Aberrant hepatic trafficking of gut-derived T cells is not specific to primary sclerosing cholangitis.

BACKGROUND AND AIMS: The "gut homing" hypothesis suggests the pathogenesis of primary sclerosing cholangitis (PSC) is driven by aberrant hepatic expression of gut adhesion molecules and subsequent recruitment of gut-derived T cells to the liver. However, inconsistencies lie within this theory including an absence of investigations and comparisons with other chronic liver diseases (CLD). Here, we examine "the gut homing theory" in patients with PSC with associated inflammatory bowel disease (PSC-IBD) and across multiple inflammatory liver diseases. APPROACH AND RESULTS: Expression of MAdCAM-1, CCL25, and E-Cadherin were assessed histologically and using RT-PCR on explanted liver tissue from patients with CLD undergoing OLT and in normal liver. Liver mononuclear cells were isolated from explanted tissue samples and the expression of gut homing integrins and cytokines on hepatic infiltrating gut-derived T cells was assessed using flow cytometry. Hepatic expression of MAdCAM-1, CCL25 and E-Cadherin was up-regulated in all CLDs compared with normal liver. There were no differences between disease groups. Frequencies of α4ß7, αEß7, CCR9, and GPR15 expressing hepatic T cells was increased in PSC-IBD, but also in CLD controls, compared with normal liver. ß7 expressing hepatic T cells displayed an increased inflammatory phenotype compared with ß7 negative cells, although this inflammatory cytokine profile was present in both the inflamed and normal liver. CONCLUSIONS: These findings refute the widely accepted "gut homing" hypothesis as the primary driver of PSC and indicate that aberrant hepatic recruitment of gut-derived T cells is not unique to PSC, but is a panetiological feature of CLD.

Characterization of a sandwich ELISA for the quantification of all human periostin isoforms.

BACKGROUND: Periostin (osteoblast-specific factor OSF-2) is a secreted protein occurring in seven known isoforms, and it is involved in a variety of biological processes in osteology, tissue repair, oncology, cardiovascular and respiratory systems or allergic manifestations. To analyze functional aspects of periostin, or the ability of periostin as potential biomarker in physiological and pathological conditions, there is the need for a precise, well-characterized assay that detects periostin in peripheral blood. METHODS: In this study the development of a sandwich ELISA using monoclonal and affinity-purified polyclonal anti-human periostin antibodies was described. Antibodies were characterized by mapping of linear epitopes with microarray technology, and by analyzing cross-reactive binding to human periostin isoforms with western blot. The assay was validated according to ICH/EMEA guidelines. RESULTS: The monoclonal coating antibody binds to a linear epitope conserved between the isoforms. The polyclonal detection antibody recognizes multiple conserved linear epitopes. Therefore, the periostin ELISA detects all known human periostin isoforms. The assay is optimized for human serum and plasma and covers a calibration range between 125 and 4000 pmol/L for isoform 1. Assay characteristics, such as precision (intra-assay: ≤3%, inter-assay: ≤6%), spike-recovery (83%-106%), dilution linearity (95%-126%), as well as sample stability meet the standards of acceptance. Periostin levels of apparently healthy individuals are 864±269 pmol/L (serum) and 817±170 pmol/L (plasma) respectively. CONCLUSION: This ELISA is a reliable and accurate tool for determination of all currently known periostin isoforms in human healthy and diseased samples.

Label-Free LC-MS/MS Proteomic Analysis of Cerebrospinal Fluid Identifies Protein/Pathway Alterations and Candidate Biomarkers for Amyotrophic Lateral Sclerosis.

Analysis of the cerebrospinal fluid (CSF) proteome has proven valuable to the study of neurodegenerative disorders. To identify new protein/pathway alterations and candidate biomarkers for amyotrophic lateral sclerosis (ALS), we performed comparative proteomic profiling of CSF from sporadic ALS (sALS), healthy control (HC), and other neurological disease (OND) subjects using label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 1712 CSF proteins were detected and relatively quantified by spectral counting. Levels of several proteins with diverse biological functions were significantly altered in sALS samples. Enrichment analysis was used to link these alterations to biological pathways, which were predominantly related to inflammation, neuronal activity, and extracellular matrix regulation. We then used our CSF proteomic profiles to create a support vector machines classifier capable of discriminating training set ALS from non-ALS (HC and OND) samples. Four classifier proteins, WD repeat-containing protein 63, amyloid-like protein 1, SPARC-like protein 1, and cell adhesion molecule 3, were identified by feature selection and externally validated. The resultant classifier distinguished ALS from non-ALS samples with 83% sensitivity and 100% specificity in an independent test set. Collectively, our results illustrate the utility of CSF proteomic profiling for identifying ALS protein/pathway alterations and candidate disease biomarkers.

Isolation of a hemidesmosome-rich fraction from a human squamous cell carcinoma cell line.

Hemidesmosomes are cell-to-matrix adhesion complexes anchoring keratinocytes to basement membranes. For the first time, we present a method to prepare a fraction from human cultured cells that are highly enriched in hemidesmosomal proteins. Using DJM-1 cells derived from human squamous cell carcinoma, accumulation of hemidesmosomes was observed when these cells were cultured for more than 10 days in a commercial serum-free medium without supplemental calcium. Electron microscopy demonstrated that numerous electron-dense adhesion structures were present along the basal cell membranes of DJM-1 cells cultured under the aforementioned conditions. After removing cellular materials using an ammonia solution, hemidesmosomal proteins and deposited extracellular matrix were collected and separated by electrophoresis. There were eight major polypeptides, which were determined to be plectin, BP230, BP180, integrin α6 and ß4 subunits, and laminin-332 by immunoblotting and mass spectrometry. Therefore, we designated this preparation as a hemidesmosome-rich fraction. This fraction contained laminin-332 exclusively in its unprocessed form, which may account for the promotion of laminin deposition, and minimal amounts of Lutheran blood group protein, a nonhemidesmosomal transmembrane protein. This hemidesmosome-rich fraction would be useful not only for biological research on hemidesmosomes but also for developing a serum test for patients with blistering skin diseases.

Nfasc155H and MAG are specifically susceptible to detergent extraction in the absence of the myelin sphingolipid sulfatide.

Mice incapable of synthesizing the myelin lipid sulfatide form paranodes that deteriorate with age. Similar instability also occurs in mice that lack contactin, contactin-associated protein or neurofascin155 (Nfasc155), the proteins that cluster in the paranode and form the junctional complex that mediates myelin-axon adhesion. In contrast to these proteins, sulfatide has not been shown to be enriched in the paranode nor has a sulfatide paranodal binding partner been identified; thus, it remains unclear how the absence of sulfatide results in compromised paranode integrity. Using an in situ extraction procedure, it has been reported that the absence of the myelin sphingolipids, galactocerebroside and sulfatide, increased the susceptibility of Nfasc155 to detergent extraction. Here, employing a similar approach, we demonstrate that in the presence of galactocerebroside but in the absence of sulfatide Nfasc155 is susceptible to detergent extraction. Furthermore, we use this in situ approach to show that stable association of myelin-associated glycoprotein (MAG) with the myelin membrane is sulfatide dependent while the membrane associations of myelin/oligodendrocyte glycoprotein, myelin basic protein and cyclic nucleotide phosphodiesterase are sulfatide independent. These findings indicate that myelin proteins maintain their membrane associations by different mechanisms. Moreover, the myelin proteins that cluster in the paranode and require sulfatide mediate myelin-axon adhesion. Additionally, the apparent dependency on sulfatide for maintaining Nfasc155 and MAG associations is intriguing since the fatty acid composition of sulfatide is altered and paranodal ultrastructure is compromised in multiple sclerosis. Thus, our findings present a potential link between sulfatide perturbation and myelin deterioration in multiple sclerosis.

Yeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidases.

Glucose oxidase (GOx) catalyzes the oxidation of glucose to form gluconic acid and hydrogen peroxide, a reaction with important applications in food preservation, the manufacture of cosmetics and pharmaceuticals, and the development of glucose monitoring devices and biofuel cells. We expressed Aspergillus niger wild type GOx and the B11 mutant, which has twice the activity of the wild type enzyme at pH 5.5, as C-terminal fusions with the Saccharomyces cerevisiae Aga2 protein, allowing the fusion proteins to be displayed on the surface of yeast EBY100 cells. After expression, we extracted the proteins from the yeast cell wall and purified them by ion-exchange chromatography and ultrafiltration. This produced a broad 100-140kDa band by denaturing SDS-PAGE and a high-molecular-weight band by native PAGE corresponding to the activity band revealed by zymography. The wild type and B11 fusion proteins had kcat values of 33.3 and 61.3s(-1) and Km values for glucose of 33.4 and 27.9mM, respectively. The pH optimum for both enzymes was 5.0. The kinetic properties of the fusion proteins displayed the same ratio as their native counterparts, confirming that yeast surface display is suitable for the high-throughput directed evolution of GOx using flow cytometry for selection. Aga2-GOx fusion proteins in the yeast cell wall could also be used as immobilized catalysts for the production of gluconic acid.

Neurofascin 186 is O-mannosylated within and outside of the mucin domain.

Protein O-mannosylation is an important modification in mammals, and deficiencies thereof lead to a variety of severe phenotypes. Although it has already been shown that the amount of O-mannosyl glycans in brain is very high, only very few proteins have been identified as O-mannosylated. Additionally, the functions of the O-mannose-based glycans are still speculative and only investigated for α-dystroglycan. In a previous study a cis-located peptide was identified, which controls O-mannosylation in mammals. A BLAST search on the basis of this peptidic determinant identified other potential O-mannosylated proteins. Among these neurofascin was chosen for further analysis as a recombinant probe (mucin domain) and as an endogenous protein from mouse brain. Mass spectrometric data for both proteins confirmed that neurofascin186 is indeed O-mannosylated. Glycopeptide analysis by liquid chromatography-tandem mass spectrometry allowed for the identification of some of the O-mannosylation sites, which are not restricted to the mucin domain but were found also within N-terminal IgG and Fibronectin domains of the protein.

Purification, crystallization and preliminary X-ray analysis of the IgV domain of human nectin-4.

Nectin-4 belongs to a family of immunoglobulin-like cell adhesion molecules and is highly expressed in cancer cells. Recently, nectin-4 was found to be a receptor of measles virus and the IgV domain sustains strong binding to measles virus H protein. In this study, the successful expression and purification of human nectin-4 V domain (nectin-4v) is reported. The purified protein was crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to 1.8 Šresolution and belonged to space group P2(1), with unit-cell parameters a = 33.1, b = 51.7, c = 56.9 Å, ß = 94.7°. Preliminary analysis of the diffraction data was also performed.

Tandem application of cationic colloidal silica and Triton X-114 for plasma membrane protein isolation and purification: towards developing an MDCK protein database.

Plasma membrane (PM) proteins are attractive therapeutic targets because of their accessibility to drugs. Although genes encoding PM proteins represent 20-30% of eukaryotic genomes, a detailed characterisation of their encoded proteins is underrepresented, due, to their low copy number and the inherent difficulties in their isolation and purification as a consequence of their high hydrophobicity. We describe here a strategy that combines two orthogonal methods to isolate and purify PM proteins from Madin Darby canine kidney (MDCK) cells. In this two-step method, we first used cationic colloidal silica (CCS) to isolate adherent (Ad) and non-adherent (nAd) PM fractions, and then subjected each fraction to Triton X-114 (TX-114) phase partitioning to further enrich for hydrophobic proteins. While CCS alone identified 255/757 (34%) membrane proteins, CCS/TX-114 in combination yielded 453/745 (61%). Strikingly, of those proteins unique to CCS/TX-114, 277/393 (70%) had membrane annotation. Further characterisation of the CCS/TX-114 data set using Uniprot and transmembrane hidden Markov model revealed that 306/745 (41%) contained one or more transmembrane domains (TMDs), including proteins with 25 and 17 TMDs. Of the remaining proteins in the data set, 69/439 (16%) are known to contain lipid modifications. Of all membrane proteins identified, 93 had PM origin, including proteins that mediate cell adhesion, modulate transmembrane ion transport, and cell-cell communication. These studies reveal that the application of CCS to first isolate Ad and nAd PM fractions, followed by their detergent-phase TX-114 partitioning, to be a powerful method to isolate low-abundance PM proteins, and a useful adjunct for in-depth cell surface proteome analyses.

The cytoskeleton-associated Ena/VASP proteins are unanticipated partners of the PMR1 mRNA endonuclease.

The PMR1 mRNA endonuclease catalyzes the selective decay of a limited number of mRNAs. It participates in multiple complexes, including one containing c-Src, its activating kinase, and one containing its substrate mRNA. This study used tandem affinity purification (TAP) chromatography to identify proteins in HeLa cell S100 associated with the mature 60-kDa form of Xenopus PMR1 (xPMR60). Unexpectedly, this identified a number of cytoskeleton-associated proteins, most notably the Ena family proteins mammalian Enabled (Mena) and vasodilator-stimulated phosphoprotein (VASP). These are regulators of actin dynamics that distribute throughout the cytoplasm and concentrate along the leading edge of the cell. xPMR60 interacts with Mena and VASP in vivo, overexpression of Mena has no impact on mRNA decay, and Mena and VASP are recovered together with xPMR60 in each of the major complexes of PMR1-mRNA decay. In a wound-healing experiment induced expression of active xPMR60 in stably transfected cells resulted in a twofold increase in cell motility compared with uninduced cells or cells expressing inactive xPMR60 degrees . Under these conditions xPMR60 colocalizes with VASP along one edge of the cell.

Local call: from integrins to actin assembly.

Integrins link the extracellular matrix to the actin cytoskeleton by triggering the assembly of different types of adhesion complex. One of their major components is filamentous actin (F-actin), and they are important signaling hubs for actin cytoskeleton reorganization in response to chemical and mechanical signals. In an exciting publication, Butler et al. have demonstrated for the first time that purified adhesion complexes possess the entire machinery necessary to actively assemble F-actin as a function of integrin activity and clustering.

Refolding, crystallization and preliminary X-ray crystallographic study of the whole extracellular regions of nectins.

The nectin family of Ca2+-independent immunoglobulin-like cell-cell adhesion molecules contains four members. Nectins, which have three Ig-like domains in their extracellular region, form cell-cell adherens junctions cooperatively with cadherins. The whole extracellular regions of nectin-1 (nectin-1-EC) and nectin-2 (nectin-2-EC) were expressed in Escherichia coli as inclusion bodies, solubilized in 8 M urea and then refolded by rapid dilution into refolding solution. The refolded proteins were subsequently purified by three chromatographic steps and crystallized using the hanging-drop vapour-diffusion method. The nectin-1-EC crystals belonged to space group P2(1)3 and the nectin-2-EC crystals belonged to space group P6(1)22 or P6(5)22.

Isolation and characterization of a large soluble form of fibronectin that stimulates adhesion, spreading, and alignment of mouse erythroleukemia cells.

Fibronectin (FN) is a major component of the extracellular matrix which plays important roles in a variety of cellular processes including cell adhesion, and migration. The soluble cellular form of FN has a monomer molecular weight of approximately 250 kDa, and generally exists as a dimer of 500 kDa. We have isolated a different form of soluble FN from mouse breast cancer cell line SC115 conditioned medium (CM) and purified it to homogeneity as evidenced by both native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate PAGE. It still exhibits a monomeric form of about 250 kDa while its form in the CM is stable and soluble with an apparent tetrameric molecular weight in the range of 800-1000 kDa. This form of FN is a potent cell adhesion factor (AF) that induces adhesion to polystyrene, elongation, spreading, alignment or "track" formation, and migration of mouse erythroleukemia cells. Column fractions homogeneous for AF protein were able to stimulate 10% cell adhesion at concentrations of 23 ng/ml and 1.9 ng/cm(2). Purified AF induced 50% cell adhesion at 94 ng/ml and 7.5 ng/cm(2). AF also increased the migration of human aortic smooth muscle and vascular endothelial cells. However, this form of FN differs from other forms as it does not bind tightly to either gelatin or heparin. Studies of this AF should shed light on adhesion of cells to extracellular matrix molecules and on cell migration, both of which are critical in several biological processes such as wound healing, metastasis, matrix formation and structure, and organ development.

Selection and identification of a new adhesion protein of Cryptosporidium parvum from a cDNA library by ribosome display.

In this study, we described a novel display method to identify surface adhesion proteins of Cryptosporidium parvum. A cDNA library of the sporozoite and oocyst stages of C. parvum was expressed on ribosome and selectively and specifically screened with intestinal epithelial cells (IECs) from newborn Cryptosporidium-free Holstein calves. Proteins were then enriched using a multi-step panning procedure. A new surface adherence protein of C. parvum was selected, named Cp20. Sequence analyses showed that Cp20 has a N-terminal signal peptide and four transmembrane regions. Indirect immunofluorescence assay (IFA) using an antibody specific for rCp20 demonstrated that the antibody specifically bound to the surface of sporozoites and oocysts. The recombinant plasmid pVAX1-Cp20 was constructed to examine the potential of the Cp20 gene as a target for specific preventive and therapeutic measures for cryptosporidiosis. The in vivo efficacies of the DNA vaccine was tested in BALB/c mice. The results indicated that the DNA vaccine elicited significant antibody responses and specific cellular responses when compared to control mice that received vector only or PBS. The DNA vaccine induced strong protective immune response against C. parvum and lower level of the oocysts shedding after challenge infection. This study suggested that Cp20 could serve as an effective target for specific preventive and therapeutic measures for cryptosporidiosis.

Cell wall glycoproteins participate in the adhesion of Sporothrix schenckii to epithelial cells.

Sporothrix schenckii is the etiologic agent of sporotrichosis. This fungal infection is an emerging disease potentially fatal in immunocompromised patients. The adhesion to host cells is a crucial early event related with the dissemination of pathogens. In order to clarify the mechanisms of adhesion of S. schenckii yeast cell to epithelial cells, we studied the biochemical basis of this process. The electrophoretic analysis of cell wall protein from S. schenckii coupled at ConA and stained with HRP, revealed nine different proteins with MW ≥ 180, 115, 90, 80, 58, 40, 36, 22 and 18 kDa. Using ligand-like assay with biotinylated S. schenckii surface proteins, five proteins with MW ≥ 190, 180, 115, 90 and 80 kDa which have affinity to epithelial cells were identified. The adhesion of yeast to epithelial monolayer was significantly inhibited when S. schenckii was pretreated with concanavalinA (ConA) and wheat germ agglutinin (WGA) lectins, alkali, periodate, trypsin, endoglycosidase H (EndoH), salt solutions and detergents. The ability of adhesion of S. schenckii yeast was recovered by blocking the lectin with sugar complementary. These data suggest that surface glycoprotein with mannose and glucose residue could be participate in the process of fungal adhesion to epithelial cells.

Preclinical Molecular PET-CT Imaging Targeting CDCP1 in Colorectal Cancer.

Colorectal cancer (CRC) is the third most common malignancy in the world, with 22% of patients presenting with metastatic disease and a further 50% destined to develop metastasis. Molecular imaging uses antigen-specific ligands conjugated to radionuclides to detect and characterise primary cancer and metastases. Expression of the cell surface protein CDCP1 is increased in CRC, and here we sought to assess whether it is a suitable molecular imaging target for the detection of this cancer. CDCP1 expression was assessed in CRC cell lines and a patient-derived xenograft to identify models suitable for evaluation of radio-labelled 10D7, a CDCP1-targeted, high-affinity monoclonal antibody, for preclinical molecular imaging. Positron emission tomography-computed tomography was used to compare zirconium-89 (89Zr)-10D7 avidity to a nonspecific, isotype control 89Zr-labelled IgGκ1 antibody. The specificity of CDCP1-avidity was further confirmed using CDCP1 silencing and blocking models. Our data indicate high avidity and specificity for of 89Zr-10D7 in CDCP1 expressing tumors at. Significantly higher levels than normal organs and blood, with greatest tumor avidity observed at late imaging time points. Furthermore, relatively high avidity is detected in high CDCP1 expressing tumors, with reduced avidity where CDCP1 expression was knocked down or blocked. The study supports CDCP1 as a molecular imaging target for CRC in preclinical PET-CT models using the radioligand 89Zr-10D7.

Proteomics of purified lamellocytes from Drosophila melanogaster HopTum-l identifies new membrane proteins and networks involved in their functions.

In healthy Drosophila melanogaster larvae, plasmatocytes and crystal cells account for 95% and 5% of the hemocytes, respectively. A third type of hemocytes, lamellocytes, are rare, but their number increases after oviposition by parasitoid wasps. The lamellocytes form successive layers around the parasitoid egg, leading to its encapsulation and melanization, and finally the death of this intruder. However, the total number of lamellocytes per larva remains quite low even after parasitoid infestation, making direct biochemical studies difficult. Here, we used the HopTum-l mutant strain that constitutively produces large numbers of lamellocytes to set up a purification method and analyzed their major proteins by 2D gel electrophoresis and their plasma membrane surface proteins by 1D SDS-PAGE after affinity purification. Mass spectrometry identified 430 proteins from 2D spots and 344 affinity-purified proteins from 1D bands, for a total of 639 unique proteins. Known lamellocyte markers such as PPO3 and the myospheroid integrin were among the components identified with specific chaperone proteins. Affinity purification detected other integrins, as well as a wide range of integrin-associated proteins involved in the formation and function of cell-cell junctions. Overall, the newly identified proteins indicate that these cells are highly adapted to the encapsulation process (recognition, motility, adhesion, signaling), but may also have several other physiological functions (such as secretion and internalization of vesicles) under different signaling pathways. These results provide the basis for further in vivo and in vitro studies of lamellocytes, including the development of new markers to identify coexisting populations and their respective origins and functions in Drosophila immunity.

Exosomal Del-1 as a Potent Diagnostic Marker for Breast Cancer: Prospective Cohort Study.

BACKGROUND: The differential diagnostic role of plasma developmental endothelial locus-1 (Del-1) was proposed in our previous study. Therefore the current study aimed to confirm the diagnostic role and explore the prognostic role of exosomal Del-1 in a prospective cohort of female patients with breast cancer. PATIENTS AND METHODS: To determine the optimal sampling time for the postoperative Del-1 measurements, blood was serially collected on days 1, 3, 5, and 7 after surgery in 22 patients (cohort 1). Thereafter, 111 female patients with breast cancer were prospectively enrolled (cohort 2) to compare exosomal Del-1 levels before and after surgery. RESULTS: Among the subsequent prospective cohort, 107 patients (96.4%) showed a high exosomal Del-1 level (optical density [OD] value > 0.5) at the time of diagnosis. Of these patients, 101 (94.6%) in this high-level group showed normalized Del-1 levels postoperatively, representing a significant difference (mean OD value, 1.232 vs. 0.196; P < .00001). High postoperative Del-1 level was significantly associated with a worse disease-free survival adjusted to the clinicopathological characteristics (hazard ratio, 24.0; P = .0011). CONCLUSION: This study confirmed the normalization of exosomal Del-1 after surgery, indicating exosomal Del-1 as a potent diagnostic biomarker for breast cancer. In addition, because a high Del-1 level after surgery was associated with early relapse, this suggests exosomal Del-1 as a potential prognostic marker by identifying the existence of residual cancer.

Heterogeneous detection of circulating tumor cells in patients with colorectal cancer by immunomagnetic enrichment using different EpCAM-specific antibodies.

BACKGROUND: Circulating tumor cells (CTC) and disseminated tumor cells (DTC) are thought to be responsible for metastasis, so the detection of CTC may serve as individual prognostic factor in patients suffering from colorectal cancer. Therefore, a series of immunomagnetic enrichment methods for CTC have been developed using a variety of monoclonal antibodies against the Epithelial Cell Adhesion Molecule (EpCAM). However, it remains unclear whether all commercially available EpCAM antibodies show the same sensitivity and specificity. Furthermore, it remains unclear which method of sample preparation and cell extraction is most suitable for immunomagnetic enrichment and detection of CTC. In this study, we aimed to investigate whether the detection of CTC by a cytokeratin 20 reverse transcriptase-polymerase chain reaction (CK20 RT-PCR) may be influenced by the use of various Epithelial Cell Adhesion Molecule (EpCAM) antibodies for immunomagnetic isolation of CTC. RESULTS: Using both EpCAM antibodies (mAb BerEP4 and mAb KS1/4) for immunomagnetic enrichment in blood samples of 39 patients with colorectal cancer we found heterogenous results in each patient with regard to tumor cell detection. In the tumor cell spiking experiments with whole blood samples the sensitivity of the CK 20 RT-PCR assay was higher using immunomagnetic beads coated with mAb KS1/4 compared to precoated mAb BerEP4 Dynabeads. Extraction of MNC fraction with Ficoll gradient centrifugation prior to immunomagnetic enrichment resulted in a higher sensitivity of the CK 20 RT-PCR assay. CONCLUSIONS: We concluded that isolation and detection of CTC with immunomagnetic enrichment methods is critically dependent on the used EpCAM clone. Further studies with a larger number of patients should clarify if the enrichment protocol influences the prognostic value of the tumor cell detection protocol.

Identification of cysteine-rich epidermal growth factor-like domain 1alpha (CRELD1alpha) as a novel alpha1A-adrenoceptor-down-regulating protein and establishment of an alpha1L-adrenoceptor-expressing cell line.

Two distinct alpha(1)-adrenoceptor phenotypes (alpha(1A)- and alpha(1L)-ARs) are known to originate from a single ADRA1A(alpha(1a)) gene by an as-yet-unknown mechanism. We hypothesized that an alpha(1a)-AR-interacting protein could generate the alpha(1L)-AR phenotype and we sought to identify such a protein and to examine its effects on the expression of alpha(1A) and alpha(1L) phenotypes. Cysteine-rich epidermal growth factor-like domain 1alpha (CRELD1alpha) was first identified using a yeast two-hybrid approach as an alpha(1a)-AR-interacting protein. Transfection of alpha(1a)-AR cDNA alone yielded Chinese hamster ovary (CHO) cells expressing alpha(1A)-ARs having a predominant high affinity site for prazosin, with a low proportion (<10%) of prazosin-low affinity sites (alpha(1L)-AR). Knockdown of endogenous CHO-CRELD1alpha [alpha(1a)-CKD(alpha(1A)-enhanced) cells] enhanced the expression of alpha(1A)-AR, whereas over-expression of CRELD1alpha reduced alpha(1A)-AR expression, yielding alpha(1a)-COE(alpha(1L)-dominant) cells expressing a high proportion (50%) of the alpha(1L)-AR phenotype. The ligand binding and functional agonist and antagonist profiles in alpha(1a)-CKD(alpha(1A)-enhanced) and alpha(1a)-COE(alpha(1L)-dominant) cell lines were entirely in accord with the alpha(1A)-AR and alpha(1L)-AR phenotypes observed in intact tissues. CRELD1alpha down-regulates expression of the alpha(1A)-AR, thereby enhancing the proportion of expression of the alpha(1L)-AR phenotype. The alpha(1L)-AR-expressing alpha(1a)-COE(alpha(1L)-dominant) cell line reflects accurately the phenotype of this AR observed in vivo and will facilitate development of alpha(1L)-AR-targeted drugs.