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PubMed2005

A method for fabricating uni-dsDNA microarray chip for analyzing DNA-binding proteins.

This paper describes an approach for preparing unimolecular double-stranded DNA (uni-dsDNA) microarray chip. In this method, the various target oligonucleotides containing a reverse complementary sequence at 5' end were firstly annealed to a same universal oligonucleotide with amino group at 5' end and immobilized on aldehyde-derivatized glass slide. An on-chip DNA polymerization reaction was then performed to elongate the universal oligonucleotides. After a denaturation and a followed intra-strand annealing, a hairpin structure was formed at the free 3' end of the immobilized oligonucleotides. Finally, another on-chip DNA polymerization was done to synthesize the uni-dsDNA microarray. Combining with a PCR amplification of chemically synthesized target oligonucleotides, this method was much cost-effective for production of the uni-dsDNA microarray. The uni-dsDNA microarray was verified applicable for detecting the presence and monitoring the DNA-binding activity of the sequence-specific DNA-binding proteins.

Cost-Benefit Analysis↗
PubMed2003

Biopsy of a biopsy: validation of immunoprofiling in gastric cancer biopsy tissue microarrays.

AIMS: Tissue microarrays offer an efficient way of examining a large number of tumour cases on a single glass slide. A major concern, however, is tumour heterogeneity. Also, the use of tissue microarrays in biopsy material is unexplored. The purpose of the present study was to assess the possibility and validity of arraying three 0.6-mm cores per case in endoscopic gastric cancer biopsies for immunophenotyping. METHODS AND RESULTS: Thirty-eight cases were studied with immunohistochemical staining for p53, CD44v6 and vascular endothelial growth factor. Full tissue sections were compared with triple core-tissue microarrays. Thirty-six cases contained three cores with tumour, one case contained two cores with tumour and one case contained only a single core with viable tumour and was excluded. Three further cores had been lost from three separate cases on the sections for immunohistochemistry. kappa values for whole-sections versus tissue microarrays ranged between 0.77 and 0.94. p53 immunohistochemical staining (interpretation as + or -) yielded the best result with only 1/37 mismatches, whereas CD44v6 (graded both for intensity and extent) showed 3/37 mismatches. The small depth of tissue in cores from biopsies necessitates all cores being arrayed flush with the face of the recipient wax block for maximizing the number of sections available. Compared with the first section over 30 additional 4- micro m sections were available before the first case (with one core left) had to be excluded and 80 sections before half the tissue cores were lost. CONCLUSIONS: It is impracticable to array more than 120-150 cores per block. Tissue microarray with three cores per case is feasible and valid for studying biopsy material.

Adult↗
PubMed2004

Expression of RON Proto-oncogene in Renal Oncocytoma and Chromophobe Renal Cell Carcinoma.

Recently, it was reported that RON proto-oncogene, encoding a receptor tyrosine kinase, was strongly expressed in renal oncocytomas but not in any renal cell carcinomas, including 5 chromophobe renal cell carcinomas, which morphologically resemble oncocytomas. To determine its diagnostic value, we studied Ron protein expression by immunohistochemistry in a larger number of renal cell neoplasms with emphasis on chromophobe renal cell carcinomas. Tissue microarrays containing 141 renal cell neoplasms, including 55 oncocytomas and 52 chromophobe renal cell carcinomas, were constructed. In addition, conventional sections from 15 cases of oncocytoma and 5 cases of chromophobe renal cell carcinoma were analyzed. Immunohistochemistry was carried out with a monoclonal mouse anti-human Ron-alpha antibody. Staining intensity was scored on a 0 to 3 scale. Ninety-nine percent of oncocytomas (69 of 70) and 96% of chromophobe renal cell carcinomas (55 of 57) showed moderate to strong, diffuse cytoplasmic Ron immunoreactivity with intensities > or =2, while only 17% of other renal cell carcinoma subtypes stained with intensities > or =2. Our study indicates that Ron immunostaining cannot be used to distinguish oncocytoma from chromophobe renal cell carcinoma.

Adenoma, Oxyphilic↗
PubMed2006

The potential allergenicity of two 2S albumins from soybean (Glycine max): a protein microarray approach.

BACKGROUND: The 2S albumins are a group of storage proteins that occur widely in seeds of dicotyledonous plants. The widespread distribution and stability to digestion of allergenic 2S albumins raise the question of why some members of this family present in important food sources, such as soybean, are not regarded as major allergens. METHODS: The pepsinolytic stability of two 2S albumins from soybean seed was determined using simulated gastric fluid. Using a new protein microarray system, IgE binding to these soybean 2S albumins was studied with the sera from 23 European individuals allergic to soybean. In order to validate the microarray result, two of the sera were selected and further tested using the micro-ELISA and UniCAP system. RESULTS: Both albumins exhibited high stability to digestion similar to other allergenic members of the 2S albumin, trypsin/amylase inhibitor and lipid transfer protein superfamily. None of the patients was found to have IgE specific to soybean 2S albumins by the microarray system, and this result was in agreement with the results from the micro-ELISA and UniCAP system. CONCLUSIONS: The results from microarray, micro-ELISA and UniCAP system suggested that the 2S albumins from soybean are not major allergens within the patient population analyzed.

Albumins↗
PubMed2005

[Effect of interleukin-8 in cell invasion and proliferation of human breast cancer].

OBJECTIVE: To identify the effect of interleukin-8 in cell progression and invasion of human breast cancer. METHODS: Human cytokine antibody arrays were applied to screen a panel of cytokine expression from 11 human breast cancer cell lines, and the mechanism of identified key factors involved in breast cancer progression was studied. RESULTS: Profiling of cytokine expression showed the expression of interleukin-8 was related to estrogen receptor status, metastasis and vimentin status in the 11 human breast cancer cell lines. Elevated expression of interleukin-8 in breast cancer cells had positive correlation with breast cancer invasion. Neutralization of antibody against interleukin-8 specifically blocked interleukin-8-mediated cell invasion. However, anti-interleukin-8 antibody did not influence the proliferation of breast cancer cells. CONCLUSION: Interleukin-8 may be the key factor involved in human breast cancer progression and invasion, and play an important role in cell invasion of breast cancer.

Breast Neoplasms↗
PubMed2002

Characterization of a human sphingosine-1-phosphate receptor gene (S1P5) and its differential expression in LGL leukemia.

Large granular lymphocyte (LGL) leukemia is a lymphoproliferative disorder often associated with autoimmune disease. A central feature of this disease is dysregulation of apoptosis. In order to identify differentially expressed genes in LGL leukemia, microarray analysis was performed. We found many differentially expressed genes including several expression sequence tags (ESTs). As a systematic study, we selected one up-regulated EST (GenBank Accession number N47089) and further investigated. An LGL leukemia library was screened using this EST as a probe and a full-length sequence for a novel gene was identified. The deduced amino acid sequence revealed that the novel gene encodes a G-protein-coupled receptor gene that exhibits 86% identity with rat sphingosine-1-phosphate receptor (edg-8/nrg-1). This gene is present in brain, spleen, and peripheral blood mononuclear cells (PBMC) and is overexpressed in leukemic LGL.

Amino Acid Sequence↗
PubMed2006

Microarray: a versatile platform for high-throughput functional proteomics.

The advent of microarray technologies has dramatically accelerated the functional study of proteins, including enzymes (catalomics) in a proteome. Herein, we review recent advances and exciting new developments of microarrays in high-throughput functional proteomics.

Oligonucleotide Array Sequence Analysis↗
PubMed2004

Gene expression profiles of periodontal ligament cells treated with enamel matrix proteins in vitro: analysis using cDNA arrays.

BACKGROUND: A number of procedures have been used to achieve periodontal regeneration. Recently, enamel matrix derivative (EMD) has been the subject of significant basic and clinical investigations. The precise molecular events involved in EMD modulation of periodontal wound healing are not completely understood; however, cDNA microarray technology may enable rapid and accurate examination of EMD-mediated changes in gene expression in periodontal ligament (PDL) cells in vitro. The present study was undertaken to explore the selective effects of EMD on the activities of 268 cytokine, growth factor, and receptor genes in PDL. METHODS: PDL cells were cultured in the absence and presence of EMD at a concentration of 100 microg/ml for 4 days. RNA was extracted and used to generate labeled cDNA probes. These were hybridized to cDNA arrays comprising 268 genes and exposed to x-ray films. Autoradiographs were digitized and analyzed. RESULTS: Forty-six percent (125 of 268) of the tested genes were found to be expressed by the PDL cells. Of these 125 genes, 38 were differentially expressed by PDL cells which had been cultured in the presence of EMD. Among the 38, 12 were found to be downregulated, notably mostly inflammatory genes, whereas 26 genes demonstrated upregulation, many of these coding for growth factors and growth factor receptors. CONCLUSIONS: The present study has shown that EMD down-regulates the expression of genes involved in the early inflammatory phases of wound healing while simultaneously upregulating genes encoding growth and repair-promoting molecules. This may partly explain the apparent efficacy of EMD application in periodontal regeneration.

Cell Culture Techniques↗
PubMed2006

Prognostic significance of expression patterns of c-erbB-2, p53, p16INK4A, p27KIP1, cyclin D1 and epidermal growth factor receptor in oesophageal adenocarcinoma: a tissue microarray study.

AIMS: To correlate immunohistochemical expression patterns and prognosis in oesophageal adenocarcinoma. METHODS: The expression of c-erbB-2, p53, p16INK4A, p27KIP1, cyclin D1 and epidermal growth factor receptor (EGFR) was studied in a series of 137 primarily resected oesophageal adenocarcinoma samples. The expression analysis on protein level was performed on routine paraffin wax-embedded material, with immunohistochemical staining of the samples, assembled on a tissue microarray. The results were correlated with clinicopathological features (pT, pN and G) and survival. RESULTS: 22 (16%) tumours showed an overexpression of the c-erbB-2 oncoprotein. Expression of EGFR was observed in 72 (55%) cases, accumulation of p53 in 68 (52%) cases and of cyclin D1 in 102 (77%) cases. Loss of p16INK4A expression was observed in 101 (76%) cases and low expression of p27KIP1 in 91 (71%) cases. Expression of these proteins did not correlate with tumour stage, grade, Lauren's or World Health Organization classification or lymph node status. On univariate survival analysis, more advanced tumour stage (p = 0.002), lymph node involvement (p = 0.003), high tumour grade (p = 0.017) and lack of EGFR expression (p = 0.034) were found to be associated with poorer survival. On multiple regression analysis, only tumour stage (p = 0.03) and lymph node involvement (p = 0.004) were shown to have an association with the survival of the patient. CONCLUSION: The immunohistochemical expression of c-erbB-2 oncoprotein, cylin D1, p16INK4A, p27KIP1, p53 and EGFR in most oesophageal adenocarcinomas suggests their implication in the pathogenesis of this entity. None of the molecular markers assessed, however, was of prognostic value. Identification of any marker superior to or even approaching the prognostic value of conventional histopathological markers (pT and pN) was therefore not possible.

Adenocarcinoma↗
PubMed2005

Comparative methods for multiplex analysis of cytokine protein expression in plasma of lipopolysaccharide-treated mice.

Changes in circulating cytokines might serve as predictors of compound-evoked inflammatory responses. CD-1 mice were treated with lipopolysaccharide (LPS; 0.2 ml of 0.25 mg/ml, intraperitoneal) for subsequent expression measurement of plasma cytokine protein expression at 24-h post-treatment using multiple antibody Western blot, and at both 2-h and 24-h post-treatment using antibody array and suspension bead array. Antibody array provided a semi-qualitative assessment and suggested significantly increased expression of GCSF at 2-h post-treatment and GCSF, IL-6, IL-12, MCP-1, MCP-5, RANTES and sTNFR1 at 24-h post-treatment. Densitometric analysis of multiple antibody Western blots provided a semi-quantitative assessment and indicated significantly increased expression of IL-6, IL-12, IL-17, GCSF, eotaxin, and MCP-2 at 24-h post-treatment. The suspension bead array yielded statistically significant cytokine protein expression increases for IL-6, IL-10, IFNgamma and TNFalpha at both 2-h and 24-h post-treatments, while significant expression at 24-h post-treatment only was noted for IL-1beta, IL-5, IL-12 and GM-CSF. Suspension bead array provided the greatest range of detection, revealing subtle increased expression of GM-CSF, IL-1beta, IL-5, IL-10, TNFalpha and IFNgamma at 24-h post-treatment, not detected by antibody array or multiple antibody Western blot. Suspension bead array proved to be the best method for detection of LPS-evoked changes in plasma cytokine levels.

Animals↗
PubMed2005

Influences of blood sample processing on low-molecular-weight proteome identified by surface-enhanced laser desorption/ionization mass spectrometry.

BACKGROUND: Profiling approaches in proteomics, such as surface-enhanced laser desorption/ionization (SELDI) mass spectrometry, are used in disease marker discovery. The aim of this study was to investigate the potential influence of selected preanalytical factors on the results obtained. METHODS: Plasma samples anticoagulated with EDTA, citrate, or heparin, and serum samples from healthy volunteers were profiled by SELDI on CM10, immobilized metal affinity capture (IMAC) array with copper, and H50 chip surfaces. Using linear mixed-effects models, we examined the influence of elapsed time between venipuncture and sample separation (immediate to 24 h) and the type of serum tube used (Greiner Vacuette activator, gel serum separator, or plain tubes). We analyzed purified platelets, as well as platelet-poor and platelet-rich plasma samples treated with calcium and/or thrombin to determine the platelet contribution, directly or via the clotting process, to the profiles generated. We then used cluster analysis to identify samples with similar peak profiles. RESULTS: Different plasma types and sera could be distinguished on the basis of cluster analyses of their spectral profiles. Elapsed time between venipuncture and separation of plasma and serum from blood samples altered the profiles obtained, particularly for serum samples and particularly on IMAC chips. The type of serum collection tube also affected the profiles because of differences in clotting time. In vitro manipulation of platelets revealed that specific peaks in IMAC profiles of serum appeared to be derived directly from platelets. Several other peaks, including some of those exhibiting time-dependent changes, arose during the clotting process. CONCLUSION: Preanalytical variables, such as sample handling, can markedly influence results.

Adult↗
PubMed2003

Tissue microarray analysis reveals prognostic significance of syndecan-1 expression in prostate cancer.

BACKGROUND: Tissue microarrays (TMA) have recently emerged as powerful tools to rapidly analyze the clinical significance of new molecular markers in human tumors. Here, we have tested several molecular markers on a prostate TMA containing 637 different specimens. METHODS: The specimens were from 551 patients with prostate cancer and long-term follow-up information on progression (median 5.3 years), tumor-specific and overall survival (median 5.9 years). Eighty-six specimens from benign prostatic hyperplasia were included as controls. Expression of Ki67, Bcl-2, p53, CD-10 (neutral endopeptidase), and syndecan-1 (CD-138) was analyzed by immunohistochemistry. RESULTS: Gleason grade and Ki67 Labeling Index (LI) were independent predictors of early recurrence and poor survival. Bcl-2 predicted early recurrence, whereas p53 was associated with poor survival. Syndecan-1 overexpression also predicted early recurrence and was significantly associated with tumor specific survival, high Gleason grade, Ki67 LI, and Bcl-2 overexpression. Neoadjuvant hormonal therapy was associated with overexpression of Bcl-2 and inhibition of Ki67 LI and CD-10, but did not affect the expression of the remaining markers. CONCLUSIONS: The results of this TMA study confirm a dominant prognostic significance of Gleason grading and Ki67 LI in prostate cancer, as compared to a less pronounced role of Bcl-2, and p53. We identified syndecan-1 as a new prognostic factor and provide evidence for an androgen-dependent regulation of CD-10 expression.

Aged↗
PubMed2005

Proteomic profiling of mature CD10+ B-cell lymphomas.

Proteomic profiling with protein-chip technology has been used successfully to discover biomarkers with potential clinical usefulness in several cancer types. Little proteomic study has been done in B-cell lymphomas. We determined whether the expression of a set of proteins by protein-chip technology coupled with new informatics tools could be used to build a model to molecularly classify B-cell lymphoma subgroups. We used surface-enhanced laser desorption/ionization time-of-flight mass spectrometry to analyze 18 CD10+ B-cell lymphomas, including 6 grade 1 (G1) follicular lymphomas (FLs), 7 grade 3 (G3) FLs, and 5 Burkitt lymphomas. We used 7 reactive follicular hyperplasia cases as a control group. By using SAX2 ProteinChip arrays (Ciphergen Biosystems, Fremont, CA), we found a unique protein expression profile for each type of lesion. Two-way hierarchical clustering analysis of these protein expression profiles differentiated reactive follicular hyperplasia, FL, and Burkitt lymphoma, with 5 major clusters of differentially expressed protein peaks. In addition, we identified histone H4 as a potential differentially expressed protein marker that seems to distinguish G1 from G3 FL. To our knowledge, this is the first proteomic study using protein-chip technology for molecular classification of B-cell lymphoma subtypes with clinical samples.

Biomarkers, Tumor↗
PubMed2004

Dysregulated expression of S100A11 (calgizzarin) in prostate cancer and precursor lesions.

S100A11 is a calcium-binding protein implicated in a variety of biologic functions such as proliferation and differentiation as well as in cancer. To further understand its role in prostate cancer, we performed immunohistochemistry on a series of benign, premalignant, malignant and metastatic prostate cancer tissues in addition to prostate cancer derived cell lines. In benign prostatic hyperplasia (n=30) and benign tissue adjacent to adenocarcinoma (n=54), S100A11 expression was significantly higher in basal cells compared with in luminal cells (P <0.001). A complete absence of staining was seen in 4/14 (29%) lesions of prostatic intraepithelial neoplasia. The majority of tumors, 39/54 (72%), showed significant overexpression of S100A11 compared with the luminal cells of adjacent benign epithelium (P <0.001), whereas 14/54 (26%) of cases showed an absence of staining. All 4 cases of metastatic cancer showed intense to moderate expression. There was a significant association between S100A11 expression and high pathologic stage (pT3b) versus lower stages (pT2a-3a; P=0.027), but not with tumor Gleason score or prostate-specific antigen levels. LNCaP, PC3, and Du145 cancer cell lines showed intense to moderate S100A11 expression by immunochemistry, which was confirmed by Western blotting and reverse-transcription polymerase chain reaction. A survey of 14 other types of normal tissues arranged on a tissue microarray showed that S100A11 is widely expressed amongst epithelia. Our finding of frequent dysregulated expression of S100A11 in cancer and precursor lesions, together with an association with high histological stage, suggests that S100A11 may be involved in prostate cancer development and progression.

Adenocarcinoma↗
PubMed2026

Immunoproteomic Profiling of Autoantibodies and Antibodies against Infectious Agents in Autoimmune Diseases.

Prior research investigated limited antibody sets within individual autoimmune diseases. Using the Nucleic-Acid Programmable Protein Array platform, we measured antibodies against 280 human, 40 viral, and 15 bacterial antigens in serum from 237 patients with 8 autoimmune diseases, including autoimmune gastritis (AG), autoimmune thyroiditis (AT), celiac disease (CD), idiopathic inflammatory myopathies (IIM), type 1 diabetes mellitus (T1D), rheumatoid arthritis (RA), Sj&#xf6;gren's disease (SjD), and systemic lupus erythematosus (SLE), and 112 controls. Candidate antibodies were identified by combining Firth logistic regression and machine learning. We identified disease-specific antibodies, ranging from 3 in IIM to 13 in SLE for IgG and 1 in CD to 13 in AG for IgA. Additionally, 63 IgG and 44 IgA antibodies were shared across two or more diseases. Notably, two IgG autoantibodies overlapped in up to five diseases: directed against STNM4 (SLE, SjD, T1D, CD, and RA) and TRIM21 (SLE, SjD, IIM, CD, and RA); and three IgA antibodies in up to seven diseases: directed against H1N1 Influenza A virus NP (IIM, SjD, AG, T1D, CD, RA, and AT) and Coxsackievirus B3MK012537 and Enterovirus C PVgp1 (IIM, SjD, AG, T1D, CD, SLE, and RA). These findings underscore the potential of antibody profiling in autoimmune disease characterization and biomarker discovery.

Humans↗