Akiko Tochimoto

Clinical manifestation and prognostic factor in anti-melanoma differentiation-associated gene 5 antibody-associated interstitial lung disease as a complication of dermatomyositis

OBJECTIVE The aim of this study is to evaluate the clinical manifestation and prognostic factors of anti-melanoma differentiation-associated gene 5 (MDA5) antibody-associated interstitial lung disease (ILD) with DM. METHODS Fourteen patients who presented with anti-MDA5 antibody and 10 patients with anti-aminoacyl-tRNA synthetase (ARS) antibody were enrolled. All patients were diagnosed as having DM with ILD. Clinical manifestations in the patients with anti-MDA5 antibody were compared with those in the patients with anti-ARS antibody. RESULTS The frequencies of acute/subacute interstitial pneumonia (A/SIP) and fatal outcome were significantly higher in the subset with anti-MDA5 antibody. The creatine kinase (CK) value was significantly lower and the gamma-glutamyl transpeptidase and ferritin values were significantly higher in the subset with anti-MDA5 antibody. Significant correlations were found between PaO(2)/F(i)O(2) and ferritin (r(s) = -0.59, P = 0.035), alveolar-arterial oxygen difference (A-aDO(2)) and KL-6 (r(s) = 0.73, P = 0.016) and A-aDO(2) and ferritin (r(s) = 0.66, P = 0.013) in the subset with anti-MDA5 antibody. The most significant prognostic factor was ferritin. The cumulative survival rate was significantly lower (P < 0.0001) in the subset with ferritin >or=1600 ng/ml than that in the subset with ferritin <1600 ng/ml in anti-MDA5 antibody-associated ILD. CONCLUSION Both serum ferritin and anti-MDA5 antibody are powerful indicators for the early diagnosis of A/SIP with DM. Ferritin also predicts disease severity and prognosis for patients with anti-MDA5 antibody. Intensive treatment should be administered to cases that have anti-MDA5 antibody-associated ILD with DM showing hyperferritinaemia, especially if the ferritin level is >or=1600 ng/ml.

Intracellular IL-1α-binding proteins contribute to biological functions of endogenous IL-1α in systemic sclerosis fibroblasts

The aberrant production of precursor IL-1α (pre-IL-1α) in skin fibroblasts that are derived from systemic sclerosis (SSc) is associated with the induction of IL-6 and procollagen, which contributes to the fibrosis of SSc. However, little is understood about how intracellular pre-IL-1α regulates the expression of the other molecules in fibroblasts. We report here that pre-IL-1α can form a complex with IL-1α-binding proteins that is translocated into the nuclei of fibroblasts. Immunoprecipitation that used anti-human IL-1α Ab and 35S-labeled nuclear extracts of fibroblasts showed three specific bands (≈31, 35, and 65 kDa). The 31-kDa molecule was identified as pre-IL-1α, and the 35- and 65-kDa molecules might be pre-IL-1α-binding proteins. A partial sequencing for the 10 aa from the N-terminals of the molecules showed 100% homology for HAX-1 (HS1-associated protein X-1) and IL-1 receptor type II (IL-1RII). Suppression of the genes of HAX-1 or IL-1RII induced the inhibitory effects of IL-1 signal transduction, including production of IL-6 and procollagen, by fibroblasts. In particular, pre-IL-1α was not translocated into the nucleus by an inhibition of HAX-1. These findings reveal that nuclear localization of pre-IL-1α depends on the binding to HAX-1 and that biological activities might be elicited by the binding to both HAX-1 and IL-1RII in SSc fibroblasts.

Clinical manifestations of neurological involvement in primary Sjögren’s syndrome.

The aim of this study was to evaluate neurological manifestations of primary Sjögren’s syndrome (pSS) and investigate the etiology and pathogenesis of peripheral and central nervous complications in pSS. Thirty-two patients with pSS were enrolled in the present study, 20 of whom had neurological involvement plus sicca symptoms. The clinical features were evaluated by neurological examinations including nerve conduction study, magnetic resonance imaging, cerebrospinal fluid, and electroencephalogram. The frequency of fever was significantly higher (P = 0.006) in pSS with neurological involvement than in pSS without neurological involvement. There was no statistical significance in other factors between the two groups. Peripheral nervous system (PNS), central nervous system (CNS), and both PNS and CNS involvements were revealed in 14, 3, and 3 patients, respectively. Optic neuritis and trigeminal neuralgia were revealed frequently in cranial neuropathy. Anti-aquaporin 4 antibody was detected in one patient with optic neuritis. Of the nine patients with polyneuropathy, eight patients presented pure sensory neuropathy including small fiber neuropathy (SFN). pSS with SFN appeared to have no clinically abnormal features, including muscle weakness and decreasing deep tendon reflex. Skin biopsy revealed epidermal nerve fiber degenerated in one pSS patient with pure sensory neuropathy who was diagnosed as having SFN. Our observations suggest that a number of mechanisms can be attributed to neurological involvements in pSS rather than just the mechanisms previously described (i.e., vasculitis and ganglioneuronitis). Presumably, specific autoantibodies may directly induce injury of the nervous system.

NOS2 polymorphisms associated with the susceptibility to pulmonary arterial hypertension with systemic sclerosis: contribution to the transcriptional activity.

Systemic sclerosis (SSc) is a connective tissue disease characterized by tissue fibrosis. One of several complications of SSc, pulmonary arterial hypertension (PAH) can be refractory to treatment, both novel and established. In the present study we investigated the ratio of circulating nitric oxide to endothelin-1 in patients with both SSc and PAH, and determined whether polymorphisms in NOS2 (the nitric oxide synthase 2 gene) are associated with susceptibility to PAH. Endothelin-1 in plasma and nitric oxide metabolites (nitrate and nitrite) in serum were measured. The nitric oxide/endothelin-1 ratio was significantly lower in patients with both SSc and PAH than in patients with SSc only or in healthy control individuals. We confirmed the presence of two single nucleotide polymorphisms at positions -1,026 and -277 and a pentanucleotide repeat (CCTTT) at -2.5 kilobases. There were significant differences in single nucleotide polymorphisms between patients with SSc who had PAH and those who did not, and between patients with both SSc and PAH and healthy control individuals. The CCTTT repeat was significantly shorter in patients with both SSc and PAH than in patients with SSc only or in healthy control individuals. Transcriptional activity were analyzed using the luciferase reporter assay. The transcriptional activity of NOS2 was much greater in fibroblasts transfected by a vector with a long allele of the CCTTT repeat than in those transfected by a vector with a short allele. Polymorphisms in the NOS2 gene are associated with transcriptional activity of the NOS2 gene and with susceptibility to SSc-related PAH.

Contribution of single nucleotide polymorphisms of the IL1A gene to the cleavage of precursor IL-1α and its transcription activity

We previously demonstrated the association of IL1A gene single nucleotide polymorphisms (SNPs) with susceptibility to systemic sclerosis (SSc) patients. In this study, we explored the effects of SNP on the transcriptional activity and processing of the precursor IL-1α (pre-IL-1α) in skin fibroblasts. Two kinds of promoter regions of the IL1A gene were prepared including C or T at −889, referred to C/IL1A and T/IL1A, and inserted into a luciferase reporter vector (pGL3). Skin fibroblasts were explanted from two SSc patients whose genomic DNA contained GG and TT genotypes at +4845 of the IL1A gene, respectively. Cell lysates were collected and reacted with various concentrations of calpain, and then the processing of pre-IL-1α was analyzed by Western blotting using monoclonal anti-IL-1α antibody. A SNP was determined by the allelic discrimination method using fluorescence-labeled Taq-Man probes. Significant differences in the luciferase activities were not detected between pGL3 (C/IL1A) and pGL3 (T/IL1A) in SSc fibroblasts. Calpain required a 100-fold higher concentration to process the pre-IL-1α containing Ala at the 114th amino acid than that to do containing Ser. The frequency of the GG genotype was significantly higher in SSc patients than that in healthy donors, whereas the frequency of TT genotype was significantly higher in RA patients than that in healthy donors. Our observation showed that the SNP at +4845 affected the enzymatic efficiency of the protease in cleaving pre-IL-1α. Pre-IL-1α with Ala, which was high in frequency in SSc patients, was more resistant to be cleaved by proteases in human sera than pro-IL-1α with Ser.

Sildenafil attenuates the fibrotic phenotype of skin fibroblasts in patients with systemic sclerosis

Systemic sclerosis (SSc) is a multi-organ fibrotic disease that affects the skin and various internal organs. Therapeutic strategies for tissue fibrosis have not been established; however, aberrantly activated fibroblasts in affected lesions are key targets for modulating fibrosis. Recently, increased intracellular cyclic GMP (cGMP) levels were demonstrated to improve fibrosis levels in various diseases. The purpose of this study was to assess the anti-fibrotic properties of cGMP in cultured fibroblasts from patients with SSc. The phosphodiesterase (PDE) 5 inhibitor sildenafil increased the intracellular cGMP levels in skin fibroblasts in a dose-dependent manner. Sildenafil treatment also significantly decreased the expression of several pro-fibrotic factors that were upregulated by TGF-β1 treatment in SSc skin fibroblasts. These inhibitory effects occurred via non-canonical TGF-β signaling. Our findings revealed that sildenafil might be a novel strategy to treat tissue fibrosis and vasculopathy in SSc.

Genetic Susceptibility to Interstitial Lung Disease Associated with Systemic Sclerosis

Systemic sclerosis (SSc) is a connective tissue disease that is characterized by tissue fibrosis, microvasculopathy, and autoimmunity. Interstitial lung disease (ILD) is a common complication of SSc and is one of the frequent causes of mortality in SSc. Although the exact etiology of SSc remains unknown, clinical and experimental investigations have suggested that genetic and environmental factors are relevant to the pathogenesis of SSc and SSc-ILD. More than 30 genes have been identified as susceptibility loci for SSc, most of which are involved in immune regulation and inflammation. It is thought that the key pathogenesis of SSc-ILD is caused by the release of profibrotic mediators such as transforming growth factor β1 and connective tissue growth factor from lung cells induced by a persistent damage. This review presents the genetic susceptibility to SSc-ILD, including human leukocyte antigen and non-human leukocyte antigen genes, especially focusing on connective tissue growth factor.

Capillary abnormalities observed by nailfold video-capillaroscopy in Japanese patients with systemic sclerosis

Nailfold capillary abnormalities are frequently observed in patients with connective tissue diseases (CTDs), especially systemic sclerosis (SSc), by dermoscopy (DS) or nailfold video-capillaroscopy (NVC). NVC and DS are widely used in Europe to distinguish SSc from primary Raynaud’s disease [1], as the capillary abnormalities of nailfolds are included in the ACR/EULAR 2013 Classification Criteria for SSc [2]. Cutolo et al. [1] proposed a reclassification of scleroderma into different subgroups by NVC: early, active and late pattern, with the frequencies of six aspects of capillary abnormalities: enlarged capillary, giant capillary, microhemorrhage, capillary loss, ramified capillary, and disorganization of normal capillary array. These specific patterns of NVC were involved in disease subtype of SSc, disease duration, and complications [3]. It has also been reported that patients with dermatomyositis/polymyositis (DM/PM) exhibit a similar capillary abnormality called “SSc-like pattern”, but it was less frequently observed compared to patients with SSc. Although extensive data have been reported from Europe, there are limited data on SSc in the Japanese population. As the clinical characteristics of SSc patients could be different among races and ethnicities [4], we designed this study to identify capillary abnormalities observed by NVC in Japanese patients with CTDs, especially SSc. Ninety-five patients with SSc (N1⁄4 39), DM/PM (N1⁄4 23), or systemic lupus erythematosus (SLE, N1⁄4 33), who entered Tokyo Women’s Medical University Hospital from June 2014 to May 2016, were enrolled. All patients with SSc and SLE fulfilled respective ACR criteria [2,5], while PM/DM was defined by Bohan and Peter’s criteria [6] or Sontheimer’s criteria for amyopathic dermatomyositis [7]. Clinical information and laboratory data were collected retrospectively by reviewing medical charts. All fingers, except for the thumbs, of both hands were examined using NVC (B-SCAN ProVR , Toku Corporation, Tokyo, Japan) with a 100 optic magnification. The evaluation of abnormal NVC images was based on qualitative parameters as previously reported: enlarged capillary, giant capillary, hemorrhage, loss of capillary, ramified capillary, and disorganization. Fingers in which we were unable to detect the shape of the nailfold capillary, due to dot-shaped capillaries, a pigmented nailfold, or a bank in the nailfold, were excluded from the analysis. Two patients with SLE, who had less than two evaluable fingers, were excluded from following analysis. The frequency of fingers with each capillary abnormality was evaluated for each disease. All patients provided written informed consent before enrollment. This study was reviewed and approved by the Institutional Review Board of Tokyo Women’s Medical University. For statistical analysis, Fisher’s exact probability test was used to compare frequencies. The Turkey–Kramer method was used for multiple comparisons of values with a normal distribution. Values that were not normally distributed were evaluated by a median test. p Values< .05 were considered significant. As a result, most patients with SSc exhibited Raynaud’s phenomenon (Table 1), and fewer fingers could be evaluated by NVC in the SSc group than in the other groups. The frequency of fingers with abnormal capillaries in patients with SSc was greater than that in the other two groups (mean ± SD: 84 ± 25% of fingers in SSc, 68 ± 33% of fingers in PM/DM, 39 ± 6% of fingers in SLE, Figure 1). Comparing capillary abnormalities among the three groups, loss of capillary was observed in most fingers of SSc patients. The patients with DM/PM showed capillary abnormalities similar to those in SSc patients, although capillary loss were less frequent than that in SSc. The cut-off value of the number of fingers with abnormal capillaries to distinguish SSc patients from non-SSc patients was estimated using the ROC curve, which was 80% or more fingers with abnormal capillaries (sensitivity 76.9% and specificity 70.4%). Among the patients with SSc, there were no significant associations between each NVC abnormalities and clinical features. We demonstrated that most fingers in patients with SSc had various NVC patterns indicating impaired microvasculature. Among five patterns of abnormal capillaries observed by NVC, capillary loss was the most specific to the impaired microvasculature of SSc. These results that we found in Japanese patients with SSc are very similar to those in European patients with SSc, as previously reported. NVC changes are useful to distinguish primary Raynaud’s disease from patients with Raynaud’s phenomenon associated with CTDs [1]. More than 90% of patients with SSc