T24 HRAS transformed NIH/3T3 mouse cells (GhrasT-NIH/3T3) in serial tumorigenic in vitro/in vivo passages give rise to increasingly aggressive tumorigenic cell lines T1-A and T2-A and metastatic cell lines T3-HA and T4-PA.

Cancer cells often arise progressively from "normal" to "pre-cancer" to "transformed" to "local metastasis" to "metastatic disease" to "aggressive metastatic disease". Recent whole genome sequencing (WGS) and spectral karyotyping (SKY) of cancer cells and tumorigenic models have shown this progression involves three major types of genome rearrangements: ordered small step-wise changes, more dramatic "punctuated evolution" (chromoplexy), and large catastrophic steps (chromothripsis) which all occur in random combinations to generate near infinite numbers of stochastically rearranged metastatic cancer cell genomes. This paper describes a series of mouse cell lines developed sequentially to mimic this type of progression. This starts with the new GhrasT-NIH/Swiss cell line that was produced from the NIH/3T3 cell line that had been transformed by transfection with HRAS oncogene DNA from the T24 human bladder carcinoma. These GhrasT-NIH/Swiss cells were injected s.c. into NIH/Swiss mice to produce primary tumors from which one was used to establish the T1-A cell line. T1-A cells injected i.v. into the tail vein of a NIH/Swiss mouse produced a local metastatic tumor near the base of the tail from which the T2-A cell line was established. T2-A cells injected i.v. into the tail vein of a nude NIH/Swiss mouse produced metastases in the liver and one lung from which the T3-HA (H=hepatic) and T3-PA (P=pulmonary) cell lines were developed, respectively. T3-HA cells injected i.v. into a nude mouse produced a metastasis in the lung from which the T4-PA cell line was established. PCR analysis indicated the human T24 HRAS oncogene was carried along with each in vitro/in vivo transfer step and found in the T2-A and T4-PA cell lines. Light photomicrographs indicate that all transformed cells are morphologically similar. GhrasT-NIH/Swiss cells injected s.c. produced tumors in 4% of NIH/Swiss mice in 6-10 weeks; T1-A cells injected s.c. produced tumors in 100% of NIH/Swiss mice in 7-10 days. T1-A, T-2A, T3-HA and T4-PA cells when injected i.v. into the tail produced local metastasis in non-nude or nude NIH/Swiss mice. T4-PA cells were more widely metastatic than T3-HA cells when injected i.v. into nude mice. Evaluation of the injected mice indicated a general increase in metastatic potential of each cell line in the progression as compared to the GhrasT-NIH/3T3 transformed cells. A new photomicrographic technique to follow growth rates within six preselected 2×2mm(2) grids per plate is described. Average doubling times of the transformed cells GhrasT-NIH/3T3 (17h), T1A (17.5h), T2A (15.5h), T3-HA (17.5h) and T4-PA (18.5h) (average 17.2h) were significantly faster (P=0.006) than NIH Swiss primary embryonic cells and NIH/3T3 cells (22 h each). This cell series is currently used in this lab for studies of cancer cell inhibitors, mitochondrial biogenesis and gene expression and is available for further study by other investigators for intra- and inter-laboratory comparisons of WGS, transcriptome sequencing, SKY and other analyses. The genome rearrangements in these cells together with their phenotypic properties may help provide more insights into how one tumorigenic progression occurred to produce the various cell lines that led to the highly metastatic T4-PA cell line.

High-frequency percussive ventilation and low tidal volume ventilation in burns: a randomized controlled trial.

OBJECTIVES: In select burn intensive care units, high-frequency percussive ventilation is preferentially used to provide mechanical ventilation in support of patients with acute lung injury, acute respiratory distress syndrome, and inhalation injury. However, we found an absence of prospective studies comparing high-frequency percussive ventilation with contemporary low-tidal volume ventilation strategies. The purpose of this study was to prospectively compare the two ventilator modalities in a burn intensive care unit setting. DESIGN: Single-center, prospective, randomized, controlled clinical trial, comparing high-frequency percussive ventilation with low-tidal volume ventilation in patients admitted to our burn intensive care unit with respiratory failure. SETTING: A 16-bed burn intensive care unit at a tertiary military teaching hospital. PATIENTS: Adult patients ≥ 18 yrs of age requiring prolonged (> 24 hrs) mechanical ventilation were admitted to the burn intensive care unit. The study was conducted over a 3-yr period between April 2006 and May 2009. This trial was registered with ClinicalTrials.gov as NCT00351741. INTERVENTIONS: Subjects were randomly assigned to receive mechanical ventilation through a high-frequency percussive ventilation-based strategy (n = 31) or a low-tidal volume ventilation-based strategy (n = 31). MEASUREMENTS AND MAIN RESULTS: At baseline, both the high-frequency percussive ventilation group and the low-tidal volume ventilation group had similar demographics to include median age (interquartile range) (28 yrs [23-45] vs. 33 yrs [24-46], p = nonsignificant), percentage of total body surface area burn (34 [20-52] vs. 34 [23-50], p = nonsignificant), and clinical diagnosis of inhalation injury (39% vs. 35%, p = nonsignificant). The primary outcome was ventilator-free days in the first 28 days after randomization. Intent-to-treat analysis revealed no significant difference between the high-frequency percussive ventilation and the low-tidal volume ventilation groups in mean (± sd) ventilator-free days (12 ± 9 vs. 11 ± 9, p = nonsignificant). No significant difference was detected between groups for any of the secondary outcome measures to include mortality except the need for "rescue" mode application (p = .02). Nine (29%) in the low-tidal volume ventilation arm did not meet predetermined oxygenation or ventilation goals and required transition to a rescue mode. By contrast, two in the high-frequency percussive ventilation arm (6%) required rescue. CONCLUSIONS: A high-frequency percussive ventilation-based strategy resulted in similar clinical outcomes when compared with a low-tidal volume ventilation-based strategy in burn patients with respiratory failure. However, the low-tidal volume ventilation strategy failed to achieve ventilation and oxygenation goals in a higher percentage necessitating rescue ventilation.

Combining early coagulation and inflammatory status improves prediction of mortality in burned and nonburned trauma patients.

BACKGROUND: After injury, there is a synergistic response between inflammation and coagulation systems. We hypothesized that combining markers of these processes and standard clinical indices would improve early prediction of in- hospital mortality in burned and nonburned trauma patients. METHODS: Patients admitted to the surgical or burn intensive care unit within 24 hours of injury with an anticipated stay >or=3 days were enrolled during a one year period. Upon admission, blood was drawn for thromboelastography, plasma-based clotting assays, and cytokine levels. Clinical indices and multiple organ dysfunction syndrome (MODS) scores were recorded. Candidate variables evaluated included age, percentage third degree burns, inhalation injury, percentage total body surface area burns, interleukin-6, tumor necrosis factor alpha, interleukin-8, prothrombin time, partial thromboplastin time (PTT), maximal amplitude reflective of clot strength, group (burn or nonburn) and admission MODS. Multiple logistic regression with stepwise selection and likelihood ratio test was performed to identify predictors for mortality. A receiver operating characteristic (ROC) curve was constructed to assess the diagnostic performance of identified predictors. Validation of the model with an additional cohort was performed. RESULTS: For model development, we enrolled 25 burned and 33 nonburned trauma patients (20 blunt and 13 penetrating injuries). Fifteen deaths occurred. Multiple logistic regression analysis identified six independent risk factors for death: age, percentage third degree burns, inhalation injury, tumor necrosis factor alpha level, maximal amplitude, and MODS score with an area under ROC curve of 0.961 (95% confidence interval: 0.891, 1.000, p < 0.05). The area under the ROC curve for the validation cohort (n = 66) was 0.936 (95% confidence interval: 0.875, 0.997, p < 0.001). CONCLUSION: Our model improves prediction of in-hospital mortality in comparison to previous methods for burn and nonburn trauma patients. Furthermore, our model is equally applicable to all patients regardless of type of traumatic injury (nonburn or burn). This improvement is because of the inclusion of patient's early coagulation and inflammatory status in addition to standard clinical indices. These data provide a baseline within which to measure incremental improvements in care.

Mitogen-activated protein kinase regulates dopamine transporter surface expression and dopamine transport capacity.

The dopamine transporter (DAT) regulates the clearance of dopamine (DA) released into the extracellular space and is an important site on which psychostimulants act to produce their effects. Here, we show that mitogen-activated protein kinase (MAPK) regulates the transport capacity and intracellular trafficking of DAT. Incubation of striatal synaptosomes or epitope-tagged human DAT (hDAT) human embryonic kidney (HEK) 293 cells with the MAPK kinase (MEK) inhibitors 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto) butadiene and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one decreased DA uptake in a concentration- and time-dependent manner. Kinetic studies revealed a decrease in the capacity of transport (Vmax) but no change in Km. Immunoblotting confirmed labeling of p42 and p44 MAPK in untreated striatal synaptosomes and HEK 293 cells, consistent with constitutive MAPK activation, and the inhibitors used decreased MAPK phosphorylation. Biotinylation and confocal imaging studies showed that MAPK inhibition promoted the clathrin-associated redistribution of hDAT from the plasma membrane to the cytosol. In contrast, transient transfection of hDAT-expressing cells with constitutively active MEK increased the Vmax of DA transport without altering Km. However, only a small increase in hDAT cell surface expression was seen. These data demonstrate an involvement of the MAPK cascade in regulating DAT transport capacity in striatum and that inhibition of this cascade decreases DAT cell surface expression in HEK 293 cells. Furthermore, they highlight the potential role of MAPK as a presynaptic mechanism that regulates DA signaling.

Elevations in inflammatory cytokines are associated with poor outcomes in mechanically ventilated burn patients.

BACKGROUND: The treatment of burn patients who undergo mechanical ventilation is complicated by many factors; patient outcomes and mortality could potentially be improved with predictive biomarkers. Severe burn provokes a systemic inflammatory response characterized by the release of a host of cytokines. Recent studies evaluated the prognostic value of temporal changes in cytokine levels in several patient populations, but few have compared differences in the cytokine profiles of survivors and nonsurvivors following severe burn. We previously compared high-frequency percussive ventilation and low-tidal-volume ventilation and found no difference in mortality or cytokine levels between the two treatments. Since it is unknown whether cytokine levels are predictive of mortality in these patients, we performed a post hoc analysis comparing cytokine levels in survivors and nonsurvivors. METHODS: We evaluated plasma levels of several cytokines (interleukin 1ß [IL-1ß], IL-6, IL-8, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor α) for their prognostic biomarker potential related to mortality at 0, 3, and 7 days in survivors and nonsurvivors of burns. RESULTS: While the majority of values for IL-1ß, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor α fell below the limit of quantification, univariate analysis demonstrated higher plasma levels of IL-6 and IL-8 in nonsurvivors on Day 7. Logistic regression revealed that elevated plasma IL-8 was independently associated with an increased likelihood of the composite end point of death or ventilator-associated pneumonia with odds ratios of 7.9, 26, and 7.3 on Days 0, 3, and 7, respectively. CONCLUSION: Early increases in plasma IL-8 are associated with a multifold increase in death or ventilator-associated pneumonia in mechanically ventilated burn patients. LEVEL OF EVIDENCE: Prognostic/epidemiologic study, level IV; therapeutic study, level IV.

Evaluation of the specificity and sensitivity of a potential rapid influenza screening system.

Influenza remains a serious worldwide health threat with numerous deaths attributed to influenza-related complications. It is likely that transmission of influenza and both the morbidity and mortality of influenza could be reduced if inexpensive but reliable influenza screening assays were more available to the general public or local medical treatment facilities. This report provides the initial evaluation of a pilot system designed by Lucigen Corp. (Middleton, WI, USA) as a potential rapid near point-of-care screening system for influenza A and influenza B. The evaluation of specificity and sensitivity was conducted on stored nasal swab samples collected from emergency department patients presenting with influenza-like symptoms at a large military academic hospital and on de-identified nasal swabs and isolated RNA from a local epidemiology laboratory. The gold standard for assessment of specificity and sensitivity was the Luminex® Respiratory Viral Panel.

Differential expression of the immunoinflammatory response in trauma patients: burn vs. non-burn.

RATIONALE: Cytokines are central mediators of the immune-inflammatory response to injury and subsequent multiple organ dysfunction syndrome (MODS). Although previous studies evaluated cytokine levels after trauma, differences between patients with burn and non-burn trauma have not been assessed systematically. METHODS: A prospective database of trauma patients admitted between May 2004 and September 2007 to the burn or surgical intensive care units within 24 h of injury with an anticipated stay of at least 72 h was analyzed. Sequential clinical and laboratory parameters were collected in the first week, including multiplex analysis data for plasma levels of inflammatory cytokines (IL-6, and IL-8). Patients with known pre-injury coagulopathy were excluded. A Marshall score of 10 or greater was defined as MODS. RESULTS: A total of 179 patients were enrolled (67 burn and 112 non-burn). Plasma IL-6 and IL-8 levels were markedly elevated in both burn and non-burn patients compared to healthy volunteers. Burn subjects had higher levels of IL-6 and IL-8 than the non-burn on days 1 through 7 after injury. Subjects with burns and at least 30% total body surface area were older and had a lower injury severity score, a higher prevalence of MODS, and correspondingly higher mortality. Multivariate analysis of injury type, MODS, and time did not demonstrate an influence of MODS. CONCLUSIONS: Burns were associated with a greater and more sustained immune-inflammatory response than non-burn trauma as evidenced by elevated plasma IL-6 and IL-8 levels during the first week. There was no association between MODS and plasma cytokine levels.

A quantitative electrochemiluminescence assay for Clostridium perfringens alpha toxin.

Described is a rapid direct sandwich format electrochemiluminescence assay for identifying and assaying Clostridium perfringens alpha toxin. Biotinylated antibodies to C. perfringens alpha toxin bound to streptavidin paramagnetic beads specifically immunoadsorbed soluble sample alpha toxin which subsequently selectively immunoadsorbed ruthenium (Ru)-labeled detection antibodies. The ruthenium chelate of detection antibodies chemically reacted in the presence of tripropylamine and upon electronic stimulation emitted photons (electrochemiluminescence) that were detected by the photodiode of the detector. Elevated toxin concentrations increased toxin immunoadsorption and the specific immunoadsorption of Ru-labeled antibodies to alpha toxin, which resulted in increased dose-dependent electrochemiluminescent signals. The standardized assay was rapid (single 2.5-h coincubation of all reagents), required no wash steps, and had a sensitivity of about 1 ng/ml of toxin. The assay had excellent accuracy and precision and was validated in buffer, serum, and urine with no apparent matrix effects.

An enzymatic electrochemiluminescence assay for the lethal factor of anthrax.

The lethal factor (LF) of anthrax toxin is the toxic component of the exotoxin (lethal toxin) secreted by toxic strains of Bacillus anthracis. The lethal factor is a zinc-dependent metalloprotease that specifically cleaves the mitogen-activated protein kinase kinase (MAPKK) family of enzymes. We took advantage of this substrate specificity to develop an electrochemiluminescence (ECL) peptide cleavage assay. The ECL assay uses the stable ruthenium (Ru) metal chelate that, in the presence of tripropylamine, generates a light reaction triggered by the application of an electric potential. The Ru label is specifically incorporated into the C-terminal CYS residue of a synthetic peptide (23mer) containing the MAPKK2 cleavage sequence of LF. Streptavidin-coated paramagnetic beads were the solid phase and facilitated separation and characterization of the enzymatic reaction products based upon N-terminal biotinylation of the peptide substrate. Intact peptide bound via the biotin moiety generated high signal due to the Ru label, whereas binding of the cleaved peptide fragment devoid of Ru label reduced the ECL signal. The proposed assay provides a novel opportunity for the screening of potential therapeutics against anthrax.