Serologic Cross-reactivity of Murine Parvovirus Capsid Antigens.

Genomic sequence analysis of autonomous parvoviruses within the genus Protoparvovirus generates 2 groups that are principally of mouse origin: the minute virus of mice (MVM) strains (MVMp, MVMi, MVMc, MVMm) and the mouse parvovirus (MPV)-like strains (MPV-1, MPV-2, MPV-3, MPV-4, MPV-5, HaPV, LuIII). Baculovirus-expressed recombinant capsid protein (rVP2) from each of these 11 parvovirus strains were produced, purified, and demonstrated to form virus-like particles. Each rVP2 preparation was then used as antigen in a multiplex fluorescent immunoassay and to immunize 5 different strains of mice. Sera from immunized mice, mice experimentally monoinfected with various MVM or MPV isolates, and mice naturally infected with murine parvoviruses were evaluated with the multiplex fluorescent immunoassay rVP2 panel. Results for sera from immunized mice indicate that homologous antigen-antisera interactions produced the strongest seroreactivity. All MVM antigens were highly cross-reactive with heterologous MVM antisera, while more variability was observed in heterologous antigen-antisera reactions among the MPV-like strains. MPV-1, MPV-3, HaPV, and LuIII were highly cross-reactive with each other, MPV-2 and MPV-5 were highly cross-reactive with each other, and MPV-4 displayed modest cross-reactivity with certain MPV-like strains. Serologic cross-reactivity patterns similar to those in immunized mice were observed in mice experimentally infected with MVMp, MVMm, MPV-1, MPV-5, or HaPV, and in sera from mice naturally infected with MVM and MPV. Serologic cross-reactivity spectrums suggest a small panel of rVP2 antigens (MVM, MPV-1, MPV-2, MPV-4) combined with the generic murine parvovirus recombinant nonstructural protein 1 (rNS1) antigen are sufficient for qualitative detection of currently known MVM and MPV-like strains.

Parallel neurodegenerative phenotypes in sporadic Parkinson's disease fibroblasts and midbrain dopamine neurons.

Understanding the mechanisms causing Parkinson's disease (PD) is vital to the development of much needed early diagnostics and therapeutics for this debilitating condition. Here, we report cellular and molecular alterations in skin fibroblasts of late-onset sporadic PD subjects, that were recapitulated in matched induced pluripotent stem cell (iPSC)-derived midbrain dopamine (DA) neurons, reprogrammed from the same fibroblasts. Specific changes in growth, morphology, reactive oxygen species levels, mitochondrial function, and autophagy, were seen in both the PD fibroblasts and DA neurons, as compared to their respective controls. Additionally, significant alterations in alpha synuclein expression and electrical activity were also noted in the PD DA neurons. Interestingly, although the fibroblast and neuronal phenotypes were similar to each other, they differed in their nature and scale. Furthermore, statistical analysis revealed potential novel associations between various clinical measures of the PD subjects and the different fibroblast and neuronal data. In essence, these findings encapsulate spontaneous, in-tandem, disease-related phenotypes in both sporadic PD fibroblasts and iPSC-based DA neurons, from the same patient, and generates an innovative model to investigate PD mechanisms with a view towards rational disease stratification and precision treatments.

Parallel Neurodegenerative Phenotypes in Sporadic Parkinson's Disease Fibroblasts and Midbrain Dopamine Neurons.

Understanding the mechanisms causing Parkinson's disease (PD) is vital to the development of much needed early diagnostics and therapeutics for this debilitating condition. Here, we report cellular and molecular alterations in skin fibroblasts of late-onset sporadic PD subjects, that were recapitulated in matched induced pluripotent stem cell (iPSC)-derived midbrain dopamine (DA) neurons, reprogrammed from the same fibroblasts. Specific changes in growth, morphology, reactive oxygen species levels, mitochondrial function, and autophagy, were seen in both the PD fibroblasts and DA neurons, as compared to their respective controls. Additionally, significant alterations in alpha synuclein expression and electrical activity were also noted in the PD DA neurons. Interestingly, although the fibroblast and neuronal phenotypes were similar to each other, they also differed in their nature and scale. Furthermore, statistical analysis revealed novel associations between various clinical measures of the PD subjects and the different fibroblast and neuronal data. In essence, these findings encapsulate spontaneous, in-tandem, disease-related phenotypes in both sporadic PD fibroblasts and iPSC-based DA neurons, from the same patient, and generates an innovative model to investigate PD mechanisms with a view towards rational disease stratification and precision treatments.

Analysis of aggregated cell-cell statistical distances within pathways unveils therapeutic-resistance mechanisms in circulating tumor cells.

MOTIVATION: As 'omics' biotechnologies accelerate the capability to contrast a myriad of molecular measurements from a single cell, they also exacerbate current analytical limitations for detecting meaningful single-cell dysregulations. Moreover, mRNA expression alone lacks functional interpretation, limiting opportunities for translation of single-cell transcriptomic insights to precision medicine. Lastly, most single-cell RNA-sequencing analytic approaches are not designed to investigate small populations of cells such as circulating tumor cells shed from solid tumors and isolated from patient blood samples. RESULTS: In response to these characteristics and limitations in current single-cell RNA-sequencing methodology, we introduce an analytic framework that models transcriptome dynamics through the analysis of aggregated cell-cell statistical distances within biomolecular pathways. Cell-cell statistical distances are calculated from pathway mRNA fold changes between two cells. Within an elaborate case study of circulating tumor cells derived from prostate cancer patients, we develop analytic methods of aggregated distances to identify five differentially expressed pathways associated to therapeutic resistance. Our aggregation analyses perform comparably with Gene Set Enrichment Analysis and better than differentially expressed genes followed by gene set enrichment. However, these methods were not designed to inform on differential pathway expression for a single cell. As such, our framework culminates with the novel aggregation method, cell-centric statistics (CCS). CCS quantifies the effect size and significance of differentially expressed pathways for a single cell of interest. Improved rose plots of differentially expressed pathways in each cell highlight the utility of CCS for therapeutic decision-making. AVAILABILITY AND IMPLEMENTATION: http://www.lussierlab.org/publications/CCS/ CONTACT: yves@email.arizona.edu or piegorsch@math.arizona.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Analysis of global and absorption, distribution, metabolism, and elimination gene expression in the progressive stages of human nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is characterized by a series of pathological changes that range from simple fatty liver to nonalcoholic steatohepatitis (NASH). The objective of this study is to describe changes in global gene expression associated with the progression of human NAFLD. This study is focused on the expression levels of genes responsible for the absorption, distribution, metabolism, and elimination (ADME) of drugs. Differential gene expression between three clinically defined pathological groups-normal, steatosis, and NASH-was analyzed. Genome-wide mRNA levels in samples of human liver tissue were assayed with Affymetrix GeneChip Human 1.0ST arrays. A total of 11,633 genes exhibited altered expression out of 33,252 genes at a 5% false discovery rate. Most gene expression changes occurred in the progression from steatosis to NASH. Principal component analysis revealed that hepatic disease status was the major determinant of differential ADME gene expression rather than age or sex of sample donors. Among the 515 drug transporters and 258 drug-metabolizing enzymes (DMEs) examined, uptake transporters but not efflux transporters or DMEs were significantly over-represented in the number of genes down-regulated. These results suggest that uptake transporter genes are coordinately targeted for down-regulation at the global level during the pathological development of NASH and that these patients may have decreased drug uptake capacity. This coordinated regulation of uptake transporter genes is indicative of a hepatoprotective mechanism acting to prevent accumulation of toxic intermediates in disease-compromised hepatocytes.

Prognostic and pathogenetic value of combining clinical and biochemical indices in patients with acute lung injury.

BACKGROUND: No single clinical or biologic marker reliably predicts clinical outcomes in acute lung injury (ALI)/ARDS. We hypothesized that a combination of biologic and clinical markers would be superior to either biomarkers or clinical factors alone in predicting ALI/ARDS mortality and would provide insight into the pathogenesis of clinical ALI/ARDS. METHODS: Eight biologic markers that reflect endothelial and epithelial injury, inflammation, and coagulation (von Willebrand factor antigen, surfactant protein D [SP-D]), tumor necrosis factor receptor-1, interleukin [IL]-6, IL-8, intercellular adhesion molecule-1, protein C, plasminogen activator inhibitor-1) were measured in baseline plasma from 549 patients in the ARDSNet trial of low vs high positive end-expiratory pressure. Mortality was modeled with multivariable logistic regression. Predictors were selected using backward elimination. Comparisons between candidate models were based on the receiver operating characteristics (ROC) and tests of integrated discrimination improvement. RESULTS: Clinical predictors (Acute Physiology And Chronic Health Evaluation III [APACHE III], organ failures, age, underlying cause, alveolar-arterial oxygen gradient, plateau pressure) predicted mortality with an area under the ROC curve (AUC) of 0.82; a combination of eight biomarkers and the clinical predictors had an AUC of 0.85. The best performing biomarkers were the neutrophil chemotactic factor, IL-8, and SP-D, a product of alveolar type 2 cells, supporting the concept that acute inflammation and alveolar epithelial injury are important pathogenetic pathways in human ALI/ARDS. CONCLUSIONS: A combination of biomarkers and clinical predictors is superior to clinical predictors or biomarkers alone for predicting mortality in ALI/ARDS and may be useful for stratifying patients in clinical trials. From a pathogenesis perspective, the degree of acute inflammation and alveolar epithelial injury are highly associated with the outcome of human ALI/ARDS.

In vivo nonmelanoma skin cancer diagnosis using Raman microspectroscopy.

BACKGROUND AND OBJECTIVES: Nonmelanoma skin cancers, including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), are the most common skin cancers, presenting nearly as many cases as all other cancers combined. The current gold-standard for clinical diagnosis of these lesions is histopathologic examination, an invasive, time-consuming procedure. There is thus considerable interest in developing a real-time, automated, noninvasive tool for nonmelanoma skin cancer diagnosis. In this study, we explored the capability of Raman microspectroscopy to provide differential diagnosis of BCC, SCC, inflamed scar tissue, and normal tissue in vivo. STUDY DESIGN: Based on the results of previous in vitro studies, we developed a portable confocal Raman system with a handheld probe for clinical study. Using this portable system, we measured Raman spectra of 21 suspected nonmelanoma skin cancers in 19 patients with matched normal skin spectra. These spectra were input into nonlinear diagnostic algorithms to predict pathological designation. RESULTS: All of the BCC (9/9), SCC (4/4), and inflamed scar tissues (8/8) were correctly predicted by the diagnostic algorithm, and 19 out of 21 normal tissues were correctly classified. This translates into a 100% (21/21) sensitivity and 91% (19/21) specificity for abnormality, with a 95% (40/42) overall classification accuracy. CONCLUSIONS: These findings reveal Raman microspectroscopy to be a viable tool for real-time diagnosis and guidance of nonmelanoma skin cancer resection.

Proteomics of transformed lymphocytes from a family with familial pulmonary arterial hypertension.

RATIONALE: Not all family members with BMPR2 mutations develop pulmonary arterial hypertension (PAH), implying that additional modifier genes or proteins are necessary for full expression of the disease. OBJECTIVES: To determine whether protein expression is altered in patients with familial PAH (FPAH) compared with obligate carriers and nondiseased control subjects. METHODS: Protein extracts from transformed blood lymphocytes from four patients with FPAH, three obligate carriers, and three married-in control subjects from one family with a known BMPR2 mutation (exon 3 T354G) were labeled with either Cy3 or Cy5. Cy3/5 pairs were separated by standard two-dimensional differential gel electrophoresis using a Cy2-labeled internal standard of all patient samples. Log volume ratios were analyzed using a linear mixed-effects model. Proteins were identified by matrix-assisted laser desorption ionization, time-of-flight mass spectrometry (MALDI-TOF MS) and tandem TOF/TOF MS/MS. MEASUREMENTS AND MAIN RESULTS: Hierarchical clustering, heat-map, and principal components analysis revealed marked changes in protein expression in patients with FPAH when compared with obligate carriers. Significant changes were apparent in expression of 16 proteins (P < 0.05) when affected patients were compared with obligates: nine showed a significant increase and seven showed a significant reduction. CONCLUSIONS: A series of novel proteins with altered expression were found that could distinguish affected patients from obligate carriers and married-in controls in a single family with a BMPR2 mutation. These differences provide new information highlighting proteins that may be involved in the mechanism(s) that differentiates those individuals with a BMPR2 mutation who develop FPAH from those who do not.

Repeatability of a reference region model for analysis of murine DCE-MRI data at 7T.

PURPOSE: To test the repeatability of a reference region (RR) model for the analysis of dynamic contrast-enhanced MRI (DCE-MRI) in a mouse model of cancer at high field. MATERIALS AND METHODS: Seven mice were injected with 10(6) 4T1 mammary carcinoma cells and imaged eight to 10 days later on a Varian 7.0T scanner. Two DCE-MRI studies were performed for each mouse (separated by 2.5 hours). The RR model was used to analyze the data, and returned estimates on the perfusion-permeability index (Ktrans) for the RR and the tissue of interest (TOI), as well as the extravascular extracellular volume fraction (ve) for the TOI. RESULTS: When the first injection was compared with the second injection, all parameters tested were highly correlated (r2=0.90, 0.62, 0.82 for the RR Ktrans, TOI Ktrans, and TOI ve, respectively, with P<0.001 for all). To observe a statistically significant change (at the 5% level) in a treatment study with seven animals in each group, log10 changes of 0.084 and 0.077 in the tumor Ktrans and ve, respectively, are required. CONCLUSION: If a reliable arterial input function (AIF) is unavailable, the RR model is a reasonable alternative to measuring MRI contrast-agent (CA) kinetics in mouse models of cancer at high field.

Urine PGE-M: A metabolite of prostaglandin E2 as a potential biomarker of advanced colorectal neoplasia.

BACKGROUND & AIMS: The enzyme cyclooxygenase-2 is expressed in a majority of colorectal carcinomas (CRCs) and is important in prostaglandin production. We have developed an accurate method to measure the urinary metabolite of prostaglandin E(2) (PGE-M) using recently developed mass spectrometric techniques. The purpose of this pre-validation study was to determine if urinary PGE-M levels can be used as a biomarker to discriminate between healthy patients and those with colorectal disease. METHODS: Urine PGE-M was assessed in a total of 228 patients with CRC, colonic adenomatous polyps, Crohn's disease, and in subjects with no endoscopically detectable disease. Thirteen rectal carcinoma patients were treated with celecoxib and urinary PGE-M was measured before and after treatment. RESULTS: Urine PGE-M levels were increased among healthy men compared with healthy women (median, 8.59 [interquartile range (IQR), 5.67-22.3] vs 4.25 [IQR, 2.35-6.03], P = .0027). Urine PGE-M levels among patients with Crohn's disease (median, 19.85 [IQR, 6.89-90.2]), CRC (median, 14.65 [IQR, 5.94-92.1]), or large adenomas greater than 1 cm in size (median, 18.85 [IQR, 11.9-25.6]) were significantly increased when compared with patients who had either small polyps less than 1 cm in size (median, 9.69 [IQR, 6.41-22.2]), or no polyps (median, 7.05 [IQR, 2.35-24.7]) (P = .0001). PGE-M levels decreased significantly after celecoxib treatment in patients with rectal cancer (median, 21.7 [IQR, 16.2-29.9] vs 9.14 [IQR, 7.14-13.2], P = .009). CONCLUSIONS: The increase in urinary PGE-M in patients with colorectal cancers and large adenomas suggests that urinary PGE-M is a potentially useful biomarker for the detection of advanced colorectal neoplasia.

Incidence and clinical complications of vancomycin-resistant enterococcus in pediatric stem cell transplant patients.

Vancomycin-resistant enterococcus (VRE) are increasingly important pathogens in stem cell transplant (SCT). In all, 61 pediatric SCT patients had surveillance stool cultures for VRE between July 1999 and November 2002. When VRE was identified, the patients were placed on strict contact isolation. VRE was detected in 15 patients (24.6%). The median age was 3.6 years (range 0.6-18.5 years). Of the 15, 13 (87%) received an allogeneic transplant (six unrelated and seven related). Five of the 15 (33%) colonized patients developed VRE bacteremia. The bacteremia resolved in all five patients after therapy with quinupristin/dalfopristin; three patients required central line removal. Four patients died (38-153 days) post-SCT due to relapse or transplant complication not related to VRE. Of the 11 surviving patients, seven cleared the colonization at a median of 144 days (range 61-198 days) postcolonization. Four patients remain colonized at 68-702 days after the first positive culture. Intestinal colonization with VRE occurred commonly in pediatric SCT patients. Although the morbidity from VRE was not substantial, transplant patients were colonized for prolonged periods. Our results indicate that surveillance for VRE is an effective way to identify colonized patients and may lead to a decrease in transmission to other patients.

Entry criteria and baseline characteristics predict outcome in acute stroke trials.

UNLABELLED: Background and Purpose-We sought to study the range of entry criteria and baseline characteristics in acute stroke trials and to understand their effects on patient outcomes. METHODS: -Randomized, placebo-controlled therapeutic trials in patients with acute ischemic stroke were identified. Entry criteria, baseline clinical characteristics, and outcome were extracted for the placebo group of each trial. The relationship between key variables was then determined. RESULTS: -Across 90 placebo groups identified, there was great variation in entry criteria and outcome measures. This was associated with divergent outcomes; for example, in some studies most placebo group patients died, while in other studies nearly all had no disability. Entry criteria were significantly correlated with outcome; for example, higher age cutoff for study entry correlated with 3-month mortality. Entry criteria also predicted baseline clinical characteristics; for example, wider time window for study entry correlated directly with time to treatment and inversely with stroke severity (initial National Institutes of Health Stroke Scale score). Baseline characteristics predicted outcome. Greater stroke severity predicted higher 3-month mortality rate; despite this, successful thrombolytic trials have enrolled more severe strokes than most trials. The mean age of enrollees also predicted 3-month mortality and was inversely related to percentage of patients with 3-month Barthel Index score >/=95. The strongest predictors of 3-month mortality were obtained with multivariate models. CONCLUSIONS: -Acute stroke studies vary widely in entry criteria and outcome measures. Across multiple studies, differences in entry criteria, and the baseline clinical characteristics they predict, influence patient outcomes along a continuum. In some studies, enrolling a specific subset of patients may have improved the chances of identifying a treatment-related effect, while in others, such chances may have been reduced. These findings may be useful in the design of future stroke therapeutic trials.

Creosotebush vegetation after 50 years of lagomorph exclusion.

In 1939, an experiment was established on the Jornada Experimental Range to evaluate the effects of shrub removal, rabbit exclusion, furrowing, and seeding in creosotebush [Larrea tridentata (DC.) Cov] vegetation. Sixteen plots (21.3×21.3 m) were laid out in four rows of four plots per row with a buffer zone of 7.6 m between plots and rows. A barbed wire fence excluded cattle and poultry wire fencing excluded lagomorphs. Treatments were factorially applied at two levels. Plant cover in the plots was sampled in 1938 (before treatment), 1947, 1956, 1960, 1967 and 1989 with randomly located, line-intercept transects. Data from all sampling dates were analyzed as a split plot in time and main effects for 1989 tested by analysis of variance for a 2×4 factorial experiment. There were significant (P<0.10) year x treatment interactions. Seeding and furrowing treatments were ineffective but lagomorph exclusion and shrub clearing treatments resulted in significant treatment differences for several species. In 1989, basal area of spike dropseed (Sporobolus contractus A.S. Hitchc.) was 30-fold greater on the lagomorph excluded than on the lagomorph unexcluded treatment. Canopy cover of honey mesquite (Prosopis glandulosa Torr. var. glandulosa), tarbush (Flourensia cernua DC.) and mariola (Parthenium incanum H.B.K.) were affected by lagomorph exclusion. None of the responses were viewed as successional in nature. They principally represented individual species sensitivities to either absence of a primary herbivore or removal of aboveground shrub biomass. Though the physical treatments could be regarded as relatively severe disturbances of the system, the impacts on community vegetation dynamics were relatively insignificant.