A novel survival-based tissue microarray of pancreatic cancer validates MUC1 and mesothelin as biomarkers

Background

One–fifth of patients with seemingly ‘curable’ pancreatic ductal adenocarcinoma (PDA) experience an early recurrence and death, receiving no definable benefit from a major operation. Some patients with advanced stage tumors are deemed ‘unresectable’ by conventional staging criteria (e.g. liver metastasis), yet progress slowly. Effective biomarkers that stratify PDA based on biologic behavior are needed. To help researchers sort through the maze of biomarker data, a compendium of ∼2500 published candidate biomarkers in PDA was compiled (PLoS Med, 2009. 6(4) p. e1000046).

Methods and Findings

Building on this compendium, we constructed a survival tissue microarray (termed s-TMA) comprised of short-term (cancer-specific death <12 months, n = 58) and long-term survivors (>30 months, n = 79) who underwent resection for PDA (total, n = 137). The s-TMA functions as a biological filter to identify bona fide prognostic markers associated with survival group extremes (at least 18 months separate survival groups). Based on a stringent selection process, 13 putative PDA biomarkers were identified from the public biomarker repository. Candidates were tested against the s-TMA by immunohistochemistry to identify the best markers of tumor biology. In a multivariate model, MUC1 (odds ratio, OR = 28.95, 3+ vs. negative expression, p = 0.004) and MSLN (OR = 12.47, 3+ vs. negative expression, p = 0.01) were highly predictive of early cancer-specific death. By comparison, pathologic factors (size, lymph node metastases, resection margin status, and grade) had ORs below three, and none reached statistical significance. ROC curves were used to compare the four pathologic prognostic features (ROC area = 0.70) to three univariate molecular predictors (MUC1, MSLN, MUC2) of survival group (ROC area = 0.80, p = 0.07).

Conclusions

MUC1 and MSLN were superior to pathologic features and other putative biomarkers as predicting survival group. Molecular assays comparing cancers from short and long survivors are an effective strategy to screen biomarkers and prioritize candidate cancer genes for diagnostic and therapeutic studies.     

Chimeric animal and plant viruses expressing epitopes of outer membrane protein F as a combined vaccine against Pseudomonas aeruginosa lung infection

Outer membrane protein F of Pseudomonas aeruginosa has vaccine efficacy against infection by P. aeruginosa as demonstrated in a variety of animal models. Through the use of synthetic peptides, three surface-exposed epitopes have been identified. These are called peptides 9 (aa 261-274 in the mature F protein, TDAYNQKLSERRAN), 10 (aa 305-318, NATAEGRAINRRVE), and 18 (aa 282-295, NEYGVEGGRVNAVG). Both the peptide 9 and 10 epitopes are protective when administered as a vaccine. In order to develop a vaccine that is suitable for use in humans, including infants with cystic fibrosis, the use of viral vector systems to present the protective epitopes has been investigated. An 11-amino acid portion of epitope 10 (AEGRAINRRVE) was successfully inserted into the antigenic B site of the hemagglutinin on the surface of influenza virus. This chimeric influenza virus protects against challenge with P. aeruginosa in the mouse model of chronic pulmonary infection. Attempts to derive a chimeric influenza virus carrying epitope 9 have been unsuccessful. A chimeric plant virus, cowpea mosaic virus (CPMV), with epitopes 18 and 10 expressed in tandem on the large coat protein subunit (CPMV-PAE5) was found to elicit antibodies that reacted exclusively with the 10 epitope and not with epitope 18. Use of this chimeric virus as a vaccine afforded protection against challenge with P. aeruginosa in the mouse model of chronic pulmonary infection. Chimeric CPMVs with a single peptide containing epitopes 9 and 18 expressed on either of the coat proteins are in the process of being evaluated. Epitope 9 was successfully expressed on the coat protein of tobacco mosaic virus (TMV), and this chimeric virus is protective when used as a vaccine in the mouse model of chronic pulmonary infection. However, initial attempts to express epitope 10 on the coat protein of TMV have been unsuccessful. Efforts are continuing to construct chimeric viruses that express both the 9 and 10 epitopes in the same virus vector system. Ideally, the use of a vaccine containing two epitopes of protein F is desirable in order to greatly reduce the likelihood of selecting a variant of P. aeruginosa that escapes protective antibodies in immunized humans via a mutation in a single epitope within protein F. When the chimeric influenza virus containing epitope 10 and the chimeric TMV containing epitope 9 were given together as a combined vaccine, the immunized mice produced antibodies directed toward both epitopes 9 and 10. The combined vaccine afforded protection against challenge with P. aeruginosa in the chronic pulmonary infection model at approximately the same level of efficacy as provided by the individual chimeric virus vaccines. These results prove in principle that a combined chimeric viral vaccine presenting both epitopes 9 and 10 of protein F has vaccine potential warranting continued development into a vaccine for use in humans.     

Resistance to extinction, generalization decrement, and conditioned reinforcement

This study investigated generalization decrement during an extinction resistance-to-change test for pigeon key pecking using a two-component multiple schedule with equal variable-interval 3-min schedules and different reinforcer amounts (one component presented 2-s access to reinforcement and the other 8s). After establishing baseline responding, subjects were assigned to one of the two extinction conditions: hopper stimuli (hopper and hopper light were activated but no food was available) or Control (inactive hopper and hopper light). Responding in the 8-s component was more resistant to extinction than responding in the 2-s component, the hopper stimuli group was more resistant to extinction compared to the Control group, and an interaction between amount of reinforcement, extinction condition, and session block was present. This finding supports generalization decrement as a factor that influences resistance to extinction. Hopper-time data (the amount of time subjects spent with their heads in the hopper) were compared to resistance-to-change data in an investigation of the role of conditioned reinforcement on resistance to change.     

PLASMOLYSIS,HECHTIAN STRAND FORMATION,AND LOCALIZED MEMBRANE‐WALL ADHESIONS IN THE DESMID,CLOSTERIUM ACEROSUM (CHLOROPHYTA)1

Closterium acerosum Ehrenberg (Chlorophyta) produced a distinct network of thin cytoplasmic strands, or Hechtian strands, upon controlled plasmolysis in a sucrose solution. The strands persisted for 30 min or longer and could be visualized with both LM and EM. Near the plasma membrane of the polar zones of plasmolyzing protoplasts, the strands formed a “lattice”‐like arrangement with interstrand spacing of 120–130 nm. The strands terminated at the fibrous zone of the inner cell wall stratum. Although actin cables could be found attached to the plasma membrane upon rhodamine phalloidin labeling of membrane ghosts, neither microfilaments nor microtubules were found in Hechtian strands at any stage of development. The formation of strands was not disrupted by centrifugation at 8000 g or by repeated cycles of plasmolysis‐deplasmolysis. Application of microtubule‐ or microfilament‐affecting agents or various proteolytic/polysaccharide‐degrading enzymes did not disrupt the formation of strands. Cold treatment of cells resulted in the formation of Hechtian strands.     

Proteomics Analysis of Bladder Cancer Exosomes

Exosomes are nanometer-sized vesicles, secreted by various cell types, present in biological fluids that are particularly rich in membrane proteins. Ex vivo analysis of exosomes may provide biomarker discovery platforms and form non-invasive tools for disease diagnosis and monitoring. These vesicles have never before been studied in the context of bladder cancer, a major malignancy of the urological tract. We present the first proteomics analysis of bladder cancer cell exosomes. Using ultracentrifugation on a sucrose cushion, exosomes were highly purified from cultured HT1376 bladder cancer cells and verified as low in contaminants by Western blotting and flow cytometry of exosome-coated beads. Solubilization in a buffer containing SDS and DTT was essential for achieving proteomics analysis using an LC-MALDI-TOF/TOF MS approach. We report 353 high quality identifications with 72 proteins not previously identified by other human exosome proteomics studies. Overrepresentation analysis to compare this data set with previous exosome proteomics studies (using the ExoCarta database) revealed that the proteome was consistent with that of various exosomes with particular overlap with exosomes of carcinoma origin. Interrogating the Gene Ontology database highlighted a strong association of this proteome with carcinoma of bladder and other sites. The data also highlighted how homology among human leukocyte antigen haplotypes may confound MASCOT designation of major histocompatability complex Class I nomenclature, requiring data from PCR-based human leukocyte antigen haplotyping to clarify anomalous identifications. Validation of 18 MS protein identifications (including basigin, galectin-3, trophoblast glycoprotein (5T4), and others) was performed by a combination of Western blotting, flotation on linear sucrose gradients, and flow cytometry, confirming their exosomal expression. Some were confirmed positive on urinary exosomes from a bladder cancer patient. In summary, the exosome proteomics data set presented is of unrivaled quality. The data will aid in the development of urine exosome-based clinical tools for monitoring disease and will inform follow-up studies into varied aspects of exosome manufacture and function.Bladder cancer is one of the eight most frequent cancers in the Western world, and the frequency of transitional cell carcinoma (TCC),¹ which accounts for 90% of bladder cancers, is second only to prostate cancer as a malignancy of the genitourinary tract. Urine cytology and cystoscopy remain the predominant clinical tools for diagnosing and monitoring the disease, but cytology is poorly sensitive, particularly for low grade tumors, and does not serve as a prognostic tool. Cystoscopy is an invasive procedure, and there is pressing need to identify informative molecular markers that can be used to replace it.Recently, small cell-derived vesicles termed exosomes that are present in body fluids (1–5) have been proposed as a potential source of diagnostic markers (2, 6–8). These nanometer-sized vesicles, which are secreted by most cell types, originate from multivesicular bodies of the endocytic tract and reflect a subproteome of the cell. Exosomes are enriched in membrane and cytosolic proteins, and this molecular repertoire appears to be of particular functional importance to the immune system (9). Exosomes also comprise an array of lipids, mRNA, and microRNA, which are likely involved in conveying intercellular communication processes (10). Importantly, many exosomal components are simply not present as free soluble molecules in body fluids, such as certain microRNA species, which are encapsulated within the exosome lumen (6, 10). Therefore, the ability to isolate exosomes from urine (2), plasma (1), saliva (11), or other physiological sources (3) holds significant potential for obtaining novel and complex sets of biomarkers in a non-invasive manner. Exosome analysis may therefore be of value in disease diagnosis and monitoring in a variety of settings (6, 7, 12–14).Exosomes as indicators of pathology were first documented in the context of renal injury where a differential proteomics approach revealed changes in urinary exosome phenotype following renal injury (7). The researchers identified exosomally expressed Fetuin-A as a marker that became elevated 50-fold within hours following nephrotoxin exposure in rodents. Exosomal Fetuin-A elevation was also apparent in patients with acute renal injury before changes in urinary creatinine were observed (7). Clinical exosome analysis may also prove useful for solid cancers, such as ovarian or lung cancer, where the quantity of epithelial cell adhesion molecule-positive serum exosomes may correlate with tumor stage/grade. Such disease-associated exosomes express microRNA species not detected in healthy subjects (6, 12), although in this respect, there is little correlation between microRNA and disease bulk (6, 12). Other recent examples include studies of urinary exosomes in prostate cancer with exosomes expressing protein markers 5T4 (15), prostate cancer gene 3 (PCA-3) (8), or mRNA (TMPRSS2-ERG) (8, 16) associated with prostate cancer. To our knowledge, exosomes have not yet been studied in the context of other urological malignancies such as renal cancer, and to date, only one report describes the urine-derived microparticles from bladder cancer patients (17). In that report, they examined the proteome of a highly complex mixture of microvesicles, exosomes, and other urinary constituents that can be pelleted by high speed ultracentrifugation, identifying eight proteins that may be elevated in cancer. However, given the nature of the sample analyzed, it is unknown whether these proteins are exosomally expressed.Identification of the principal and most relevant molecular markers in these and other clinical scenarios remains a major challenge. In part, this is because exosomes present within complex body fluids originate from heterogeneous cell types. For example, plasma exosomes may be derived from platelets, lymphocytes, or endothelial cells (1), and a proportion may arise from well perfused organs such as the liver (18) and likely other organs as well (16). Similarly, exosomes present in urine arise from urothelial cells of the kidney and downstream of the renal tract (2, 8, 15).Importantly, all proteomics studies of exosomes isolated from body fluids are unavoidably complicated by the presence of high abundance non-exosomal proteins contaminating the preparations. Examples include albumin, immunoglobulin, and complement components present in exosomes prepared from malignant effusions (5) and Tamm-Horsfall protein present in exosomes purified from urine (2). As such, great care must be taken in the interpretation of the large data sets produced by proteomics studies, requiring careful validation of the proteins of interest. The protein composition of exosomes using a single homogenous cell type is one approach that may be used to uncover the protein components of exosomes produced by various cell types.There remain two major issues in the realm of exosome proteomics that complicate our interpretation of lists of identified proteins. Foremost are the diverse methods chosen for exosome purification that in some studies have involved attempts to remove contaminants through a key biophysical property of the vesicles, i.e. their capacity to float on sucrose (19, 20) or other dense media (21). Not all published studies, however, have taken such steps, preferring a far simpler pellet (or pellet and wash) approach. These latter preparations may be significantly contaminated by components of the cellular secretome, cell fragments, and other components. All of these factors could lead to false positive identifications of exosome proteins. The second key issue centers on the MS approaches utilized in various exosome proteomics studies. Many early examples relied only on a peptide mass fingerprinting approach, lacking robust peptide sequence data (22, 23), and more recently, search criteria that are generally recommended for MS-derived sequence data have not been specified in all studies. In this study, we have listed only those proteins identified by good quality MS/MS data for two or more peptides. Variability in the robustness and bias in bioinformatics analysis of data sets and in the steps taken to validate identified proteins is an additional factor that impacts the confidence in the identification lists produced.In this study, we aimed to perform the first proteomics analysis of human bladder cancer exosomes. We took extensive steps to produce high purity and quality-assured exosome preparations prior to beginning proteomics workflows. Solubilizing the sample with SDS and a reducing agent (DTT) was a critical step that allowed for global protein identification using nanoscale liquid chromatography followed by MALDI-TOF/TOF mass spectrometry. In this study, we present the identification of a significant number of exosomally expressed proteins (353 in total) of unrivaled quality. Critical manual examination of these identifications revealed issues with multiple (physiologically impossible) MHC Class I identifications that were attributed to a misdesignation of nomenclature by MASCOT due to peptide (and target protein) homology. The data were subjected to unbiased overrepresentation analysis (examining ExoCarta and Gene Ontology databases) to reveal a proteome consistent with exosomes, particularly of carcinoma origin. Validation of several identified proteins, by combining ultracentrifugation on a linear sucrose gradient with Western blotting and/or analysis of exosome-coated latex beads, demonstrated correct surface orientation of several MS-identified membrane proteins at densities consistent with exosomes.The robust approaches taken emphasize our confidence in the validity of the identifications generated and highlight that 72 (of 353) proteins have not been previously shown to be exosomally expressed by other human proteomics studies. The data will be useful for future studies in this underinvestigated disease and will form a platform not only for future clinical validation of some of these putative markers but also to aid further investigations into novel aspects of exosome function and manufacture.     

The endosymbiont Wolbachia pipientis induces the expression of host antioxidant proteins in an Aedes albopictus cell line

Wolbachia are obligate intracellular bacteria which commonly infect arthropods. They are maternally inherited and capable of altering host development, sex determination, and reproduction. Reproductive manipulations include feminization, male-killing, parthenogenesis, and cytoplasmic incompatibility. The mechanism by which Wolbachia avoid destruction by the host immune response is unknown. Generation of antimicrobial peptides (AMPs) and reactive oxygen species (ROS) by the host are among the first lines of traditional antimicrobial defense. Previous work shows no link between a Wolbachia infection and the induction of AMPs. Here we compare the expression of protein in a cell line naturally infected with Wolbachia and an identical cell line cured of the infection through the use of antibiotics. Protein extracts of each cell line were analyzed by two dimensional gel electrophoresis and LC/MS/MS. Our results show the upregulation of host antioxidant proteins, which are active against ROS generated by aerobic cell metabolism and during an immune response. Furthermore, flow cytometric and microscopic analysis demonstrates that ROS production is significantly greater in Wolbachia-infected mosquito cells and is associated with endosymbiont-containing vacuoles located in the host cell cytoplasm. This is the first empirical data supporting an association between Wolbachia and the insect antioxidant system.     

Mapping Brucellosis Increases Relative to Elk Density Using Hierarchical Bayesian Models

The relationship between host density and parasite transmission is central to the effectiveness of many disease management strategies. Few studies, however, have empirically estimated this relationship particularly in large mammals. We applied hierarchical Bayesian methods to a 19-year dataset of over 6400 brucellosis tests of adult female elk (Cervus elaphus) in northwestern Wyoming. Management captures that occurred from January to March were over two times more likely to be seropositive than hunted elk that were killed in September to December, while accounting for site and year effects. Areas with supplemental feeding grounds for elk had higher seroprevalence in 1991 than other regions, but by 2009 many areas distant from the feeding grounds were of comparable seroprevalence. The increases in brucellosis seroprevalence were correlated with elk densities at the elk management unit, or hunt area, scale (mean 2070 km²; range  = [95–10237]). The data, however, could not differentiate among linear and non-linear effects of host density. Therefore, control efforts that focus on reducing elk densities at a broad spatial scale were only weakly supported. Additional research on how a few, large groups within a region may be driving disease dynamics is needed for more targeted and effective management interventions. Brucellosis appears to be expanding its range into new regions and elk populations, which is likely to further complicate the United States brucellosis eradication program. This study is an example of how the dynamics of host populations can affect their ability to serve as disease reservoirs.     

Zebrafish aussicht mutant embryos exhibit widespread overexpression of ace (fgf8) and coincident defects in CNS development

During the development of the zebrafish nervous system both noi, a zebrafish pax2 homolog, and ace, a zebrafish fgf8 homolog, are required for development of the midbrain and cerebellum. Here we describe a dominant mutation, aussicht (aus), in which the expression of noi and ace is upregulated. In aus mutant embryos, ace is upregulated at many sites in the embryo, while noi expression is only upregulated in regions of the forebrain and midbrain which also express ace. Subsequent to the alterations in noi and ace expression, aus mutants exhibit defects in the differentiation of the forebrain, midbrain and eyes. Within the forebrain, the formation of the anterior and postoptic commissures is delayed and the expression of markers within the pretectal area is reduced. Within the midbrain, En and wnt1 expression is expanded. In heterozygous aus embryos, there is ectopic outgrowth of neural retina in the temporal half of the eyes, whereas in putative homozygous aus embryos, the ventral retina is reduced and the pigmented retinal epithelium is expanded towards the midline. The observation that aus mutant embryos exhibit widespread upregulation of ace raised the possibility that aus might represent an allele of the ace gene itself. However, by crossing carriers for both aus and ace, we were able to generate homozygous ace mutant embryos that also exhibited the aus phenotype. This indicated that aus is not tightly linked to ace and is unlikely to be a mutation directly affecting the ace locus. However, increased Ace activity may underly many aspects of the aus phenotype and we show that the upregulation of noi in the forebrain of aus mutants is partially dependent upon functional Ace activity. Conversely, increased ace expression in the forebrain of aus mutants is not dependent upon functional Noi activity. We conclude that aus represents a mutation involving a locus normally required for the regulation of ace expression during embryogenesis.     

Relationship of hydrogen peroxide production by Mycoplasma pulmonis to virulence for catalase-deficient mice

Mycoplasma pulmonis, an etiological agent of murine pneumonia, produced about 0.065 mumoles of hydrogen peroxide (H(2)O(2)) per hr per 10(10) colony-forming units. When glucose was present at a concentration of 0.01 m, H(2)O(2) production was increased by 50%. To determine if H(2)O(2) production by M. pulmonis could be correlated with virulence, normal, acatalasemic, and acatalatic mice were infected with the organism. Three days after infection with M. pulmonis significantly more acatalatic mice had pneumonia than did normal or acatalasemic mice. The pneumonia in acatalatic mice was also more severe than in the other two groups. Five days after infection, pneumonia in the acatalatic mice was resolved, whereas normal mice were severely affected. The presence of pneumonia and the severity were correlated with the recovery of M. pulmonis from the lesions. In vitro studies of the effect of catalase on M. pulmonis showed that exogenously supplied catalase stimulated the growth of M. pulmonis at 37 C and prolonged its survival at 25 C. Hemolysis of sheep blood, guinea pig blood, rabbit blood, and normal and acatalasemic mouse blood by M. pulmonis was inversely related to the catalase activity of the erythrocytes. These findings suggest that H(2)O(2) secretion contributes to the virulence of M. pulmonis and to the death of the microorganism in the absence of host catalase.