Potential biomarkers of spinal dural arteriovenous fistula: C4BPA and C1QA.

BACKGROUND AND PURPOSE: A major challenge in spinal dural arteriovenous fistula (SDAVF) is timely diagnosis, but no specific predictive biomarkers are known. METHODS: In the discovery cohort (case, n = 8 vs. control, n = 8), we used cerebrospinal fluid (CSF) and paired plasma samples to identify differentially expressed proteins by label-free quantitative proteomics. Further bioinformatics enrichment analyses were performed to screen target proteins. Finally, it was validated by ELISA in two of the new cohorts (case, n = 17 vs. control, n = 9), and univariate analysis, simple linear regression, and receiver operator characteristic (ROC) curve analysis were performed to evaluate the diagnostic potential. RESULTS: In the discovery cohort, the most overexpressed proteins were APOB and C4BPA in CSF samples of patients. The GO/KEGG enrichment analysis indicated that the upregulated proteins were mainly involved in the acute inflammatory response and complement activation. Hub-gene analysis revealed that APP might be the key protein in the molecular interaction network. In the validation cohort, C4BPA and C1QA were significantly overexpressed in the CSF of patients, averaging 3046.9 ng/ml and 2167.2 ng/ml, respectively. Simple linear regression demonstrated that levels of C1QA and C4 were positively correlated with total protein in CSF (R2 = 0.8021, p = 0.0005; R2 = 0.7447, p = 0.0013). The areas under the ROC curves of C4BPA and C1QA were 0.86 and 1.00, respectively. CONCLUSIONS: This study was the first to identify C4BPA and C1QA as potential biomarkers for the diagnosis of SDAVF and revealed that complement pathway activation might be one of the molecular mechanisms for venous hypertension myelopathy.

Immunoexpression of C4 Binding Protein in Oral Leukoplakia and Oral Squamous Cell Carcinoma.

BACKGROUND: The immune evasion of dysplastic cells plays an important role in suppressing the immune response and progression of malignancy. The role of the complement inhibitors in the development of oral epithelial dysplastic lesions and squamous cell carcinoma (SCC) is still unclear. OBJECTIVE: This study aimed to assess the expression of C4 binding protein (C4BP) as a complement inhibitor in oral squamous cell carcinoma and leukoplakia. METHODS: In this study, 94 samples were classified into four groups: leukoplakia with mild to moderate dysplasia, leukoplakia with severe dysplasia or carcinoma in situ, early invasive SCC, and invasive SCC. The expression of C4BP marker was evaluated by immunohistochemistry (IHC) and real-time PCR. The results were analyzed by the Kruskal-Wallis, Bonferroni adjusted Dunn's multiple comparison, and one-way ANOVA tests. RESULTS: The results of IHC revealed the expression patterns of C4BP in oral dysplasia and SCC, and indicated that the C4BP expression was not significantly different between different histopathological grades in epithelial cells and vessels (P=0.157 and P=0.123, respectively) but, it was significantly different in fibroblasts and lymphocytes (P=0.017 and P=0.043, respectively). The real-time PCR showed a significant correlation between the dysplasia grade and expression of C4BP (P<0.05). CONCLUSION: According to the results, C4BP is expressed in the cancerous tissue by the tumor cells and their surrounding stroma. In addition, upregulation of the C4BP gene as an inhibitor of the complement system is a possible strategy adopted by the tumor cells to evade the immune system.

UPLC-MS/MS based diagnostics for epithelial ovarian cancer using fully sialylated C4-binding protein.

Serum levels of fully sialylated C4-binding protein (FS-C4BP) are remarkably elevated in patients with epithelial ovarian cancer (EOC) and can be used as a marker to distinguish ovarian clear cell carcinoma from endometrioma. This study aimed to develop a stable, robust and reliable liquid chromatography-hybrid mass spectrometry (UPLC-MS/MS) based diagnostic method that would generalize FS-C4BP as a clinical EOC biomarker. Glycopeptides derived from 20 µL of trypsin-digested serum glycoprotein were analyzed via UPLC equipped with an electrospray ionization time-of-flight mass spectrometer. This UPLC-MS/MS-based diagnostic method was optimized for FS-C4BP and validated using sera from 119 patients with EOC and 127 women without cancer. A1958 (C4BP peptide with two fully sialylated biantennary glycans) was selected as a marker of FS-C4BP because its level in serum was highest among FS-C4BP family members. Preparation and UPLC-MS/MS were optimized for A1958, and performance and robustness were significantly improved relative to our previous method. An area under the curve analysis of the FS-C4BP index receiver operating characteristic curve revealed that the ratio between A1958 and A1813 (C4BP peptide with two partially sialylated biantennary glycans) reached 85%. A combination of the FS-C4BP index and carbohydrate antigen-125 levels further enhanced the sensitivity and specificity.

Identification of a novel serum biomarker for pancreatic cancer, C4b-binding protein α-chain (C4BPA) by quantitative proteomic analysis using tandem mass tags.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) remains a devastating disease due to the lack of specific early diagnostic markers. To improve the outcomes, proteomic approaches are being developed for the discovery of novel biomarkers of PDAC. METHODS: Using tandem mass tag labelling and LC-MS/MS, we performed comparative analyses of pre- and postoperative sera from PDAC patients to identify specific serum biomarkers for PDAC. In validation studies, we evaluated the discriminatory power of candidate proteins. RESULTS: Among the 302 proteins analysed, 20 were identified as potential biomarkers, with C4b-binding protein α-chain (C4BPA) and polymeric immunoglobulin receptor (PIGR) being selected for further analysis. The sera levels of C4BPA and PIGR were significantly higher in the preoperative PDAC patients than in the postoperative ones (P<0.008, P<0.036, respectively). In addition, serum C4BPA levels, but not PIGR, in patients with PDAC were significantly higher than those in healthy controls as well as in patients with pancreatitis and other malignancies including biliary tract cancers (BTC) (P<0.001). The respective area under the receiver operator characteristics (ROC) curve (AUC) was 0.860 for C4BPA, 0.846 for CA19-9 and 0.930 for the combination of C4BPA and CA19-9 in PDAC vs non-cancer individuals. The respective AUC was 0.912 for C4BPA, 0.737 for CA19-9 in Stages I and II of PDAC, 0.854 for C4BPA and 0.264 for CA19-9 in PDAC vs BTC. CONCLUSIONS: We have demonstrated that C4BPA is a novel serum biomarker for detecting early stage PDAC, as well as for distinguishing PDAC from other gastroenterological cancers. Further analysis in large cohort studies will warrant C4BPA as a promising biomarker of PDAC in clinical use.

Quantitative proteomics revealed novel proteins associated with molecular subtypes of breast cancer.

UNLABELLED: The early diagnosis and successful treatment of breast cancer (BC) is still a challenging task due to the diverse origin and functional heterogeneity of cancer cells. The heterogeneity of BC may likely to explained by molecular BC subtypes, comprises Luminal-A (LA), Luminal-B (LB), Triple-negative (TN) and HER2-positive (HP), which are governed by a variety of cancer associated pathways. To identify protein signatures in different BC subtypes, we performed isobaric tag for absolute and relative quantitation (iTRAQ) of enriched blood plasma samples of BC subtypes (N=32) and healthy subjects (N=8). After analyses of data, 58 proteins were found to be modulated in BC subtypes from healthy subjects (p<0.05) and among these; Fibronectin (FN1), Alpha-2-macroglobulin (A2M), and Complement component-4-binding protein-alpha (C4BPA) and Complement factor-B (CFB) were selected for validation in BC subtypes and healthy subjects in the independent set of blood plasma (N=100) and tissue samples (N=25). Statistical analysis showed the significant modulation of FN1 and C4BPA in LB, and A2M in TN patients in both plasma as well as tissues comparatively control (p<0.05). Further, FN1 and C4BPA in LB subtype revealed a good diagnostic accuracy in plasma level validation. The receiver operating characteristic (ROC) curve and regression analysis demonstrated that these proteins with associated criterion of expression could act as discriminating signatures among BC subtypes with diagnostic and prognostic relevance. SIGNIFICANCE: The heterogeneity of breast cancer (BC) has gained many challenges for successful management of BC, thus, the delineating proteomic alterations BC subtypes may provide great clinical values in diagnostic, prognostic and therapeutics of BC. The findings from the present quantitative proteomic study have deciphered the altered proteomic patterns and their possible molecular interactions in each BC subtype. The study showed a strong association of FN1, A2M, C4BPA and CFB in molecular subtypes of BC, in which, C4BPA and A2M demonstrated a potent signature in blood plasma and tissue samples of LB and TN subtypes in BC patients, respectively. The findings also revealed the altered level expressions of these selected proteins could classify BC subtypes through plasma and tissue based expression analysis in patients and control samples. Hence, these proteins could have clinical importance for the diagnosis and prognosis purposes among molecular BC subtypes.

C4b-Binding Protein Deposition is Induced in Diseased Aortic Heart Valves, Coinciding with C3d.

BACKGROUND AND AIM OF THE STUDY: It has been found recently that activated complement is more widespread in diseased aortic valves compared to the endogenous complement inhibitors C1-inhibitor and clusterin. Previously, another endogenous inhibitor of complement, C4b-binding protein (C4BP) has been described in atherosclerotic diseased coronary arteries. The study aim was to analyze C4BP levels in diseased aortic valves. METHODS: Aortic valve tissue was derived from surgical procedures and classified as 'degenerative', 'atherosclerotic' or 'atherosclerotic with bacterial infection'. Valves were stained with specific antibodies against C4BP, C3d and caspase-3. Areas of positivity were then quantified using computer- assisted morphometry. RESULTS: In atherosclerotic valves, the areas of C4BP and C3d positivity (38.8 +/- 0.4% versus 32.7 +/- 1.0%, respectively) were significantly higher compared to the degenerative and control groups. In atherosclerotic valves with bacterial infection, the area of positivity for C4BP was even further increased compared to atherosclerotic valves (65.1 +/- 1.2%; 70.1 +/- 1.9% for C3d). The areas of C4BP and C3d positivity were not significantly different in all groups. Caspase-3 was only present in <10% of endothelial cells in the atherosclerotic valves without bacterial infection and in neutrophilic granulocytes in atherosclerotic valves, with and without bacterial infection. CONCLUSION: It has been shown for the first time that C4BP is deposited in the diseased aortic valve, coinciding with C3d. The area of C4BP positivity was more extensive compared to the areas of other endogenous complement inhibitors (C1-inhibitor and clusterin).

Baseline plasma proteomic analysis to identify biomarkers that predict radiation-induced lung toxicity in patients receiving radiation for non-small cell lung cancer.

PURPOSE: To identify new plasma proteomic markers before radiotherapy start to predict later grade ≥2 radiation-induced lung toxicity (RILT2). METHODS: Fifty-seven patients with non-small cell lung cancer received radiotherapy (RT) were eligible. Forty-eight patients with minimum follow-up of 1 year, nine with RILT2 with tumor stage matched to 39 without RILT2, were enrolled for this analysis. Platelet-poor plasma was obtained within 2 weeks before radiotherapy. The plasma proteomes were compared using a multiplexed quantitative proteomics approach involving ExacTag labeling, reverse-phase high-performance liquid chromatography, and nano liquid chromatography electrospray ionization tandem mass spectrometry. Z scores and Bonferroni-adjusted p values for the two-sample mean comparison were used to identify the differential protein expression between patients with and without RILT2. RESULTS: More than 200 proteins were identified and quantified. After excluding proteins that were not detected in at least 40% of the 48 patient samples, C4b-binding protein alpha chain and vitronectin had significantly higher (p < 0.001 and p = 0.02) expression levels in patients with RILT2 compared with patients without RILT2. These two proteins were validated by Western blot. Ingenuity pathway analysis revealed that they both play important roles in the inflammatory response and are associated with the known pathways of radiation-induced lung damage. CONCLUSIONS: This proteomic approach demonstrates new plasma protein biomarkers before treatment for future studies on RILT2 prediction.

Plasma proteomic analysis may identify new markers for radiation-induced lung toxicity in patients with non-small-cell lung cancer.

PURPOSE: To study whether radiation induces differential changes in plasma proteomics in patients with and without radiation-induced lung toxicity (RILT) of Grade >/=2 (RILT2). METHODS AND MATERIALS: A total of 57 patients with NSCLC received radiation therapy (RT) were eligible. Twenty patients, 6 with RILT2 with tumor stage matched to 14 without RILT2, were enrolled for this analysis. Platelet-poor plasma was obtained before RT, at 2, 4, 6 weeks during RT, and 1 and 3 months after RT. Plasma proteomes were compared using a multiplexed quantitative proteomics approach involving ExacTag labeling, reverse-phase high-performance liquid chromatography and nano-LC electrospray tandem mass spectrometry. Variance components models were used to identify the differential protein expression between patients with and without RILT2. RESULTS: More than 100 proteins were identified and quantified. After excluding proteins for which there were not at least 2 subjects with data for at least two time points, 76 proteins remained for this preliminary analysis. C4b-binding protein alpha chain, Complement C3, and Vitronectin had significantly higher expression levels in patients with RILT2 compared with patients without RILT2, based on both the data sets of RT start to 3 months post-RT (p < 0.01) and RT start to the end of RT (p < 0.01). The expression ratios of patients with RILT2 vs. without RILT2 were 1.60, 1.36, 1.46, and 1.66, 1.34, 1.46, for the above three proteins, respectively. CONCLUSIONS: This proteomic approach identified new plasma protein markers for future studies on RILT prediction.

Complement activation and plasma levels of C4b-binding protein in critical limb ischemia patients.

OBJECTIVE: Critical limb ischemia (CLI) is a peripheral arterial disease manifested by drastically diminished blood flow to the legs, pain at rest, nonhealing wounds, and gangrene caused by atherosclerosis. Significant tissue necrosis is associated with late stage CLI and the patients have a poor prognosis. Necrotic and apoptotic cells activate complement and bind complement inhibitor C4b-binding protein (C4BP). The major isoform of C4BP is composed of seven identical alpha-chains and one beta-chain, here termed C4BP(beta), whereas upon inflammation a normally less abundant isoform is upregulated that is exclusively composed of alpha-chains. Measuring the alpha-chains of C4BP includes both isoforms and is termed total C4BP (C4BP(tot)). The hypothesis of this study was that levels of complement activation and C4BP are predictive for the severity of the disease and that their measurement might be of clinical advantage. METHODS: This was a prospective, single-center study of 259 consecutive patients with CLI admitted to a secondary referral center for vascular diseases. Interventions included evaluation of soluble terminal complement complexes (C5b-9), C4BP(tot) and C4BP(beta), lipid levels, the inflammatory mediators tumor necrosis factor-alpha, interleukin-6, 8-iso-prostaglandin F(2alpha), high-sensitivity C-reactive protein, neopterin, plasma homocysteine, and plasma endothelin-1 in plasma as well as resistance to activated protein C and ankle blood pressure. All data were compared with an age-matched population based control group of 219 currently healthy individuals. RESULTS: The data are presented as mean +/- SEM/median. CLI patients showed systemic complement activation (1.17 +/- 0.06/1.13 AU/mL vs 0.69 +/- 0.07/0.59 AU/mL in healthy controls, P < .0001), which was even higher in patients with gangrene (1.33 +/- 0.11/1.28 AU/mL vs 1.1 +/- 0.08/1.0 AU/mL, P = .0264), who also showed increased C4BP levels (421 +/- 28.6/386 microg/mL vs 341 +/- 10.8/318 microg/mL for C4BP(tot), P = .0248; 374 +/- 25.4/332 microg/mL vs 305 +/- 9.5/285 microg/mL for C4BP(beta), P = .0581). C4BP plasma levels were significantly elevated in CLI patients in comparison to healthy controls (351 +/- 8.1/322 microg/mL vs 297 +/- 8.0/288 microg/mL for C4BP(tot), P = .0001; 314 +/- 7.0/287 microg/mL vs 265 +/- 7.0/263 microg/mL for C4BP(beta), P = .0004) and correlated to levels of interleukin-6 (P(tot/beta) = .0048/.0019), high-sensitivity C-reactive protein (P < .0001), leukocyte (P(tot/beta) = .0086/.0043) and platelet count (P = .0001), LDL/HDL ratio (P(tot) = .0151) and HDL (P(tot/beta) = .0047/.0177), but not to tumor necrosis factor-alpha. CONCLUSIONS: Increased complement activation and C4BP plasma levels are related to the degree of tissue necrosis and disease severity of critical limb ischemia. This knowledge in combination with the found correlations to other biomarkers is useful for understanding the pathophysiology of the disease.

Acquired protein S deficiency leads to obliterative portal venopathy and to compensatory nodular regenerative hyperplasia in HIV-infected patients.

OBJECTIVE: To identify the mechanism of nodular regenerative hyperplasia in HIV-infected patients. DESIGN: Case-control study. SETTING: The hepatology and the infectious disease units of two tertiary care centers in France. PATIENTS: We compared 13 consecutive HIV-positive patients with unexplained nodular regenerative hyperplasia to 16 consecutive HIV-positive patients without nodular regenerative hyperplasia, to eight HIV-negative patients with nodular regenerative hyperplasia from an identified cause and to 10 anonymous healthy blood donors. MAIN OUTCOME MEASURE: Patients and controls were screened for diminished protein S activity and antiprotein S immunoglobulin G (IgG) antibodies. The antiprotein S activity of purified IgG from patients and controls was assessed in a functional test of activation of protein C in which protein S serves as a cofactor. A full liver CT portography was realized on the liver explant of a case patient. RESULTS: The CT portography disclosed diffuse obliterative portal venopathy. Levels of protein S activity were lower among patients with HIV-associated nodular regenerative hyperplasia when compared with HIV-positive patients without nodular regenerative hyperplasia and when compared with HIV-negative patients with nodular regenerative hyperplasia (P < 0.005 for all comparisons). HIV-positive patients with nodular regenerative hyperplasia had significantly higher levels of antiprotein S IgG than HIV-positive patients without nodular regenerative hyperplasia and healthy controls. Purified IgG from patients with HIV-associated nodular regenerative hyperplasia specifically inhibited the protein S-dependent protein C activation. CONCLUSION: Acquired autoimmune protein S paucity and secondary thrombophilia appear to be causes of obliterative portal venopathy and compensatory nodular regenerative hyperplasia in HIV-positive patients.

Regulatory mechanisms of C4b-binding protein (C4BP)alpha and beta expression in rat hepatocytes by lipopolysaccharide and interleukin-6.

BACKGROUND: C4b-binding protein (C4BP), a multimeric protein structurally composed of alpha chains (C4BPalpha) and a beta chain (C4BPbeta), regulates the anticoagulant activity of protein S (PS). Patients with sepsis have increased levels of plasma C4BP, which appears to be induced by interleukin (IL)-6. However, it is not fully understood how lipopolysaccharide (LPS) and IL-6 affect the plasma C4BP antigen level and C4BPalpha and C4BPbeta expression in hepatocytes. OBJECTIVES: To assess the effect of LPS and IL-6 on plasma C4BP, PS-C4BP complex levels, PS activity, and C4BP expression by rat liver in vivo and on C4BP expression by isolated rat hepatocytes in vitro. RESULTS: Plasma C4BP antigen level transiently decreased from 2 to 12 h after LPS (2 mg kg(-1)) injection, and then it abruptly increased up to 24 h after LPS injection. Plasma C4BP antigen level increased until 8 h after IL-6 (10 microg kg(-1)) injection, and then gradually decreased up to 24 h after IL-6 injection. LPS significantly decreased the protein and mRNA expression of both C4BPalpha and C4BPbeta in rat hepatocytes, and this effect was inhibited by NFkappaB and MEK/ERK inhibitors. IL-6 mediated increase in C4BPbeta expression in rat hepatocytes, which leads to increased plasma PS-C4BP complex level and to decreased plasma PS activity, was inhibited by inhibition of STAT-3. CONCLUSION: LPS decreases both C4BPalpha and C4BPbeta expression via the NFkappaB and MEK/ERK pathways, whereas IL-6 specifically increases C4BPbeta expression via the STAT-3 pathway, causing an increase in plasma PS-C4BP complex, and thus decreasing the anticoagulant activity of PS.

Hypercomplementemia in adult patients with IgA nephropathy.

IgA nephropathy (IgAN) is the most common form of chronic glomerulonephritis. Although glomerular deposition of complement components is well known, the evidence of serological complement activation in IgAN is inconclusive. We hypothesized that serum levels of complement components and regulatory proteins in patients with IgAN are correlated with its pathogenesis. In the present study we measured complement components in 50 patients with IgAN and 50 healthy volunteers. C5, C1 inhibitor, factor B, C4 binding protein, factor H, and factor I were measured with the use of single radial immunodiffusion. Mannose-binding lectin (MBL) and properdin (P) were measured by enzyme-linked immunosorbent assay (ELISA). The correlations among complements in the sera of patients with clinical gradings for IgAN (i.e., the good prognosis group, relatively good prognosis group, relatively poor prognosis group, and poor prognosis group) were evaluated. CH50, C4, factor B, P, factor I, and factor H were significantly higher in IgAN patients than in healthy controls. There were significant correlations between C5 and C4 binding protein, between C3 and C5, or between C4 and factor B in patients with IgAN. In the poor prognosis group, C4 binding protein was significantly higher than in the other groups of IgAN patients. hypercomplementemia occurs in IgAN and is associated with an increase in complement regulatory protein (CRP). C4 binding protein analyses can be used to predict disease prognosis.

Molecular bases of type II protein S deficiency: the I203-D204 deletion in the EGF4 domain alters GLA domain function.

OBJECTIVE: To characterize the first type II protein S (PS) deficiency affecting the epidermal growth factor (EGF)4 domain, a calcium-binding module with a poorly defined functional role. PATIENTS: The proband suffered from recurrent deep vein thrombosis and showed reduced PS anticoagulant activity (31%), and total, free PS antigen and C4bBP levels in the normal range. RESULTS: Reverse transcription-polymerase chain reaction analysis showed the presence of the IVSg-2A/T splicing mutation that, by activating a cryptic splice site, causes the deletion of codons Ile203 and Asp204. Free PS, immunopurified from proband's plasma, showed an altered electrophoretic pattern in native condition or in the presence of Ca2+. The recombinant PS (rPS) mutant showed reduced anticoagulant (<10%) and activated protein C-independent activities (24-38%) when compared with wild-type rPS (rPSwt). Binding of the rPS variant to phospholipid vesicles (Kd 235.7 +/- 30.8 nM, rPSwt; Kd 15.2 +/- 0.9 nM) as well as to Ca2+-dependent conformation-specific monoclonal antibodies for GLA domain was significantly reduced. CONCLUSIONS: These data aid in the characterization of the functional role of the EGF4 domain in the anticoagulant activities of PS and in defining the thrombophilic nature of type II PS deficiency.