Elevated serum levels of sialyl Lewis X (sLeX) and inflammatory mediators in patients with breast cancer.

PURPOSE: The carbohydrate sialyl LewisX (sLeX) mediates cell adhesion, is critical in the normal function of immune cells, and is frequently over-expressed on cancer cells. We assessed the association, differential levels, and prognostic value of sLeX and inflammatory cytokines/chemokines in breast cancer sera. METHODS: We retrospectively measured sLeX and a panel of cytokines/chemokines in the sera of 26 non-invasive ductal carcinoma in situ (DCIS), 154 invasive non-metastatic breast cancer (non-MBC), 63 metastatic breast cancer (MBC) patients, and 43 healthy controls. Differences in sLeX and inflammatory cytokines among and between patient groups and healthy controls were assessed with nonparametric tests and we performed survival analysis for the prognostic potential of sLeX using a cut-off of 8 U/mL as previously defined. RESULTS: Median serum sLeX was significantly higher than controls for invasive breast cancer patients (MBC and non-MBC) but not DCIS. In univariate analysis, we confirmed patients with serum sLeX > 8 U/mL have a significantly shorter progression-free survival (PFS) (P = 0.0074) and overall survival (OS (P = 0.0003). Similarly, patients with high serum MCP-1 and IP-10 had shorter OS (P = 0.001 and P < 0.001, respectively) and PFS (P = 0.010 and P < 0.001, respectively). sLeX, MCP-1 and IP-10 remained significant in multivariate survival analysis. CONCLUSION: Elevated serum sLeX was associated with invasive cancer but not DCIS. High serum sLeX levels were associated with inflammatory mediators and may play a role in facilitating local invasion of breast tumor. Furthermore, serum MCP-1, IP-10 and sLeX may have prognostic value in breast cancer.

Fetal Bone Formation Is Decreased from Middle Pregnancy to Birth.

Fetal bone development is a complex process that is regulated and maintained by minerals, hormones, and growth factors delivered from the mother via the placenta. Various biochemical markers of fetal bone development have been identified. However, many aspects of this process remain unclear. The aim of the study was to measure the activities of serum tartrate-resistant acid phosphatase type 5b (TRACP 5b) as a bone resorption marker and bone alkaline phosphatase (BAP) as a bone formation marker in preterm and term neonates, and to investigate fetal bone development in middle and late pregnancy. The study included 111 neonates (87 preterm and 24 term) born at Dokkyo Medical University Hospital. Neonates with illnesses and maternal diseases were excluded. Serum samples were collected within 3 hours after birth and stored at -80°C. Univariate and multivariate linear regression analyses were performed. The 111 neonates (median birth weight, 1,510 g) were born at a median of 31.3 weeks of gestation, and had TRACP 5b and BAP activities of 10.9 ± 4.0 U/L and 127.5 ± 49.2 U/L, respectively. TRACP 5b activity showed a tendency to be higher in term neonates, while BAP activity tended to be lower in term neonates. Importantly, TRACP 5b activity was positively correlated with gestational age and birth weight, and BAP activity was negatively correlated with gestational age, rate of born small-for-gestational-age neonates, and birth weight. These results suggest that bone formation during fetal growth is gradually decreased from middle pregnancy to birth, whereas bone resorption is gradually increased.

Serum anti-Ku86 is a potential biomarker for early detection of hepatitis C virus-related hepatocellular carcinoma.

Hepatocellular carcinoma (HCC), the predominant form of primary liver cancer, is one of the most common cancers worldwide and the third most common cause of cancer-related death. Imaging studies including ultrasound and computed tomography are recommended for early detection of HCC, but they are operator dependent, costly and involve radiation. Therefore, there is a need for simple and sensitive serum markers for the early detection of hepatocellular carcinoma (HCC). In our recent proteomic studies, a number of proteins overexpressed in HCC tissues were identified. We thought if the serum autoantibodies to these overexpressed proteins were detectable in HCC patients. Of these proteins, we focused on Ku86, a nuclear protein involved in multiple biological processes and aimed to assess the diagnostic value of serum anti-Ku86 in the early detection of HCC. Serum samples were obtained prior to treatment from 58 consecutive patients with early or relatively early hepatitis C virus (HCV)-related HCC and 137 patients with HCV-related liver cirrhosis without evidence of HCC. Enzyme immunoassays were used to measure serum levels of autoantibodies. Serum levels of anti-Ku86 antibodies were significantly elevated in HCC patients compared to those in liver cirrhosis patients (0.41±0.28 vs. 0.18±0.08Abs at 450nm, P<0001). Setting the cut-off level to give 90% specificity, anti-Ku86 was positive in 60.7% of stage I solitary tumor <2cm in diameter, whereas the sensitivities of alpha-fetoprotein (AFP) and protein induced by vitamin K absence or antagonist II (PIVKA-II) were 17.8% and 21.4%, respectively. The results of ROC analyses indicated the better performance of anti-Ku86 for early detection of HCC. Serum anti-Ku86 levels decreased after surgical resection of the tumors in the 12 HCC cases tested, Elevation of anti-Ku86 in solid tumors other than liver was minimal. Serum anti-Ku86 is a potential biomarker for early detection of HCV-related HCC. Further studies in a larger number of HCC patients with various etiologies are needed to further evaluate the diagnostic and pathophysiological roles of elevation of serum anti-Ku86 in early HCC.

Development of a sandwich ELISA for the 5.9-kDa fibrinogen alpha C chain fragment detected by serum proteome analysis.

PURPOSE: We previously identified novel biomarker candidates in heavy consumers of alcohol using serum proteome analysis. Among several candidates, a 5.9 kDa peptide identified as a fragment of the fibrinogen alpha C chain (FIC5.9) was the most promising. To move FIC5.9 toward potential diagnostic use, we developed an enzyme immunoassay that enables measurement of serum FIC5.9 levels. EXPERIMENTAL DESIGN: Two monoclonal antibodies specific to the N and C-termini of the 5.9-kDa peptide were used to develop a FIC5.9 sandwich ELISA. The assay was evaluated by comparing the results with those obtained by the stable isotope-labeled dilution mass spectrometry (SID-MS) using the ClinProt™ system. RESULTS: The ELISA results correlated with the SID-MS findings (slope=0.795, intercept=-0.011, r(2) =0.908) and the performance of the ELISA was satisfactory in terms of recovery (98.5-103.0%) and within-run (1.4-4.7%) and between-day (2.8-8.4%) reproducibility. The assay was capable of detecting changes in FIC5.9 during abstinence from drinking in patients with alcohol dependency (p<0.0001). CONCLUSIONS AND CLINICAL RELEVANCE: The sandwich ELISA developed in this study will be useful for validation of the diagnostic significance of serum FIC5.9 levels in various pathological conditions, including alcoholism.

Adsorption of urinary proteins on the conventionally used urine collection tubes: possible effects on urinary proteome analysis and prevention of the adsorption by polymer coating.

One possible factor determining recovery of trace amount of protein biomarker candidates during proteome analyses could be adsorption on urine tubes. This issue, however, has not been well addressed so far. Recently, a new technical device of surface coating by poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)) (poly(MPC-co-BMA)) has been developed mainly to prevent the adsorption of plasma proteins. We assessed whether conventionally used urine tubes adsorb trace amount of urinary proteins and, if any, whether the surface coating by poly(MPC-co-BMA) can minimize the adsorption. Proteinuric urine samples were kept in poly(MPC-co-BMA)-coated and noncoated urine tubes for 15 min and possibly adsorbed proteins and/or peptides onto urine tubes were analyzed by SDS-PAGE, 2-DE, and the MALDI-TOF MS. It was found that a number of proteins and/or peptides adsorb on the conventionally used urine tubes and that surface coating by poly(MPC-co-BMA) can minimize the adsorption without any significant effects on routine urinalysis test results. Although it remains to be clarified to what extent the protein adsorption can modify the results of urinary proteome analyses, one has to consider this possible adsorption of urinary proteins when searching for trace amounts of protein biomarkers in urine.

Tartrate-resistant acid phosphatase 5a and 5b contain distinct sugar moieties.

Tartrate-resistant acid phosphatase (TRAP) consists of 2 structurally related isoforms, 5a and 5b, and the latter has been characterized as containing a sugar moiety devoid of sialic acid(s). We provide evidence of a greater difference between the sugar moieties of the two isoforms than the presence of sialic acid(s) in TRAP 5a. TRAP 5a and 5b were highly purified from human blood and femur extracts, respectively, and after purifying the TRAPs by successive chromatography on a series of lectin affinity columns, we used the chromatographs obtained to estimate the composition of their sugar chain structure and found that both TRAP 5a and 5b had contained the heterogenous sugar chains. More specifically, TRAP 5a mainly contained a high-mannose-type sugar chain, while TRAP 5b had multi-antennary complex-type sugar chain. These results indicate that the differences between TRAP 5a and 5b consist not only of a difference in modification by sialic acid but differences in other sugar chain structures. These differences are involved in an extraordinary enzyme maturation process since the carbohydrate chains cover the repressive loop where the proteolytic site is located.

Generation of a monoclonal antibody specific for tissue-nonspecific alkaline phosphatase and its use in a clinical diagnostic study.

Tissue-nonspecific alkaline phosphatase (TNSALP) in serum comprises liver alkaline phosphatase (liver-ALP) and bone alkaline phosphatase (bone-ALP). Liver-ALP is a marker of liver disease; thus a specific method for its measurement would be useful. Measurement of ALP by electrophoresis is difficult, although all of the isozymes can be assessed simultaneously. Total ALP can also be measured by automated analyzer, but it is difficult to determine the cause of a high ALP value because bone-, intestine-, placenta-, and tumor-ALP are measured together. Thus, anti-TNSALP monoclonal antibodies that can resolve these problems are needed. Here we have generated an anti-TNSALP monoclonal antibody, 3-29-3R. This clone has specificity to liver-ALP rather than to bone-ALP. In electrophoresis, 3-29-3R reacted with TNSALP and shifted the bands. The use of 3-29-3R enabled easy interpretation of the results. Furthermore, we tested 3-29-3R by developing an immunocapture enzymatic assay (IEA). Preliminary results of the IEA show that this method is effective for measurement of liver-ALP. Thus, the monoclonal antibody that we have established may be a useful tool for clinical diagnosis.

Development of an ELISA method for detecting immune complexes between tissue-nonspecific alkaline phosphatase and immunoglobulin G.

A convenient method for measuring immune complexes between tissue-nonspecific alkaline phosphatase (TNSALP) and immunoglobulin G (IgG) (i.e., TNSALP-IgG) would be highly useful for routine analyses. Here, we identified a surface-active agent that would dissolve membrane but not dissociate TNSALP-IgG complexes. Next, we developed an enzyme-linked immunosorbent assay (ELISA) method for detecting TNSALP-IgG complexes with two monoclonal antibodies (MoAbs): 3-29-3R was coated on assay plates and captured TNSALP-IgG from a specimen; an horseradish peroxidase (HRP)-conjugated anti-human IgG1 then reacted with captured TNSALP-IgG to form an "immunocomplex sandwich." The immunocomplex was detected via the absorbance of an HRP substrate, resulting in a semiquantitative assay. The mean absorbance of 0.195 (n=5) measured in sera from healthy donors was designated as an arbitrary unit (AU/mL) of TNSALP-IgG concentration. The ELISA values of patient sera known to contain TNSALP-IgG complexes were greater than those of normal sera (normal, 1.86 plusmn; 0.61; patient, 9.30 plusmn; 5.44), and these data were confirmed by electrophoresis. Thus, the ELISA could detect TNSALP-IgG complexes. The intraassay coefficient of variation (CV) was within 7.4% and analytical recovery was excellent. There was no significant interference from hemolytic, lipemic, or icteric serum. In summary, an ELISA using 3-29-3R MoAb and HRP-conjugated anti-human IgG1 constitutes a reliable and convenient method for the semiquantitative detection of TNSALP-IgG complexes in human serum.

[The mechanism of TRACP 5b maturation].

Serum tartrate resistant acid phosphatase 5b (TRACP 5b) is an isozyme of osteoclast origin. Indeed, measurement of TRACP 5b activity is used as an index of osteoclast activity. However, the precise mechanism of TRACP 5b maturation is unclear. This study aimed to clarify the mechanism of generation of TRACP 5b. We used a highly sensitive fiber-type DNA chip to investigate the mechanism of generation of TRACP 5b at the genetic level. Genes derived from three related cell types (monocytes, macrophages and osteoclasts) were compared. In addition, at the protein level, posttranscriptional modification was tested by Western blotting using an antiserum specific for the flexible loop region of TRACP 5. Our DNA chip study shows that genes implicated in oligosaccharide construction do not show significant differences in expression levels between the cell types under investigation. Strongly expressed Cathepsin K was observed in osteoclasts. Western blotting demonstrated that TRACP undergoes unique partial degradation during bone resorption, such that serum TRACP 5b lacks the flexible loop found in TRACP 5a. In conclusion, TRACP 5b generated by a specific posttranscriptional modification pathway undergoes partial digestion in resorption lacunae or inside osteoclasts. Serum TRACP 5b lacking the flexible loop differs from TRACP 5a in terms of optimum pH, isoelectric point, sugar chain and antigenicity. The measurement of TRACP 5b could therefore be of great use for monitoring of osteoporosis, rheumatoid arthritis and bone metastasis.

Development of a novel fragments absorbed immunocapture enzyme assay system for tartrate-resistant acid phosphatase 5b.

BACKGROUND: Osteoclastic activity is mainly assessed by measuring urinary markers. To correct for differences in renal clearance, the levels of urinary markers are usually corrected by the urine creatinine concentration. Therefore, alternative serum markers to evaluate osteoclastic activity are required. We developed a novel system for the determination of serum tartrate-resistant acid phosphatase 5b (TRACP5b) activity to evaluate osteoclastic activity. METHODS: Two unique monoclonal antibodies were generated and the specificity was tested using a surface enhanced laser desorption/ionization time-of-flight mass spectroscopy (SELDI TOF-MS). A novel fragments absorbed immunocapture enzymatic assay (FAICEA) method was developed using 2 monoclonal antibodies. RESULTS: FAICEA gave a sensitivity 0.1 U/l, linearity of 0.1-28 U/l, recovery 92-103%, inter-assay CV 2.95% and intra-assay CV 2.15%. Unlike other TRACP5b assay systems, FAICEA avoided interference from TRACP 5a. CONCLUSIONS: According to the FAICEA, postmenopausal women had higher TRACP5b concentrations than younger women. The results show that TRACP5b is a novel bone resorption marker in serum.

Development and characterization of novel monoclonal antibodies against tartrate-resistant acid phosphatase 5.

Serum band 5 tartrate-resistant acid phosphatase (TRACP 5; EC 3.1.3.2) is a glycoprotein that exists as two very similar isoforms, TRACP 5a and TRACP 5b. The similarity of these two isoforms has made it difficult to establish monoclonal antibodies specific for either isoform. We report here the development of a monoclonal antibody with high specificity for TRACP 5b. We prepared TRACP 5b antigens from four sources: TRACP 5b purified from human bone, recombinant TRACP 5 from Escherichia coli, recombinant TRACP 5 from insect cells, and a synthetic TRACP 5b peptide. Thirty-seven mice were each immunized with 1 of the 4 different TRACP antigens to generate 473 antibody-producing clones. Three of these clones, Trk27, Trk49, and Trk62, reacted with TRACP 5b. These three clones were all established from mice exposed to native bone TRACP 5b antigen. In fact, none of the other antigens were able to generate anti-TRACP 5b monoclonal antibodies in mice. Furthermore, Trk62 interacted more strongly with TRACP 5b than with TRACP 5a. These results suggested that although recombinant proteins can be effective antigens, the native TRACP 5 protein might be more effective at generating monoclonal antibodies of greater specificity due to its more faithful representation of the native three-dimensional structure of the protein.