Identification of prostate cancer antigens by automated high-throughput filter immunoscreening.

There is a need for earlier and more accurate cancer diagnostics as well as new targets for cancer immunotherapy. To this end, it is important to identify sets of tumour antigens specific for different cancer forms. Several methods that identify potential tumour antigens in an arrayed and high-throughput format have been developed during the last years of SEREX (serological identification of antigens by recombinant expression cloning) related research. Such techniques may hold the potential to describe the complete immunogenic part of the cancer proteome, also called the cancer immunoproteome. We have developed a powerful platform for automated serological high-throughput filter screening of tumour cDNA libraries. The screening format of this method is 18,000 single cDNAs clones, which is superior to other high-throughput methods described. The output is antigens, which are potential diagnostic cancer markers and vaccine targets. We present here the results from the screening of a prostate tumour cDNA library with autologous patient antibodies. We first demonstrated the feasibility of the automated high-throughput filter immunoscreening method by use of the NY-ESO-1sv (NY-ESO-1 splice variant) antigen. We then screened 18,000 cDNA clones from a phage display selected prostate tumour cDNA library with autologous patient antibodies and identified several relevant antigens such as NY-ESO-1, XAGE-1, DJ-1 and transcription factor 25 (TCF25). The present high-throughput immunoscreening method has the potential to identify both patient-specific and disease-specific antigens for use in diagnostics and therapy.

Wnt3A activates canonical Wnt signalling in acute lymphoblastic leukaemia (ALL) cells and inhibits the proliferation of B-ALL cell lines.

Acute lymphoblastic leukaemia (ALL) is the most common malignancy in children. Recently, there has been a growing interest in Wnt signalling in several aspects of cellular development, including cancer formation. Little is known about Wnt signalling in B-ALL. We investigated whether activation of canonical Wnt signalling could occur in B-ALL cells and thereby play a potential role in cellular growth and/or survival. This study found that Wnt3A induced beta-catenin accumulation in both primary B-ALL cells and B-ALL leukaemia cell lines. Further, Wnt3A was shown to induce nuclear translocation of beta-catenin and TCF/Lef-1 dependent transcriptions in the B-ALL cell line Nalm-6. Examination of the mRNA expression pattern of WNT ligands, FZD receptors and WNT antagonists in Nalm-6 cells identified a set of ligands and receptors available for signalling, as well as antagonists potentially available for modulating the response. Functional analyses showed that Wnt3A inhibited the proliferation of several, but not all, B-ALL cell lines studied. Finally, microarray analysis was used to identify several Wnt3A target genes involved in a diverse range of cellular activities, which are potential mediators of the Wnt3A-restrained proliferation.

Wnt expression and canonical Wnt signaling in human bone marrow B lymphopoiesis.

BACKGROUND: The early B lymphopoiesis in mammals is regulated through close interactions with stromal cells and components of the intracellular matrix in the bone marrow (BM) microenvironment. Although B lymphopoiesis has been studied for decades, the factors that are implicated in this process, both autocrine and paracrine, are inadequately explored. Wnt signaling is known to be involved in embryonic development and growth regulation of tissues and cancer. Wnt molecules are produced in the BM, and we here ask whether canonical Wnt signaling has a role in regulating human BM B lymphopoiesis. RESULTS: Examination of the mRNA expression pattern of Wnt ligands, Fzd receptors and Wnt antagonists revealed that BM B progenitor cells and stromal cells express a set of ligands and receptors available for induction of Wnt signaling as well as antagonists for fine tuning of this signaling. Furthermore, different B progenitor maturation stages showed differential expression of Wnt receptors and co-receptors, beta-catenin, plakoglobin, LEF-1 and TCF-4 mRNAs, suggesting canonical Wnt signaling as a regulator of early B lymphopoiesis. Exogenous Wnt3A induced stabilization and nuclear accumulation of beta-catenin in primary lineage restricted B progenitor cells. Also, Wnt3A inhibited B lymphopoiesis of CD133+CD10- hematopoietic progenitor cells and CD10+ B progenitor cells in coculture assays using a supportive layer of stromal cells. This effect was blocked by the Wnt antagonists sFRP1 or Dkk1. Examination of early events in the coculture showed that Wnt3A inhibits cell division of B progenitor cells. CONCLUSION: These results indicate that canonical Wnt signaling is involved in human BM B lymphopoiesis where it acts as a negative regulator of cell proliferation in a direct or stroma dependent manner.

CD10+ stromal cells form B-lymphocyte maturation niches in the human bone marrow.

In adult mammals, early B-lymphopoiesis takes place in the bone marrow in close association with stromal cells. Both the phenotype of the stromal cells and the molecules involved in this essential interaction are as yet inadequately described. In this study, all benign, differentiating B-cells (Pax-5+ lymphoid cells) are shown, by using two-colour immunohistochemistry on biopsies from human bone marrow, to be in close contact with scant dendritic CD10+ stromal cells until they leave via the sinusoids. This CD10+ stromal cell population does not fully overlap with the VCAM-1+ stromal cell population. Furthermore, using a set of B-cell differentiation markers (TdT, Pax-5, and CD20), B-cell development is shown to be spatially oriented, with maturation progressing towards bone marrow sinusoids. In conclusion, CD10+ stromal cells form distinct B-lymphocyte maturation niches in the human bone marrow.

[Clinical use of haematopoietic stem cells from cord blood]. / Klinisk bruk av hematopoetiske stamceller fra navlestrengsblod.

A group of experts appointed by the Norwegian Centre for Health Technology Assessment (SMM) has undertaken a systematic review of available literature on the clinical effectiveness of transplanting haematopoietic stem cells from cord blood. A total of 17 studies form the documentary basis of the review. Autologous transplants of stem cells from cord blood have not been published. Retrospective studies suggest that the clinical effect of allogeneic cord blood transplants, at least in children, is comparable to transplants with allogeneic stem cells from bone marrow or peripheral blood cells. This review demonstrates the need for prospective studies comparing transplantations of cord blood with bone marrow or peripheral blood stem cells.

Serological cloning of cancer/testis antigens expressed in prostate cancer using cDNA phage surface display.

Serological cloning of tumor-associated antigens (TAAs) using patient autoantibodies and tumor cDNA expression libraries (SEREX) has identified a wide array of tumor proteins eliciting B-cell responses in patients. However, alternative cloning strategies with the possibility of high throughput analysis of patient sera and tumor libraries may be of interest. We explored the pJuFo phage surface display system, allowing display of recombinant tumor proteins on the surface of M13 filamentous phage, for cloning of TAAs in prostate cancer (PC). Control experiments established that after a few rounds of selection on immobilized specific IgG, a high degree of enrichment of seroreactive clones was achieved. With an increasing number of selection rounds, a higher yield of positive clones was offset by an apparent loss of diversity in the repertoire of selected clones. Using autologous patient serum IgG in a combined biopanning and immunoscreening approach, we identified 13 different TAAs. Three of these (NY-ESO-1, Lage-1, and Xage-1) were known members of the cancer/testis family of TAAs, and one other protein had previously been isolated by SEREX in cancer types other than PC. Specific IgG responses against NY-ESO-1 were found in sera from 4/20 patients with hormone refractory PC, against Lage-1 in 3/20, and Xage-1 in 1/20. No reactivity against the remaining proteins was detected in other PC patients, and none of the TAAs reacted with serum from healthy subjects. The results demonstrate that phage surface display combined with postselection immunoscreening is suitable for cloning a diverse repertoire of TAAs from tumor tissue cDNA libraries. Furthermore, candidate TAAs for vaccine development of PC were identified.

Characterization of the novel human transmembrane protein 9 (TMEM9) that localizes to lysosomes and late endosomes.

We have identified and characterized the novel human transmembrane protein 9 (TMEM9). TMEM9 encodes a 183 amino-acid protein that contains an N-terminal signal peptide, a single transmembrane region, three potential N-glycosylation sites, and three conserved cys-rich domains in the N-terminus, but no hitherto known functional domains. The protein is highly conserved between species from Caenorhabditis elegans to man and belongs to a novel family of transmembrane proteins. The TMEM9 gene consists of at least 6 exons and is localized to chromosome 1q41. TMEM9 mRNA is expressed in a wide range of tissues and cells. COS-1 cells transfected with a TMEM9 expression plasmid gave three bands of about 28, 31, and 33kDa representing glycosylated forms of TMEM9 with a protein backbone of about 26kDa. In COS-1 cells transfected with a TMEM9-GFP expression construct,TMEM9-GFP is co-expressed with LAMP1 on late endosomes and lysosomes as well as on ER. Thus, TMEM9 is a phylogenetically conserved, widely expressed transmembrane protein with a potential, but unknown function in intracellular transport.