TMPRSS2-ERG gene fusions occur in 50% of prostate cancers and result in the overexpression of a chimeric fusion transcript that encodes a truncated ERG product. Previous attempts to detect truncated ERG products have been hindered by a lack of specific antibodies. Here, we characterize a rabbit anti-ERG monoclonal antibody (clone EPR 3864; Epitomics, Burlingame, CA) using immunoblot analysis on prostate cancer cell lines, synthetic TMPRSS2-ERG constructs, chromatin immunoprecipitation, and immunofluorescence. We correlated ERG protein expression with the presence of ERG gene rearrangements in prostate cancer tissues using a combined immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) analysis. We independently evaluated two patient cohorts and observed ERG expression confined to prostate cancer cells and high-grade prostatic intraepithelial neoplasia associated with ERG-positive cancer, as well as vessels and lymphocytes (where ERG has a known biologic role). Image analysis of 131 cases demonstrated nearly 100% sensitivity for detecting ERG rearrangement prostate cancer, with only 2 (1.5%) of 131 cases demonstrating strong ERG protein expression without any known ERG gene fusion. The combined pathology evaluation of 207 patient tumors for ERG protein expression had 95.7% sensitivity and 96.5% specificity for determining ERG rearrangement prostate cancer. In conclusion, this study qualifies a specific anti-ERG antibody and demonstrates exquisite association between ERG gene rearrangement and truncated ERG protein product expression. Given the ease of performing IHC versus FISH, ERG protein expression may be useful for molecularly subtyping prostate cancer based on ERG rearrangement status and suggests clinical utility in prostate needle biopsy evaluation.

Background The impact of anesthetic technique on perioperative outcomes remains controversial. We studied a large national sample of primary joint arthroplasty recipients and hypothesized that neuraxial anesthesia favorably influences perioperative outcomes. Methods Data from approximately 400 hospitals between 2006 and 2010 were accessed. Patients who underwent primary hip or knee arthroplasty were identified and subgrouped by anesthesia technique: general, neuraxial, and combined neuraxial–general. Demographics, postoperative complications, 30-day mortality, length of stay, and patient cost were analyzed and compared. Multivariable analyses were conducted to identify the independent impact of choice of anesthetic on outcomes. Results Of 528,495 entries of patients undergoing primary hip or knee arthroplasty, information on anesthesia type was available for 382,236 (71.4%) records. Eleven percent were performed under neuraxial, 14.2% under combined neuraxial– general, and 74.8% under general anesthesia. Average age and comorbidity burden differed modestly between groups. When neuraxial anesthesia was used, 30-day mortality was significantly lower (0.10, 0.10, and 0.18%; P < 0.001), as was the incidence of prolonged (>75th percentile) length of stay, increased cost, and in-hospital complications. In the multivariable regression, neuraxial anesthesia was associated with the most favorable complication risk profile. Thirty-day mortality remained significantly higher in the general compared with the neuraxial or neuraxial–general group for total knee arthroplasty (adjusted odds ratio [OR] of 1.83, 95% CI 1.08–3.1, P = 0.02; OR of 1.70, 95% CI 1.06–2.74, P = 0.02, respectively). Conclusions The utilization of neuraxial versus general anesthesia for primary joint arthroplasty is associated with superior perioperative outcomes. More research is needed to study potential mechanisms for these findings.

Candidate HIV-1 vaccines currently being evaluated in clinical trials are designed to elicit HIV-1-specific cellular immunity. Intracellular cytokine staining (ICS) assays allow sensitive, quantitative ex vivo assessments of antigen-specific T cells including immunophenotyping of responding cells and measurement of multiple effector functions. Additionally, the use of banked cryopreserved PBMC samples makes this assay attractive in the setting of large efficacy trials where it is less feasible to perform immunoassays on freshly isolated samples. Here we describe extensive studies to optimize and quantitatively validate the 8-color ICS assay for use in clinical trials of candidate vaccines, which includes measurement of viable IFN-gamma, IL-2, TNF-alpha and IL-4 producing CD4+ and CD8+ T cells. We show that omission of viability dye staining results in an over-estimate of the true antigen-specific T cell response by up to two-fold. After optimization, the 8-color assay was validated for specificity, precision, linearity, limit of quantitation and robustness. The assay has a lower quantitation limit generally below 0.04%, depending on the cytokine subset. Additionally, with appropriate gating, the 8-color assay gives comparable cytokine-positive responses to those observed with the conventional 4-color assay. In conclusion, we provide the first description of a quantitatively validated ICS assay, which permits quantitative and qualitative evaluation of vaccine-induced immunogenicity and analysis of immune correlates of protection.

Interferon-gamma (IFN-gamma) ELISpot and intracellular cytokine staining (ICS) assays are routinely employed in clinical HIV vaccine trials to identify antigen-specific T cells in cryopreserved peripheral blood mononuclear cells (PBMC). Several parameters involved in blood collection, processing and shipping may influence immunological function of the resulting cells, including anticoagulant type, time from venipuncture to PBMC isolation/cryopreservation, method of PBMC isolation and procedure for sample shipping. We examined these parameters in single and multiple site studies, and found the length of time from venipuncture to cryopreservation is the most important parameter affecting performance of T cells in immunological assays. Comparing blood processed at 24 h after venipuncture with that processed within 8 h, we observed on average a modest reduction in PBMC viability ( approximately 8% decrease), a greater loss in cell recovery ( approximately 32%), and between 36-56% loss in IFN-gamma T cell frequencies by ELISpot assay. We also describe three cold shipping methods that maintain immunological function in appropriately cryopreserved PBMC. These data indicate that cryopreservation of PBMC should occur within 8 h of venipuncture for optimal performance. This narrow window for specimen processing has important implications in selecting and monitoring clinical sites with laboratory capacity to perform these procedures in future clinical trials.

In cultured shrimp, w h~t e spot syndrome 'baculovirus' (WSBV) Infection IS characterized by a wide range of target tissues, rapid disease onset and high mortality Dunng the viremic phase of infection, the virus is present in many organs However, the s~tuation in the natural environment remalns unclear To identify the pattern of the t~s s u e t r o p~s m of WSBV infection in adult Penaeus monodon (black tiger shnmp) of wild ongin, w e conducted a c o m b~n e d study using currently available nucleic acid diagnostic tools and conventional histological observat~ons uslng light (LM) and transnxssion electron (TEM) mlcroscopy to examine the sites for virus mult~plication S~x t e e n parts excised from shnmp specimens were examined pleopods, gills, stomach, abdominal muscle, hemolymph. m~d g u t , heart, pereiopods lymphoid organs, integument, nervous tissue, hepatopancreas, testes, ovaries, spermatophores, and eye stalks All these tissues/organs were found to support WSBV replic a t~o n For the f~r s t t~m e in s~t u hybridization and TEM showed evidence of WSBV in reproduct~ve organs of black tiger shrlmp In testes, WSBV-positive cells were located in the connective Ussue layer surrounding the seminiferous tubules and no germ cells were found to b e infected In the spermatophore only muscle and connective t~s s u e cells were WSBV posltive In the ovary, foll~cle cells oogonla oocytes and connective tissue cells were WSBV positlve However, the fact that w e were unable to find any Infected mature eggs suggested that infected e g g cells were killed by the virus before maturation

SPOP is mutated in 4.6% to 14.4% of patients with prostate cancer across different ethnic and demographic backgrounds. There was no significant association between SPOP mutations with ethnicity, clinical, or pathologic parameters. Mutual exclusivity of SPOP mutation with ERG rearrangement as well as a high association with CHD1 deletion reinforces SPOP mutation as defining a distinct molecular subclass of prostate cancer.

Object. Evidence-based guidelines recommend intracranial pressure (ICP) monitoring for patients with severe traumatic brain injury (TBI), but there is limited evidence that monitoring and treating intracranial hypertension reduces mortality. This study uses a large, prospectively collected database to examine the effect on 2-week mortality of ICP reduction therapies administered to patients with severe TBI treated either with or without an ICP monitor.Methods. From a population of 2134 patients with severe TBI (Glasgow Coma Scale [GCS] Score < 9), 1446 patients were treated with ICP-lowering therapies. Of those, 1202 had an ICP monitor inserted and 244 were treated without monitoring. Patients were admitted to one of 20 Level I and two Level II trauma centers, part of a New York State quality improvement program administered by the Brain Trauma Foundation between 2000 and 2009. This database also contains information on known independent early prognostic indicators of mortality, including age, admission GCS score, pupillary status, CT scanning findings, and hypotension.Results. Age, initial GCS score, hypotension, and CT scan findings were associated with 2-week mortality. In addition, patients of all ages treated with an ICP monitor in place had lower mortality at 2 weeks (p = 0.02) than those treated without an ICP monitor, after adjusting for parameters that independently affect mortality.Conclusions. In patients with severe TBI treated for intracranial hypertension, the use of an ICP monitor is associated with significantly lower mortality when compared with patients treated without an ICP monitor. Based on these findings, the authors conclude that ICP-directed therapy in patients with severe TBI should be guided by ICP monitoring. (http://thejns.org/doi/abs/10.3171/2012.7.JNS111816) KeY WorDs • intracranial pressure • mortality rate • traumatic brain injury • evidence-based guidelines 729Abbreviations used in this paper: AANS = American Association of Neurological Surgeons; BTF = Brain Trauma Foundation; CNS = Congress of Neurological Surgeons; GCS = Glasgow Coma Scale; ICP = intracranial pressure; TBI = traumatic brain injury.

Object In spite of evidence that use of the Brain Trauma Foundation Guidelines for the Management of Severe Traumatic Brain Injury (Guidelines) would dramatically reduce morbidity and mortality, adherence to these Guidelines remains variable across trauma centers. The authors analyzed 2-week mortality due to severe traumatic brain injury (TBI) from 2001 through 2009 in New York State and examined the trends in adherence to the Guidelines. Methods The authors calculated trends in adherence to the Guidelines and age-adjusted 2-week mortality rates between January 1, 2001, and December 31, 2009. Univariate and multivariate logistic regression analyses were performed to evaluate the effect of time period on case-fatality. Intracranial pressure (ICP) monitor insertion was modeled in a 2-level hierarchical model using generalized linear mixed effects to allow for clustering by different centers. Results From 2001 to 2009, the case-fatality rate decreased from 22% to 13% (p < 0.0001), a change that remained significant after adjusting for factors that independently predict mortality (adjusted OR 0.52, 95% CI 0.39–0.70; p < 0.0001). Guidelines adherence increased, with the percentage of patients with ICP monitoring increasing from 56% to 75% (p < 0.0001). Adherence to cerebral perfusion pressure treatment thresholds increased from 15% to 48% (p < 0.0001). The proportion of patients having an ICP elevation greater than 25 mm Hg dropped from 42% to 29% (p = 0.0001). Conclusions There was a significant reduction in TBI mortality between 2001 and 2009 in New York State. Increase in Guidelines adherence occurred at the same time as the pronounced decrease in 2-week mortality and decreased rate of intracranial hypertension, suggesting a causal relationship between Guidelines adherence and improved outcomes. Our findings warrant future investigation to identify methods for increasing and sustaining adherence to evidence-based Guidelines recommendations.