Does Eta Protein Differentiate Rheumatoid Arthritis from Psoriatic Arthritis?

AIM: The clinical symptoms and laboratory markers of Rheumatoid Arthritis (RA) and Psoriatic Arthritis (PsA) can be very similar, so making a differential diagnosis between these two diseases is often difficult. Serological parameters to be used in differential diagnosis can guide the clinician. This study aimed to investigate the usability of 14-3-3η (eta) protein as a biomarker in the differential diagnosis of PsA and RA, and the relationships between eta protein and disease activity scores and joint erosions in PsA and RA. METHODS: 54 PsA patients, 53 RA patients, and 56 healthy individuals were included in this study. The ELISA (Enzyme-Linked ImunoSorbent Assay) kit was used as a quantitative sandwich enzyme immunoassay technique to detect human eta protein levels. Receiver- operating Characteristic (ROC) curves analysis was used to determine the sensitivity and specificity of the eta protein. RESULTS: Eta protein levels were found to be significantly higher in the RA group than in the PsA [B: -0.341, OR (95% CI): 0.711 (0.556-0.909), p: 0.007] and control [B: -0.225, OR (95% CI): 0.798 (0.641-0.995), p: 0.045] groups. Eta protein median values were significantly higher in patients with joint erosion than in those without [ß= 0.151, OR (95% CI): 1.163 (1.003-1.349), p: 0.046]. CONCLUSION: Eta protein levels are higher in the serum of RA patients than PsA and are associated with joint erosion. Eta protein may be a potential biomarker in the differential diagnosis of RA and PsA. It may represent a possible therapeutic step in the pathophysiological pathways in the development of joint erosion.

Prevalence and significance of serum 14-3-3η in juvenile idiopathic arthritis.

BACKGROUND: Prompt diagnosis of juvenile idiopathic arthritis (JIA) is important to avoid long term complications. Elevated serum 14-3-3η levels improve the diagnostic sensitivity of rheumatoid factor (RF) and cyclic citrullinated peptide (CCP) antibody in adult rheumatoid arthritis (RA), and have been associated with more severe phenotype. We investigated the prevalence and clinical significance of serum 14-3-3η in different types of JIA. METHODS: JIA patients (n = 151) followed by the Pediatric Rheumatology Core at Children's Hospital of Los Angeles were categorized into 5 groups: polyarticular JIA RF+ (PJIA RF+; n = 39), PJIA RF- (n = 39), psoriatic arthritis (PsA; n = 19), enthesitis-related arthritis (ERA; n = 18), and oligoarticular JIA (OJIA [control group]; n = 36). RF, CCP antibody, and 14-3-3η were measured for all patients. 14-3-3η serum levels > 0.2 ng/mL were considered positive. Disease activity was assessed by the Juvenile Arthritis Disease Activity Score-71 (JADAS-71). RESULTS: Elevated 14-3-3η levels were detected in 34/151 (23%) patients, and across all groups tested. Most patients with 14-3-3η had titers ≥4 times above the cutoff value. The majority (22, 65%) of 14-3-3η-positive patients were also positive for RF or CCP antibodies, 16 (47%) were positive for all 3, and 12 (35%) were single-positive for 14-3-3η. The highest prevalence of 14-3-3η was in PJIA RF+ patients (49%), followed by OJIA (22%). Positivity for 14-3-3η was not significantly associated with disease activity or age at diagnosis. CONCLUSION: Serum 14-3-3η can be detected in all forms of JIA tested but appears to be most common in PJIA RF+. 14-3-3η does not appear to correlate with disease activity in JIA.

The diagnostic value of 14-3-3η protein levels in patients with rheumatoid arthritis.

14-3-3η may represent a useful diagnostic biomarker for rheumatoid arthritis (RA). We assessed the prevalence and serum levels of 14-3-3η in patients with RA and in patients with other rheumatic diseases. Serum levels of 14-3-3η were measured in 96 patients with RA, in 101 patients with other rheumatic diseases, and in 66 healthy subjects. All of the sera samples were evaluated by JOINT stat 14-3-3η ELISA test kits (Augurex Life Sciences Corp.). Median (IQR) 14-3-3η levels were significantly higher in the early RA group [0.25 ng/ml (0.075-3.11)] and in patients with established RA [0.15 ng/ml (0.08-1.26)] than in healthy subjects [0 ng/ml (0-0)] and disease controls: SLE [0.01 ng/ml (0-0.055)], AS [0.05 ng/ml (0-0.255)], and PsA [0.01 ng/ml (0-0.065)]. The prevalence of 14-3-3η positivity in patients with early RA was 58%, significantly higher than that in disease controls and healthy subjects (p < 0.001). In patients with established RA, this prevalence was 43%, and it was significantly higher than that in patients with other rheumatic diseases and healthy subjects (p < 0.05), excluding the AS group (p = 0.054). In the early RA cohort, the positivity for 14-3-3η, RF, and anti-CCP was 58%, 67%, and 71%, respectively. Eighty-two percent of the patients in this cohort were positive for at least one of these biomarkers. The concentration of 14-3-3η protein may be used to distinguish between patients with early RA and patients with other rheumatic diseases.

Identification of 14­3­3ζ as a potential biomarker in gastric cancer by proteomics­based analysis.

The identification of tumor biomarkers to support early diagnosis and tumor progression monitoring may potentially reduce the mortality of gastric cancer (GC). The present study aimed to detect novel tumor­associated antigens from the AGS GC cell line, and to identify their associated autoantibodies in sera from patients with GC by proteomics­based approaches. Proteins from AGS cell lysates were isolated using two­dimensional polyacrylamide gel electrophoresis, and western blotting was subsequently performed, to determine autoantibody responses in sera derived from patients with GC and healthy individuals. Positive protein spots were removed from gels stained with Coomassie blue, and were then evaluated by liquid chromatography­tandem mass spectrometry. Sera from patients with GC produced numerous spots, one of which was identified as 14­3­3ζ. Autoantibody frequency to 14­3­3ζ was 17.6% (15/85) in patients with GC, which was significantly higher than that in healthy control individuals (2.4%; 2/85; P<0.01). These results suggested that the autoantibody against 14­3­3ζ may be a potential serological biomarker for the detection and diagnosis of GC.

Clinical significance of serum 14-3-3 beta in patients with hepatocellular carcinoma.

OBJECTIVE: The 14-3-3 family of conserved regulatory proteins comprises the isoforms beta (ß), gamma (γ), zeta (ζ), sigma (ε), tau (η), and delta (σ), which are overexpressed and associated with a high risk of metastasis and poor survival in hepatocellular carcinoma (HCC). In the present study, we investigated whether serum 14-3-3 isoforms are related to HCC progression and patient survival. METHODS: Serum samples from 63 HCC patients who underwent surgical reSection 104 HCC patients who received non-surgical anti-HCC treatments, 50 patients with liver cirrhosis alone, 45 patients with chronic hepatitis alone, and 50 healthy subjects were collected between January 2006 and December 2010. Serum levels of 14-3-3 (ß, ε, γ, σ, and ζ) isoforms were measured by ELISA. The correlation between 14-3-3 (ß and σ) isoforms and clinicopathological factors was examined by logistic regression analysis. The feasibility of serum 14-3-3 ß for discriminating HCC patients was assessed by ROC curve analysis. Patient survival analyses were performed by Kaplan-Meier analyses and Cox regression models. RESULTS: Serum levels of 14-3-3 (ß and σ) were significantly higher in HCC patients than in those with liver cirrhosis, chronic hepatitis, and healthy subjects (p< 0.05). There was no difference in the serum levels of 14-3-3 ε, γ, and ζ between HCC and the other groups (p> 0.05). High levels of serum 14-3-3 ß were associated with vascular invasion (p= 0.016), TNM stage (p= 0.012), BCLC stage (p= 0.01), and early recurrence (p= 0.013). Patients with high levels of serum 14-3-3 ß had a poor prognosis. There was no significant association between 14-3-3 σ levels and clinicopathological parameters. A significant independent association between serum 14-3-3 ß and HCC was observed by univariate and multivariate analysis (p< 0.05). Serum 14-3-3 ß could effectively discriminate HCC patients at a cut-off point of 18.7 ng/mL, with 91.4% sensitivity and 75.3% specificity. CONCLUSIONS: Serum 14-3-3 ß is a potential biomarker for the diagnosis of early-stage HCC, and high levels of serum 14-3-3 ß were associated with metastasis and poor prognosis in HCC.

Detection of 14-3-3 sigma (σ) promoter methylation as a noninvasive biomarker using blood samples for breast cancer diagnosis.

As a tumor suppressor gene, 14-3-3 σ has been reported to be frequently methylated in breast cancer. However, the clinical effect of 14-3-3 σ promoter methylation remains to be verified. This study was performed to assess the clinicopathological significance and diagnostic value of 14-3-3 σ promoter methylation in breast cancer. 14-3-3 σ promoter methylation was found to be notably higher in breast cancer than in benign lesions and normal breast tissue samples. We did not observe that 14-3-3 σ promoter methylation was linked to the age status, tumor grade, clinic stage, lymph node status, histological subtype, ER status, PR status, HER2 status, or overall survival of patients with breast cancer. The combined sensitivity, specificity, AUC (area under the curve), positive likelihood ratios (PLR), negative likelihood ratios (NLR), diagnostic odds ratio (DOR), and post-test probability values (if the pretest probability was 30%) of 14-3-3 σ promoter methylation in blood samples of breast cancer patients vs. healthy subjects were 0.69, 0.99, 0.86, 95, 0.31, 302, and 98%, respectively. Our findings suggest that 14-3-3 σ promoter methylation may be associated with the carcinogenesis of breast cancer and that the use of 14-3-3 σ promoter methylation might represent a useful blood-based biomarker for the clinical diagnosis of breast cancer.

Elevated levels of 14-3-3 proteins, serotonin, gamma enolase and pyruvate kinase identified in clinical samples from patients diagnosed with colorectal cancer.

BACKGROUND: Colorectal cancer (CRC), a heterogeneous disease that is common in both men and women, continues to be one of the predominant cancers worldwide. Lifestyle, diet, environmental factors and gene defects all contribute towards CRC development risk. Therefore, the identification of novel biomarkers to aid in the management of CRC is crucial. The aim of the present study was to identify candidate biomarkers for CRC, and to develop a better understanding of their role in tumourogenesis. METHODS: In this study, both plasma and tissue samples from patients diagnosed with CRC, together with non-malignant and normal controls were examined using mass spectrometry based proteomics and metabolomics approaches. RESULTS: It was established that the level of several biomolecules, including serotonin, gamma enolase, pyruvate kinase and members of the 14-3-3 family of proteins, showed statistically significant changes when comparing malignant versus non-malignant patient samples, with a distinct pattern emerging mirroring cancer cell energy production. CONCLUSION: The diagnosis and management of CRC could be enhanced by the discovery and validation of new candidate biomarkers, as found in this study, aimed at facilitating early detection and/or patient stratification together with providing information on the complex behaviour of cancer cells.

Multiplex targeted proteomic assay for biomarker detection in plasma: a pancreatic cancer biomarker case study.

Biomarkers are most frequently proteins that are measured in the blood. Their development largely relies on antibody creation to test the protein candidate performance in blood samples of diseased versus nondiseased patients. The creation of such antibody assays has been a bottleneck in biomarker progress due to the cost, extensive time, and effort required to complete the task. Targeted proteomics is an emerging technology that is playing an increasingly important role to facilitate disease biomarker development. In this study, we applied a SRM-based targeted proteomics platform to directly detect candidate biomarker proteins in plasma to evaluate their clinical utility for pancreatic cancer detection. The characterization of these protein candidates used a clinically well-characterized cohort that included plasma samples from patients with pancreatic cancer, chronic pancreatitis, and healthy age-matched controls. Three of the five candidate proteins, including gelsolin, lumican, and tissue inhibitor of metalloproteinase 1, demonstrated an AUC value greater than 0.75 in distinguishing pancreatic cancer from the controls. In addition, we provide an analysis of the reproducibility, accuracy, and robustness of the SRM-based proteomics platform. This information addresses important technical issues that could aid in the adoption of the targeted proteomics platform for practical clinical utility.

Identification of 14-3-3ß in human gastric cancer cells and its potency as a diagnostic and prognostic biomarker.

Gastric cancer is the second most common cause of cancer deaths worldwide and due to its poor prognosis, it is important that specific biomarkers are identified to enable its early detection. Through 2-D gel electrophoresis and MALDI-TOF-TOF-based proteomics approaches, we found that 14-3-3ß, which was one of the proteins that were differentially expressed by 5-fluorouracil-treated gastric cancer SC-M1 cells, was upregulated in gastric cancer cells. 14-3-3ß levels in tissues and serum were further validated in gastric cancer patients and controls. The results showed that 14-3-3ß levels were elevated in tumor tissues (n=40) in comparison to normal tissues (n=40; p<0.01), and serum 14-3-3ß levels in cancer patients (n=145) were also significantly higher than those in controls (n=63; p<0.0001). Elevated serum 14-3-3ß levels highly correlated with the number of lymph node metastases, tumor size and a reduced survival rate. Moreover, overexpression of 14-3-3ß enhanced the growth, invasiveness and migratory activities of tumor cells. Twenty-eight proteins involved in anti-apoptosis and tumor progression were also found to be differentially expressed in 14-3-3ß-overexpressing gastric cancer cells. Overall, these results highlight the significance of 14-3-3ß in gastric cancer cell progression and suggest that it has the potential to be used as a diagnostic and prognostic biomarker in gastric cancer.

14-3-3 protein beta/alpha as a urinary biomarker for renal cell carcinoma: proteomic analysis of cyst fluid.

Although various samples, including tissue, cells, serum, and urine, from patients with renal cell carcinoma (RCC) have been analyzed, biomarkers with diagnostic value have yet to be identified. We used a proteomics approach to analyze cyst fluid in cases of cyst-associated RCC to identify accessible and abundant proteins that are overexpressed and/or secreted by RCC cells. Proteins in the cyst fluid were separated by reverse-phase high-performance liquid chromatography and agarose two-dimensional gel electrophoresis and were identified by tandem mass spectrometry. We conducted a National Center for Biotechnology Information search and a MEDLINE search to predict the function of these identified proteins and to select a tumor-marker candidate protein. Our search resulted in the identification and selection of the differentially regulated protein known as 14-3-3 protein beta/alpha, which was overexpressed in cyst fluid from cyst-associated RCC but has not been previously associated with RCC. We then measured its incidence through Western blotting of various normal and RCC samples (serum, urine, tissue, and cyst fluid). The expression levels of 14-3-3 protein beta/alpha were higher in urine samples from patients with RCC than in samples from healthy volunteers. Receiver operating characteristic (ROC) curve analyses were performed to assess this potential biomarker; these data (area under the ROC curve value was 0.8813) indicate a high degree of accuracy for this screening method. 14-3-3 Protein beta/alpha may be a diagnostically useful biomarker for early diagnosis of RCC.

Serum 14-3-3; matrix metalloproteinases and their inhibitors' levels before and after cardiovascular surgery in diabetic and non-diabetic patients.

AIMS: We sought to assess the serum levels of the main extracellular matrix components before and after surgery in order to differentiate the response of diabetic patients to acute wounds from that of non-diabetic patients. METHODS: The serum levels of 14-3-3, Pro-MMP-1, MMP-3, and TIMP-1 were measured in diabetics (18 patients) and non-diabetics (22 patients) in samples obtained before a coronary artery bypass grafting operation and on the 1st, 3rd, and 5th postoperative days. RESULTS: The diabetics had higher serum levels of 14-3-3 both in the pre- and postoperative phases. Nevertheless, there was a postoperative drop in these amounts in all the patients. There was no difference in the serum levels of Pro-MMP-1 between the two groups. In addition, the serum levels of MMP-3 on the 3rd and 5th postoperative days and also TIMP-1 (inhibitor of both MMPs) on all postoperative days were higher in the diabetics. CONCLUSIONS: There was perfect synchronicity between the changes in the serum levels of these proteins and their functional nature in the injured tissue. Furthermore, the diabetic patients exhibited more changes in the levels of some of their extracellular enzymes in the wake of acute wounds; these changes were also traceable in the serum.

LC-MS/MS analysis of ovarian cancer metastasis-related proteins using a nude mouse model: 14-3-3 zeta as a candidate biomarker.

Peritoneal implantation is the most common metastatic pattern of epithelial ovarian cancer, and the five-year survival rate of patients is dramatically decreased when large-scale peritoneal metastasis occurs. This study aimed to determine serum proteins that could be used to detect early peritoneal metastasis of ovarian cancer. The secreted (or shed) proteins of the ovarian cancer cell line SKOV-3 were analyzed using LC-MS/MS, and 97 proteins were identified in the SKOV-3 culture supernatant. After the SKOV-3 cells were xenografted into the peritoneal cavities of nude mice, 3 of the 97 proteins were detected in animal sera. Following enzyme-linked immunosorbent assay (ELISA)-based screening of clinical blood samples, one of the three proteins, 14-3-3 zeta, was identified as a candidate biomarker. The average serum levels of 14-3-3 zeta in patients with epithelial ovarian cancer and benign gynecological diseases were significantly different. The expression of 14-3-3 zeta was associated with the degree of cancer peritoneal metastasis, the emergence of ascites, bilateral involvement, and the clinical stage and substage. Using 14-3-3 zeta, the overall diagnostic accuracy for ovarian cancer was greatly improved. Furthermore, siRNA-based experiments demonstrated that 14-3-3 zeta was responsible for approximately 62, 65, and 30% of the migratory, invasive, and implantation abilities of SKOV-3 cells, respectively. The present results demonstrated that the nude mouse xenograft model is an efficient system for performing function-oriented biomarker discovery, which can be used for a variety of research tasks in future molecular diagnoses, targeted therapies, and ovarian cancer vaccine development.