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2.  Differential Accessibility of a Rotavirus VP6 Epitope in Trimers Comprising Type I, II, or III Channels as Revealed by Binding of a Human Rotavirus VP6-Specific Antibody 
Journal of Virology  2014;88(1):469-476.
Previous human antibody studies have shown that the human VH1–46 antibody variable gene segment encodes much of the naturally occurring human B cell response to rotavirus and is directed to virus protein 6 (VP6). It is currently unknown why some of the VH1-46-encoded human VP6 monoclonal antibodies inhibit viral transcription while others do not. In part, there are affinity differences between antibodies that likely affect inhibitory activity, but we also hypothesize that there are differing modes of binding to VP6 that affect the ability to block the transcriptional pore on double-layered particles. Here, we used a hybrid method approach for antibody epitope mapping, including single-particle cryo-electron microscopy (cryo-EM) and enhanced amide hydrogen-deuterium exchange mass spectrometry (DXMS) to determine the location and mode of binding of a VH1-46-encoded antibody, RV6-25. The structure of the RV6-25 antibody–double-layered particle (DLP) complex indicated a very complex binding pattern that revealed subtle differences in accessibility of the VP6 epitope depending on its position in the type I, II, or III channels. These subtle variations in the presentation or accessibility of the RV VP6 capsid layer led to position-specific differences in occupancy for binding of the RV6-25 antibody. The studies also showed that the location of binding of the noninhibitory antibody RV6-25 on the apical surface of RV VP6 head domain does not obstruct the transcription pore upon antibody binding, in contrast to binding of an inhibitory antibody, RV6-26, deeper in the transcriptional pore.
PMCID: PMC3911710  PMID: 24155406
3.  Different rectal toxicity tolerance with and without simultaneous conventionally-fractionated pelvic lymph node treatment in patients receiving hypofractionated prostate radiotherapy 
To investigate added morbidity associated with the addition of pelvic elective nodal irradiation (ENI) to hypofractionated radiotherapy to the prostate.
Methods and materials
Two-hundred twelve patients, treated with hypofractionated radiotherapy to the prostate between 2004 and 2011, met the inclusion criteria for the analysis. All patients received 70 Gy to the prostate delivered over 28 fractions and 103 (49%) received ENI consisting of 50.4 Gy to the pelvic lymphatics delivered simultaneously in 1.8 Gy fractions. The mean dose-volume histograms were compared between the two subgroups defined by use of ENI, and various dose-volume parameters were analyzed for effect on late lower gastrointestinal (GI) and genitourinary (GU) toxicity.
Acute grade 2 lower GI toxicity occurred in 38 (37%) patients receiving ENI versus 19 (17%) in those who did not (p = 0.001). The Kaplan-Meier estimate of grade ≥ 2 lower GI toxicity at 3 years was 15.3% for patients receiving ENI versus 5.3% for those who did not (p = 0.026). Each rectal isodose volume was increased for patients receiving ENI up to 50 Gy (p ≤ 0.021 for each 5 Gy increment). Across all patients, the absolute V70 of the rectum was the only predictor of late GI toxicity. When subgroups, defined by the use of ENI, were analyzed separately, rectal V70 was only predictive of late GI toxicity for patients who received ENI. For patients receiving ENI, V70 > 3 cc was associated with an increased risk of late GI events.
Elective nodal irradiation increases the rates of acute and late GI toxicity when delivered simultaneously with hypofractioanted prostate radiotherapy. The use of ENI appears to sensitize the rectum to hot spots, therefore we recommend added caution to minimize the volume of rectum receiving 100% of the prescription dose in these patients.
PMCID: PMC4060093  PMID: 24893842
Prostate cancer; Rectal toxicity; Hypofractionation; Pelvic lymph node irradiation
4.  Characterizing the Complexity of Medication Safety using a Human Factors Approach: An Observational Study in Two Intensive Care Units 
BMJ quality & safety  2013;23(1):56-65.
To examine medication safety in two ICUs and to assess the complexity of medication errors and adverse drug events (ADEs) in ICUs across the stages of the medication-management process.
Four trained nurse data collectors gathered data on medication errors and ADEs between October 2006 and March 2007. Patient care documents (e.g., medication order sheets, notes) and incident reports were used to identify medication errors and ADEs in a 24-bed adult medical/surgical ICU and an 18-bed cardiac ICU in a tertiary care, community teaching hospital. In this cross-sectional study, a total of 630 consecutive ICU patient admissions were assessed to produce data on the number, rates and types of potential and preventable ADEs across stages of the medication-management process.
An average of 2.9 preventable or potential ADEs occurred in each admission, i.e., 0.4 events per patient-day. Preventable or potential ADEs occurred in 2.6% of the medication orders. The rate of potential ADEs per 1,000 patient-days was 276, whereas the rate of preventable ADEs per 1,000 patient-days was 9.2. Most medication errors occur at the ordering (32%) and administration stages (39%). In 16–24% of potential and preventable ADEs, clusters of errors occurred either as sequence of errors (e.g., delay in medication dispensing leading to delay in medication administration) or grouped errors (e.g., route and frequency errors in the order for a medication). Many of the sequences led to administration errors that were caused by errors earlier in the medication-management process.
Understanding the complexity of the vulnerabilities of the medication-management process is important to devise solutions to improve patient safety. Electronic health record technology with computerized physician order entry may be one step necessary to improve medication safety in ICUs. Solutions that target multiple stages of the medication-management process are necessary to address sequential errors.
PMCID: PMC3938094  PMID: 24050986
medication safety; medication errors; adverse drug events; intensive care unit; human factors engineering
5.  Human Rotavirus VP6-Specific Antibodies Mediate Intracellular Neutralization by Binding to a Quaternary Structure in the Transcriptional Pore 
PLoS ONE  2013;8(5):e61101.
Several live attenuated rotavirus (RV) vaccines have been licensed, but the mechanisms of protective immunity are still poorly understood. The most frequent human B cell response is directed to the internal protein VP6 on the surface of double-layered particles, which is normally exposed only in the intracellular environment. Here, we show that the canonical VP6 antibodies secreted by humans bind to such particles and inhibit viral transcription. Polymeric IgA RV antibodies mediated an inhibitory effect against virus replication inside cells during IgA transcytosis. We defined the recognition site on VP6 as a quaternary epitope containing a high density of charged residues. RV human mAbs appear to bind to a negatively-charged patch on the surface of the Type I channel in the transcriptionally active particle, and they sterically block the channel. This unique mucosal mechanism of viral neutralization, which is not apparent from conventional immunoassays, may contribute significantly to human immunity to RV.
PMCID: PMC3650007  PMID: 23671563
6.  An Intrinsically Disordered Region of the Adenovirus Capsid Is Implicated in Neutralization by Human Alpha Defensin 5 
PLoS ONE  2013;8(4):e61571.
Human α-defensins are proteins of the innate immune system that suppress viral and bacterial infections by multiple mechanisms including membrane disruption. For viruses that lack envelopes, such as human adenovirus (HAdV), other, less well defined, mechanisms must be involved. A previous structural study on the interaction of an α-defensin, human α-defensin 5 (HD5), with HAdV led to a proposed mechanism in which HD5 stabilizes the vertex region of the capsid and blocks uncoating steps required for infectivity. Studies with virus chimeras comprised of capsid proteins from sensitive and resistant serotypes supported this model. To further characterize the critical binding site, we determined subnanometer resolution cryo-electron microscopy (cryoEM) structures of HD5 complexed with both neutralization-sensitive and -resistant HAdV chimeras. Models were built for the vertex regions of these chimeras with monomeric and dimeric forms of HD5 in various initial orientations. CryoEM guided molecular dynamics flexible fitting (MDFF) was used to restrain the majority of the vertex model in well-defined cryoEM density. The RGD-containing penton base loops of both the sensitive and resistant virus chimeras are predicted to be intrinsically disordered, and little cryoEM density is observed for them. In simulations these loops from the sensitive virus chimera, interact with HD5, bridge the penton base and fiber proteins, and provides significant stabilization with a three-fold increase in the intermolecular nonbonded interactions of the vertex complex. In the case of the resistant virus chimera, simulations revealed fewer bridging interactions and reduced stabilization by HD5. This study implicates a key dynamic region in mediating a stabilizing interaction between a viral capsid and a protein of the innate immune system with potent anti-viral activity.
PMCID: PMC3631211  PMID: 23620768
7.  Derepression of Cancer/Testis Antigens in cancer is associated with distinct patterns of DNA Hypomethylation 
BMC Cancer  2013;13:144.
The Cancer/Testis Antigens (CTAs) are a heterogeneous group of proteins whose expression is typically restricted to the testis. However, they are aberrantly expressed in most cancers that have been examined to date. Broadly speaking, the CTAs can be divided into two groups: the CTX antigens that are encoded by the X-linked genes and the non-X CT antigens that are encoded by the autosomes. Unlike the non-X CTAs, the CTX antigens form clusters of closely related gene families and their expression is frequently associated with advanced disease with poorer prognosis. Regardless however, the mechanism(s) underlying their selective derepression and stage-specific expression in cancer remain poorly understood, although promoter DNA demethylation is believed to be the major driver.
Here, we report a systematic analysis of DNA methylation profiling data from various tissue types to elucidate the mechanism underlying the derepression of the CTAs in cancer. We analyzed the methylation profiles of 501 samples including sperm, several cancer types, and their corresponding normal somatic tissue types.
We found strong evidence for specific DNA hypomethylation of CTA promoters in the testis and cancer cells but not in their normal somatic counterparts. We also found that hypomethylation was clustered on the genome into domains that coincided with nuclear lamina-associated domains (LADs) and that these regions appeared to be insulated by CTCF sites. Interestingly, we did not observe any significant differences in the hypomethylation pattern between the CTAs without CpG islands and the CTAs with CpG islands in the proximal promoter.
Our results corroborate that widespread DNA hypomethylation appears to be the driver in the derepression of CTA expression in cancer and furthermore, demonstrate that these hypomethylated domains are associated with the nuclear lamina-associated domains (LADS). Taken together, our results suggest that wide-spread methylation changes in cancer are linked to derepression of germ-line-specific genes that is orchestrated by the three dimensional organization of the cancer genome.
PMCID: PMC3618251  PMID: 23522060
DNA hypomethylation; Cancer/Testis antigens; Lamina attachment domains; Insulator regions
8.  Optimal Planning Target Volume Margins for Elective Pelvic Lymphatic Radiotherapy in High-Risk Prostate Cancer Patients 
ISRN Oncology  2013;2013:941269.
Purpose. High-risk prostate cancer patients often receive radiotherapy (RT) to pelvic lymphatics (PLs). The aim of this study was to determine the safety margin around clinical target volume for PL (PL-CTV) to construct planning target volume for PL (PL-PTV) and for planning elective PL irradiation. Methods and Materials. Six patients who received RT to PL as part of prostate cancer treatment were identified. To determine average daily shifts of PL, the right and left IVs were contoured at 3 predetermined slices on the daily MV scans and their daily shifts were measured at these 3 levels using a measuring tool. Results. A total of 1,932 observations were made. Daily shifts of IV were random in distribution, and the largest observed shift was 13.6 mm in lateral and 15.4 mm in AP directions. The mean lateral and AP shifts of IV were 2.1 mm (±2.2) and 3.5 mm (±2.7), respectively. The data suggest that AP and lateral margins of 8.9 mm and 6.5 mm are necessary. Conclusions. With daily alignment to the prostate, we recommend an additional PL-CTV to PL-PTV conversion margin of 9 mm (AP) and 7 mm (lateral) to account for daily displacement of PL relative to the prostate.
PMCID: PMC3606762  PMID: 23533814
9.  Cost-effective therapeutic hypothermia treatment device for hypoxic ischemic encephalopathy 
Despite recent advances in neonatal care and monitoring, asphyxia globally accounts for 23% of the 4 million annual deaths of newborns, and leads to hypoxic-ischemic encephalopathy (HIE). Occurring in five of 1000 live-born infants globally and even more in developing countries, HIE is a serious problem that causes death in 25%–50% of affected neonates and neurological disability to at least 25% of survivors. In order to prevent the damage caused by HIE, our invention provides an effective whole-body cooling of the neonates by utilizing evaporation and an endothermic reaction. Our device is composed of basic electronics, clay pots, sand, and urea-based instant cold pack powder. A larger clay pot, lined with nearly 5 cm of sand, contains a smaller pot, where the neonate will be placed for therapeutic treatment. When the sand is mixed with instant cold pack urea powder and wetted with water, the device can extract heat from inside to outside and maintain the inner pot at 17°C for more than 24 hours with monitoring by LED lights and thermistors. Using a piglet model, we confirmed that our device fits the specific parameters of therapeutic hypothermia, lowering the body temperature to 33.5°C with a 1°C margin of error. After the therapeutic hypothermia treatment, warming is regulated by adjusting the amount of water added and the location of baby inside the device. Our invention uniquely limits the amount of electricity required to power and operate the device compared with current expensive and high-tech devices available in the United States. Our device costs a maximum of 40 dollars and is simple enough to be used in neonatal intensive care units in developing countries.
PMCID: PMC3540914  PMID: 23319871
therapeutic hypothermia; evaporative cooling; hypoxic ischemic encephalopathy; birth asphyxia; neuroprotection
10.  Cancer/testis antigens and urological malignancies 
Nature reviews. Urology  2012;9(7):386-396.
Cancer/testis antigens (CTAs) are a group of tumour-associated antigens (TAAs) that display normal expression in the adult testis—an immune-privileged organ—but aberrant expression in several types of cancers, particularly in advanced cancers with stem cell-like characteristics. There has been an explosion in CTA-based research since CTAs were first identified in 1991 and MAGE-1 was shown to elicit an autologous cytotoxic T-lymphocyte (CTL) response in a patient with melanoma. The resulting data have not only highlighted a role for CTAs in tumorigenesis, but have also underscored the translational potential of these antigens for detecting and treating many types of cancers. Studies that have investigated the use of CTAs for the clinical management of urological malignancies indicate that these TAAs have potential roles as novel biomarkers, with increased specificity and sensitivity compared to those currently used in the clinic, and therapeutic targets for cancer immunotherapy. Increasing evidence supports the utilization of these promising tools for urological indications.
PMCID: PMC3477645  PMID: 22710665
11.  A new metabolomic assay to examine inflammation and redox pathways following LPS challenge 
Shifts in intracellular arginine (Arg) and sulfur amino acid (SAA) redox metabolism modulate macrophage activation, polarization and phenotype. Despite their importance in inflammation and redox regulatory pathways, comprehensive analysis of these metabolic networks was not previously possible with existing analytical methods.
The Arg/thiol redox LC-MS/MS metabolomics assay permits simultaneous assessment of amino acids and derivative products generated from Arg and SAA metabolism. Using this assay, LPS-induced changes in macrophage amino acid metabolism were monitored to identify pathway shifts during activation and their linkage to cellular redox regulation.
Metabolite concentrations most significantly changed after treatment of a macrophage-like cell line (RAW) with LPS for 24 hrs were citrulline (Cit) (48-fold increase), ornithine (Orn) (8.5-fold increase), arginine (Arg) (66% decrease), and aspartic acid (Asp) (73% decrease). The ratio Cit + Orn/Arg + Asp (CO/AA) was more sensitive to LPS stimulation than other amino acid ratios commonly used to measure LPS-dependent inflammation (e.g., SAM/SAH, GSH/GSSG) and total media NOx. The CO/AA ratio was also the first ratio to change significantly after LPS treatment (4 hrs). Changes in the overall metabolomic profile over time indicated that metabolic pathways shifted from Arg catabolism to thiol oxidation.
Simultaneous quantification of Arg and SAA metabolic pathway shifts following LPS challenge of macrophage indicate that, in this system, the Arg-Citrulline/NO cycle and arginase pathways are the amino acid metabolic pathways most sensitive to LPS-challenge. The cellular (Cit + Orn)/(Arg + Asp) ratio, which summarizes this pathway, was more responsive to lower concentrations of LPS and responded earlier than other metabolic biomarkers of macrophage activation including GSH redox. It is suggested that the CO/AA ratio is a redox- independent early biomarker of macrophage activation. The ability to measure both the CO/AA and GSH-redox ratios simultaneously permits quantification of the relative effects of LPS challenge on macrophage inflammation and oxidative stress pathways. The use of this assay in humans is discussed, as are clinical implications.
PMCID: PMC3507808  PMID: 23036094
Macrophage; Lipopolysaccharide; Sub-clinical inflammation; Arginine metabolism; Redox regulation; Biomarker
12.  Hypofractionated Prostate Radiotherapy with or without Conventionally Fractionated Nodal Irradiation: Clinical Toxicity Observations and Retrospective Daily Dosimetry 
Prostate Cancer  2012;2012:546794.
Purpose. To evaluate toxicity associated with the addition of elective nodal irradiation (ENI) to a hypofractionated regimen for the treatment of prostate cancer. Methods and Materials. Fifty-seven patients received pelvic image-guided IMRT to 50.4 Gy in 28 fractions with a hypofractionated simultaneous boost to the prostate to 70 Gy. Thirty-one patients received prostate-only treatment to 70 Gy in 28 fractions. Results. Median followup was 41.1 months. Early grade ≥2 urinary toxicity rates were 49% (28 of 57) for patients receiving ENI and 58% (18 of 31) for those not (P = 0.61). Early grade ≥2 rectal toxicity rates were 40% (23 of 57) and 23% (7 of 31), respectively (P = 0.09). The addition of ENI resulted in a 21% actuarial rate of late grade ≥2 rectal toxicity at 4 years, compared to 0% for patients treated to the prostate only (P = 0.02). Retrospective daily dosimetry of patients experiencing late rectal toxicity revealed an average increase of 2.67% of the rectal volume receiving 70 Gy compared to the original plan. Conclusions. The addition of ENI resulted in an increased risk of late rectal toxicity. Grade ≥2 late rectal toxicity was associated with worse daily rectal dosimetry compared to the treatment plan.
PMCID: PMC3394386  PMID: 22966463
13.  Spread of the Emerging Viral Hemorrhagic Septicemia Virus Strain, Genotype IVb, in Michigan, USA 
Viruses  2012;4(5):734-760.
In 2003, viral hemorrhagic septicemia virus (VHSV) emerged in the Laurentian Great Lakes causing serious losses in a number of ecologically and recreationally important fish species. Within six years, despite concerted managerial preventive measures, the virus spread into the five Great Lakes and to a number of inland waterbodies. In response to this emerging threat, cooperative efforts between the Michigan Department of Natural Resources (MI DNR), the Michigan State University Aquatic Animal Health Laboratory (MSU-AAHL), and the United States Department of Agriculture-Animal and Plant Health Inspection Services (USDA-APHIS) were focused on performing a series of general and VHSV-targeted surveillances to determine the extent of virus trafficking in the State of Michigan. Herein we describe six years (2005–2010) of testing, covering hundreds of sites throughout Michigan’s Upper and Lower Peninsulas. A total of 96,228 fish representing 73 species were checked for lesions suggestive of VHSV and their internal organs tested for the presence of VHSV using susceptible cell lines. Of the 1,823 cases tested, 30 cases from 19 fish species tested positive for VHSV by tissue culture and were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). Gene sequence analyses of all VHSV isolates retrieved in Michigan demonstrated that they belong to the emerging sublineage “b” of the North American VHSV genotype IV. These findings underscore the complexity of VHSV ecology in the Great Lakes basin and the critical need for rigorous legislation and regulatory guidelines in order to reduce the virus spread within and outside of the Laurentian Great Lakes watershed.
PMCID: PMC3386630  PMID: 22754647
Michigan; viral hemorrhagic septicemia virus; Laurentian Great Lakes; emerging disease
14.  Assessment of endoplasmic reticulum glutathione redox status is confounded by extensive ex vivo oxidation 
Antioxidants & redox signaling  2008;10(5):963-972.
Glutathione (GSH) and glutathione disulfide (GSSG) form the principal thiol redox couple in the endoplasmic reticulum (ER); however, few studies have attempted to quantify GSH redox status in this organelle. To address this gap, GSH and GSSG levels and the extent of protein glutathionylation were analyzed in rat liver microsomes. Because of the likelihood of artifactual GSH oxidation during the lengthy microsomal isolation procedure, iodoacetic acid (IAA) was used to preserve the physiological thiol redox state. Non-IAA-treated microsomes exhibited a GSH:GSSG ratio between 0.7:1 to 1.2:1 compared to IAA-treated microsomes that yielded a GSH:GSSG redox ratio between 4.7:1 and 5.5:1. The majority of artifactual oxidation occurred within the first 2 h of isolation. Thus, the ER GSH redox ratio is subject to extensive ex vivo oxidation and when controlled, the microsomal GSH redox state is significantly higher than previously believed. Moreover, in vitro studies showed that PDI reductase activity was markedly increased at this higher thiol redox ratio versus previously reported GSH:GSSG ratios for the ER. Lastly, we show by both HPLC and Western blot analysis that ER proteins are highly resistant to glutathionylation. Together, these results may necessitate a re-evaluation of GSH and its role in ER function.
PMCID: PMC3220945  PMID: 18205546
15.  Biobanking after robotic-assisted radical prostatectomy: a quality assessment of providing prostate tissue for RNA studies 
RNA quality is believed to decrease with ischaemia time, and therefore open radical prostatectomy has been advantageous in allowing the retrieval of the prostate immediately after its devascularization. In contrast, robotic-assisted laparoscopic radical prostatectomies (RALP) require the completion of several operative steps before the devascularized prostate can be extirpated, casting doubt on the validity of this technique as a source for obtaining prostatic tissue. We seek to establish the integrity of our biobanking process by measuring the RNA quality of specimens derived from robotic-assisted laparoscopic radical prostatectomy.
We describe our biobanking process and report the RNA quality of prostate specimens using advanced electrophoretic techniques (RNA Integrity Numbers, RIN). Using multivariate regression analysis we consider the impact of various clinicopathological correlates on RNA integrity.
Our biobanking process has been used to acquire 1709 prostates, and allows us to retain approximately 40% of the prostate specimen, without compromising the histopathological evaluation of patients. We collected 186 samples from 142 biobanked prostates, and demonstrated a mean RIN of 7.25 (standard deviation 1.64) in 139 non-stromal samples, 73% of which had a RIN ≥ 7. Multivariate regression analysis revealed cell type - stromal/epithelial and benign/malignant - and prostate volume to be significant predictors of RIN, with unstandardized coefficients of 0.867(p = 0.001), 1.738(p < 0.001) and -0.690(p = 0.009) respectively. A mean warm ischaemia time of 120 min (standard deviation 30 min) was recorded, but multivariate regression analysis did not demonstrate a relationship with RIN within the timeframe of the RALP procedure.
We demonstrate the robustness of our protocol - representing the concerted efforts of dedicated urology and pathology departments - in generating RNA of sufficient concentration and quality, without compromising the histopathological evaluation and diagnosis of patients. The ischaemia time associated with our prostatectomy technique using a robotic platform does not negatively impact on biobanking for RNA studies.
PMCID: PMC3161873  PMID: 21791045
biobanking; prostate collection; ischaemia time; robotic-assisted radical prostatectomy; RNA quality; RIN
16.  Clinical assay of four thiol amino acid redox couples by LC-MS/MS: utility in thalassemia 
The total concentrations of four sulfur amino acid (SAA) metabolite redox couples (reduced and oxidized forms of homocysteine, cysteine, glutathione, and cysteinylglycine) in human blood are assayed with a simple and sensitive method by liquid chromatography-electrospray positive ionization tandem mass spectrometry. To prevent ex vivo thiol oxidation, iodoacetamide (IAM) is used immediately following the blood draw. To selectively enrich for S-carboxyamidomethylated SAA, and other cationic amino acids metabolites, proprietary strong cation-exchange solid phase extraction tips are used. Analytes are further derivatized with isopropylchloroformate (IPCF) to esterify the amino and the carboxylic groups. Double derivatization with IAM and IPCF improves the reverse phase liquid chromatography separation of SAA metabolites. The use of detection mode of multiple-reaction monitoring (MRM) allows sensitive and specific simultaneous detection of SAA. The internal standards used to account for the matrix effects of human plasma and erythrocytes were plant glutathione analogue, homoglutathione, and stable isotopes of cystine and homocystine. The method was validated for its linearity, accuracy, and precision. Excellent linearity of detection (r2 > 0.98) was observed over relevant ranges for plasma and erythrocyte samples, and the limits of detection was established to be between 5 and 20 nM. Relative standard deviations were <9% for within-day variations and <15% for between day variations. The method was used to assess thiol redox states in plasma and erythrocytes isolated from healthy subjects and thalassemia patients.
PMCID: PMC3077474  PMID: 19616487
Thiol amino acids; LC-MS/MS; Thiol Redox; Oxidative Stress; Hemoglobinopathy
17.  RHAMM (CD168) Is Overexpressed at the Protein Level and May Constitute an Immunogenic Antigen in Advanced Prostate Cancer Disease1 
Neoplasia (New York, N.Y.)  2009;11(9):956-963.
Localized prostate cancer (CaP) can be cured using several strategies. However, the need to identify active substances in advanced tumor stages is tremendous, as the outcome in such cases is still disappointing. One approach is to deliver human tumor antigen-targeted therapy, which is recognized by T cells or antibodies. We used data mining of the Cancer Immunome Database (CID), which comprises potential immunologic targets identified by serological screening of expression libraries. Candidate antigens were screened by DNA microarrays. Genes were then validated at the protein level by tissue microarrays, representing various stages of CaP disease. Of 43 targets identified by CID, 10 showed an overexpression on the complementary DNA array in CaP metastases. The RHAMM (CD168) gene, earlier identified by our group as an immunogenic antigen in acute and chronic leukemia, also showed highly significant overexpression in CaP metastases compared with localized disease and benign prostatic hyperplasia. At the protein level, RHAMM was highest in metastatic tissue samples and significantly higher in neoplastic localized disease compared with benign tissue. High RHAMM expression was associated with clinical parameters known to be linked to better clinical outcome. Patients with high RHAMM expression in the primaries had a significantly lower risk of biochemical failure. The number of viable cells in cell cultures was reduced in blocking experiments using hormone-sensitive and hormone-insensitive metastatic CaP cell lines. Acknowledging the proven immunogenic effects of RHAMM in leukemia, this antigen is intriguing as a therapeutic target in far-advanced CaP.
PMCID: PMC2735808  PMID: 19724689
18.  Testing a Multigene Signature of Prostate Cancer Death in the Swedish Watchful Waiting Cohort 
While prostate cancer is a leading cause of cancer death, most men die with and not from their disease, underscoring the urgency to distinguish potentially lethal from indolent prostate cancer. We tested the prognostic value of a previously identified multigene signature of prostate cancer progression to predict cancer-specific death. The Örebro Watchful Waiting Cohort included 172 men with localized prostate cancer of whom 40 died of prostate cancer. We quantified protein expression of the markers in tumor tissue by immunohistochemistry, and stratified the cohort by quintiles according to risk classification. We accounted for clinical parameters (age, Gleason, nuclear grade, tumor volume) using Cox regression, and calculated Receiver Operator Curves to compare discriminatory ability. The hazard ratio of prostate cancer death increased with increasing risk classification by the multigene model, with a 16-fold greater risk comparing highest versus lowest risk strata, and predicted outcome independent of clinical factors (p=0.002). The best discrimination came from combining information from the multigene markers and clinical data, which perfectly classified the lowest risk stratum where no one developed lethal disease; using the two lowest risk groups as referent, the hazard ratio (95% confidence interval) was 11.3 (4.0–32.8) for the highest risk group and difference in mortality at 15 years was 60% (50–70%). The combined model provided greater discriminatory ability (AUC 0.78) than the clinical model alone (AUC 0.71), p=0.04. Molecular tumor markers can add to clinical parameters to help distinguish lethal and indolent prostate cancer, and hold promise to guide treatment decisions.
PMCID: PMC2536630  PMID: 18583469
19.  Identification of Leukocyte E-selectin Ligands, P-selectin Glycoprotein Ligand-1 and E-selectin Ligand-1, on Human Metastatic Prostate Tumor Cells 
Cancer research  2005;65(13):5750-5760.
Prostate tumor cells, which characteristically metastasize to bone, initiate binding interactions with bone marrow endothelium under blood flow conditions through binding interactions with E-selectin. We hypothesized that E-selectin ligands on prostate tumor cells are directly associated with bone-metastatic potential. In this report, we elucidate the identity of E-selectin ligands on human metastatic prostate tumor cells and examine their association with prostate tumor progression and metastasis in vivo. To our surprise, we found that the E-selectin-binding form of P-selectin glycoprotein ligand-1 (PSGL-1) is expressed on the human bone-metastatic prostate tumor MDA PCa 2b cell line. Interestingly, we also found that human prostate tumor cells derived from bone, lymph node and brain metastases expressed another leukocyte E-selectin ligand, E-selectin ligand-1 (ESL-1). Immunohistochemical analysis of PSGL-1 and ESL-1 in normal prostate tissue and in localized and metastatic prostate tumors revealed that ESL-1 was principally localized to intracellular cell membrane and expressed on all normal and malignant prostate tissue, whereas PSGL-1 was notably detected on the surfaces of bone-metastatic prostate tumor cells. These findings implicate a functional role of PSGL-1 in the bone tropism of prostate tumor cells and establish a new perspective into the molecular mechanism of human prostate tumor metastasis.
PMCID: PMC1472661  PMID: 15994950
Metastasis; E-selectin Ligands; Prostate Cancer; Bone Marrow; Homing
20.  Defining Aggressive Prostate Cancer Using a 12-Gene Model1 
Neoplasia (New York, N.Y.)  2006;8(1):59-68.
The critical clinical question in prostate cancer research is: How do we develop means of distinguishing aggressive disease from indolent disease? Using a combination of proteomic and expression array data, we identified a set of 36 genes with concordant dysregulation of protein products that could be evaluated in situ by quantitative immunohistochemistry. Another five prostate cancer biomarkers were included using linear discriminant analysis, we determined that the optimal model used to predict prostate cancer progression consisted of 12 proteins. Using a separate patient population, transcriptional levels of the 12 genes encoding for these proteins predicted prostate-specific antigen failure in 79 men following surgery for clinically localized prostate cancer (P = .0015). This study demonstrates that cross-platform models can lead to predictive models with the possible advantage of being more robust through this selection process.
PMCID: PMC1584291  PMID: 16533427
Metastasis; cancer; proteomics; prostate cancer; bioinformatics
21.  Internet-based profiler system as integrative framework to support translational research 
BMC Bioinformatics  2005;6:304.
Translational research requires taking basic science observations and developing them into clinically useful tests and therapeutics. We have developed a process to develop molecular biomarkers for diagnosis and prognosis by integrating tissue microarray (TMA) technology and an internet-database tool, Profiler. TMA technology allows investigators to study hundreds of patient samples on a single glass slide resulting in the conservation of tissue and the reduction in inter-experimental variability. The Profiler system allows investigator to reliably track, store, and evaluate TMA experiments. Here within we describe the process that has evolved through an empirical basis over the past 5 years at two academic institutions.
The generic design of this system makes it compatible with multiple organ system (e.g., prostate, breast, lung, renal, and hematopoietic system,). Studies and folders are restricted to authorized users as required. Over the past 5 years, investigators at 2 academic institutions have scanned 656 TMA experiments and collected 63,311 digital images of these tissue samples. 68 pathologists from 12 major user groups have accessed the system. Two groups directly link clinical data from over 500 patients for immediate access and the remaining groups choose to maintain clinical and pathology data on separate systems. Profiler currently has 170 K data points such as staining intensity, tumor grade, and nuclear size. Due to the relational database structure, analysis can be easily performed on single or multiple TMA experimental results. The TMA module of Profiler can maintain images acquired from multiple systems.
We have developed a robust process to develop molecular biomarkers using TMA technology and an internet-based database system to track all steps of this process. This system is extendable to other types of molecular data as separate modules and is freely available to academic institutions for licensing.
PMCID: PMC1343596  PMID: 16364175

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