A high-sensitivity ELISA for detection of human FGF18 in culture supernatants from tumor cell lines.

Fibroblast growth factor 18 (FGF18) is elevated in several human cancers, such as gastrointestinal and ovarian cancers, and stimulates the proliferation of tumor cells. This suggests that FGF18 may be a promising candidate biomarker in cancer patients. However, the lack of a high-sensitivity enzyme-linked immunosorbent assay (ELISA) does not permit testing of this possibility. In this study, we generated monoclonal antibodies against human FGF18 and developed a high-sensitivity ELISA to measure human FGF18 at concentrations as low as 10 pg/mL. Of the eight tumor cell lines investigated, we detected human FGF18 in culture supernatants from four tumor cell lines, including HeLa, OVCAR-3, BxPC-3, and SW620 cells, albeit the production levels were relatively low in the latter two cell lines. Moreover, the in-house ELISA could detect murine FGF18 in sera from mice overexpressing murine Fgf18 in hepatocytes, although the sensitivity in detecting murine FGF18 was relatively low. This FGF18 ELISA could be a valuable tool to validate FGF18 as a potential biomarker for cancer patients and to test the contribution of FGF18 for various disease models invivo and in vitro.

Structural basis for antigen recognition by methylated lysine-specific antibodies.

Proteins are modulated by a variety of posttranslational modifications including methylation. Despite its importance, the majority of protein methylation modifications discovered by mass spectrometric analyses are functionally uncharacterized, partly owing to the difficulty in obtaining reliable methylsite-specific antibodies. To elucidate how functional methylsite-specific antibodies recognize the antigens and lead to the development of a novel method to create such antibodies, we use an immunized library paired with phage display to create rabbit monoclonal antibodies recognizing trimethylated Lys260 of MAP3K2 as a representative substrate. We isolated several methylsite-specific antibodies that contained unique complementarity determining region sequence. We characterized the mode of antigen recognition by each of these antibodies using structural and biophysical analyses, revealing the molecular details, such as binding affinity toward methylated/nonmethylated antigens and structural motif that is responsible for recognition of the methylated lysine residue, by which each antibody recognized the target antigen. In addition, the comparison with the results of Western blotting analysis suggests a critical antigen recognition mode to generate cross-reactivity to protein and peptide antigen of the antibodies. Computational simulations effectively recapitulated our biophysical data, capturing the antibodies of differing affinity and specificity. Our exhaustive characterization provides molecular architectures of functional methylsite-specific antibodies and thus should contribute to the development of a general method to generate functional methylsite-specific antibodies by de novo design.

Impact of isolated tumor cells in sentinel lymph nodes detected by immunohistochemical staining.

AIM: Isolated tumor cells (ITCs) in lymph nodes are defined histologically as node-negative. The clinical impact of ITCs in sentinel lymph nodes (SLNs) remains unclear. We report the prognosis of breast cancer patients with ITC-positive SLNs detected by immunohistochemical staining. PATIENTS AND METHODS: One hundred and sixty-five breast cancer patients with histologically negative SLNs were seen between January 1998 and December 2000. In 69 patients, sentinel node biopsy (SNB) was immediately followed by axillary lymph node dissection, and 96 had undergone SNB alone. Permanent sections of 301 SLNs were re-examined after hematoxylin-eosin staining and cytokeratin 19 immunohistochemical staining. RESULTS: ITCs were found in 18 SLNs of 17 patients and a micrometastasis was found in one SLN of one patient. As of November 2005, only one patient with ITCs in one SLN had supraclavicular lymph node recurrence. In contrast, 18 of the 147 patients with negative SLNs had tumor recurrence. Surgical management of the axilla had no influence on recurrence-free survival in all of the patients. CONCLUSION: This study shows that breast cancer patients with ITC-positive SLNs should be clinically managed as node-negative patients.

Omental cyst: report of a case.

We report the case of an omental cyst, a rare type of abdominal cystic lesion that is difficult to diagnose preoperatively. A 43-year-old man with no clinical symptoms was admitted to our hospital for investigation of an abdominal cyst detected by ultrasonography (US). We performed diagnostic examinations including US, computed tomography, and magnetic resonance imaging. An omental cyst was diagnosed because of its position and connection to the surrounding tissues. Pathological examination of the surgical specimen revealed endothelial cells on its internal wall and colonies of lymphocytes, confirming a diagnosis of lymphangioma, which is the most common type of omental cyst.

Measuring respiration of cultured cell with oxygen electrode as a metabolic indicator for drug screening.

New trend in methods for assessing pharmacological action to bacteria and cell is to measure their metabolic activities induced, while the conventional methods used population growth. We focused on respiration volume as an indicator of cell metabolism, and developed inexpensive disposable oxygen electrode sensor and multi-channel dissolved oxygen meters (DOX-10 and DOX-96KB). Using these instruments, cytotoxicity was measured for 48 hrs and the method showed superior features to conventional methods in its handiness of one step assay, and excellent adaptability to automated systems. Total usability of this oxygen electrode method is being evaluated in bacterial drug susceptibility test, anticancer drug susceptibility test, and alternatives to animal experiment.

Induction of murine gamma delta T cells cytotoxic for xenogeneic rat cells.

C57BL/6 mice deprived or nondeprived of CD4+ and CD8+ T cells by mAbs were challenged with a rat T cell line, W7TM-1. Spleen cells obtained from CD4- and CD8-depleted animals rejecting W7TM-1 were examined by cytofluorometry, which demonstrated the presence of highly increased gamma delta type CD4-CD8- T cell population (30 to 50% of entire T cells). In vitro sensitization of these spleen cells with W7TM-1 generated a mixture of gamma delta and alpha beta type CD4-CD8- CTL for W7TM-1. Repeated stimulation of these cells with W7TM-1 resulted in a gamma delta-type T cell population with more than 95% purity by day 45. In contrast, alpha beta type CD8+ CTL for W7TM-1 were induced from mice nondeprived of CD4+ and CD8+ T cells. Both gamma delta-type CD4-CD8- CTL and alpha beta type CD8+ CTL were cytotoxic for rat cells in a species-specific manner. However, only reactivity of gamma delta type CD4-CD8- CTL, but not alpha beta type CTL, was inhibited by a mAb for TCR-gamma delta. The gamma delta type CD4-CD8- CTL clones were also prepared from spleen cells derived from CD4- and CD8-depleted mice. They were also reactive for xenogeneic cells in a species-specific manner. Spleen cells derived from CD4- and CD8-depleted mice rejecting the whole-layer rat skin grafts were in vitro sensitized with rat spleen cells, which also generated gamma delta type CD4-CD8- CTL specific for rat cells. V gamma 1 was detected as a major V gamma gene expressed in this gamma delta population by reverse transcriptase-PCR. Cytotoxicity for xenogeneic cells may represent one of major function of gamma delta T cells.