Charles E. Birse

Decreased Survival of B Cells of HIV-viremic Patients Mediated by Altered Expression of Receptors of the TNF Superfamily

Human immunodeficiency virus (HIV) infection leads to numerous perturbations of B cells through mechanisms that remain elusive. We performed DNA microarray, phenotypic, and functional analyses in an effort to elucidate mechanisms of B cell perturbation associated with ongoing HIV replication. 42 genes were up-regulated in B cells of HIV-viremic patients when compared with HIV-aviremic and HIV-negative patients, the majority of which were interferon (IFN)-stimulated or associated with terminal differentiation. Flow cytometry confirmed these increases and indicated that CD21low B cells, enhanced in HIV-viremic patients, were largely responsible for the changes. Increased expression of the tumor necrosis factor (TNF) superfamily (TNFSF) receptor CD95 correlated with increased susceptibility to CD95-mediated apoptosis of CD21low B cells, which, in turn, correlated with HIV plasma viremia. Increased expression of BCMA, a weak TNFSF receptor for B lymphocyte stimulator (BLyS), on CD21low B cells was associated with a concomitant reduction in the expression of the more potent BLyS receptor, BAFF-R, that resulted in reduced BLyS binding and BLyS-mediated survival. These findings demonstrate that altered expression of genes associated with IFN stimulation and terminal differentiation in B cells of HIV-viremic patients lead to an increased propensity to cell death, which may have substantial deleterious effects on B cell responsiveness to antigenic stimulation.

An integrated functional genomics screening program reveals a role for BMP-9 in glucose homeostasis.

A coordinated functional genomics program was implemented to identify secreted polypeptides with therapeutic applications in the treatment of diabetes. Secreted factors were predicted from a diverse expressed-sequence tags (EST) database, representing >1,000 cDNA libraries, using a combination of bioinformatic algorithms. Subsequently, ∼8,000 human proteins were screened in high-throughput cell-based assays designed to monitor key physiological transitions known to be centrally involved in the physiology of type 2 diabetes. Bone morphogenetic protein-9 (BMP-9) gave a positive response in two independent assays: reducing phosphoenolpyruvate carboxykinase (PEPCK) expression in hepatocytes and activating Akt kinase in differentiated myotubes. Purified recombinant BMP-9 potently inhibited hepatic glucose production and activated expression of key enzymes of lipid metabolism. In freely fed diabetic mice, a single subcutaneous injection of BMP-9 reduced glycemia to near-normal levels, with maximal reduction observed 30 hours after treatment. BMP-9 represents the first hepatic factor shown to regulate blood glucose concentration. Using a combination of bioinformatic and high-throughput functional analyses, we have identified a factor that may be exploited for the treatment of diabetes.

Expansion of CD133+ colon cancer cultures retaining stem cell properties to enable cancer stem cell target discovery

Background:Despite earlier studies demonstrating in vitro propagation of solid tumour cancer stem cells (CSCs) as non-adherent tumour spheres, it remains controversial as to whether CSCs can be maintained in vitro. Additional validation of the CSC properties of tumour spheres would support their use as CSC models and provide an opportunity to discover additional CSC cell surface markers to aid in CSC detection and potential elimination.Methods:Primary tumour cells isolated from 13 surgically resected colon tumour specimens were propagated using serum-free CSC-selective conditions. The CSC properties of long-term cultured tumour spheres were established and mass spectrometry-based proteomics performed.Results:Freshly isolated CD133+ colorectal cancer cells gave rise to long-term tumour sphere (or spheroids) cultures maintaining CD133 expression. These spheroid cells were able to self-renew and differentiate into adherent epithelial lineages and recapitulate the phenotype of the original tumour. Relative to their differentiated progeny, tumour spheroid cells were more resistant to the chemotherapeutic irinotecan. Finally, CD44, CD166, CD29, CEACAM5, cadherin 17, and biglycan were identified by mass spectrometry to be enriched in CD133+ tumour spheroid cells.Conclusion:Our data suggest that ex vivo-expanded colon CSCs isolated from clinical specimens can be maintained in culture enabling the identification of CSC cell surface-associated proteins.

Use of an Immunoaffinity-Mass Spectrometry-based Approach for the Quantification of Protein Biomarkers from Serum Samples of Lung Cancer Patients

It is a challenging task to verify and quantify potential biomarkers expressed at elevated levels in sera from cancer patients. An immunoaffinity-mass spectrometry-based approach has been developed using antibodies to enrich proteins of interest from sera followed by mass spectrometry-based quantification. Antibodies specific to the protein of interest were immobilized to hydrazide resin via the carbohydrate moiety on the Fc region of the antibody. Captured proteins were eluted, reduced, alkylated, and digested with trypsin. Peptides were analyzed by LC coupled with multiple reaction monitoring approach, and quantification was achieved by the addition of stable isotope-labeled (heavy) standard peptides. Using this methodology, we were able to achieve a linear response from 15 to 250 ng/ml for carcinoembryonic antigen (CEA), a known tumor biomarker. Moreover we observed elevated levels of CEA in sera samples from lung cancer patients that to our knowledge is the first time that circulating CEA has been detected by mass spectrometry-based analysis. This approach was further applied to potential protein biomarkers discovered from tumor cell lines and tumor tissues. A linear response was obtained from a multiplex spiking experiment in normal human sera for secretory leukocyte peptidase inhibitor (4–500 ng/ml), tissue factor pathway inhibitor (TFPI) (42–1000 ng/ml), tissue factor pathway inhibitor 2 (TFPI2) (2–250 ng/ml), and metalloproteinase inhibitor 1 (TIMP1) (430–1000 ng/ml). A replicate experiment for a single concentration value yielded a relative coefficient of variation better than 11% for TFPI, secretory leukocyte peptidase inhibitor, and TFPI2. The expression level of the proteins in lung cancer patient sera was assayed by an immunoaffinity-multiple reaction monitoring method, and the results were comparable with those obtained from ELISA. This immunoaffinity-mass spectrometry-based quantification approach thus provides a specific and accurate assay for verifying the expression of potential biomarkers in patient serum samples especially for those proteins for which the necessary reagents for ELISA development are unavailable.

Antibacterial properties of the sperm-binding proteins and peptides of human epididymis 2 (HE2) family; salt sensitivity, structural dependence and their interaction with outer and cytoplasmic membranes of Escherichia coli.

During passage through the epididymis, sperm interact with secreted epididymal proteins that promote maturation, including the acquisition of motility and fertilization competence. Viewed previously as distinct from sperm maturation, host defence capabilities are now recognized functions of the human epididymis 2 (HE2) family of sperm-binding proteins. We analysed the potent dose and time-dependent bactericidal activity of recombinant HE2alpha, HE2beta1 and HE2beta2 and found that the full-length proteins (10 microg/ml or approximately 1 microM) caused more than a 50% decrease in Escherichia coli colony forming units within 15 min. By contrast, human beta-defensin-1, at a similar concentration, required more than 90 min to exhibit similar antibacterial activity. The epididymis-specific lipocalin, LCN6, failed to kill bacteria. Higher concentrations (25-100 microg/ml) of HE2 proteins and a longer duration of treatment resulted in near total inhibition of bacterial growth. The C-terminal peptides of HE2alpha, HEbeta1 and HEbeta2 proteins exhibited antibacterial activity similar to their full-length counterparts, indicating that the antibacterial activity of HE2 proteins resides in these C-terminal regions. Antibacterial activities of HE2 proteins and peptides were slightly inhibited by NaCl concentrations of up to 150 mM, while human beta-defensin-1 activity was nearly eliminated. Reduction and alkylation of disulphide bonds in HE2 proteins and their C-terminal peptides abolished their antibacterial activity. Consistent with the ability to kill bacteria, full-length HE2 proteins and C-terminal peptides caused rapid dose-dependent permeabilization of outer and cytoplasmic E. coli membranes. A much longer exposure time was required for human beta-defensin-1-mediated permeabilization of membranes, suggesting a possible difference in mode of action compared with the HE2 antibacterial peptides.

LCN6, a novel human epididymal lipocalin

BackgroundThe lipocalin (LCN) family of structurally conserved hydrophobic ligand binding proteins is represented in all major taxonomic groups from prokaryotes to primates. The importance of lipocalins in reproduction and the similarity to known epididymal lipocalins prompted us to characterize the novel human epididymal LCN6.Methods and ResultsLCN6 cDNA was identified by database analysis in a comprehensive human library sequencing program. Macaca mulatta (rhesus monkey) cDNA was obtained from an epididymis cDNA library and is 93% homologous to the human. The gene is located on chromosome 9q34 adjacent LCN8 and LCN5. LCN6 amino acid sequence is most closely related to LCN5, but the LCN6 beta-barrel structure is best modeled on mouse major urinary protein 1, a pheromone binding protein. Northern blot analysis of RNAs isolated from 25 human tissues revealed predominant expression of a 1.0 kb mRNA in the epididymis. No other transcript was detected except for weak expression of a larger hybridizing mRNA in urinary bladder. Northern hybridization analysis of LCN6 mRNA expression in sham-operated, castrated and testosterone replaced rhesus monkeys suggests mRNA levels are little affected 6 days after castration. Immunohistochemical staining revealed that LCN6 protein is abundant in the caput epithelium and lumen. Immunofluorescent staining of human spermatozoa shows LCN6 located on the head and tail of spermatozoa with the highest concentration of LCN6 on the post-acrosomal region of the head, where it appeared aggregated into large patches.ConclusionsLCN6 is a novel lipocalin closely related to Lcn5 and Lcn8 and these three genes are likely products of gene duplication events that predate rodent-primate divergence. Predominant expression in the epididymis and location on sperm surface are consistent with a role for LCN6 in male fertility.

SOSTDC1 differentially modulates Smad and beta-catenin activation and is down-regulated in breast cancer

Sclerostin domain containing 1 (SOSTDC1) protein regulates processes from development to cancer by modulating activity of bone morphogenetic protein (BMP) and wingless/int (Wnt) signaling pathways. As dysregulation of both BMP and Wnt signaling has been observed in breast cancer, we investigated whether disruption of SOSTDC1 signaling occurs in breast cancer. SOSTDC1 mRNA expression levels in breast tissue were examined using a dot blot. Affymetrix microarray data on SOSTDC1 levels were correlated with breast cancer patient survival using Kaplan–Meier plots. Correlations between SOSTDC1 protein levels and clinical parameters were assessed by immunohistochemistry of a breast cancer tissue microarray. SOSTDC1 secretion and BMP and Wnt signaling were investigated using immunoblotting. We found that SOSTDC1 is expressed in normal breast tissue and this expression is reduced in breast cancer. High levels of SOSTDC1 mRNA correlated with increased patient survival; conversely, SOSTDC1 protein levels decreased as tumor size and disease stage increased. Treatment of breast cancer cells with recombinant SOSTDC1 or Wise, a SOSTDC1 orthologue, demonstrated that SOSTDC1 selectively blocks BMP-7-induced Smad phosphorylation without diminishing BMP-2 or Wnt3a-induced signaling. In conclusion, SOSTDC1 mRNA and protein are reduced in breast cancer. High SOSTDC1 mRNA levels correlate with increased distant metastasis-free survival in breast cancer patients. SOSTDC1 differentially affects Wnt3a, BMP-2, and BMP-7 signaling in breast cancer cells. These results identify SOSTDC1 as a clinically important extracellular regulator of multiple signaling pathways in breast cancer.

RNAi Validation of Pancreatic Cancer Antigens Identified by Cell Surface Proteomics

Pancreatic cancer is one of the most aggressive malignancies that is often diagnosed at a late stage following tumor metastasis. Given the low response rate to current therapies, there is a clear and urgent need for new therapeutic and diagnostic approaches. Proteomics is an emerging tool that offers the opportunity to discover novel antigens elevated in cancer. Considering the large number of marketed drugs that target plasma membrane proteins, and given their amenability to both small molecule and antibody-based technologies, an approach that combines a cell membrane labeling strategy with proteomic (LC-MS/MS) methodology would enable novel cell surface antigens that can act as novel biomarkers and/or targets for drug development to be identified. One major consideration when selecting targets for therapeutics is whether or not the antigen plays a functional role in a disease context. RNA interference (RNAi) permits the downregulation of any given gene, and therefore allows the identification of functionality in the target disease. As such, this tool permits the selection of targets prior to investment in a therapeutic antibody program or an extensive small molecule–screening program. In this chapter, we describe the utility of the cell membrane labeling strategy and LC-MS/MS method that was developed at Celera. Subsequent application of RNAi allowed us to assign functionality to cell surface antigens we identified that were upregulated in pancreatic cancer. By using a combination of a proteomics and an RNAi platform, we demonstrate that it is possible to discover novel therapeutic and diagnostic targets for this devastating disease.