3 we found significant associations between the levels of mitochondrial point mutations and DAS28-CRP (P = 0.01; r = 0.83), CRP (P = 0.02; r = 0.77) and ESR (P = 0.04; r = 0.73).

Discussion

Chronic inflammatory arthropathies, such as RA and PsA, are characterised by complex chronic inflammatory processes. Oxygen metabolism is important in synovitis and joint destruction [23]. ROS stimulates synovial fibroblasts to secrete matrix metalloproteinases, inhibits cartilage proteoglycan synthesis and accelerates bone resorption [24,25]. Previously, we have demonstrated profoundly hypoxic synovial environment of the inflamed joint (approximately 3%) [26]. Furthermore, we have shown that biologic anti-TNF-α therapy significantly increased the synovial in vivo tpO 2 levels only in those patients who respond to anti-TNF-α therapy [17].  In this study we examine the effect of TNF-blocking therapy on mitochondrial mutagenesis and synovial oxidative stress profiles. We report for the first time that the increase in tpO 2 levels observed in responders is associated with significant decrease and strong inverse correlation of synovial lipid peroxidation. In addition, increases in tpO 2 significantly reduces the levels of random mitochondrial mutations, presumably as a result of decreased oxidative stress profile. TNF-α affects many cellular processes, such as activation of phospholipases [27], proteases [28] and DNA damage [29]. Mitochondrially derived ROS are strongly implicated in TNF-α cytotoxicity and may mediate the activation of transcriptional factor NF-B, which in turn can stimulate mitochondrial NADPH oxidase [15,30]. Inhibition of ETC complex III by antimycin A increases ROS and inhibits TNF-α triggered NF-B activation, highlighting the importance of the ETC in TNF-α cytotoxicity [31]. Recently, we have shown that hypoxia is an important stimulus of TNF-α secretion, where higher levels of synovial fluid TNF-α were detected in patients with synovial tpO 2 less than 20 mmHg than in those with tpO 2 more than 20 mmHg [26]. Figure 2 Effects of anti TNF-a therapy on the levels of mitochondrial point mutation and disease activity (DAS28-CRP). Patients were categorised into two groups according to their in vivo tissue oxygen tension (tpO 2 ) changes from baseline (T0 -white boxes) to three months after anti TNF-α therapy (T3 -grey boxes). (a) Group 1 represents patients whose tpO 2 levels improved at T3 in comparison with T0 (n = 4; P < 0.05). (b) Increase in tpO 2 was associated with significantly reduced frequency of mitochondrial point mutations at T3 in comparison with baseline levels (P < 0.05). (c) It was also associated with significantly lower DAS28-CRP scores at T3 than at T0 (P < 0.05). (d) Group 2 represents patients whose in vivo oxygen levels remained the same or reduced at T3 in comparison with T0 (n = 4; P < 0.05). (e) No significant changes in the pre/post levels of mtDNA mutations were observed in patients having more hypoxic synovium at T3 than at T0 (NS). (f) No significant changes in the pre/post levels of DAS28-CRP were found in patients who were more hypoxic at T3 than at T0 (NS). Boxes represent the 25th to 75th percentiles, lines within the boxes represent the median, and lines outside the boxes represent the 10th and 90th percentiles. Oxidative stress arising from overproduction of ROS leads to formation of reactive aldehydes such as 4-HNE. Mitochondrial are primed for attack by 4-HNE and formation of adducts between 4-HNE and mitochondrial components. Detection of 4-HNE-mitochondrial protein adducts can reflect mitochondrial dysfunction and oxidative stress [32]. We have previously assessed the expression of synovial lipid peroxidation in IA patients and demonstrated a significant inverse correlation between 4-HNE expression and oxygen tension of the inflamed join, probably reflecting mitochondrial damage [20]. Mitochondrial membrane components are targets for 4-HNE modification and the adenine nucleotide translocator in the inner mitochondrial membrane is affected by lipid peroxidation [33]. This study in the first to show that patients who respond to TNF-blocking therapy show a significant increase in tpO 2 and this is associated with reduced 4-HNE levels. In contrast, in non-responders there is no change in in vivo oxygen levels and subsequently no change in 4-HNE levels. These data suggest that as the joint tissue becomes less hypoxic, a corresponding reduction in oxidative stress is affected. Previous studies have demonstrated positive effects of anti-TNF-α treatment on oxidative damage in RA, where urinary levels of oxidative DNA damage and lipid peroxidation were significantly reduced at three months therapy [34]. However, our study considerably extends the above reports and shows direct evidence of a significant reduction of oxidative stress in relation to in vivo hypoxia measurements.

We have recently demonstrated that increased tpO 2 levels after successful anti-TNF biologic therapy is associated with reduced disease activity and macroscopic vascularity [17]. Furthermore, we have also reported that high synovial 4-HNE levels positively correlated with clinical disease activity scores in patients prior to receiving TNF-α blocking therapy [20]. In this study the same parameters were assessed in patients after anti-TNF-α treatment and we found significant positive association between synovial 4-HNE expression and clinical measures of arthritis.

Several cellular and environmental sources of synovial oxidative stress have been proposed, including activated neutrophils, monocytes and macrophages, hypoxia and vascular changes. Furthermore, studies by Remans et al. indicated synovial T lymphocytes as the main generators of intracellular free radicals in RA patients [35]. We demonstrate a correlation between oxidative stress, inflammation and angiogenesis, where increase in tpO 2 and reduce oxidative stress observed in responders is associated with lower microscopic scores of T-cells (CD4 and CD8), B-cells (CD20), macrophages (CD68) and angiogenesis (VEGF). Experiments using 4-HNEmodified antigens of T and B cells showed rapid autoimmune response, suggesting that B and T cell modification by 4-HNE may result in the onset of autoimmune reactions or even autoimmune disease processes [36]. The link between oxidative lipid modifications and activation of the inflammatory potential of macrophages has been also suggested [37]. In human osteoarthritic chondrocytes 4-HNE induces prostaglandin E release and cyclooxygenase-2 (COX-2) expression, providing evidence for the role of 4-HNE as redox-sensitive signalling mechanisms of inflammatory response [38]. Furthermore, 4-HNE elevated VEGF secretion has been shown in retinal pigment epithelial cells [39] and vascular smooth muscle cells [40]. This correlation of VEGF expression and 4-HNE supports our current findings.

RA has many features of autoimmune disease; however, some studies suggest inflammation-independent joint destruction [41]. It has been shown that elevated production of ROS at the sites of chronic inflammation has genotoxic effects and increases the likelihood of mutagenic events. In RA, local exposure to oxidative stress was found to induce genetic changes and was proposed as a mechanism that permanently alters and imprints synovial cells [42,43]. Furthermore, oxidative stress can suppress expression of DNA repair enzymes in inflamed synovium such as DNA mismatch repair system that might potentially limit the accumulation of mutations [44]. Other extensive studies demonstrated synovial p53 mutations, which are characteristic DNA damage caused by oxidative stress. High expression of p53 was found in synovial tissue from longstanding RA patients and lower in early RA patients, osteoarthritis (OA) and reactive arthritis patients [45]. This oxidative DNA damage of p53 gene is likely to promote neoplastic transformation of synovial cells that may subsequently contribute to disease progression and joint destruction.

Oxidative stress may also contribute to somatic mtDNA mutation. mtDNA mutations were known to have a key role in ageing-related diseases and carcinogenesis. Currently, there is a growing body of evidence suggesting the role of mitochondrial alterations in rheumatoid disorders [46]. Recent studies showed higher accumulation of mtDNA damage in chondrocytes from OA patients compared with those from normal donors [47]. Higher incidence of mtDNA somatic mutations has also been detected in synoviocytes and synovial tissue of RA than OA controls [48]; however, the frequency of mitochondrial mutations has not been examined. Recently, using synovial tissue of baseline IA patients, we have screened a large number of mtDNA molecules for the presence of unexpanded random mutations, which may be crucial in driving disease progression. We demonstrated, for the first time that greater levels of mtDNA point mutations were significantly associated with higher hypoxia in vivo, oxidative stress and disease activity [49].

TNF-α was demonstrated to induce in vitro mitochondrial ROS release and DNA damage in human chondrocytes and overexpression of the DNA repair enzyme prevents mtDNA alterations following TNF-α exposure [50]. In this study, we determined whether TNF therapy affect the levels of mtDNA mutations. We observed that the increase in tpO 2 after treatment was associated with significant decrease in the levels of mtDNA mutations and reduction of disease activity scores DAS28-CRP. Contrary, no significant improvements in the levels of mtDNA mutations and DAS28-CRP were found in patients who had more hypoxic synovium after receiving TNF blocking treatment. Our findings strongly support the hypothesis that an increase in mutation frequency is a consequence of elevated hypoxia and oxidative damage to the mitochondrial genome. Furthermore, our results are in agreement with another report indicating the role of oxidative stress and diminished mtDNA integrity in the progression of OA, where high levels of mutagenesis following exposure to ROS were associated with reduced mtDNA capacity and cell viability [47]. In addition, our study is the first to show that successful anti-TNF-α therapy reduces the frequency of mitochondrial synovial mutagenesis in IA. It may suggest a central role of mitochondrial mutagenesis in the cellular mechanism of anti-TNF-α response or resistance to the treatment

Conclusions

We have clearly demonstrated a close association between oxidative stress, mitochondrial mutagenesis and clinical responses to TNF-blocking therapy in IA patients. The greater mitochondrial mutation burden in synovial tissue is associated with higher hypoxia levels in vivo and these significant mitochondrial genome alterations are rescued following successful anti-TNF treatment.

Figure 1
1Representative pre/post immunohistochemical images of 4-HNE expression and their graphical representation. (a to c) Responders. (d to f) Non-responders. T0 is time at baseline; T3 is three months after anti-TNF-α treatment. (a to b) Biologic therapy responders showed lower synovial 4-hydroxy-2-nonenal (4-HNE) expression at (b) T3 compared with their (a) T0 levels. (c) Graphical illustration of synovial 4-HNE levels at T0 and T3 (P < 0.05). (d to e) No significant 4-HNE changes were seen between (d) T0 and (e) T3 in patients who did not respond to therapy. (f) Graphical representation of synovial 4-HNE levels in non-responders at T0 and T3.

We found significant positive correlations between levels of 4-HNE and DAS28-CRP (P = 0.02; r = 0.53), 4-HNE and tender joint count (TJC)-28 (P = 0.03; r = 0.49), 4-HNE and swollen joint count (SJC)-28 (P = 0.03; r = 0.50), 4-HNE and VAS (P = 0.04; r = 0.48). These results demonstrate a link between oxidative stress and clinical parameters of disease activity and suggest that microscopically assessed levels of 4-HNE may closely reflect clinical scores of IA. Synovial levels of oxidative stress, inflammation and angiogenesis pre/post biologic therapy Levels of lipid peroxidation were correlated with specific markers of T-cells (CD4 and CD8), B-cells (CD20), and macrophages (CD68). Table 2 demonstrates significant positive associations between the number of 4-HNE positive cells and CD4 + cells (P = 0.04; r = 0.60), CD8 + cells (P = 0.001; r = 0.70), CD20 + cells (P = 0.04; r = 0.68) and CD68 + cells (P = 0.04; r = 0.47). Furthermore, high 4-HNE expression correlates with high level of VEGF angiogenic marker (P = 0.01; r = 0.63;

Table 1
1Spearman's rank test correlations of 4-HNE microscopic scores in synovial tissue pre/post anti TNF-a therapy with clinical parameters4-HNE
r-value
P value

tpO 2
-0.60
0.008

DAS28-CRP
0.53
0.02

TJC-28
0.49
0.03

SJC-28
0.50
0.03

VAS
0.48
0.04

DAS28-CRP, 28-joint count disease activity score using C-reactive protein; 4-
HNE, 4-hydroxy-2-nonenal; SJC-28, swollen joint count for 28 joints; TJC-28,
tender joint count for 28 joints; tpO 2 , in vivo tissue oxygen tension; VAS, visual
analogue scale.

Table 2
2Spearman's rank test correlations of 4-HNE
synovial tissue pre/post anti TNF-a therapy with synovial
inflammation and angiogenesis

4-HNE
r-value
P value

CD4 ll
0.60
0.04

CD8 sl
0.70
0.001

CD20 sl
0.68
0.04

CD68 ll
0.47
0.04

VEGF bv
0.63
0.01

bv, blood vessel; CD4 and CD8, T-cell markers; CD20, B-cell marker; CD68,
marker of macrophages; 4-HNE, 4-hydroxy-2-nonenal; ll, lining layer; sl,
sublining layer; VEGF, vascular endothelial growth factor.

Table 3
3Spearman's rank test correlations of mitochondrial point mutations pre/post anti TNF-a therapy with clinical parametersMitochondrial point mutations
r-value
P value

DAS28-CRP
0.83
0.01

CRP (mmg/L)
0.77
0.02

ESR (mm/hr)
0.73
0.04

CRP, C-reactive protein; DAS28-CRP, 28-joint count disease activity score using
C-reactive protein; ESR, erythrocyte sedimentation rate.

Biniecka et al. Arthritis Research & Therapy 2011, 13:R121 http://arthritis-research.com/content/13/4/R121 Page 3 of 9
Biniecka et al. Arthritis Research & Therapy 2011, 13:R121 http://arthritis-research.com/content/13/4/R121 Page 7 of 9
AcknowledgementsThis work was funded by the Health Research Board of Ireland (R10238 and JRFC-05-01).Authors' contributions MB conducted most of the experiments and analysis of data. AK, CTN, TCC, EB, EF and UF performed some of the experiments. JNO, UF, DV and MB participated in the data analysis and manuscript preparation and final approval of the version to be published. JNO, UF and DV participated in the study design and supervised the research. DV and CTN recruited all patients, performed the arthroscopies and oxygen measurements and provided all clinical information. All authors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.
Mitochondrial reactive oxygen species regulate hypoxic signaling. R B Hamanaka, N S Chandel, Curr Opin Cell Biol. 21Hamanaka RB, Chandel NS: Mitochondrial reactive oxygen species regulate hypoxic signaling. Curr Opin Cell Biol 2009, 21:894-899.

The causes of cancer revisited: "mitochondrial malignancy" and ROS-induced oncogenic transformation -why mitochondria are targets for cancer therapy. S J Ralph, S Rodriguez-Enriquez, J Neuzil, E Saavedra, R Moreno-Sanchez, Mol Aspects Med. 31Ralph SJ, Rodriguez-Enriquez S, Neuzil J, Saavedra E, Moreno-Sanchez R: The causes of cancer revisited: "mitochondrial malignancy" and ROS-induced oncogenic transformation -why mitochondria are targets for cancer therapy. Mol Aspects Med 2010, 31:145-170.

Mitochondria and hypoxic signaling: a new view. R O Poyton, P R Castello, K A Ball, D K Woo, N Pan, Ann N Y Acad Sci. 1177Poyton RO, Castello PR, Ball KA, Woo DK, Pan N: Mitochondria and hypoxic signaling: a new view. Ann N Y Acad Sci 2009, 1177:48-56.

Oxidative stress, inflammation, and cancer: how are they linked? Free Radic. S Reuter, S C Gupta, M M Chaturvedi, B B Aggarwal, Biol Med. 49Reuter S, Gupta SC, Chaturvedi MM, Aggarwal BB: Oxidative stress, inflammation, and cancer: how are they linked? Free Radic Biol Med 2010, 49:1603-1616.

The mitochondrial genome in human adaptive radiation and disease: on the road to therapeutics and performance enhancement. D C Wallace, Gene. 354Wallace DC: The mitochondrial genome in human adaptive radiation and disease: on the road to therapeutics and performance enhancement. Gene 2005, 354:169-180.

Mitochondrial DNA mutations in human disease. R W Taylor, D M Turnbull, Nat Rev Genet. 6Taylor RW, Turnbull DM: Mitochondrial DNA mutations in human disease. Nat Rev Genet 2005, 6:389-402.

Lipid peroxidation of membrane phospholipids generates hydroxy-alkenals and oxidized phospholipids active in physiological and/or pathological conditions. A Catala, Chem Phys Lipids. 157Catala A: Lipid peroxidation of membrane phospholipids generates hydroxy-alkenals and oxidized phospholipids active in physiological and/or pathological conditions. Chem Phys Lipids 2009, 157:1-11.

Lipid peroxidation and cell cycle signaling: 4-hydroxynonenal, a key molecule in stress mediated signaling. Y Yang, R Sharma, A Sharma, S Awasthi, Y C Awasthi, Acta Biochim Pol. 50Yang Y, Sharma R, Sharma A, Awasthi S, Awasthi YC: Lipid peroxidation and cell cycle signaling: 4-hydroxynonenal, a key molecule in stress mediated signaling. Acta Biochim Pol 2003, 50:319-336.

4-Hydroxynonenal-protein adducts: A reliable biomarker of lipid oxidation in liver diseases. G Poli, F Biasi, G Leonarduzzi, Mol Aspects Med. 29Poli G, Biasi F, Leonarduzzi G: 4-Hydroxynonenal-protein adducts: A reliable biomarker of lipid oxidation in liver diseases. Mol Aspects Med 2008, 29:67-71.

Reactive species and mitochondrial dysfunction: mechanistic significance of 4-hydroxynonenal. J R Roede, D P Jones, Environ Mol Mutagen. 51Roede JR, Jones DP: Reactive species and mitochondrial dysfunction: mechanistic significance of 4-hydroxynonenal. Environ Mol Mutagen 2010, 51:380-390.

Lipid peroxidation-induced DNA damage in cancer-prone inflammatory diseases: a review of published adduct types and levels in humans. U Nair, H Bartsch, J Nair, Free Radic Biol Med. 43Nair U, Bartsch H, Nair J: Lipid peroxidation-induced DNA damage in cancer-prone inflammatory diseases: a review of published adduct types and levels in humans. Free Radic Biol Med 2007, 43:1109-1120.

Molecular and cellular basis of rheumatoid joint destruction. E Karouzakis, M Neidhart, R E Gay, S Gay, Immunol Lett. 106Karouzakis E, Neidhart M, Gay RE, Gay S: Molecular and cellular basis of rheumatoid joint destruction. Immunol Lett 2006, 106:8-13.

NF-kappaB: a key role in inflammatory diseases. P P Tak, G S Firestein, J Clin Invest. 107Tak PP, Firestein GS: NF-kappaB: a key role in inflammatory diseases. J Clin Invest 2001, 107:7-11.

Tumor necrosis factor-alpha generates reactive oxygen species via a cytosolic phospholipase A2-linked cascade. C H Woo, Y W Eom, M H Yoo, H J You, H J Han, W K Song, Y J Yoo, J S Chun, J H Kim, J Biol Chem. 275Woo CH, Eom YW, Yoo MH, You HJ, Han HJ, Song WK, Yoo YJ, Chun JS, Kim JH: Tumor necrosis factor-alpha generates reactive oxygen species via a cytosolic phospholipase A2-linked cascade. J Biol Chem 2000, 275:32357-32362.

NF-kappaB inhibits TNF-induced accumulation of ROS that mediate prolonged MAPK activation and necrotic cell death. S Sakon, X Xue, M Takekawa, T Sasazuki, T Okazaki, Y Kojima, J H Piao, H Yagita, K Okumura, T Doi, H Nakano, EMBO J. 22Sakon S, Xue X, Takekawa M, Sasazuki T, Okazaki T, Kojima Y, Piao JH, Yagita H, Okumura K, Doi T, Nakano H: NF-kappaB inhibits TNF-induced accumulation of ROS that mediate prolonged MAPK activation and necrotic cell death. EMBO J 2003, 22:3898-3909.

Reduction of oxidative stress marker levels by anti-TNF-alpha antibody, infliximab, in patients with rheumatoid arthritis. Y Kageyama, M Takahashi, T Ichikawa, E Torikai, A Nagano, Clin Exp Rheumatol. 26Kageyama Y, Takahashi M, Ichikawa T, Torikai E, Nagano A: Reduction of oxidative stress marker levels by anti-TNF-alpha antibody, infliximab, in patients with rheumatoid arthritis. Clin Exp Rheumatol 2008, 26:73-80.

. Biniecka, Arthritis Research & Therapy. 13Biniecka et al. Arthritis Research & Therapy 2011, 13:R121 http://arthritis-research.com/content/13/4/R121

TNF blocking therapy alters joint inflammation and hypoxia. A Kennedy, C T Ng, T C Chang, M Biniecka, J N O'sullivan, E Heffernan, U Fearon, D J Veale, Arthritis Rheum. 63Kennedy A, Ng CT, Chang TC, Biniecka M, O'Sullivan J N, Heffernan E, Fearon U, Veale DJ: TNF blocking therapy alters joint inflammation and hypoxia. Arthritis Rheum 2011, 63:923-932.

Classification of clinical subsets in psoriatic arthritis. D Veale, S Rogers, O Fitzgerald, Br J Rheumatol. 33Veale D, Rogers S, Fitzgerald O: Classification of clinical subsets in psoriatic arthritis. Br J Rheumatol 1994, 33:133-138.

The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. F C Arnett, S M Edworthy, D A Bloch, D J Mcshane, J F Fries, N S Cooper, L A Healey, S R Kaplan, M H Liang, H S Luthra, Arthritis Rheum. 31Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, Healey LA, Kaplan SR, Liang MH, Luthra HS: The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988, 31:315-324.

Oxidative damage in synovial tissue is associated with in vivo hypoxic status in the arthritic joint. M Biniecka, A Kennedy, U Fearon, C T Ng, D J Veale, O' Sullivan, J N , Ann Rheum Dis. 69Biniecka M, Kennedy A, Fearon U, Ng CT, Veale DJ, O'Sullivan JN: Oxidative damage in synovial tissue is associated with in vivo hypoxic status in the arthritic joint. Ann Rheum Dis 2010, 69:1172-1178.

Quantitative microscopic analysis of inflammation in rheumatoid arthritis synovial membrane samples selected at arthroscopy compared with samples obtained blindly by needle biopsy. P P Youssef, M Kraan, F Breedveld, B Bresnihan, N Cassidy, G Cunnane, P Emery, O Fitzgerald, D Kane, S Lindblad, R Reece, D Veale, P P Tak, Arthritis Rheum. 41Youssef PP, Kraan M, Breedveld F, Bresnihan B, Cassidy N, Cunnane G, Emery P, Fitzgerald O, Kane D, Lindblad S, Reece R, Veale D, Tak PP: Quantitative microscopic analysis of inflammation in rheumatoid arthritis synovial membrane samples selected at arthroscopy compared with samples obtained blindly by needle biopsy. Arthritis Rheum 1998, 41:663-669.

Quantification of random mutations in the mitochondrial genome. M Vermulst, J H Bielas, L A Loeb, Methods. 46Vermulst M, Bielas JH, Loeb LA: Quantification of random mutations in the mitochondrial genome. Methods 2008, 46:263-268.

The role of reactive oxygen species in immunopathogenesis of rheumatoid arthritis. A Mirshafiey, M Mohsenzadegan, Iran J Allergy Asthma Immunol. 7Mirshafiey A, Mohsenzadegan M: The role of reactive oxygen species in immunopathogenesis of rheumatoid arthritis. Iran J Allergy Asthma Immunol 2008, 7:195-202.

The role of reactive oxygen species in homeostasis and degradation of cartilage. Y E Henrotin, P Bruckner, J P Pujol, Osteoarthritis Cartilage. 11Henrotin YE, Bruckner P, Pujol JP: The role of reactive oxygen species in homeostasis and degradation of cartilage. Osteoarthritis Cartilage 2003, 11:747-755.

Bone loss caused by iron overload in a murine model: importance of oxidative stress. J Tsay, Z Yang, F P Ross, S Cunningham-Rundles, H Lin, R Coleman, P Mayer-Kuckuk, S B Doty, R W Grady, P J Giardina, A L Boskey, M G Vogiatzi, Blood. 116Tsay J, Yang Z, Ross FP, Cunningham-Rundles S, Lin H, Coleman R, Mayer- Kuckuk P, Doty SB, Grady RW, Giardina PJ, Boskey AL, Vogiatzi MG: Bone loss caused by iron overload in a murine model: importance of oxidative stress. Blood 2010, 116:2582-2589.

Synovial tissue hypoxia and inflammation in vivo. C T Ng, M Biniecka, A Kennedy, J Mccormick, O Fitzgerald, B Bresnihan, D Buggy, C T Taylor, J O'sullivan, U Fearon, D J Veale, Ann Rheum Dis. 69Ng CT, Biniecka M, Kennedy A, McCormick J, Fitzgerald O, Bresnihan B, Buggy D, Taylor CT, O'Sullivan J, Fearon U, Veale DJ: Synovial tissue hypoxia and inflammation in vivo. Ann Rheum Dis 2010, 69:1389-1395.

Tumour-necrosis-factor-mediated cytotoxicity is correlated with phospholipase-A2 activity, but not with arachidonic acid release per se. P Suffys, R Beyaert, De Valck, D Vanhaesebroeck, B Van Roy, F Fiers, W , Eur J Biochem. 195Suffys P, Beyaert R, De Valck D, Vanhaesebroeck B, Van Roy F, Fiers W: Tumour-necrosis-factor-mediated cytotoxicity is correlated with phospholipase-A2 activity, but not with arachidonic acid release per se. Eur J Biochem 1991, 195:465-475.

Protection from tumor necrosis factor cytotoxicity by protease inhibitors. V Ruggiero, S E Johnson, C Baglioni, Cell Immunol. 107Ruggiero V, Johnson SE, Baglioni C: Protection from tumor necrosis factor cytotoxicity by protease inhibitors. Cell Immunol 1987, 107:317-325.

DNA fragmentation and cytotoxicity caused by tumor necrosis factor is enhanced by interferongamma. G B Dealtry, M S Naylor, W Fiers, F R Balkwill, Eur J Immunol. 17Dealtry GB, Naylor MS, Fiers W, Balkwill FR: DNA fragmentation and cytotoxicity caused by tumor necrosis factor is enhanced by interferon- gamma. Eur J Immunol 1987, 17:689-693.

Enhanced mitochondrial radical production in patients which rheumatoid arthritis correlates with elevated levels of tumor necrosis factor alpha in plasma. R Miesel, M P Murphy, H Kroger, Free Radic Res. 25Miesel R, Murphy MP, Kroger H: Enhanced mitochondrial radical production in patients which rheumatoid arthritis correlates with elevated levels of tumor necrosis factor alpha in plasma. Free Radic Res 1996, 25:161-169.

Depletion of the mitochondrial electron transport abrogates the cytotoxic and gene-inductive effects of TNF. K Schulze-Osthoff, R Beyaert, V Vandevoorde, G Haegeman, W Fiers, EMBO J. 12Schulze-Osthoff K, Beyaert R, Vandevoorde V, Haegeman G, Fiers W: Depletion of the mitochondrial electron transport abrogates the cytotoxic and gene-inductive effects of TNF. EMBO J 1993, 12:3095-3104.

A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling. K S Echtay, T C Esteves, J L Pakay, M B Jekabsons, A J Lambert, M Portero-Otin, R Pamplona, A J Vidal-Puig, S Wang, S J Roebuck, M D Brand, EMBO J. 22Echtay KS, Esteves TC, Pakay JL, Jekabsons MB, Lambert AJ, Portero-Otin M, Pamplona R, Vidal-Puig AJ, Wang S, Roebuck SJ, Brand MD: A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling. EMBO J 2003, 22:4103-4110.

Inhibition of adenine nucleotide translocator by lipid peroxidation products. J J Chen, H Bertrand, B P Yu, Free Radic Biol Med. 19Chen JJ, Bertrand H, Yu BP: Inhibition of adenine nucleotide translocator by lipid peroxidation products. Free Radic Biol Med 1995, 19:583-590.

Etanercept reduces the oxidative stress marker levels in patients with rheumatoid arthritis. Y Kageyama, M Takahashi, T Nagafusa, E Torikai, A Nagano, Rheumatol Int. 28Kageyama Y, Takahashi M, Nagafusa T, Torikai E, Nagano A: Etanercept reduces the oxidative stress marker levels in patients with rheumatoid arthritis. Rheumatol Int 2008, 28:245-251.

Intracellular free radical production in synovial T lymphocytes from patients with rheumatoid arthritis. P H Remans, M Van Oosterhout, T J Smeets, M Sanders, W M Frederiks, K A Reedquist, P P Tak, F C Breedveld, J M Van Laar, Arthritis Rheum. 52Remans PH, van Oosterhout M, Smeets TJ, Sanders M, Frederiks WM, Reedquist KA, Tak PP, Breedveld FC, van Laar JM: Intracellular free radical production in synovial T lymphocytes from patients with rheumatoid arthritis. Arthritis Rheum 2005, 52:2003-2009.

Autoimmunity and oxidatively modified autoantigens. B T Kurien, R H Scofield, Autoimmun Rev. 7Kurien BT, Scofield RH: Autoimmunity and oxidatively modified autoantigens. Autoimmun Rev 2008, 7:567-573.

A lipid peroxidation-derived inflammatory mediator: identification of 4-hydroxy-2-nonenal as a potential inducer of cyclooxygenase-2 in macrophages. T Kumagai, N Matsukawa, Y Kaneko, Y Kusumi, M Mitsumata, K Uchida, J Biol Chem. 279Kumagai T, Matsukawa N, Kaneko Y, Kusumi Y, Mitsumata M, Uchida K: A lipid peroxidation-derived inflammatory mediator: identification of 4- hydroxy-2-nonenal as a potential inducer of cyclooxygenase-2 in macrophages. J Biol Chem 2004, 279:48389-48396.

Alterations of metabolic activity in human osteoarthritic osteoblasts by lipid peroxidation end product 4-hydroxynonenal. Q Shi, F Vaillancourt, V Cote, H Fahmi, P Lavigne, H Afif, Di Battista, J A Fernandes, J C Benderdour, M , Arthritis Res Ther. 8159Shi Q, Vaillancourt F, Cote V, Fahmi H, Lavigne P, Afif H, Di Battista JA, Fernandes JC, Benderdour M: Alterations of metabolic activity in human osteoarthritic osteoblasts by lipid peroxidation end product 4- hydroxynonenal. Arthritis Res Ther 2006, 8:R159.

Induction of vascular endothelial growth factor by 4-hydroxynonenal and its prevention by glutathione precursors in retinal pigment epithelial cells. S P Ayalasomayajula, U B Kompella, Eur J Pharmacol. 449Ayalasomayajula SP, Kompella UB: Induction of vascular endothelial growth factor by 4-hydroxynonenal and its prevention by glutathione precursors in retinal pigment epithelial cells. Eur J Pharmacol 2002, 449:213-220.

Induction of vascular endothelial growth factor in balloon-injured baboon arteries. A novel role for reactive oxygen species in atherosclerosis. J Ruef, Z Y Hu, L Y Yin, Y Wu, S R Hanson, A B Kelly, L A Harker, G N Rao, M S Runge, C Patterson, Circ Res. 81Ruef J, Hu ZY, Yin LY, Wu Y, Hanson SR, Kelly AB, Harker LA, Rao GN, Runge MS, Patterson C: Induction of vascular endothelial growth factor in balloon-injured baboon arteries. A novel role for reactive oxygen species in atherosclerosis. Circ Res 1997, 81:24-33.

Clinical improvement and radiological deterioration in rheumatoid arthritis: evidence that the pathogenesis of synovial inflammation and articular erosion may differ. D Mulherin, O Fitzgerald, B Bresnihan, Br J Rheumatol. 35Mulherin D, Fitzgerald O, Bresnihan B: Clinical improvement and radiological deterioration in rheumatoid arthritis: evidence that the pathogenesis of synovial inflammation and articular erosion may differ. Br J Rheumatol 1996, 35:1263-1268.

Somatic mutations in the p53 tumor suppressor gene in rheumatoid arthritis synovium. G S Firestein, F Echeverri, M Yeo, N J Zvaifler, D R Green, Proc Natl Acad Sci. 94Firestein GS, Echeverri F, Yeo M, Zvaifler NJ, Green DR: Somatic mutations in the p53 tumor suppressor gene in rheumatoid arthritis synovium. Proc Natl Acad Sci USA 1997, 94:10895-10900.

Rheumatoid arthritis and p53: how oxidative stress might alter the course of inflammatory diseases. P P Tak, N J Zvaifler, D R Green, G S Firestein, Immunol Today. 21Tak PP, Zvaifler NJ, Green DR, Firestein GS: Rheumatoid arthritis and p53: how oxidative stress might alter the course of inflammatory diseases. Immunol Today 2000, 21:78-82.

Microsatellite instability and suppressed DNA repair enzyme expression in rheumatoid arthritis. S H Lee, D K Chang, A Goel, C R Boland, W Bugbee, D L Boyle, G S Firestein, J Immunol. 170Lee SH, Chang DK, Goel A, Boland CR, Bugbee W, Boyle DL, Firestein GS: Microsatellite instability and suppressed DNA repair enzyme expression in rheumatoid arthritis. J Immunol 2003, 170:2214-2220.

p53 overexpression in synovial tissue from patients with early and longstanding rheumatoid arthritis compared with patients with reactive arthritis and osteoarthritis. P P Tak, T J Smeets, D L Boyle, M C Kraan, Y Shi, S Zhuang, N J Zvaifler, F C Breedveld, G S Firestein, Arthritis Rheum. 42Tak PP, Smeets TJ, Boyle DL, Kraan MC, Shi Y, Zhuang S, Zvaifler NJ, Breedveld FC, Firestein GS: p53 overexpression in synovial tissue from patients with early and longstanding rheumatoid arthritis compared with patients with reactive arthritis and osteoarthritis. Arthritis Rheum 1999, 42:948-953.

Somatic mutations in mitochondria: the chicken or the egg?. C Ospelt, S Gay, Arthritis Res Ther. 7Ospelt C, Gay S: Somatic mutations in mitochondria: the chicken or the egg? Arthritis Res Ther 2005, 7:179-180.

Diminished mitochondrial DNA integrity and repair capacity in OA chondrocytes. V I Grishko, R Ho, G L Wilson, Pearsall Awt, Osteoarthritis Cartilage. 17Grishko VI, Ho R, Wilson GL, Pearsall AWt: Diminished mitochondrial DNA integrity and repair capacity in OA chondrocytes. Osteoarthritis Cartilage 2009, 17:107-113.

Somatic mutations in the mitochondria of rheumatoid arthritis synoviocytes. T R Da Sylva, A Connor, Y Mburu, E Keystone, G E Wu, Arthritis Res Ther. 7Da Sylva TR, Connor A, Mburu Y, Keystone E, Wu GE: Somatic mutations in the mitochondria of rheumatoid arthritis synoviocytes. Arthritis Res Ther 2005, 7:R844-851.

M Biniecka, E Fox, W Gao, C T Ng, D J Veale, U Fearon, O' Sullivan, J , Hypoxia induces mitochondrial mutagenesis and dysfunction in inflammatory arthritis. Biniecka M, Fox E, Gao W, Ng CT, Veale DJ, Fearon U, O'Sullivan J: Hypoxia induces mitochondrial mutagenesis and dysfunction in inflammatory arthritis. Arthritis Rheum 2011.

Grishko V: Mitochondrial DNA damage is involved in apoptosis caused by proinflammatory cytokines in human OA chondrocytes. J Kim, M Xu, R Xo, A Mates, G L Wilson, Pearsall Awt, 10.1186/ar3424Osteoarthritis Cartilage. 18Kim J, Xu M, Xo R, Mates A, Wilson GL, Pearsall AWt, Grishko V: Mitochondrial DNA damage is involved in apoptosis caused by pro- inflammatory cytokines in human OA chondrocytes. Osteoarthritis Cartilage 2010, 18:424-432. doi:10.1186/ar3424

Successful tumour necrosis factor (TNF) blocking therapy suppresses oxidative stress and hypoxia-induced mitochondrial mutagenesis in inflammatory arthritis. Biniecka, Arthritis Research & Therapy. 13121Cite this article asCite this article as: Biniecka et al.: Successful tumour necrosis factor (TNF) blocking therapy suppresses oxidative stress and hypoxia-induced mitochondrial mutagenesis in inflammatory arthritis. Arthritis Research & Therapy 2011 13:R121.


Risk Factors for Nipah Virus Encephalitis in Bangladesh 1
American Society of Tropical Medicine and HygieneCopyright American Society of Tropical Medicine and HygieneOctober 2008 1. 2004 Nov 7-11. October 2008

Joel M Montgomery firstname.lastname@example.com
Current affi liation: US Naval Medical Research Center Detachment
International Centre for Diarrheal Diseases Research
US Naval Medical Research Center Detachment
3230, 20521-3230Lima, Dhaka, Lima Place, WashingtonDCPeru., USA

Mohamed J Hossain
E Gurley
D S Carroll
A Croisier
E Bertherat
N Asgari
P Formenty
N Keeler
J Comer
M R Bell
K Akram
A R Molla
K Zaman
Mohamed R Islam
K Wagoner
J N Mills
P E Rollin
T G Ksiazek
R F Breiman
M R Comer
K Bell
J N Wagoner
P E Mills
T G Rollin
Ksiazek
Bangladesh
M J Hossain
E Gurley
R F Breiman
; World
Health Organization
Geneva
Switzerland
A Croisier
E Bertherat
N Asgari
P Formenty
; World
Health Organization
( K Dhaka
K Akram
Zaman
Joel M Montgomery

FloridaUSA

Risk Factors for Nipah Virus Encephalitis in Bangladesh 1

Miami, 1532 Emerging Infectious Diseases • www.cdc.gov/eid •
American Society of Tropical Medicine and Hygiene1410October 2008 1. 2004 Nov 7-11. October 200810.3201/eid1410.060507RESEARCH 1526 Emerging Infectious Diseases • www. Author affi liations: Centers for Disease Control and Prevention, At-lanta, Georgia, USA (J.M. Montgomery, D.S. Carroll, N. Keeler, J. and Institute of Epidemiology Disease Control and Re-search; Dhaka (A.R. Molla, M.R. Islam) Address for correspondence: Use of trade names is for identifi cation only and does not imply endorsement by the Public Health Service or by the U.S. Department of Health and Human Services.
Nipah virus (NiV) is a paramyxovirus that causes severe encephalitis in humans. During January 2004, twelve patients with NiV encephalitis (NiVE) were identifi ed in westcentral Bangladesh. A case-control study was conducted to identify factors associated with NiV infection. NiVE patients from the outbreak were enrolled in a matched case-control study. Exact odds ratios (ORs) and 95% confi dence intervals (CIs) were calculated by using a matched analysis. Climbing trees (83% of cases vs. 51% of controls, OR 8.2, 95% CI 1.25-∞) and contact with another NiVE patient (67% of cases vs. 9% of controls, OR 21.4, 95% CI 2.78-966.1) were associated with infection. We did not identify an increased risk for NiV infection among persons who had contact with a potential intermediate host. Although we cannot rule out person-to-person transmission, case-patients were likely infected from contact with fruit bats or their secretions.

H enipaviruses (family Paromyxoviridae, genus Henipavirus) are enveloped RNA viruses that cause respiratory illness in pigs and horses and respiratory illness and encephalitis in humans (1)(2)(3)(4)(5)(6). After a 4-to 18-day incubation period, human disease can rapidly progress from mild illness (fever, headache, myalgia) to coma and death within 10 days; the case-fatality ratio is 40%-76% (3,(7)(8)(9)(10). The fi rst recognized human Henipavirus infections occurred in 1994 in Australia, where a respiratory disease among horses was associated with illness in 2 humans (11). The etiologic agent, Hendra virus, was subsequently isolated from asymptomatic fl ying foxes (fruit bats of the family Pteropodidae) (12). Field et al. (2) suggested that horses, identifi ed as the intermediate hosts linked to human illness, may have become infected through indirect contact with fruit bats (e.g., infected fetal bat tissues or fl uids).

The fi rst reported human epidemic of encephalitis caused by another Henipavirus, Nipah virus (NiV), occurred between September 1998 and April 1999 in Malaysia and Singapore and was associated with an outbreak of severe respiratory illness in pigs (13)(14)(15). Most (86%-93%) human NiV encephalitis (NiVE) infections during this outbreak involved occupational exposure to pigs, implicating these animals as an intermediate host for NiV (15)(16)(17)(18). Outbreaks of NiVE occurred in Bangladesh during 2001 and 2003, in areas where NiV antibody-positive fruit bats have been identifi ed (19). These reports, in addition to ecologic surveys conducted in Cambodia, have strengthened evidence that pteropid bats are the reservoir for Hendra and Nipah viruses (12,(20)(21)(22)(23)(24)(25).

An outbreak of encephalitis in Bangladesh was recognized on January 21, 2004; it affected 2 villages of Goalando township, Rajbari District, Dhaka Division, 70 km west of the city of Dhaka (Figure 1). Ten deaths were reported among 12 ill persons with symptoms compatible with NiVE, resulting in a case-fatality ratio of 83% (9,23). Although previous outbreaks of NiVE outside Bangladesh involved primarily men and women >25 years of age (5,16,17,19,26), most (75%) patients in this outbreak were boys <15 years of age. We describe a matched case-control study that was conducted to characterize the epidemiology of NiVE and, specifi cally, to determine if risk for NiVE was associated with contact with animals; an environmental exposure, activity, or behavior; or contact with other NiVE patients during the 2004 NiVE outbreak in Goalando township.

Study Participants, Materials, and Methods

A matched case-control study was conducted in Goalando, Bangladesh (Figure 1), February [18][19][20][21][22]2004. Hypotheses tested in this study, as mentioned above (e.g., increased risk for NiV infection caused by contact with animals, environmental exposure, contact with fruit in season) were based upon factors associated with previous outbreaks of NiVE in Malaysia, Singapore, and Bangladesh.

Case Defi nition

A confi rmed NiVE case-patient was defi ned as any patient with fever and symptoms compatible with encephalitis after December 15, 2003, with NiV-specifi c immunoglobulin M antibodies in cerebrospinal fl uid (CSF) or serum by enzyme immunoassay (EIA). A probable case of NiVE was defi ned as a patient with a diagnosis of encephalitis in whom fever developed and who was living in the same village as a patient with a confi rmed case of NiVE after December 15,2003. Cases remained in the probable category if the patient died and a specimen for laboratory confi rmation could not be obtained.

We conducted a population census of the affected area in February 2004; this census was the basis for selecting controls. We identifi ed 3 controls for each case-patient. The controls were selected randomly from the population and then matched to each case-patient on the basis of gender and age group. All households identifi ed during the census, including houses of case-patients and controls, were mapped by Global Positioning System, and data were uploaded into ERDAS Imagine 8.5 (Leica Geosystems, Atlanta, GA, USA) and merged with a November 2000 IKONOS Geo 1-m satellite image of the outbreak area (Space Imaging, Thornton, CO, USA).

Participation was strictly voluntary, and written informed consent was obtained for all participants; for those <18 years of age, individual and parental consent was obtained. The Bangladesh Ministry of Health and Family Welfare that requested this investigation reviewed and approved all protocols.

Study Population

Probable and confi rmed cases identifi ed in 2 contiguous villages of Goalando township ( Figure 1) were included in this study. Seven of the 12 cases were clustered within 3 households. Of these 7 clustered cases, 3 occurred in 1 household, and the remaining 4 were distributed in 2 separate homes (Figures 1, 2). Therefore, we conducted 2 separate analyses to assess the effect of case clustering on results. The fi rst analysis contained the complete dataset of 12 cases and 36 controls; the subanalysis consisted of 8 cases (we randomly selected 1 case/household) and 24 matched controls. Similar results (proportions, odds ratios [ORs], 95% confi dence intervals [CIs]) were obtained from both analyses. Thus, data presented in this article, including all tables, are derived from the complete dataset.

Specimen Collection and Testing

Serum samples and CSF were tested as previously described (27). When possible, a serum specimen was collected from controls.  Figure 2. Map also shows extreme habitat disturbance; areas under cultivation (for rice, sugar cane) are highlighted with "C," and remaining trees (fruit trees and bamboo stands) with "T." Bottom: Location of outbreak village.

Data Collection and Interviews

After informed consent was obtained, case-patients and controls were interviewed at home by trained interviewers, in their native Bengali language, with a standardized questionnaire. Information such as demographics, types of animal exposures, environmental and occupational exposures, exposure to ill persons, and history of illness was obtained. Proxy interviews of family members and/or friends were conducted for deceased patients. To minimize interview bias, proxy interview methods were also used for all controls that were matched to deceased case-patients.

Statistical Analysis

Exact ORs and 95% CIs were calculated by using a matched univariate logistic regression analysis in SAS version 9.0 (SAS Institute Inc, Cary, NC, USA) (28). Associations were considered statistically signifi cant at p<0.05.

Results

Descriptive Characteristics

Four (33%) cases were confi rmed by EIA; the remaining 8 (67%) case-patients, from whom a diagnostic specimen was not available, were considered probable cases. Among all 13 (36%) controls who consented to blood collection, results of serologic tests for NiV-specifi c antibodies were negative. Furthermore, none of the controls reported having had a perceived fever or symptoms compatible with NiVE from December 15, 2003, through the week the study was conducted (February [18][19][20][21][22]2004). In addition, an antibody prevalence study conducted among persons (n = 300) living in the outbreak site showed no evidence of asymptomatic or mild infection, which suggested that controls entered into the study were likely uninfected (A. Croisier, unpub. data). Proxy interviews were administered to equal proportions of case-patients (83%) and controls ( Table 1). The median age of case-patients included in the study was 11.5 years (range 2-28 years); 9 (75%) were male, and 11 (91%) were <15 years of age ( Table 1). Residences of all case-patients and controls were located within the affected villages, an area with a radius of ≈800 m ( Figure 1).

Animal Exposures

In the matched case-control analysis, a greater percentage of case-patients (60%) than controls (34%) had observed or touched dead animals, although this fi nding was not statistically signifi cant ( Table 2). We observed no differences between case-patients and controls with respect to contact with ill animals ( Table 2), including pigs, ruminants, and fruit bats. Chickens and ducks were often slaughtered for religious purposes or for consumption; however, close contact with these animals and their bodily fl uids (e.g., blood, saliva) during this process was not associated with NiV infection ( Table 2). None of the case-patients or controls had known contact with pigs (healthy or ill) or pig excreta ( Table 2). Four (36%) of 11 case-patients and 7 (19%) of the controls observed fruit bats around their household during the night (OR 4.1, p = 0.49; Table 2). However, some proxy family members and/or friends answering on behalf of patients who had died were unable to answer specifi c questions (e.g., Did you observe fruit bats around your house during the night?).

Environmental and Behavioral Exposures

A greater proportion of case-patients (83%) than controls (51%) reported having climbed trees between December 15, 2003, and February 3, 2004 (OR 8.2, p = 0.025; Table 2). No statistically signifi cant differences were observed between case-patients and controls with respect to outdoor activities such as hunting, fi shing, or playing outdoor games (e.g., hide-and-seek, cricket, soccer). Eating fruit that was locally available (on trees or collected from fruit trees locally) between December and February  (17) was not associated with illness, regardless of how the fruit was collected (from the ground, picked from tree, from the market) (Table 2). Although a greater proportion of casepatients reported environmental exposures (drinking raw date palm sap, harvesting date palm sap, having someone in the household who collects date palm sap, or drinking sap directly from the collection vessel), these differences were not statistically signifi cant (Table 2).

NiVE Case Exposure

There were strong associations between illness and 1) visiting a hospital and/or 2) having had contact with a probable or confi rmed NiVE patient ( Table 2). In one 2-case family cluster, a mother (26 years of age) and her infant son (2 years of age) both became ill and died. The child became symptomatic 2 days before the mother's illness onset (Figure 2; household 4). Among the other affected family clusters, the patients became ill within 3 days of one another (Figure 2; households 1 and 5); all persons in these 2 clusters reported a history of climbing fruit trees. There was no evidence of contact of persons between case households during their illness.

Discussion and Conclusions

In contrast to the patients in the Malaysian and Singapore outbreaks, which occurred primarily among adults, a preponderance of the NiV patients in the January/February 2004 Bangladesh outbreak were young boys. These fi ndings, in the absence of high infection rates among adults or evidence of antibodies to NiV in the general population (investigation team, unpub. data), suggest an association between NiV infection and some childhood activity or specifi c behavior. The odds of NiV infection were significantly elevated among persons who climbed trees, an activity observed almost exclusively among boys <15 years of age. This behavior is quite common among children because they gather fruit from trees. Therefore, these children may have had contact with partially eaten fruit from fruit bats or the secretions/excretions of these animals. Or, the children may have contacted contaminated fruit bat guano or urine in the trees. The percentages of case-patients playing hideand-seek, hunting, and fi shing-all of which were typical behaviorial traits of local boys-were not signifi cantly different than those for controls. These activities generally occur outdoors; however, they do not place a child in direct contact with bat excretions or secretions, as may be true for tree climbing. Therefore, infection was apparently related to a specifi c behavior, tree climbing, rather than age or outdoor activities in general. Furthermore, although other exposures that may have placed persons in closer contact with bat secretions (e.g., collecting fruit or palm sap from trees, drinking palm sap directly from collection vessel) were observed more often among case-patients than controls, these fi ndings were not statistically signifi cant; perhaps because of the small sample size. Nonetheless, our fi ndings can and have been used to help guide NiV outbreak investigations, leading investigators to similar conclusions as ours (29).

Fruit bats forage at night in various trees that are producing ripe fruit and often drink from palm sap collection vessels (30). Fruits are also a major food source for many villagers and, as a result of environmental disturbances (31) in the form of crop development (e.g., jute, rice, and sugar cane), the few remaining fruit trees grow only in close proximity to human dwellings ( Figure 1). This in turn creates a situation in which fruit bats are forced into close proximity with humans, especially while these mammals are foraging and feeding. In addition, date palm sap is routinely collected in rural areas of Bangladesh between December and May. According to villagers, including palm sap harvesters, dead fruit bats are occasionally found in the collection vessels. Local villagers reported that they often observed fruit bats feeding from palm sap collection vessels, and some collectors place cloth over the opening of the vessel to prevent this (investigational team observation). In fact, a greater proportion of case-patients in our study collected palm sap, drank from the palm sap collection vessel, or had a family member who collected palm sap; however, these differences were not statistically signifi cant. The power of our study to detect exposure risks was limited by the outbreak size. Therefore, until additional data are available, remaining cautious of date palm sap collection vessels, especially those visibly contaminated with fruit bat excreta or carcasses, would be prudent.

Numerous investigators have found serologic evidence suggesting that fruit bats of the genus Pteropus are the reservoir hosts for NiV (23,24), and there are reports of NiV isolation from bat urine (20,25) and partially eaten fruit (20). Unpublished laboratory data from the Bangladesh investigation have not supported the presence of an intermediate or primary reservoir host other than P. giganteus. Available data from this study, therefore, suggest direct transmission of NiV to humans through contact with bat secretions or excretions (saliva, urine, guano, partially eaten fruit) during fruit-tree climbing.

Although indirect contact with bats may have been the primary means of infection for this outbreak, Hsu and others (19) demonstrated that contact with ill cows was associated with an increased risk for NiV infection during the 2001 Bangladesh NiV outbreak. Therefore, intermediated hosts should be considered in future NiV outbreaks in Bangladesh.

In contrast to the patients in the Malaysia and Singapore outbreaks (5,16,17,25,26), most of the Bangladesh population (and all of the case-patients included in this study; data not shown) are practicing Muslims who do not consume pork and who avoid contact with pigs. None of the case-patients and controls in our study population reported any contact with pigs or pig excreta, so it is unlikely that these animals played a role in this outbreak.

Clustering of cases within households was a prominent feature of this outbreak ( Figure 2); 1 household contained 3 case-patients, all brothers of ages 7-15 years. However, the longest estimated incubation periods (duration from symptom onset to fi rst known exposure to a NiVE family member) within the clusters reported here were less than the currently recognized 4-day minimum (7). This fi nding suggests that the family clustering may have resulted from a common source of infection (e.g., a specifi c tree they climbed, fruit they consumed, or palm sap collection vessel they were in contact with) rather than person-to-person transmission. Our data also show strong associations between NiV infection and visiting a hospital. However, because the participants were asked if they had visited a hospital within a range of dates (December 15, 2003-February 3, 2004 and not a specifi c date, we were unable to determine if they were ill with NiV before visiting the hospital or whether they acquired their infection there. Some accounts in the literature suggest person-to-person transmission of NiV; therefore, it is plausible that someone could acquire, through contact with a patient's secretions or excretions, an NiV infection while visiting a hospital (6,10,20). Nevertheless, the most probable explanation for the observed association is that NiV encephalitis patients during this outbreak were severely ill, requiring hospitalization.

Although person-to-person transmission may have occurred in this outbreak, the initial infection (index case) may have occurred through contact with bat secretions rather than contact with an intermediate host. A limitation of our study is that we were unable to identify a specifi c mechanism by which person-to-person transmission may have occurred. NiV has been isolated from the respiratory secretions and urine of patients in the Malaysia, Singapore, and current Bangladesh outbreaks (3,8,32,33), which suggests a potential for NiV to be transmitted from person to person. Data based upon chain-of-transmission events and clustering of cases during other 2003 and May 2004 Bangladesh outbreaks led investigators to conclude that human-to-human transmission may have occurred (3,19). Therefore, given the potential for household or nosocomial transmission, we recommend the use of personal protective equipment (i.e., gloves, masks, gowns, and eye protection); strict hand hygiene and surface disinfection during and after contact with an NiVE patient; isolation of patients with confi rmed or suspected NiV infection; and proper disposal of potentially contaminated materials.

In summary, tree climbing, a behavior largely engaged in by young boys, was associated with an increased risk for NiV infection; although the exact mode of transmission is unclear. Our data do not rule out the potential for person-toperson transmission. If person-to-person transmission were extremely effi cient, the conditions and population density of Bangladesh (≈1,000 persons/km 2 ; total population 141 million/144,000/km 2 ) may have resulted in a much larger outbreak. Indeed, a study among health workers in Bangladesh did not fi nd evidence of incidental transmission to persons caring for patients hospitalized with Nipah-related illnesses (34). Bat-to-human was the most probable route of transmission in Goalando; however, some undetermined intermediate or incidental hosts cannot be ruled out. Periodic introductions of NiV to human populations in this region may continue to occur because of the overlapping nature of human and pteropid bat habitats. Moreover, bathuman interactions are likely to increase due to bat habitat loss because the few fruit trees that remain will likely be found in close proximity to human dwellings ( Figure 1).

As a prevention measure, we recommend avoiding contact with fruit bats and their secretions/excretions. We also encourage persons to wash and/or peel fruit, in addition to washing their hands, before preparing meals or consuming fruit. Greater understanding of the relationships between pteropid fruit bats, NiV, and its transmission to humans might offer additional strategies for safe coexistence and disease prevention for Bangladesh and other countries where fruit bats reside. Finally, because the geographic range of this highly lethal pathogen may correspond to the distribution of the genus Pteropus, including parts of China and Australia, most of the Indian subcontinent, and Southeast Asia (12,30), factors that promote transmission from bats to humans need to be defi ned and the role of person-toperson transmission needs to be better characterized.

Figure 1 .
1Top: Distribution of Nipah virus case (n = 12) and control (n = 36) households within the outbreak/study site of Goalando township, Bangladesh, January 2004. Number in the yellow triangle corresponds to household no. in

1528Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 10, October 2008Figure 2. Epidemic curve of Nipah virus outbreak in Goalando,
Bangladesh, in 2004, demonstrating household clustering.
Households 1 and 4 each had 2 cases, household 5 had 3 cases,
and all other households, single cases.

Table 1. Descriptive characteristics of Nipah virus case-patients
and controls, Bangladesh, January 2004
No. (%)
Characteristic
Case-patients, n = 12
Controls, n = 36
Sex
M
9 (75)
27 (75)
F
3 (25)
9 (25)
Age group, y
1-5
1 (8)
3 (8)
6-10
4 (33)
12 (33)
11-15
6 (50)
18 (50)
16-20
0
0
21-25
0
0
26-30
1 (8)
3 (8)
Interview type
Proxy
10 (83)
30 (83)
Self
2 (17)
6

Table 2 .
2Exposures and activities associated with Nipah virus infection, Bangladesh, December 2003-January 2004*No. (%) study participants with reported exposure or activity †

The Centers for Disease Control and Prevention, Department of Health and Human Services, US Public Health Service, provided fi nancial support for this research. Dr Montgomery is an infectious disease epidemiologist, who completed his training with the CDC Epidemic Intelligence Service in 2004. His main research interests are infectious diseases of public health importance, especially those of zoonotic origin. He is currently detailed from CDC to the US Naval Medical Research Center Detachment in Lima, Peru, and is director of their Emerging Infectious Diseases Program and Outbreak Investigation Response Team.
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 10, October 2008
AcknowledgmentsWe thank the staff of the Bangladesh Ministry of Health, the International Centre for Diarrhoeal Disease Research, Bangladesh; the Institute of Epidemiology, Disease Control and Research; the World Health Organization (WHO) in Dhaka, Bangladesh, the WHO Communicable Disease Surveillance and Response, Geneva; and M. Niezgoda and I. Kuzmin for their collaborative support. We also acknowledge the contributions of those who conducted the Nipah virus antibody assays, including J.L. Betts, D.L. Cannon, K.A. Slaughter, T.L. Stevens, and P.C. Stockton. Many thanks to S. Luby, J. Woodward, J. Robertson, and K. Montgomery for helpful reviews of the manuscript. Finally, we respectfully acknowledge the many Nipah virus-infected patients, their families, and the healthcare providers who cared for them in Bangladesh.
Nipah virus: a recently emergent deadly paramyxovirus. K B Chua, W J Bellini, P A Rota, B H Harcourt, A Tamin, S K Lam, 10.1126/science.288.5470.1432Science. 288Chua KB, Bellini WJ, Rota PA, Harcourt BH, Tamin A, Lam SK, et al. Nipah virus: a recently emergent deadly paramyxovirus. Science. 2000;288:1432-5. DOI: 10.1126/science.288.5470.1432

The natural history of Hendra and Nipah viruses. H Field, P Young, J M Yob, J Mills, L Hall, J Mackenzie, 10.1016/S1286-4579(01)01384-3Microbes Infect. 3Field H, Young P, Yob JM, Mills J, Hall L, Mackenzie J. The natural history of Hendra and Nipah viruses. Microbes Infect. 2001;3:307- 14. DOI: 10.1016/S1286-4579(01)01384-3

Person-to-person transmission of Nipah virus in a Bangladeshi community. E S Gurley, J M Montgomery, M J Hossain, M Bell, A K Azad, M R Islam, Emerg Infect Dis. 13Gurley ES, Montgomery JM, Hossain MJ, Bell M, Azad AK, Islam MR, et al. Person-to-person transmission of Nipah virus in a Bangla- deshi community. Emerg Infect Dis. 2007;13:1031-7.

Molecular characterization of Nipah virus, a newly emergent paramyxovirus. B H Harcourt, A Tamin, T G Ksiazek, P E Rollin, L J Anderson, W J Bellini, DOI: 10.1006/ viro.2000.0340Virology. 271Harcourt BH, Tamin A, Ksiazek TG, Rollin PE, Anderson LJ, Bellini WJ, et al. Molecular characterization of Nipah virus, a newly emer- gent paramyxovirus. Virology. 2000;271:334-49. DOI: 10.1006/ viro.2000.0340

Case-control study of risk factors for human infection with a new zoonotic paramyxovirus, Nipah virus, during a 1998-1999 outbreak of severe encephalitis in Malaysia. U D Parashar, L M Sunn, F Ong, A W Mounts, M T Arif, T G Ksiazek, 10.1086/315457J Infect Dis. 181Parashar UD, Sunn LM, Ong F, Mounts AW, Arif MT, Ksiazek TG, et al. Case-control study of risk factors for human infection with a new zoonotic paramyxovirus, Nipah virus, during a 1998-1999 outbreak of severe encephalitis in Malaysia. J Infect Dis. 2000;181:1755-9. DOI: 10.1086/315457

Nipah virus infection: pathology and pathogenesis of an emerging paramyxoviral zoonosis. K T Wong, W J Shieh, S Kumar, K Norain, W Abdullah, J Guarner, Am J Pathol. 161Wong KT, Shieh WJ, Kumar S, Norain K, Abdullah W, Guarner J, et al. Nipah virus infection: pathology and pathogenesis of an emerg- ing paramyxoviral zoonosis. Am J Pathol. 2002;161:2153-67.

Nipah virus outbreak in Malaysia. K B Chua, 10.1016/S1386-6532(02)00268-8J Clin Virol. 26Chua KB. Nipah virus outbreak in Malaysia. J Clin Virol. 2003;26:265-75. DOI: 10.1016/S1386-6532(02)00268-8

Clinical features of Nipah virus encephalitis among pig farmers in Malaysia. K J Goh, C T Tan, N K Chew, P S Tan, A Kamarulzaman, S A Sarji, DOI: 10.1056/ NEJM200004273421701N Engl J Med. 342Goh KJ, Tan CT, Chew NK, Tan PS, Kamarulzaman A, Sarji SA, et al. Clinical features of Nipah virus encephalitis among pig farm- ers in Malaysia. N Engl J Med. 2000;342:1229-35. DOI: 10.1056/ NEJM200004273421701

. Emerging Infectious Diseases • www.cdc.gov/eid •. 1410Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 10, October 2008

Nipah encephalitis outbreak over wide area of western Bangladesh. Health and Science Bulletin. 2International Centre for Diarrhoeal Disease ResearchInternational Centre for Diarrhoeal Disease Research, Bangladesh. Nipah encephalitis outbreak over wide area of western Bangladesh, 2004. Health and Science Bulletin. 2004;2:7-11.

Nipah virus outbreak(s) in Bangladesh. Wkly Epidemiol Rec. 79World Health OrganizationWorld Health Organization. Nipah virus outbreak(s) in Bangladesh, January-April 2004. Wkly Epidemiol Rec. 2004;79:85-92.

A novel morbillivirus pneumonia of horses and its transmission to humans. K Murray, R Rogers, L Selvey, P Selleck, A Hyatt, A Gould, Emerg Infect Dis. 1Murray K, Rogers R, Selvey L, Selleck P, Hyatt A, Gould A, et al. A novel morbillivirus pneumonia of horses and its transmission to humans. Emerg Infect Dis. 1995;1:31-3.

Isolation of Hendra virus from pteropid bats: a natural reservoir of Hendra virus. K Halpin, P L Young, H E Field, J S Mackenzie, J Gen Virol. 81Halpin K, Young PL, Field HE, Mackenzie JS. Isolation of Hendra virus from pteropid bats: a natural reservoir of Hendra virus. J Gen Virol. 2000;81:1927-32.

Fatal encephalitis due to Nipah virus among pig-farmers in Malaysia. K B Chua, K J Goh, K T Wong, A Kamarulzaman, P S Tan, T G Ksiazek, 10.1016/S0140-6736-(99Lancet. 354Chua KB, Goh KJ, Wong KT, Kamarulzaman A, Tan PS, Ksiazek TG, et al. Fatal encephalitis due to Nipah virus among pig-farmers in Malaysia. Lancet. 1999;354:1257-9. DOI: 10.1016/S0140-6736- (99)04299-3

Nipah virus infection of pigs in peninsular Malaysia. Revue Scientifi que et Technique (International Offi ce of Epizootics). Mohd Nor, M N Gan, C H Ong, B L , 19Mohd Nor MN, Gan CH, Ong BL. Nipah virus infection of pigs in peninsular Malaysia. Revue Scientifi que et Technique (International Offi ce of Epizootics). 2000;19:160-5.

First case of Nipah virus encephalitis in Singapore. P A Tambyah, J H Tan, Bkc Ong, K H Ho, K P Chan, 10.1111/j.1444-0903.2001.00032.xIntern Med J. 31Tambyah PA, Tan JH, Ong BKC, Ho KH, Chan KP. First case of Nipah virus encephalitis in Singapore. Intern Med J. 2001;31:132-3. DOI: 10.1111/j.1444-0903.2001.00032.x

A survey of Nipah virus infection among various risk groups in Singapore. K P Chan, P E Rollin, T G Ksiazek, Y S Leo, K T Goh, N I Paton, Epidemiol Infect. 128Chan KP, Rollin PE, Ksiazek TG, Leo YS, Goh KT, Paton NI, et al. A survey of Nipah virus infection among various risk groups in Singapore. Epidemiol Infect. 2002;128:93-8.

Risk factors for Nipah virus infection among abattoir workers in Singapore. Mhl Chew, P M Arguin, D K Shay, K J Goh, P E Rollin, W-J Shieh, 10.1086/315443J Infect Dis. 181Chew MHL, Arguin PM, Shay DK, Goh KJ, Rollin PE, Shieh W-J, et al. Risk factors for Nipah virus infection among abattoir workers in Singapore. J Infect Dis. 2000;181:1760-3. DOI: 10.1086/315443

Outbreak of Nipah-virus infection among abattoir workers in Singapore. N I Paton, Y S Leo, S R Zaki, A P Auchus, K E Lee, A E Ling, 10.1016/S0140-6736(99)04379-2Lancet. 354Paton NI, Leo YS, Zaki SR, Auchus AP, Lee KE, Ling AE, et al. Out- break of Nipah-virus infection among abattoir workers in Singapore. Lancet. 1999;354:1253-6. DOI: 10.1016/S0140-6736(99)04379-2

Nipah virus encephalitis reemergence. V P Hsu, M J Hossain, U D Parashar, M M Ali, T G Ksiazek, I Kuzmin, Bangladesh. Emerg Infect Dis. 10Hsu VP, Hossain MJ, Parashar UD, Ali MM, Ksiazek TG, Kuzmin I, et al. Nipah virus encephalitis reemergence, Bangladesh. Emerg Infect Dis. 2004;10:2082-7.

Isolation of Nipah virus from Malaysian Island fl ying-foxes. K B Chua, C L Koh, P S Hooi, K F Wee, J H Khong, B H Chua, 10.1016/S1286-4579(01)01522-2Microbes Infect. 4Chua KB, Koh CL, Hooi PS, Wee KF, Khong JH, Chua BH, et al. Iso- lation of Nipah virus from Malaysian Island fl ying-foxes. Microbes Infect. 2002;4:145-51. DOI: 10.1016/S1286-4579(01)01522-2

Identifi cation of likely natural hosts for equine morbillivirus. K Halpin, P Young, H Field, Commun Dis Intell. 20476Halpin K, Young P, Field H. Identifi cation of likely natural hosts for equine morbillivirus. Commun Dis Intell. 1996;20:476.

Newly discovered viruses of fl ying foxes. K Halpin, P L Young, H Field, J S Mackenzie, Vet Microbiol. 6899Halpin K, Young PL, Field H, Mackenzie JS. Newly discovered vi- ruses of fl ying foxes. Vet Microbiol. 1999;68:83-7. DOI: 10.1016/ S0378-1135(99)00063-2

Nipah virus infection in bats (order Chiroptera) in peninsular Malaysia. J M Yob, H Field, A M Rashdi, C Morrissy, B Van Der Heide, P Rota, Emerg Infect Dis. 7Yob JM, Field H, Rashdi AM, Morrissy C, van der Heide B, Rota P, et al. Nipah virus infection in bats (order Chiroptera) in peninsular Malaysia. Emerg Infect Dis. 2001;7:439-41.

Antibodies to Nipah-like virus in bats (Pteropus lylei), Cambodia. J G Olson, C Rupprecht, P E Rollin, An Us, M Niezgoda, T Clemins, Emerg Infect Dis. 8Olson JG, Rupprecht C, Rollin PE, An US, Niezgoda M, Clemins T, et al. Antibodies to Nipah-like virus in bats (Pteropus lylei), Cambo- dia. Emerg Infect Dis. 2002;8:987-8.

Nipah virus in Lyle's fl ying foxes. J M Reynes, D Counor, S Ong, C Faure, V Seng, S Molia, Cambodia. Emerg Infect Dis. 11Reynes JM, Counor D, Ong S, Faure C, Seng V, Molia S, et al. Nipah virus in Lyle's fl ying foxes, Cambodia. Emerg Infect Dis. 2005;11:1042-7.

Nipah virus infection among abattoir workers in Malaysia, 1998-1999. M Sahani, U D Parashar, A Roslinah, P Das, M S Lye, M M Isa, DOI: 10.1093/ ije/30.5.1017Int J Epidemiol. 30Sahani M, Parashar UD, Roslinah A, Das P, Lye MS, Isa MM, et al. Nipah virus infection among abattoir workers in Malaysia, 1998-1999. Int J Epidemiol. 2001;30:1017-20. DOI: 10.1093/ ije/30.5.1017

Laboratory diagnosis of Nipah and Hendra virus infections. P Daniels, T Ksiazek, B T Eaton, 10.1016/S1286-4579(01)01382-XMicrobes Infect. 3Daniels P, Ksiazek T, Eaton BT. Laboratory diagnosis of Nipah and Hendra virus infections. Microbes Infect. 2001;3:289-95. DOI: 10.1016/S1286-4579(01)01382-X

Logistic regression: a self-learning text. D G Kleinbaum, M Klein, Spring-VerlagNew York2nd edKleinbaum DG, Klein M. Logistic regression: a self-learning text. 2nd ed. New York: Spring-Verlag; 2002.

Foodborne transmission of Nipah virus. S P Luby, M Rahman, M J Hossain, L S Blum, M M Husain, E Gurley, Bangladesh. Emerg Infect Dis. 12Luby SP, Rahman M, Hossain MJ, Blum LS, Husain MM, Gurley E, et al. Foodborne transmission of Nipah virus, Bangladesh. Emerg Infect Dis. 2006;12:1888-94.

Walker's bats of the world. Baltimore (MD). R Nowak, The Johns Hopkins University PressNowak R. Walker's bats of the world. Baltimore (MD). The Johns Hopkins University Press; 1994. p. 1-287.

United Nations Environment Programme. BangladeshThe ProgrammeUnited Nations Environment Programme. State of the environment, Bangladesh. The Programme; 2001 [cited 2008 Aug 10]. Available from http://www.rrcap.unep.org/pub/soe/bangladeshsoe.cfm

The presence of Nipah virus in respiratory secretions and urine of patients during an outbreak of Nipah virus encephalitis in Malaysia. K B Chua, S K Lam, K J Goh, P S Hooi, T Ksiazek, A Kamarulzaman, 10.1053/jinf.2000.0782J Infect. 42Chua KB, Lam SK, Goh KJ, Hooi PS, Ksiazek T, Kamarulzaman A, et al. The presence of Nipah virus in respiratory secretions and urine of patients during an outbreak of Nipah virus encephalitis in Malaysia. J Infect. 2001;42:40-3. DOI: 10.1053/jinf.2000.0782

A cohort study of health care workers to assess nosocomial transmissibility of Nipah virus, Malaysia. A W Mounts, H Kaur, U D Parashar, T Ksiazek, D Cannon, J T Arokiasamy, 10.1086/318822J Infect Dis. 183Mounts AW, Kaur H, Parashar UD, Ksiazek T, Cannon D, Aroki- asamy JT, et al. A cohort study of health care workers to assess noso- comial transmissibility of Nipah virus, Malaysia, 1999. J Infect Dis. 2001;183:810-3. DOI: 10.1086/318822

Risk of nosocomial transmission of Nipah virus in a Bangladesh hospital. E S Gurley, J M Montgomery, M J Hossain, M R Islam, M A Molla, S M Shamsuzzaman, 10.1086/516665Infect Control Hosp Epidemiol. 28Gurley ES, Montgomery JM, Hossain MJ, Islam MR, Molla MA, Shamsuzzaman SM, et al. Risk of nosocomial transmission of Nipah virus in a Bangladesh hospital. Infect Control Hosp Epidemiol. 2007;28:740-2. DOI: 10.1086/516665