Immune response to dermatomyositis-specific autoantigen, transcriptional intermediary factor 1γ can result in experimental myositis.

OBJECTIVES: To investigate whether autoimmunity to transcriptional intermediary factor 1 (TIF1)γ, a ubiquitous nuclear autoantigen for myositis-specific autoantibodies detected in patients with dermatomyositis (DM) is pathogenetic for inflammatory myopathy. METHODS: Wild-type, ß2-microglobulin-null, perforin-null, Igµ-null and interferon α/ß receptor (IFNAR)-null mice were immunised with recombinant human TIF1γ whole protein. A thymidine incorporation assay was performed using lymph node T cells from TIF1γ-immunised mice. Plasma was analysed using immunoprecipitation followed by western blot analysis and enzyme-linked immunosorbent assays. Femoral muscles were histologically and immunohistochemically evaluated. CD8+ or CD4+ T cells isolated from lymph node T cells or IgG purified from plasma were adoptively transferred to naïve mice. TIF1γ-immunised mice were treated with anti-CD8 depleting antibody and a Janus kinase inhibitor, tofacitinib. RESULTS: Immunisation with TIF1γ-induced experimental myositis presenting with necrosis/atrophy of muscle fibres accompanied by CD8+ T cell infiltration successfully in wild-type mice, in which TIF1γ-specific T cells and antihuman and murine TIF1γ IgG antibodies were detected. The incidence and severity of myositis were significantly lower in ß2-microglobulin-null, perforin-null, CD8-depleted or IFNAR-null mice, while Igµ-null mice developed myositis normally. Adoptive transfer of CD8+ T cells induced myositis in recipients, while transfer of CD4+ T cells or IgG did not. Treatment with tofacitinib inhibited TIF1γ-induced myositis. CONCLUSIONS: Here we show that TIF1γ is immunogenic enough to cause experimental myositis, in which CD8+ T cells and type I interferons, but not CD4+ T cells, B cells or antibodies, are required. This murine model would be a tool for understanding the pathologies of DM.

Improved quantification of a commercial enzyme-linked immunosorbent assay kit for measuring anti-MDA5 antibody.

OBJECTIVES: To compare the quantitative performance for measuring anti-MDA5 antibody titer of two enzyme-linked immunosorbent assay (ELISA) systems: an in-house ELISA and the commercial MESACUPTM anti-MDA5 test. METHODS: Anti-MDA5 antibody titer was measured in sera from 70 patients with dermatomyositis using an in-house ELISA and the MESACUPTM anti-MDA5 test side-by-side. For the commercial ELISA kit, serum samples diluted 1:101 were used according to the manufacturer's protocol, but serial dilutions of sera were also examined to identify the optimal serum dilution for quantification. RESULTS: The anti-MDA5 antibody titers measured by the in-house and commercial ELISAs were positively correlated with each other (r = 0.53, p = .0001), but the antibody titer measured by the commercial ELISA was less sensitive to change after medical treatment, and 37 (80%) of 46 anti-MDA5-positive sera had antibody titer exceeding the quantification range specified by the manufacturer (≥150 index). Experiments using diluted serum samples revealed that diluting the sera 1:5050 improved the quantitative performance of the MESACUPTM anti-MDA5 test, including a better correlation with the in-house ELISA results and an increased sensitivity to change. CONCLUSION: We improved the ability of the commercial ELISA kit to quantify anti-MDA5 antibody titer by altering its protocol.

Decreased carbonyl reductase 1 expression promotes tumor growth via epithelial mesenchymal transition in uterine cervical squamous cell carcinomas.

PURPOSE: Carbonyl reductase 1 (CBR1) is involved in cancer progression. Recently, the authors reported that the loss of CBR1 expression is associated with a poor prognosis in uterine cervical cancer. Here, we investigated whether the decreased CBR1 expression promotes cancer progression by inducing the epithelial mesenchymal transition (EMT). METHODS: Antisense constructs of CBR1 complementary DNA (antisense clones) and the empty vectors (control clones) were transfected into human uterine cervical squamous cell carcinoma cell lines (SKG II and SiHa) and the proliferation and EMT marker expression of these clones were analyzed in vitro. In an in vivo study, 107 cells of the antisense and control clones were subcutaneously injected into nude mice and the tumorigenesis was observed for 8 weeks. RESULTS: With the decreased CBR1 expression, the proliferation of the antisense clones increased, accompanied by a decrease in epithelial markers (E-cadherin and cytokeratin) and an increase in mesenchymal markers (fibronectin, alpha-smooth muscle actin, and N-cadherin), which suggests EMT induction. In the in vivo study, the tumor volume in the antisense group was significantly larger than that in the control group. CONCLUSION: Decreased CBR1 expression promotes tumor growth by inducing EMT in uterine cervical squamous cell carcinomas.

Functional significance of transgelin-2 in uterine cervical squamous cell carcinoma.

AIM: Transgelin-2 (TAGLN2) has previously been found to be highly expressed in uterine cervical squamous cell carcinoma (SCC) tissues by proteomic analyses. The present study investigated the role of TAGLN2 in the malignant behaviors of cervical SCC cells in vitro and in vivo, and the clinical significance of TAGLN2 using immunohistochemistry for human cervical SCC tissues. METHODS: Antisense (AS) constructs of TAGLN2 cDNA (AS clones) and the empty vector (control clone) were transfected into a human uterine SCC cell line (SKG IIIa), and malignant behaviors were analyzed in vitro. In an in vivo experiment, 10(7) cells of the AS and control clones were subcutaneously inoculated into female BALB/c nude mice. In immunohistochemistry with anti-TAGLN2 antibodies for human cervical SCC, FIGO stage IA and IB (n = 75), the expression patterns of TAGLN2 were divided into two groups: weak and strong. The relation between expression pattern and prognosis was analyzed. RESULTS: Suppression of TAGLN2 inhibited cancer cell migration and secretion of matrix metalloproteinases. Tumors in the control clone group continued to grow, whereas those in the AS clone group clearly stopped growing. Six weeks after injection, the tumor size was significantly smaller in the AS clone group than in the control clone group. Immunohistochemistry revealed that the strong pattern was associated with poor overall survival compared with the weak pattern by the Kaplan-Meier method. CONCLUSION: TAGLN2 plays functional roles in the progression of cervical SCC. Suppression of TAGLN2 may be a new strategy for the treatment of cervical SCC.

Heat shock protein 70 is involved in malignant behaviors and chemosensitivities to cisplatin in cervical squamous cell carcinoma cells.

AIM: Heat shock protein 70 (HSP70) was previously found to be highly expressed in cervical squamous cell carcinoma (SCC) tissues compared with normal tissues by proteomic analyses. The present study investigated the roles of HSP70 in malignant behaviors and chemosensitivity to cisplatin in cervical SCC cells in vitro. METHODS: Effects of HSP70 knockdown on activities of cell migration, cell invasion and cell proliferation, cell cycle status, and apoptosis were examined using siRNA in two human cervical cancer cells (SiHa and SKG II). Furthermore, the effect of HSP70 knockdown on the apoptotic effect by cisplatin was examined. RESULTS: HSP70 knockdown significantly suppressed activities of cell migration, cell invasion and cell proliferation. The cell cycle was arrested at the S- and G2/M-phases with the increase in apoptosis by HSP70 knockdown. Although cisplatin had no apoptotic effect in the control cell, it showed a dose-dependent apoptotic effect in the HSP70 knockdown cells in SiHa. In SKG II, 5 µg/mL of cisplatin induced apoptosis, and the apoptotic effect by cisplatin was enhanced by HSP70 knockdown. CONCLUSION: It was suggested that HSP70 played functional roles in the progression of uterine cervical SCC, and HSP70 knockdown enhanced chemosensitivities to cisplatin in cervical SCC cells.

Clinical impact of systematic pelvic and para-aortic lymphadenectomy for pT1 and pT2 ovarian cancer: a retrospective survey by the Sankai Gynecology Study Group.

BACKGROUND: The therapeutic value of systematic lymphadenectomy for early-stage epithelial ovarian cancer (EOC) is controversial. This study evaluates the survival impact and adverse events of systematic pelvic and para-aortic lymphadenectomy in patients with pT1 and pT2 EOC. METHODS: A retrospective investigation was performed using data from patients with pT1 and pT2 EOC at multi-institutions belonging to the Sankai Gynecologic Study Group (SGSG). We selected patients who had undergone systematic pelvic and para-aortic lymphadenectomy (Group LA) (n = 284) and patients who had not undergone lymph node resection (Group no-LA) (n = 138). Outcomes for patients and peri-operative adverse events were compared between the two groups. RESULTS: The median operation time was significantly longer in Group LA (288 min) than in Group no-LA (128 min) (P < 0.0001). Total blood loss was significantly higher in Group LA, 43.7 % of patients receiving blood transfusions. There were no significant differences between the treatment groups for progression-free survival (PFS) or overall survival (OS). However, for patients with pT2, PFS was significantly longer in Group LA than in Group no-LA (P = 0.0150). Lymph node metastases were detected in 23 cases (8.1 %) and these patients had significantly shorter PFS than those without metastasis (P = 0.0409). The outcome for patients who underwent chemotherapy after surgery was significantly improved in the Group no-LA, but no improvement was observed in Group LA. CONCLUSIONS: Systematic lymphadenectomy may improve outcomes only in pT2 EOC patients with acceptable peri-operative events. Furthermore, accurate surgical staging may avoid unnecessary adjuvant chemotherapy in selected early-stage cases.

Anti-Myxovirus Resistance Protein-1 Immunoglobulin A Autoantibody in Idiopathic Pulmonary Fibrosis.

Background: We have previously analysed serum autoantibody levels in patients with idiopathic pulmonary fibrosis (IPF), idiopathic nonspecific interstitial pneumonia (iNSIP), and healthy controls and identified the autoantibody against anti-myxovirus resistance protein-1 (MX1) to be a specific autoantibody in iNSIP. We found that a higher anti-MX1 autoantibody level was a significant predictor of a good prognosis in patients with non-IPF idiopathic interstitial pneumonias. In this retrospective study, we sought to clarify the prognostic significance of the anti-MX1 autoantibody in IPF. Methods: We measured anti-MX1 immunoglobulin (Ig) G, IgA, and IgM autoantibody levels by enzyme-linked immunosorbent assay in serum collected at the time of diagnosis from 71 patients with IPF diagnosed according to the 2018 IPF guideline. The gender-age-physiology (GAP) index was calculated in each case. Results: The study population (59 men and 12 women) had a median age of 67 years. Serum anti-MX1 IgG and IgA autoantibody levels correlated positively with GAP stage (p < 0.05). Univariate Cox proportional hazards regression analysis did not identify an elevated anti-MX1 IgG, IgA, or IgM autoantibody level as a significant prognostic factor; however, a higher anti-MX1 IgA autoantibody level heralded significantly poorer survival after adjustment for GAP stage (p=0.030) and for percent forced vital capacity and modified Medical Research Council score (p=0.018). Neither the anti-MX1 IgG autoantibody nor the IgM autoantibody could predict survival after these adjustments. Conclusions: The serum anti-MX1 IgA autoantibody level is a significant prognostic factor in IPF. Further studies are needed to clarify the pathophysiological role of this autoantibody in IPF.

Identification of a novel SEREX antigen family, ECSA, in esophageal squamous cell carcinoma.

BACKGROUND: Diagnosis of esophageal squamous cell carcinoma (SCC) may improve with early diagnosis. Currently it is difficult to diagnose SCC in the early stage because there is a limited number of tumor markers available. RESULTS: Fifty-two esophageal SCC SEREX antigens were identified by SEREX (serological identification of antigens by recombinant cDNA expression cloning) using a cDNA phage library and sera of patients with esophageal SCC. Sequence analysis revealed that three of these antigens were similar in amino acid sequences, and they were designated as ECSA (esophageal carcinoma SEREX antigen)-1, -2 and -3. The ECSA family was also similar to an EST clone, hepatocellular carcinoma-associated antigen 25a (HCA25a). Serum antibody levels to ECSA-1, -2 and -3 were significantly higher in patients with esophageal SCC than in healthy donors. Based on the conserved amino acid sequences, three peptides were synthesized and used for enzyme-linked immunosorbent assays (ELISA). The serum antibody levels against one of these peptides were significantly higher in patients with esophageal SCC. This peptide sequence was also conserved in FAM119A, GOSR1 and BBS5, suggesting that these are also ECSA family members. Reverse transcription followed by quantitative PCR analysis showed that the mRNA expression levels of ECSA-1, -2 and -3 and FAM119A but not of HCA25a, GOSR1 and BBS5 were frequently elevated in esophageal SCC tissues. CONCLUSIONS: We have identified a new gene family designated ECSA. Serum antibodies against the conserved domain of the ECSA family may be a promising tumor marker for esophageal SCC.

In vivo assay of human NK-dependent ADCC using NOD/SCID/gammac(null) (NOG) mice.

Monoclonal antibodies are essential to the success of molecularly targeted therapies. Recently, numerous therapeutic antibodies have been developed for various diseases, including cancer and autoimmune diseases. Experimental systems to effectively evaluate these candidate antibodies are urgently needed. One of the mechanisms used by antibodies to kill tumor cells is antibody-dependent cellular cytotoxicity (ADCC), in which natural killer cells (NK) are the main mediator. The capacity to induce ADCC has conventionally been assessed in the human-mouse xeno-graft model, in which human peripheral blood mononuclear cells (PBMC), containing NK cells along with antibodies, are administered to tumor-bearing immunodeficient mice. However, contamination from other cellular populations often affects tumor growth, making it difficult to evaluate the antibody's effect. In this study, we established a new NK-dependent ADCC assay model using a supra-immunodeficient strain of mice, NOD/SCID/gammac(null) (NOG). Our model system simply consisted of three elements: isolated human NK cells, a Burkitt's lymphoma cell line (Daudi), and an anti-CD20 antibody (Rituximab). In this experimental setting, human NK cells from healthy donors retained their killing activity and suppressed the growth of Daudi cells in NOG mice when they were administered along with Rituximab. This system, therefore, is useful for evaluating the in vivo function of human NK cells.

Overexpression of carbonyl reductase 1 inhibits malignant behaviors and epithelial mesenchymal transition by suppressing TGF-ß signaling in uterine leiomyosarcoma cells.

Carbonyl reductase 1 (CBR1) has been reported to be involved in cancer progression. Recently, we found that CBR1 overexpression inhibited malignant behaviors and the epithelial mesenchymal transition (EMT) in uterine cervical cancer. It remained unclear whether this was also the case in uterine leiomyosarcoma (uLMS), which is derived from mesenchymal cells and is a much more malignant gynecological tumor. A number of previous studies suggested that malignant behaviors are associated with EMT, even in mesenchymal malignant tumors. In the present study, we investigated whether CBR1 inhibits malignant behaviors and EMT in uLMS. We established clones of uLMS cells (SKN cells) and uterine sarcoma cells (MES-SA cells) that overexpressed CBR1. Cell proliferative, migratory and invasive activities were suppressed by CBR1 overexpression, accompanied by increases in the expressions of epithelial markers (E-cadherin and cytokeratin) and decreases in the expressions of mesenchymal markers (N-cadherin and fibronectin), suggesting that CBR1 overexpression inhibits malignant behaviors and EMT in uLMS cells. In addition, transforming growth factor-ß (TGF-ß) production and the subsequent signaling and phosphorylation of Smad were suppressed in the clones. To investigate the association between TGF-ß and EMT, SKN cells were treated with TGF-ß or a TGF-ß receptor blocker (SB431542). EMT was promoted by TGF-ß and inhibited by SB431542. In conclusion, this is the first study, to the best of the authors' knowledge, showing that CBR1 overexpression inhibits malignant behaviors and EMT in uLMS cells. The present study provided novel insight demonstrating that the suppressive effect of CBR1 is mediated through TGF-ß signaling.

Relationship between decreased expression of squamous cell carcinoma antigen 2 and E-cadherin in primary cervical cancer lesions and lymph node metastasis.

Squamous cell carcinoma antigen (SCCA) has been used for the management of squamous cell carcinoma, especially in order to evaluate therapeutic effects and monitor recurrence. Recent studies have shown that SCCA performs several biological functions and can influence the behavior of cancer cells. It is well known that altered expression of E-cadherin is involved in the process of cancer invasion and metastasis. The present study was therefore undertaken to investigate the relationship between the expression of SCCA, E-cadherin and lymph node metastasis in advanced cervical squamous cell carcinoma patients. We studied 70 patients who had undergone radical hysterectomy and pelvic lymphadenectomy for stage IB, IIA and IIB of the disease, without pretreatments. Immunohistochemistry, using monoclonal antibodies against SCCA2 and E-cadherin, was performed to examine the relationship between SCCA2 and E-cadherin expression patterns in primary cancer lesions and lymph node metastasis. There was a significant positive relationship between the two expression patterns in primary cancer lesions (p<0.01). Both exhibited a heterogeneous expression pattern in the primary tumor which indicated a significant relationship with lymph node metastasis (p<0.01). Our data clearly show that SCCA2 expression is significantly related to E-cadherin expression and that the heterogeneous pattern of SCCA and E-cadherin in primary lesions is strongly associated with the high incidence of lymph node metastasis in cervical squamous cell carcinoma. These findings suggest that SCCA2 may be involved in cancer behavior such as metastasis, and as such can be a useful marker in predicting lymph node metastasis.

E-cadherin increases squamous cell carcinoma antigen expression through phosphatidylinositol-3 kinase-Akt pathway in squamous cell carcinoma cell lines.

Squamous cell carcinoma antigen (SCCA) has been used for the management of squamous cell carcinoma, especially for evaluating therapeutic effects and monitoring recurrence. It has been reported that SCCA has several biological activities and influences behavior of cancer cells. E-cadherin is a cell adhesion molecule and plays important roles in the process of cancer invasion and metastasis. Our previous studies have shown that blockage of E-cadherin action by anti-E-cadherin antibody treatment suppresses SCCA production in squamous cell carcinoma cells. This finding strongly suggests that E-cadherin regulates SCCA expression. The present study was, therefore, undertaken to investigate the correlation between E-cadherin and SCCA2. For this purpose, E-cadherin cDNA was transfected into squamous cell carcinoma cell lines, SiHa and SKG IIIa. Overexpression of E-cadherin increased SCCA2 expression together with cell aggregation. We also examined the involvement of phosphatidylinositol 3-kinase (PI3K)-Akt pathway, which is one of major signaling pathways from E-cadherin. E-cadherin transfection increased phosphorylated Akt expression concomitantly with the increase in SCCA2 expression, and the increased SCCA2 expression was inhibited by a PI3K inhibitor. In conclusion, SCCA2 is up-regulated by E-cadherin through PI3K-Akt pathway, suggesting that SCCA2, as well as E-cadherin, may be involved in the regulation of cancer behavior.

Clinical Utility of YKL-40 in Polymyositis/dermatomyositis-associated Interstitial Lung Disease.

OBJECTIVE: Interstitial lung disease (ILD) is involved in polymyositis/dermatomyositis (PM/DM), a disease associated with poor prognoses. Chitinase-3-like-1 protein (YKL-40) has pleiotropic biological activities involved in inflammation, cell proliferation, and tissue remodeling; however, the clinical application of YKL-40 remains limited. We investigated the clinical significance of YKL-40 in PM/DM-ILD. METHODS: Sixty-nine consecutive patients with PM/DM-ILD and 34 healthy controls were analyzed. We measured baseline and followup serum YKL-40 using an ELISA, evaluated the association of YKL-40 with clinical variables and survival, and examined YKL-40 expression in lung specimens from patients with PM/DM-ILD using immunohistochemistry. RESULTS: Serum YKL-40 levels were significantly greater in patients with PM/DM-ILD compared with healthy controls (p < 0.0001). Serum YKL-40 was correlated with arterial oxygen pressure (r = -0.40, p < 0.001) and percent-predicted DLCO (r = -0.41, p = 0.01) in patients with PM/DM-ILD. Multivariate Cox hazard analysis demonstrated that higher serum YKL-40 and lower percent-predicted forced vital capacity were independently associated with a poor prognosis. Immunohistochemistry analysis demonstrated that YKL-40 expression was enhanced in aggregated intraalveolar macrophages and hyperproliferative alveolar epithelial cells in patients with PM/DM-ILD. CONCLUSION: YKL-40 is a promising biomarker for evaluating PM/DM-ILD activity/severity and predicting disease prognosis. Insights into YKL-40 might help elucidate the pathogenesis of PM/DM-ILD.

Classification of idiopathic interstitial pneumonias using anti-myxovirus resistance-protein 1 autoantibody.

Chronic fibrosing idiopathic interstitial pneumonia (IIP) can be divided into two main types: idiopathic pulmonary fibrosis (IPF), a steroid-resistant and progressive disease with a median survival of 2-3 years, and idiopathic non-specific interstitial pneumonia (INSIP), a steroid-sensitive and non-progressive autoimmune disease. Although the clinical courses of these two diseases differ, they may be difficult to distinguish at diagnosis. We performed a comprehensive analysis of serum autoantibodies from patients definitively diagnosed with IPF, INSIP, autoimmune pulmonary alveolar proteinosis, and sarcoidosis. We identified disease-specific autoantibodies and enriched KEGG pathways unique to each disease, and demonstrated that IPF and INSIP are serologically distinct. Furthermore, we discovered a new INSIP-specific autoantibody, anti-myxovirus resistance-1 (MX1) autoantibody. Patients positive for anti-MX1 autoantibody constituted 17.5% of all cases of chronic fibrosing IIPs. Notably, patients rarely simultaneously carried the anti-MX1 autoantibody and the anti-aminoacyl-transfer RNA synthetase autoantibody, which is common in chronic fibrosing IIPs. Because MX1 is one of the most important interferon-inducible anti-viral genes, we have not only identified a new diagnostic autoantibody of INSIP but also obtained new insight into the pathology of INSIP, which may be associated with viral infection and autoimmunity.

Promoter analyses of SCC antigen genes.

SCC antigen (SCCA) has been used as a tumor marker for squamous cell carcinoma. Analyses of the SCCA1 and SCCA2 genes, which are almost identical, and their promoters have been reported. Recently it was found that both SCCAs were stimulated by interleukin (IL)-4 and IL-13. Here we analyzed the promoter activity of both SCCAs in the 5'-flanking region, exon 1, and intron 1 to evaluate a putative STAT6 binding site. The addition of intron 1 to the luciferase assay constructs including the 5'-flanking region significantly augmented the promoter activity of both SCCA1 and SCCA2. Furthermore, deletion analyses of intron 1 revealed that a 50-bp fragment of intron 1 that includes putative STAT6 binding site was responsible for the increased promoter activity. Although the sequences of SCCA1 and SCCA2 are very similar in the 5'-flanking region, the analysis of the -337 single nucleotide polymorphism of SCCA2 indicated that this polymorphism may underlie the difference in promoter activity between SCCA1 and SCCA2.

Suppression of SCC antigen promotes cancer cell invasion and migration through the decrease in E-cadherin expression.

Squamous cell carcinoma antigen (SCCA) is a useful tumor marker for diagnosis and management of squamous cell carcinoma. Recent studies have shown that SCCA can influence the behavior of cancer cells. It is well known that cell-cell adhesion is an important factor for the progression of cancer. The present study, therefore, was undertaken to investigate the effect of SCCA2 on the cell adhesion related molecule, E-cadherin, and cancer cell behavior. For this purpose, antisense SCCA2 cDNA was transfected into human uterine cancer cell lines, SKG IIIa and SiHa, which express SCCA2. Suppression of SCCA2 expression by antisense SCCA2 cDNA transfection decreased E-cadherin expression and promoted cell migration and invasion as well as the blockage of E-cadherin function by anti-E-cadherin antibody administration. In conclusion, SCCA2 regulates cell migration and invasion via E-cadherin expression, suggesting that SCCA2 may be involved in cancer behavior such as invasion or metastasis.

Different expression patterns of intact forms of squamous cell carcinoma antigens between normal and malignant cervical squamous epithelial tissues: nondenaturing polyacrylamide gel electrophoretic analysis.

Squamous cell carcinoma antigen (SCCA), a 45-kDa tumor-associated serpin, mainly consists of two highly homologous molecules, SCCA1 and SCCA2, which possess unique proteinase inhibitory properties. Importantly, our previous study demonstrated that an intact structure of SCCAs, and not a cleaved form yielded by interacting with target proteinase, is essential for their function as a serpin. The aim of this study is therefore, to develop a simple method of analyzing expression patterns of intact forms of SCCAs (functional SCCAs) in cervical squamous epithelial tissues and to investigate whether there are any differences in the expression of intact forms of SCCAs between normal and malignant cervical squamous epithelial tissues. We used nondenaturing polyacrylamide gel electrophoresis (PAGE) with immunoblotting. The newly generated antibody, Pab Y2, recognizes only intact form of SCCAs, while the conventional antibody, Mab 27, reacts with the cleaved form of SCCA1 as well as intact forms of SCCAs. Nondenaturing PAGE using Pab Y2 showed that an intact form of SCCAs in the heat-treated tissue extract at 60 degrees C for 2 h was separated into at least five bands, termed as bands A-E from cathode to anode. By comparison with two-dimensional electrophoresis patterns of SCCAs, it was found that the first three bands, i.e. bands A-C, are derived from the intact form of SCCA1, while the other two bands, i.e. band D and E are from the intact form of SCCA2. Specifically, band E, but not band D, of SCCA2 is apparently increased in squamous cell carcinomas compared with normal squamous epithelium. In conclusion, this novel analytical approach will be useful for investigating the different expression patterns of functional SCCAs between normal and malignant cervical squamous epithelial tissues.

Enzyme-linked immunosorbent assays for detection of anti-transcriptional intermediary factor-1 gamma and anti-Mi-2 autoantibodies in dermatomyositis.

BACKGROUND: Autoantibodies against transcriptional intermediary factor 1 (TIF1) and Mi-2 are selectively detected in patients with dermatomyositis (DM). To measure these antibodies readily, the development of reliable ELISA systems has been needed. OBJECTIVE: This study aimed to establish enzyme-linked immunosorbent assays (ELISAs) for anti-TIF1γ and anti-Mi-2ß antibodies (Abs) and to assess their utility. METHODS: Serum samples were obtained from 104 patients with classic DM, 68 with clinically amyopathic DM (CADM) and 70 with polymyositis, who were followed up at 8 medical centers across Japan. Serum samples from 190 patients with other connective tissue diseases (CTDs) and 123 healthy individuals were also assessed. Serum antibody levels were examined by ELISAs coated with full-length TIF1γ or Mi-2ß proteins produced by a baculovirus expression system. To assess the cross-reactivity, partial-length Mi-2ß proteins with or without mutations were produced and examined for reactivity. RESULTS: When compared with immunoprecipitation assay, anti-TIF1γ Ab ELISA showed 100% sensitivity and 100% specificity, while anti-Mi-2ß Ab ELISA showed 100% sensitivity and 99.6% specificity. Anti-TIF1γ Ab was positive in 30 (28.8%) with classic DM and 4 (5.9%) with CADM, whereas 14 (13.5%) with classic DM, but none with CADM, were positive for anti-Mi-2ß Ab. Of 30 anti-TIF1γ Ab-positive DM patients, 23 (67.6%) had malignancy. Anti-Mi-2ß Ab-positive serum samples exhibited modest cross-reactivity with the TIF1γ protein due to the homologous amino acid sequence containing cysteines in their plant homeodomains. CONCLUSION: The current study demonstrates the utility of newly established ELISAs for anti-TIF1γ and anti-Mi-2ß Abs, which can serve as easier detection systems for routine testing.

Clinical Utility of an Enzyme-Linked Immunosorbent Assay for Detecting Anti-Melanoma Differentiation-Associated Gene 5 Autoantibodies.

OBJECTIVE: Autoantibodies to melanoma differentiation-associated gene 5 (MDA5) are specifically expressed in patients with dermatomyositis (DM) and are associated with a subset of DM patients with rapidly progressive interstitial lung disease (RP-ILD). Here, we examined the clinical utility of a newly developed enzyme-linked immunosorbent assay (ELISA) system for detecting these antibodies. METHODS: Here we developed an improved ELISA for detecting anti-MDA5 antibodies. We then performed a multicenter clinical study involving 8 medical centers and enrolled 242 adult patients with polymyositis (PM)/DM, 190 with non-PM/DM connective tissue disease (CTD), 154 with idiopathic interstitial pneumonia (IIP), and 123 healthy controls. Anti-MDA5 antibodies in the patients' serum samples were quantified using our newly developed ELISA, and the results were compared to those obtained using the gold-standard immunoprecipitation (IP) assay. In addition, correlations between the ELISA-quantified anti-MDA5 antibodies and clinical characteristics were evaluated. RESULTS: In patients with PM/DM, the anti-MDA5 antibody measurements obtained from the ELISA and IP assay were highly concordant; the ELISA exhibited an analytical sensitivity of 98.2%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 99.5% (compared to the IP assay). Anti-MDA5 antibodies were detected in 22.7% of the DM patients, but not in any of the patients with PM, non-PM/DM CTD, or IIP. Clinically amyopathic DM, RP-ILD, arthritis, and fever were more prevalent in DM patients who were anti-MDA5 antibody-positive than in those who were antibody-negative (P ≤ 0.0002 for all comparisons). In addition, anti-MDA5 antibody-positive patients with RP-ILD exhibited higher antibody levels than those without RP-ILD (P = 0.006). CONCLUSION: Our newly developed ELISA can detect anti-MDA5 antibodies as efficiently as the gold standard IP assay and has the potential to facilitate the routine clinical measurement of anti-MDA5 antibodies in patients who suspected to have DM.

Comprehensive assessment of myositis-specific autoantibodies in polymyositis/dermatomyositis-associated interstitial lung disease.

OBJECTIVES: Myositis-specific autoantibodies (MSAs) are associated with clinical phenotypes in polymyositis/dermatomyositis (PM/DM). No study has investigated the clinical features based on comprehensive MSA assessment in PM/DM-associated interstitial lung disease (ILD). We aimed to determine the practical significance of MSAs in PM/DM-ILD. METHODS: Sixty consecutive PM/DM-ILD patients were retrospectively analysed. Serum MSAs were comprehensively measured using immunoprecipitation assay. Clinical features and prognosis were compared among MSA subgroups. RESULTS: Twenty-six (43.3%) PM/DM-ILD patients were anti-aminoacyl tRNA-synthetase antibody-positive (anti-ARS-positive), 15 (25.0%) were anti-melanoma differentiation-associated gene 5 antibody-positive (anti-MDA5-positive), 3 (5%) were anti-signal recognition particle antibody-positive, 1 (1.7%) was anti-transcriptional intermediary factor 1-gamma antibody-positive, and 15 (25%) were MSA-negative. There were significant differences in clinical features, including ILD form, serum ferritin and surfactant protein-D levels at ILD diagnosis, and high-resolution CT pattern among the anti-ARS-positive, anti-MDA5-positive and MSA-negative groups. The anti-MDA5-positive group showed the lowest 90-day survival rate (66.7%, anti-MDA5-positive; 100%, anti-ARS-positive; 100%, MSA-negative; P < 0.01). The anti-ARS-positive group had the highest 5-year survival rate (96%, anti-ARS-positive; 66.7%, anti-MDA5-positive; 68.3%, MSA-negative, P = 0.02). Univariate analysis revealed that anti-ARS antibody was associated with better prognosis (HR = 0.45; 95% CI, 0.18-0.89; P = 0.02), whereas anti-MDA5 antibody was associated with poorer prognosis (HR = 1.90; 95% CI, 1.02-3.39; P = 0.04). CONCLUSIONS: The comprehensive MSA assessment demonstrated that anti-ARS and anti-MDA5 antibodies were two major MSAs, and the clinical features differed depending on MSA status in PM/DM-ILD. Assessment of anti-ARS and anti-MDA5 antibodies is practically useful for predicting clinical course and prognosis in PM/DM-ILD patients.