Cost analysis of noninvasive blood-based microRNA testing versus CT scans for follow-up in patients with testicular germ-cell tumors

BACKGROUND: Our group has developed a noninvasive blood-based microRNA (miRNA) test for improving diagnosis, disease monitoring, and relapse detection in malignant testicular germ-cell tumors (TGCTs). Performance analysis suggests the test is likely to have comparable sensitivity and specificity in detecting TGCT as computed tomography (CT), thus reducing the need for serial CT scans for follow-up monitoring, with associated reductions in cumulative radiation burden and second cancer risk. To facilitate clinical adoption, we undertook a cost analysis to identify the budget impact of replacing CT scans with miRNA testing within health care systems. METHODS: The TGCT aftercare pathway was mapped out using National Comprehensive Cancer Network guidelines. A Markov model was built to simulate the impact of the miRNA test on TGCT aftercare costs. Incidence, treatment probabilities, relapse rate, and death rate data were collected from published studies to populate the model. RESULTS: Applying our model to the US health care system, the miRNA test has the potential to save up to $69 million per year in aftercare expenses related to TGCT treatment, with exact savings depending on the adoption rate and test price. CONCLUSION: This analysis demonstrates the potential positive budget impact of adopting miRNA testing in place of CT scans in the clinical management of TGCTs

A BAC-Based Transgenic Mouse Specifically Expresses an Inducible Cre in the Urothelium

Cre-loxp mediated conditional knockout strategy has played critical roles for revealing functions of many genes essential for development, as well as the causal relationships between gene mutations and diseases in the postnatal adult mice. One key factor of this strategy is the availability of mice with tissue- or cell type-specific Cre expression. However, the success of the traditional molecular cloning approach to generate mice with tissue specific Cre expression often depends on luck. Here we provide a better alternative by using bacterial artificial chromosome (BAC)-based recombineering to insert iCreERT2 cDNA at the ATG start of the Upk2 gene. The BAC-based transgenic mice express the inducible Cre specifically in the urothelium as demonstrated by mRNA expression and staining for LacZ expression after crossing with a Rosa26 reporter mouse. Taking into consideration the size of the gene of interest and neighboring genes included in a BAC, this method should be widely applicable for generation of mice with tissue specific gene expression or deletions in a more specific manner than previously reported

Klebsiella pneumoniae induces host metabolic stress that promotes tolerance to pulmonary infection

K. pneumoniae sequence type 258 (Kp ST258) is a major cause of healthcare-associated pneumonia. However, it remains unclear how it causes protracted courses of infection in spite of its expression of immunostimulatory lipopolysaccharide, which should activate a brisk inflammatory response and bacterial clearance. We predicted that the metabolic stress induced by the bacteria in the host cells shapes an immune response that tolerates infection. We combined in situ metabolic imaging and transcriptional analyses to demonstrate that Kp ST258 activates host glutaminolysis and fatty acid oxidation. This response creates an oxidant-rich microenvironment conducive to the accumulation of anti-inflammatory myeloid cells. In this setting, metabolically active Kp ST258 elicits a disease-tolerant immune response. The bacteria, in turn, adapt to airway oxidants by upregulating the type VI secretion system, which is highly conserved across ST258 strains worldwide. Thus, much of the global success of Kp ST258 in hospital settings can be explained by the metabolic activity provoked in the host that promotes disease tolerance. Keywords: immunometabolism, Klebsiella pneumoniae, immunosuppressive, anti-inflammatory, itaconate, Type Six Secretion Syste

iCreERT2 activity in <i>TgUICBAC</i> mice in vivo.

<p><b>A</b>) Eight week old <i>TgUICBAC;Rosa26</i> mice were injected with Tamoxifen for 3 days (TAM) or equal volume of sunflower oil (OIL, control group). Mice were sacrificed one week after injection, bladder sections were obtained and X-gal staining was performed. Note that X-gal blue staining is observed in the epithelial cells of the bladder in mice injected with TAM, whereas no staining is observed in the bladder of control mice. Bars correspond to 100 µm. <b>B)</b> Representative immunofluorescence analysis of Upk2 (green) and ß-galactosidase (red) co-expression in bladder urothelium of <i>TgUICBAC;Rosa26</i> mice injected with Tamoxifen. Note that most of the Upk2-positive urothelial cells show a doted staining for ß-galactosidase. Bars correspond to 100 µm. <b>C)</b> Quantification of percentage of recombination in the analyzed bladder sections from 2 female and 2 male transgenic mice illustrated by mean ± SD.</p

Generation of mice carrying insertion of <i>iCreERT2</i> into the <i>ATG</i> of <i>Upk2</i> gene.

<p><b>A)</b> The BAC clone bMQ-343M5 contains the <i>Upk2</i> gene flanked by parts of <i>Foxr1</i> and <i>Blr1</i> genes, which together with <i>Upk2</i> gene are all transcribed from reverse strand, and <i>Bcl9l</i>, which is from forward strand. The size of the BAC clone insert is 82951 bp (from 44267673 to 44184722 in mouse chromosome 9). <i>iCreERT2-pA</i> is inserted into the <i>ATG</i> start of <i>Upk2</i> gene. ScaI sites and probes for southern blot (P5) are shown. Arrows indicate primers used for PCR genotyping. <b>B)</b> Mouse genotyping by PCR (upper panel) and Southern blot (lower panel). Positive founder lines (#1 and #5) are marked with circles. <i>UIC</i> stands for the <i>TgUICBAC</i> transgene.</p

Tissue-specific activity of iCReERT2 in vivo.

<p>Eight week old <i>TgUICBAC;Rosa26</i> mice were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035243#pone-0035243-g003" target="_blank">Figure 3</a>, sections of other organs were obtained and X-gal staining was performed. Note that X-gal blue staining is observed only in the epithelial cells of the renal pelvis, ureter (indicated with arrows) in mice injected with TAM, whereas no staining is observed in control mice or other organs of TAM-injected mice. Bars correspond to 100 µm.</p

Primer sequences for generation and characterization of <i>TgUICBAC</i> mice.

<p>Primer sequences for generation and characterization of <i>TgUICBAC</i> mice.</p

iCreERT2 expression in <i>TgUICBAC</i> mice.

<p>Total RNA from various organs of male (M) and female (F) mice were extracted, and subjected to RT-PCR analysis of iCreERT2 expression. Note that iCreERT2 expression is observed only in kidney (renal pelvis), ureter and bladder, revealing that it is specific of organs coated with urothelium. Mouse Gapdh served as a control.</p