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1.  APOE-ε2 and APOE-ε4 Correlate with Increased Amyloid Accumulation in Cerebral Vasculature 
The APOE ε4 allele correlates with increased risk of Alzheimer disease (AD) and increased parenchymal amyloid plaques. We tested how the APOE genotype correlated with cerebral amyloid angiopathy (CAA) by analyzing 371 brains for parenchymal and meningeal CAA in 4 brain regions (frontal, parietal, temporal, and occipital neocortex). The overall severity of CAA was highest in the occipital lobe. APOE-ε4/4 brains (n = 22) had the highest levels of CAA across regions. In the occipital lobe, nearly all APOE-ε4/4 cases were scored with the highest level of CAA (meninges, 95% of cases; parenchyma, 81%). In this brain region as in others, APOE-ε3/4 brains (n = 115) showed consistently less CAA that APOE-ε4/4 brains (meninges, 43%; parenchyma, 43%). APOE-ε3/3 brains (n = 182) showed even less CAA (meninges, 19%; parenchyma, 19%). Interestingly, APOE-ε2/3 cases (n = 42) had more CAA than APOE-ε3/3 (meninges, 44%; parenchyma, 32%), despite a reduced risk for AD in the APOE-ε2/3 individuals. APOE-ε4/4 brains also had the fewest regions without CAA, whereas APOE-ε3/3 brains had the most. Ordinal regression analyses demonstrated significant impacts of APOE-ε2 and APOE-ε4 on CAA in at least some brain region. These data demonstrate that APOE genotype correlations with Ab deposition in CAA only incompletely correspond to other AD-linked brain pathologies.
PMCID: PMC3715146  PMID: 23771217
Alzheimer disease; Apolipoprotein; CAA; Dementia; Hemorrhagic stroke; Risk factor
2.  Prognostic Value of ERG Oncoprotein in Prostate Cancer Recurrence and Cause-Specific Mortality 
The Prostate  2013;73(9):905-912.
ETS-related gene (ERG) protein is present in 40–70% of prostate cancer and is correlated with TMPRSS2-ERG gene rearrangements. This study evaluated ERG expression at radical prostatectomy to determine whether it was predictive of earlier relapse or prostate cancer-specific mortality (PCSM).
One hundred patients who underwent radical prostatectomy at Virginia Mason in Seattle between 1991 and 1997 were identified. Recurrence was confirmed by tissue diagnosis or radiographic signs. PCSM was confirmed by death certificates. Thirty-three patients with metastases or PCSM were matched to patients without recurrence at a 1:2 ratio. Paraffin embedded tissue was stained with two anti-ERG monoclonal antibodies, EPR3864 and 9FY. Nuclear expression intensity was evaluated as present/absent, on a 4-point relative intensity scale, and as a composite score (0–300).
Mean follow-up was 10.26 years. The two antibodies were highly correlated (P < 0.0001). Patients with higher ERG expression intensity and composite scores were significantly more likely to develop biochemical relapse, metastases, and PCSM. Kaplan–Meier survival curve analysis for the composite score of ERG expression revealed a significant association between higher ERG expression (EPR3864) and shorter PCa-specific survival (P = 0.047).
While the presence of ERG expression at the time of surgery was not predictive of earlier relapse or PCSM, the relative intensity and composite score for ERG expression was prognostic for the development of biochemical relapse, metastases, and PCSM. Quantitative ERG scoring may be useful to identify patients who would benefit from adjuvant treatment or closer follow-up, allowing more accurate individual patient treatment plans.
PMCID: PMC3677047  PMID: 23334893
TMPRSS2-ERG fusion protein; biomarker; metastasis; survival
3.  PPP2R2C loss promotes castration-resistance and is associated with increased prostate cancer-specific mortality 
Molecular cancer research : MCR  2013;11(6):568-578.
Metastatic prostate cancers generally rely on androgen receptor (AR) signaling for growth and survival, even following systemic androgen deprivation therapy (ADT). However, recent evidence suggests that some advanced prostate cancers escape ADT by utilizing signaling programs and growth factors that bypass canonical AR ligand-mediated mechanisms. We utilized an in vitro high-throughput RNAi screen to identify pathways in androgen-dependent prostate cancer cell lines whose loss of function promotes androgen ligand-independent growth. We identified 40 genes where knockdown promoted proliferation of both LNCaP and VCaP prostate cancer cells in the absence of androgen. Of these, 14 were down-regulated in primary and metastatic prostate cancer, including two subunits of the protein phosphatase 2 (PP2A) holoenzyme complex: PPP2R1A, a structural subunit with known tumor-suppressor properties in several tumor types; and PPP2R2C, a PP2A substrate-binding regulatory subunit that has not been previously identified as a tumor suppressor. We demonstrate that loss of PPP2R2C promotes androgen ligand depletion-resistant prostate cancer growth without altering AR expression or canonical AR-regulated gene expression. Furthermore, cell proliferation induced by PPP2R2C loss was not inhibited by the AR antagonist MDV3100, indicating that PPP2R2C loss may promote growth independently of known AR-mediated transcriptional programs. Immunohistochemical analysis of PPP2R2C protein levels in primary prostate tumors determined that low PPP2R2C expression significantly associated with an increased likelihood of cancer recurrence and cancer-specific mortality. These findings provide insights into mechanisms by which prostate cancers resist AR-pathway suppression, and support inhibiting PPP2R2C complexes or the growth pathway(s) activated by PPP2R2C as a therapeutic strategy.
PMCID: PMC3687002  PMID: 23493267
PP2A; PPP2R2C; castration-resistant prostate cancer; androgen-pathway independence
4.  Urinary TMPRSS2:ERG and PCA3 in an active surveillance cohort: results from a baseline analysis in the Canary Prostate Active Surveillance Study 
Active surveillance is used to manage low risk prostate cancer. Both PCA3 and TMRPSS2-ERG are promising biomarkers that may be associated with aggressive disease. This study examines the correlation of these biomarkers with higher cancer volume and grade determined at the time of biopsy in an active surveillance cohort.
Experimental Design
Post-DRE urine was collected prospectively as part of the multi-institutional Canary Prostate Active Surveillance Study (PASS). PCA3 and TMPRSS2-ERG levels were analyzed in urine collected at study entry. Biomarker scores were correlated to clinical and pathologic variables.
In 387 men, both PCA3 and TMPRSS2-ERG scores were significantly associated with higher volume disease. For a negative repeat biopsy, and 1–10%, 11–33%, ≥34% positive cores, median PCA3 and TMPRSS2-ERG scores increased incrementally (P < 0.005). Both PCA3 and TMPRSS2-ERG scores were also significantly associated with presence of high grade disease. For a negative repeat biopsy, Gleason 6 and Gleason ≥7 cancers, the median PCA3 and TMPRSS2-ERG scores also increased incrementally (P = 0.02 and P = 0.001, respectively). Using the marker scores as a continuous variables, the odds ratio for a biopsy in which cancer was detected versus a negative repeat biopsy (ref) on modeling was 1.41 (95% CI 1.07–1.85), P = 0.01 for PCA3 and 1.28 (95% CI 1.10–1.49), P = 0.001 for TMPRSS2-ERG.
For men on active surveillance both PCA3 and TMPRSS2-ERG appear to stratify risk of having aggressive cancer as defined by tumor volume or Gleason score.
PMCID: PMC3674574  PMID: 23515404
prostate cancer; active surveillance; biomarkers
5.  Using mice to model Alzheimer's dementia: an overview of the clinical disease and the preclinical behavioral changes in 10 mouse models 
The goal of this review is to discuss how behavioral tests in mice relate to the pathological and neuropsychological features seen in human Alzheimer's disease (AD), and present a comprehensive analysis of the temporal progression of behavioral impairments in commonly used AD mouse models that contain mutations in amyloid precursor protein (APP). We begin with a brief overview of the neuropathological changes seen in the AD brain and an outline of some of the clinical neuropsychological assessments used to measure cognitive deficits associated with the disease. This is followed by a critical assessment of behavioral tasks that are used in AD mice to model the cognitive changes seen in the human disease. Behavioral tests discussed include spatial memory tests [Morris water maze (MWM), radial arm water maze (RAWM), Barnes maze], associative learning tasks (passive avoidance, fear conditioning), alternation tasks (Y-Maze/T-Maze), recognition memory tasks (Novel Object Recognition), attentional tasks (3 and 5 choice serial reaction time), set-shifting tasks, and reversal learning tasks. We discuss the strengths and weaknesses of each of these behavioral tasks, and how they may correlate with clinical assessments in humans. Finally, the temporal progression of both cognitive and non-cognitive deficits in 10 AD mouse models (PDAPP, TG2576, APP23, TgCRND8, J20, APP/PS1, TG2576 + PS1 (M146L), APP/PS1 KI, 5×FAD, and 3×Tg-AD) are discussed in detail. Mouse models of AD and the behavioral tasks used in conjunction with those models are immensely important in contributing to our knowledge of disease progression and are a useful tool to study AD pathophysiology and the resulting cognitive deficits. However, investigators need to be aware of the potential weaknesses of the available preclinical models in terms of their ability to model cognitive changes observed in human AD. It is our hope that this review will assist investigators in selecting an appropriate mouse model, and accompanying behavioral paradigms to investigate different aspects of AD pathology and disease progression.
PMCID: PMC4005958  PMID: 24795750
Alzheimer's disease; mouse models; neuropsychological assessment; behavior; cognition; APP mice; APP/PS1 mice; 3×TG-AD mice
6.  Neuroinflammatory phenotype in early Alzheimer’s disease 
Neurobiology of aging  2012;34(4):1051-1059.
Alzheimer’s disease (AD) involves progressive neurodegeneration in the presence of misfolded proteins and poorly-understood inflammatory changes. However, research has shown that AD is genetically, clinically and pathologically heterogeneous.
In frozen brain samples of frontal cortex (diseased) and cerebellum (non-diseased) from the University of Kentucky Alzheimer’s Disease Center autopsy cohort, we performed gene expression analysis for genes categorizing inflammatory states (termed M1 and M2) from early and late stage AD, and age-matched non-demented controls. We performed analysis of the serum samples for a profile of inflammatory proteins and examined the neuropathological data on these samples.
Striking heterogeneity was found in early AD. Specifically, early-stage AD brain samples indicated apparent polarization toward either the M1 or M2 brain inflammatory states when compared to age-matched non-disease control tissue. This polarization was observed in the frontal cortex and not in cerebellar tissue. We were able to detect both differences in AD neuropathology, and changes in serum proteins that distinguished the individuals apparent M1 versus M2 brain inflammatory polarization.
PMCID: PMC3579221  PMID: 23062700
7.  CSF1R mutations link POLD and HDLS as a single disease entity 
Neurology  2013;80(11):1033-1040.
Pigmented orthochromatic leukodystrophy (POLD) and hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS) are rare neurodegenerative disorders characterized by cerebral white matter abnormalities, myelin loss, and axonal swellings. The striking overlap of clinical and pathologic features of these disorders suggested a common pathogenesis; however, no genetic or mechanistic link between POLD and HDLS has been established. Recently, we reported that mutations in the colony-stimulating factor 1 receptor (CSF1R) gene cause HDLS. In this study, we determined whether CSF1R mutations are also a cause of POLD.
We performed sequencing of CSF1R in 2 pathologically confirmed POLD families. For the largest family (FTD368), a detailed case report was provided and brain samples from 2 affected family members previously diagnosed with POLD were re-evaluated to determine whether they had HDLS features. In vitro functional characterization of wild-type and mutant CSF1R was also performed.
We identified CSF1R mutations in both POLD families: in family 5901, we found c.2297T>C (p.M766T), previously reported by us in HDLS family CA1, and in family FTD368, we identified c.2345G>A (p.R782H), recently reported in a biopsy-proven HDLS case. Immunohistochemical examination in family FTD368 showed the typical neuronal and glial findings of HDLS. Functional analyses of CSF1R mutant p.R782H (identified in this study) and p.M875T (previously observed in HDLS), showed a similar loss of CSF1R autophosphorylation of selected tyrosine residues in the kinase domain for both mutations when compared with wild-type CSF1R.
We provide the first genetic and mechanistic evidence that POLD and HDLS are a single clinicopathologic entity.
PMCID: PMC3653204  PMID: 23408870
8.  Stromal Targeting of Sodium Iodide Symporter Using Mesenchymal Stem Cells Allows Enhanced Imaging and Therapy of Hepatocellular Carcinoma 
Human Gene Therapy  2013;24(3):306-316.
The tumor-homing property of mesenchymal stem cells (MSC) has lead to their use as delivery vehicles for therapeutic genes. The application of the sodium iodide symporter (NIS) as therapy gene allows noninvasive imaging of functional transgene expression by 123I-scintigraphy or PET-imaging, as well as therapeutic application of 131I or 188Re. Based on the critical role of the chemokine RANTES (regulated on activation, normal T-cell expressed and presumably secreted)/CCL5 secreted by MSCs in the course of tumor stroma recruitment, use of the RANTES/CCL5 promoter should allow tumor stroma-targeted expression of NIS after MSC-mediated delivery. Using a human hepatocellular cancer (HCC) xenograft mouse model (Huh7), we investigated distribution and tumor recruitment of RANTES-NIS-engineered MSCs after systemic injection by gamma camera imaging. 123I-scintigraphy revealed active MSC recruitment and CCL5 promoter activation in the tumor stroma of Huh7 xenografts (6.5% ID/g 123I, biological half-life: 3.7 hr, tumor-absorbed dose: 44.3 mGy/MBq). In comparison, 7% ID/g 188Re was accumulated in tumors with a biological half-life of 4.1 hr (tumor-absorbed dose: 128.7 mGy/MBq). Administration of a therapeutic dose of 131I or 188Re (55.5 MBq) in RANTES-NIS-MSC-treated mice resulted in a significant delay in tumor growth and improved survival without significant differences between 131I and 188Re. These data demonstrate successful stromal targeting of NIS in HCC tumors by selective recruitment of NIS-expressing MSCs and by use of the RANTES/CCL5 promoter. The resulting tumor-selective radionuclide accumulation was high enough for a therapeutic effect of 131I and 188Re opening the exciting prospect of NIS-mediated radionuclide therapy of metastatic cancer using genetically engineered MSCs as gene delivery vehicles.
Knoop and colleagues demonstrate selective recruitment of mesenchymal stem cells (MSCs) expressing sodium iodide symporter (NIS) under the control of the RANTES/CCL5 promoter (RANTES-NIS-MSCs) into xenografted tumors in a human hepatocellular cancer mouse model. Administration of a therapeutic dose of either 131I or 188Re radionuclide results in significantly delayed tumor growth and improves survival in mice treated with RANTES-NIS-MSCs.
PMCID: PMC3609610  PMID: 23402366
9.  Renal dendritic cells: an update 
Nephron. Experimental nephrology  2009;111(3):e67-e71.
Discovery into the role of renal dendritic cells (rDCs) in health and disease of the kidney is rapidly accelerating. Progress in deciphering DC precursors and the heterogeneity of monocyte subsets in mice and humans are providing insights into the biology of rDCs. Recent findings have extended knowledge of the origins, anatomy, and function of the rDC network at steady-state and during periods of injury to the renal parenchyma. This brief review highlights these new findings and provides an update on the study of rDCs.
PMCID: PMC3932375  PMID: 19276627
10.  CD33 Alzheimer's Risk-Altering Polymorphism, CD33 Expression, and Exon 2 Splicing 
The Journal of Neuroscience  2013;33(33):13320-13325.
Genome-wide association studies are identifying novel Alzheimer's disease (AD) risk factors. Elucidating the mechanism underlying these polymorphisms is critical to the validation process and, by identifying rate-limiting steps in AD risk, may yield novel therapeutic targets. Here, we elucidate the mechanism of action of the AD-associated polymorphism rs3865444 in the promoter of CD33, a member of the sialic acid-binding Ig-superfamily of lectins (SIGLECs). Immunostaining established that CD33 is expressed in microglia in human brain. Consistent with this finding, CD33 mRNA expression correlated well with expression of the microglial genes CD11b and AIF-1 and was modestly increased with AD status and the rs3865444C AD-risk allele. Analysis of CD33 isoforms identified a common isoform lacking exon 2 (D2-CD33). The proportion of CD33 expressed as D2-CD33 correlated robustly with rs3865444 genotype. Because rs3865444 is in the CD33 promoter region, we sought the functional polymorphism by sequencing CD33 from the promoter through exon 4. We identified a single polymorphism that is coinherited with rs3865444, i.e., rs12459419 in exon 2. Minigene RNA splicing studies in BV2 microglial cells established that rs12459419 is a functional single nucleotide polymorphism (SNP) that modulates exon 2 splicing efficiency. Thus, our primary findings are that CD33 is a microglial mRNA and that rs3865444 is a proxy SNP for rs12459419 that modulates CD33 exon 2 splicing. Exon 2 encodes the CD33 IgV domain that typically mediates sialic acid binding in SIGLEC family members. In summary, these results suggest a novel model wherein SNP-modulated RNA splicing modulates CD33 function and, thereby, AD risk.
PMCID: PMC3742922  PMID: 23946390
11.  APOE ε4 Increases Risk for Dementia in Pure Synucleinopathies 
JAMA neurology  2013;70(2):223-228.
To test for an association between the apolipoprotein E (APOE) ε4 allele and dementias with synucleinopathy.
Genetic case-control association study.
Academic research.
Autopsied subjects were classified into 5 categories: dementia with high-level Alzheimer disease (AD) neuropathologic changes (NCs) but without Lewy body disease (LBD) NCs (AD group; n=244), dementia with LBDNCs and high-level ADNCs (LBD-AD group; n=224), dementia with LBDNCs and no or low levels of ADNCs (pure DLB [pDLB] group; n=91), Parkinson disease dementia (PDD) with no or low levels of ADNCs (n=81), and control group (n=269).
Main Outcome Measure
The APOE allele frequencies.
The APOE ε4 allele frequency was significantly higher in the AD (38.1%), LBD-AD (40.6%), pDLB (31.9%), and PDD (19.1%) groups compared with the control group (7.2%; overall χ42=185.25; P=5.56×10−39), and it was higher in the pDLB group than the PDD group (P=.01). In an age-adjusted and sex-adjusted dominant model, ε4 was strongly associated with AD (odds ratio, 9.9; 95% CI, 6.4–15.3), LBD-AD (odds ratio, 12.6; 95% CI, 8.1–19.8), pDLB (odds ratio, 6.1; 95% CI, 3.5–10.5), and PDD (odds ratio, 3.1; 95% CI, 1.7–5.6).
The APOE ε4 allele is a strong risk factor across the LBD spectrum and occurs at an increased frequency in pDLB relative to PDD. This suggests that ε4 increases the likelihood of presenting with dementia in the context of a pure synucleinopathy. The elevated ε4 frequency in the pDLB and PDD groups, in which the overall brain neuritic plaque burden was low, indicates that apoE might contribute to neurodegeneration through mechanisms unrelated to amyloid processing.
PMCID: PMC3580799  PMID: 23407718
12.  A Novel 4-color Fluorescence in Situ Hybridization Assay for Detection of TMPRSS2 and ERG Rearrangements in Prostate Cancer 
Cancer genetics  2013;206(0):1-11.
Since the identification of TMPRSS2/ERG rearrangement as the most common fusion event in prostate cancer, various methods have been developed to detect this rearrangement and to study its prognostic significance. We hereby report a novel 4-color fluorescence in situ hybridization (FISH) assay that not only detects the typical TMPRSS2:ERG fusion but also alternative rearrangements of either the TMPRSS2 or ERG gene.
Experimental design
We validated this assay on fresh, frozen, or formalin-fixed paraffin-embedded prostate cancer specimens including cell lines, primary prostate cancer, xenograft tissues derived from metastatic prostate cancer, and metastatic tissues from castration-resistant prostate cancer (CRPC) patients.
When compared with RT-PCR or Gen-Probe method as the technical reference, the 4-color FISH assay demonstrated an analytical sensitivity of 94.5% (95% Confidence Interval [CI] 0.80-0.99) and specificity of 100% (95% CI 0.89-1.00) for detecting TMPRSS2:ERG fusion. TMPRSS2:ERG fusion was detected at 41% and 43% in primary prostate cancer (n = 59) and CRPC tumors (n = 82), respectively. Alternative rearrangements other than the typical TMPRSS2:ERG fusion were confirmed by karyotype analysis and shown present in 7% primary cancer and 13% CRPC tumors. Successful karyotype analysis is reported for the first time on four of the xenograft samples, complementing the FISH results.
This 4-color FISH assay provides sensitive detection of TMPRSS2 and ERG gene rearrangements in prostate cancer.
PMCID: PMC3632390  PMID: 23352841
Prostate cancer; TMPRSS2; ERG; FISH
13.  A model for the design and construction of a resource for the validation of prognostic prostate cancer biomarkers: the Canary Prostate Cancer Tissue Microarray 
Advances in anatomic pathology  2013;20(1):39-44.
Tissue microarrays provide unique resources for rapid evaluation and validation of tissue biomarkers. The Canary Foundation Retrospective Prostate Tissue Microarray Resource used a rigorous statistical design, quota sampling, a variation of the case-cohort study, to select patients for inclusion in a multicenter, retrospective prostate cancer tissue microarray cohort. The study is designed to definitively validate tissue biomarkers of prostate cancer recurrence after radical prostatectomy. Tissue samples from over 1,000 participants treated for prostate cancer with radical prostatectomy between 1995 and 2004 were selected at six participating institutions in the United States and Canada. This design captured the heterogeneity of screening and clinical practices in the contemporary North American population. Standardized clinical data were collected in a centralized database. The project has been informative in several respects. The scale and complexity of assembling tissue microarrays (TMAs) with over 200 cases at each of six sites involved unanticipated levels of effort and time. Our statistical design promises to provide a model for outcome-based studies where tissue localization methods are applied to high-density tissue microarrays.
PMCID: PMC3535290  PMID: 23232570
Prostate Cancer; Prognosis; Tissue Microarray; quota sampling
14.  A Study of Small RNAs from Cerebral Neocortex of Pathology-Verified Alzheimer’s Disease, Dementia with Lewy Bodies, Hippocampal Sclerosis, Frontotemporal Lobar Dementia, and Non-Demented Human Controls 
MicroRNAs (miRNAs) are small (20–22 nucleotides) regulatory non-coding RNAs that strongly influence gene expression. Most prior studies addressing the role of miRNAs in neurodegenerative diseases (NDs) have focused on individual diseases such as Alzheimer’s disease (AD), making disease-to-disease comparisons impossible. Using RNA deep sequencing, we sought to analyze in detail the small RNAs (including miRNAs) in the temporal neocortex gray matter from non-demented controls (n = 2), AD (n = 5), dementia with Lewy bodies (n = 4), hippocampal sclerosis of aging (n = 4), and frontotemporal lobar dementia (FTLD) (n = 5) cases, together accounting for the most prevalent ND subtypes. All cases had short postmortem intervals, relatively high-quality RNA, and state-of-the-art neuropathological diagnoses. The resulting data (over 113 million reads in total, averaging 5.6 million reads per sample) and secondary expression analyses constitute an unprecedented look into the human cerebral cortical miRNome at single nucleotide resolution. While we find no apparent changes in isomiR or miRNA editing patterns in correlation with ND pathology, our results validate and extend previous miRNA profiling studies with regard to quantitative changes in NDs. In agreement with this idea, we provide independent cohort validation for changes in miR-132 expression levels in AD (n = 8) and FTLD (n = 14) cases when compared to controls (n = 8). The identification of common and ND-specific putative novel brain miRNAs and/or short-hairpin molecules is also presented. The challenge now is to better understand the impact of these and other alterations on neuronal gene expression networks and neuropathologies.
PMCID: PMC3753694  PMID: 23403535
Alzheimer’s disease; deep sequencing; dementia with Lewy bodies; frontotemporal lobar dementia; hippocampal sclerosis; isomiR; microRNA; progressive supranuclear palsy
15.  Tmprss2 Is Essential for Influenza H1N1 Virus Pathogenesis in Mice 
PLoS Pathogens  2013;9(12):e1003774.
Annual influenza epidemics and occasional pandemics pose a severe threat to human health. Host cell factors required for viral spread but not for cellular survival are attractive targets for novel approaches to antiviral intervention. The cleavage activation of the influenza virus hemagglutinin (HA) by host cell proteases is essential for viral infectivity. However, it is unknown which proteases activate influenza viruses in mammals. Several candidates have been identified in cell culture studies, leading to the concept that influenza viruses can employ multiple enzymes to ensure their cleavage activation in the host. Here, we show that deletion of a single HA-activating protease gene, Tmprss2, in mice inhibits spread of mono-basic H1N1 influenza viruses, including the pandemic 2009 swine influenza virus. Lung pathology was strongly reduced and mutant mice were protected from weight loss, death and impairment of lung function. Also, after infection with mono-basic H3N2 influenza A virus body weight loss and survival was less severe in Tmprss2 mutant compared to wild type mice. As expected, Tmprss2-deficient mice were not protected from viral spread and pathology after infection with multi-basic H7N7 influenza A virus. In conclusion, these results identify TMPRSS2 as a host cell factor essential for viral spread and pathogenesis of mono-basic H1N1 and H3N2 influenza A viruses.
Author Summary
Seasonal influenza epidemics and pandemics represent a serious health threat to the human population. Resistance to presently available anti-viral drugs is frequently observed. Therefore, identification of new targets for anti-viral therapy is an urgent need. Host proteases are required for processing of the virus hemagglutinin and may thus represent a suitable target for intervention. Here, we report that the deletion of a single host protease gene, Tmprss2, in mice protects the host against viral spread in infected lungs. Only very mild pathogenesis was observed in Tmprss2 mutant mice after infection with H1N1 virus and less severe pathogenesis was observed after infection with H3N2 virus. Thus, our results suggest that the host protease TMPRSS2 may be a prime target for antiviral intervention.
PMCID: PMC3857797  PMID: 24348248
16.  Prostate cancer cell phenotypes based on AGR2 and CD10 expression 
The combination of expression patterns of AGR2 and CD10 by prostate cancer provided four phenotypes that correlated with clinical outcome. Based on immunophenotyping, CD10lowAGR2high, CD10highAGR2high, CD10lowAGR2low, and CD10highAGR2low were distinguished. AGR2+ tumors were associated with longer recurrence-free survival and CD10+ tumors with shorter recurrence-free survival. In high-stage cases, the CD10lowAGR2high phenotype was associated with a 9-fold higher recurrence-free survival than the CD10highAGR2low phenotype. The CD10highAGR2high and CD10lowAGR2low phenotypes were intermediate. The CD10highAGR2low phenotype was most frequent in high-grade primary tumors. Conversely, bone and other soft tissue metastases, and derivative xenografts, expressed more AGR2 and less CD10. AGR2 protein was readily detected in tumor metastases. The CD10highAGR2low phenotype in primary tumors is predictive of poor outcome; however, the CD10lowAGR2high phenotype is more common in metastases. It appears that AGR2 has a protective function in primary tumors but may have a role in the distal spread of tumor cells.
PMCID: PMC3638070  PMID: 23348903
Prostate cancer; AGR2; CD10; cancer cell phenotypes; patient stratification; bone and soft tissue metastases; xenografts
17.  Digital Pathology and Image Analysis for Robust High-Throughput Quantitative Assessment of Alzheimer Disease Neuropathologic Changes 
Quantitative neuropathologic methods provide information that is important for both research and clinical applications. The technological advancement of digital pathology and image analysis offers new solutions to enable valid quantification of pathological severity that is reproducible between raters regardless of experience. Using an Aperio ScanScope XT and its accompanying image analysis software, we designed algorithms for quantitation of amyloid and tau pathologies on 65 β-amyloid (6F/3D antibody) and 48 phospho-tau (PHF-1)-immunostained sections of human temporal neocortex. Quantitative digital pathologic data were compared with manual pathology counts. There were excellent correlations between manually counted and digitally analyzed neuropathologic parameters (R2 values 0.56-0.72). Data were highly reproducible among 3 participants with varying degrees of expertise in neuropathology (Intra-class correlation coefficient values >0.910). Digital quantification also provided additional parameters, including average plaque area, which show statistically significant differences when samples are stratified according to APOE allele status (average plaque area 380.9 μm2 in ApoE ε4 carriers vs. 274.4 μm2 for non-carriers, p < 0.001). Thus, digital pathology offers a rigorous and reproducible method for quantifying AD neuropathologic changes and may provide additional insight into morphologic characteristics that were previously more challenging to assess due to technical limitations.
PMCID: PMC3511606  PMID: 23147505
Alzheimer disease; Autopsy; Digital pathology; Image analysis; Neuropathology
18.  Canary Prostate Active Surveillance Study (PASS); Design of a Multi-institutional Active Surveillance Cohort and Biorepository 
Urology  2009;75(2):10.1016/j.urology.2009.05.050.
Active surveillance is a management plan for localized prostate cancer that offers selective delayed intervention upon indication of disease progression, allowing patients to delay or avoid treatment and associated side-effects. Outcomes from centers that promote active surveillance are favorable, with high rates of disease-specific survival. However, there remains a need for prognostic variables or biomarkers that distinguish with high specificity the aggressive cancers that progress on surveillance from the indolent cancers. The Canary Prostate Active Surveillance Study(PASS) is a multicenter study and biorepository that will discover and confirm biomarkers of aggressive disease as defined by histologic, PSA, or clinical criteria.
PMCID: PMC3827723  PMID: 19758683
Prostate cancer; Active Surveillance; Clinical Trial
19.  GBA mutations increase risk for Lewy body disease with and without Alzheimer disease pathology 
Neurology  2012;79(19):1944-1950.
Mutations in the GBA gene occur in 7% of patients with Parkinson disease (PD) and are a well-established susceptibility factor for PD, which is characterized by Lewy body disease (LBD) neuropathologic changes (LBDNCs). We sought to determine whether GBA influences risk of dementia with LBDNCs, Alzheimer disease (AD) neuropathologic changes (ADNCs), or both.
We screened the entire GBA coding region for mutations in controls and in subjects with dementia and LBDNCs and no or low levels of ADNCs (pure dementia with Lewy bodies [pDLB]), LBDNCs and high-level ADNCs (LBD-AD), and high-level ADNCs but without LBDNCs (AD).
Among white subjects, pathogenic GBA mutations were identified in 6 of 79 pDLB cases (7.6%), 8 of 222 LBD-AD cases (3.6%), 2 of 243 AD cases (0.8%), and 3 of 381 controls (0.8%). Subjects with pDLB and LBD-AD were more likely to carry mutations than controls (pDLB: odds ratio [OR] = 7.6; 95% confidence interval [CI] = 1.8–31.9; p = 0.006; LBD-AD: OR = 4.6; CI = 1.2–17.6; p = 0.025), but there was no significant difference in frequencies between the AD and control groups (OR = 1.1; CI = 0.2–6.6; p = 0.92). There was a highly significant trend test across groups (χ2(1) = 19.3; p = 1.1 × 10−5), with the likelihood of carrying a GBA mutation increasing in the following direction: control/AD < LBD-AD < pDLB.
GBA is a susceptibility gene across the LBD spectrum, but not in AD, and appears to convey a higher risk for PD and pDLB than for LBD-AD. PD and pDLB might be more similar to one another in genetic determinants and pathophysiology than either disease is to LBD-AD.
PMCID: PMC3484986  PMID: 23035075
20.  The Monoamine Oxidase A Gene Promoter Repeat and Prostate Cancer Risk 
The Prostate  2012;72(15):1622-1627.
Amine catabolism by Monoamine Oxidase A (MAOA) contributes to oxidative stress, which plays a role in prostate cancer (PCa) development and progression. An upstream variable-number tandem repeat (uVNTR) in the MAOA promoter influences gene expression and activity, and may thereby affect PCa susceptibility.
Caucasian (n=2,572) men from two population-based case-control studies of PCa were genotyped for the MAOA-VNTR. Logistic regression was used to assess PCa risk in relation to genotype.
Common alleles of the MAOA-VNTR were not associated with the relative risk of PCa, nor did the relationship differ by clinical features of the disease. The rare 5-copy variant (frequency: 0.5% in cases; 1.8% in controls), however, was associated with a reduced PCa risk (odds ratio, OR=0.30, 95% CI 0.13–0.71).
A rare polymorphism of the MAOA promoter previously shown to confer low expression was associated with a reduced risk of developing prostate cancer. This novel finding awaits confirmation in other study populations.
PMCID: PMC3532925  PMID: 22473857
MAOA; polymorphism; prostate cancer
21.  The MAPT H1 haplotype is associated with tangle-predominant dementia 
Acta neuropathologica  2012;124(5):693-704.
Tangle-predominant dementia (TPD) patients exhibit cognitive decline that is clinically similar to early to moderate-stage Alzheimer disease (AD), yet autopsy reveals neurofibrillary tangles in the medial temporal lobe composed of the microtubule-associated protein tau without significant amyloid-beta (Aβ)-positive plaques. We performed a series of neuropathological, biochemical and genetic studies using autopsy brain tissue drawn from a cohort of 34 TPD, 50 AD and 56 control subjects to identify molecular and genetic signatures of this entity. Biochemical analysis demonstrates a similar tau protein isoform composition in TPD and AD, which is compatible with previous histological and ultrastructural studies. Further, biochemical analysis fails to uncover elevation of soluble Aβ in TPD frontal cortex and hippocampus compared to control subjects, demonstrating that non-plaque-associated Aβ is not a contributing factor. Unexpectedly, we also observed high levels of secretory amyloid precursor protein α (sAPPα) in the frontal cortex of some TPD patients compared to AD and control subjects, suggesting differences in APP processing. Finally, we tested whether TPD is associated with changes in the tau gene (MAPT). Haplotype analysis demonstrates a strong association between TPD and the MAPT H1 haplotype, a genomic inversion associated with some tauopathies and Parkinson disease (PD), when compared to age-matched control subjects with mild degenerative changes, i.e., successful cerebral aging. Next-generation resequencing of MAPT followed by association analysis shows an association between TPD and two polymorphisms in the MAPT 3′ untranslated region (UTR). These results support the hypothesis that haplotype-specific variation in the MAPT 3′ UTR underlies an Aβ-independent mechanism for neurodegeneration in TPD.
PMCID: PMC3608475  PMID: 22802095
Dementia; Neurofibrillary tangle; Tau; Amyloid; MAPT; 3′ Untranslated region; Aging; Alzheimer’s disease; sAPPα
22.  Retinoids Augment the Expression of Podocyte Proteins by Glomerular Parietal Epithelial Cells in Experimental Glomerular Disease 
Nephron. Experimental nephrology  2012;121(1-2):e23-e37.
A decrease in glomerular podocyte number in membranous nephropathy and FSGS ultimately precipitates glomerulosclerosis and the decrease in kidney function. Recent studies have shown that in these diseases, glomerular parietal epithelial cells begin to express proteins considered unique to podocytes, and that these glomerular epithelial transition cells might serve as podocyte progenitors. Because retinoids improve many forms of experimental glomerular disease characterized by podocyte injury and loss, we asked if ATRA induces parietal epithelial cells to express podocyte proteins.
ATRA or vehicle was administered to rats with experimental membranous nephropathy (PHN model) and mice with experimental FSGS (anti-glomerular antibody model) following the onset of proteinuria. Immunohistochemistry staining of PAX2 (parietal epithelial cell marker), WT-1 (podocyte cell marker), and Ki-67 (proliferation marker) were performed on kidney tissues.
Compared to diseased animals receiving vehicle, ATRA statistically significantly increased the number of glomerular transition cells, defined as cells double staining for PAX2 and WT-1, in membranous nephropathy at weeks 2, 5 and 16, and in FSGS at weeks 1 and 2. This was accompanied by an increase in the number of podocytes compared to diseased controls receiving vehicle.
ATRA increases the number of glomerular epithelial transition cells in experimental proteinuric glomerular diseases. Thus, ATRA may provide a useful pharmacologic approach to decipher the mechanisms underlying the possible progenitor role of parietal epithelial cells.
PMCID: PMC3574166  PMID: 23107969
all-trans retinoic acid; proteinuria; passive Heymann nephritis; membranous nephropathy; focal segmental glomerulosclerosis
23.  Membrane-anchored chemokine fusion proteins 
Oncoimmunology  2013;2(11):e26619.
Adoptive or active cancer immunotherapy can fail owing to the inefficient recruitment of effector leukocytes to malignant lesions. The intratumoral injection of recombinant proteins comprising a chemokine-derived domain linked to the mucin stalk of chemokine (C-X3-C motif) ligand 1 (CX3CL1) and a glycosylphosphatidylinositol anchor can specifically enhance the recruitment of effector cell subsets to solid tumors.
PMCID: PMC3887060  PMID: 24427544
active immunotherapy; adoptive immunotherapy; cancer therapy; chemokine; endothelial anergy; GPI anchor; leukocyte recruitment
24.  Design of the Nephrotic Syndrome Study Network (NEPTUNE) to evaluate primary glomerular nephropathy by a multi-disciplinary approach 
Kidney international  2013;83(4):749-756.
The Nephrotic Syndrome Study Network (NEPTUNE) is a North American multi-center collaborative consortium established to develop a translational research infrastructure for Nephrotic Syndrome. This includes a longitudinal observational cohort study, a pilot and ancillary studies program, a training program, and a patient contact registry. NEPTUNE will enroll 450 adults and children with minimal change disease, focal segmental glomerulosclerosis and membranous nephropathy for detailed clinical, histopathologic, and molecular phenotyping at the time of clinically-indicated renal biopsy. Initial visits will include an extensive clinical history, physical examination, collection of urine, blood and renal tissue samples, and assessments of quality of life and patient-reported outcomes. Follow-up history, physical measures, urine and blood samples, and questionnaires will be obtained every 4 months in the first year and bi-annually, thereafter. Molecular profiles and gene expression data will be linked to phenotypic, genetic, and digitalized histologic data for comprehensive analyses using systems biology approaches. Analytical strategies were designed to transform descriptive information to mechanistic disease classification for Nephrotic Syndrome and to identify clinical, histological, and genomic disease predictors. Thus, understanding the complexity of the disease pathogenesis will guide further investigation for targeted therapeutic strategies.
PMCID: PMC3612359  PMID: 23325076
25.  A Three-Marker FISH Panel Detects More Genetic Aberrations of AR, PTEN and TMPRSS2/ERG in Castration-Resistant or Metastatic Prostate Cancers than in Primary Prostate Tumors 
PLoS ONE  2013;8(9):e74671.
TMPRSS2/ERG rearrangement, PTEN gene deletion, and androgen receptor (AR) gene amplification have been observed in various stages of human prostate cancer. We hypothesized that using these markers as a combined panel would allow better differentiation between low-risk and high-risk prostate cancer. We analyzed 110 primary prostate cancer samples, 70 metastatic tumor samples from 11 patients, and 27 xenograft tissues derived from 22 advanced prostate cancer patients using fluorescence in situ hybridization (FISH) analysis with probes targeting the TMPRSS2/ERG, PTEN, and AR gene loci. Heterogeneity of the aberrations detected was evaluated. Genetic patterns were also correlated with transcript levels. Among samples with complete data available, the three-marker FISH panel detected chromosomal abnormalities in 53% of primary prostate cancers and 87% of metastatic (Met) or castration-resistant (CRPC) tumors. The number of markers with abnormal FISH result had a different distribution between the two groups (P<0.001). At the patient level, Met/CRPC tumors are 4.5 times more likely to show abnormalities than primary cancer patients (P<0.05). Heterogeneity among Met/CRPC tumors is mostly inter-patient. Intra-patient heterogeneity is primarily due to differences between the primary prostate tumor and the metastases while multiple metastatic sites show consistent abnormalities. Intra-tumor variability is most prominent with the AR copy number in primary tumors. AR copy number correlated well with the AR mRNA expression (rho = 0.52, P<0.001). Especially among TMPRSS2:ERG fusion-positive CRPC tumors, AR mRNA and ERG mRNA levels are strongly correlated (rho = 0.64, P<0.001). Overall, the three-marker FISH panel may represent a useful tool for risk stratification of prostate cancer patients.
PMCID: PMC3787014  PMID: 24098661

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