Identification of an EGFRvIII-JNK2-HGF/c-Met-Signaling Axis Required for Intercellular Crosstalk and Glioblastoma Multiforme Cell Invasion.

Glioblastoma multiforme (GBM) is the most aggressive and common form of adult brain cancer. Current therapeutic strategies include surgical resection, followed by radiotherapy and chemotherapy. Despite such aggressive multimodal therapy, prognosis remains poor, with a median patient survival of 14 months. A proper understanding of the molecular drivers responsible for GBM progression are therefore necessary to instruct the development of novel targeted agents and enable the design of effective treatment strategies. Activation of the c-Jun N-terminal kinase isoform 2 (JNK2) is reported in primary brain cancers, where it associates with the histologic grade and amplification of the epidermal growth factor receptor (EGFR). In this manuscript, we demonstrate an important role for JNK2 in the tumor promoting an invasive capacity of EGFR variant III, a constitutively active mutant form of the receptor commonly found in GBM. Expression of EGFR variant III induces transactivation of JNK2 in GBM cells, which is required for a tumorigenic phenotype in vivo. Furthermore, JNK2 expression and activity is required to promote increased cellular invasion through stimulation of a hepatocyte growth factor-c-Met signaling circuit, whereby secretion of this extracellular ligand activates the receptor tyrosine kinase in both a cell autonomous and nonautonomous manner. Collectively, these findings demonstrate the cooperative and parallel activation of multiple RTKs in GBM and suggest that the development of selective JNK2 inhibitors could be therapeutically beneficial either as single agents or in combination with inhibitors of EGFR and/or c-Met.

High-throughput direct fecal PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in sheep and cattle.

Johne's disease (JD) is a chronic enteric disease caused by Mycobacterium avium subsp. paratuberculosis that affects ruminants. Transmission occurs by the fecal-oral route. A commonly used antemortem diagnostic test for the detection of M. avium subsp. paratuberculosis in feces is liquid culture; however, a major constraint is the 2- to 3-month incubation period needed for this method. Rapid methods for the detection of M. avium subsp. paratuberculosis based on PCR have been reported, but comprehensive validation data are lacking. We describe here a new test, the high-throughput-Johnes (HT-J), to detect M. avium subsp. paratuberculosis in feces. Its diagnostic accuracy was compared with that of liquid radiometric (Bactec) fecal culture using samples from cattle (1,330 samples from 23 herds) and sheep (596 samples from 16 flocks). The multistage protocol involves the recovery of M. avium subsp. paratuberculosis cells from a fecal suspension, cell rupture by bead beating, extraction of DNA using magnetic beads, and IS900 quantitative PCR. The limit of detection of the assay was 0.0005 pg, and the limit of quantification was 0.005 pg M. avium subsp. paratuberculosis genomic DNA. Only M. avium subsp. paratuberculosis was detected from a panel of 51 mycobacterial isolates, including 10 with IS900-like sequences. Of the 549 culture-negative fecal samples from unexposed herds and flocks, 99% were negative in the HT-J test, while 60% of the bovine- and 84% of the ovine-culture-positive samples were positive in the HT-J test. As similar total numbers of samples from M. avium subsp. paratuberculosis-exposed animals were positive in culture and HT-J tests in both species, and as the results of a McNemar's test were not significant, these methods probably have similar sensitivities, but the true diagnostic sensitivities of these tests are unknown. These validation data meet the consensus-based reporting standards for diagnostic test accuracy studies for paratuberculosis and the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (S. A. Bustin et al., Clin. Chem. 55:611-622, 2009, doi:10.1373/clinchem.2008.112797). The HT-J assay has been approved for use in JD control programs in Australia and New Zealand.

Culture phenotypes of genomically and geographically diverse Mycobacterium avium subsp. paratuberculosis isolates from different hosts.

Mycobacterium avium subsp. paratuberculosis causes paratuberculosis (Johne's disease) in ruminants in most countries. Historical data suggest substantial differences in culturability of M. avium subsp. paratuberculosis isolates from small ruminants and cattle; however, a systematic comparison of culture media and isolates from different countries and hosts has not been undertaken. Here, 35 field isolates from the United States, Spain, Northern Ireland, and Australia were propagated in Bactec 12B medium and Middlebrook 7H10 agar, genomically characterized, and subcultured to Lowenstein-Jensen (LJ), Herrold's egg yolk (HEY), modified Middlebrook 7H10, Middlebrook 7H11, and Watson-Reid (WR) agars, all with and without mycobactin J and some with sodium pyruvate. Fourteen genotypes of M. avium subsp. paratuberculosis were represented as determined by BstEII IS900 and IS1311 restriction fragment length polymorphism analysis. There was no correlation between genotype and overall culturability, although most S strains tended to grow poorly on HEY agar. Pyruvate was inhibitory to some isolates. All strains grew on modified Middlebrook 7H10 agar but more slowly and less prolifically on LJ agar. Mycobactin J was required for growth on all media except 7H11 agar, but growth was improved by the addition of mycobactin J to 7H11 agar. WR agar supported the growth of few isolates. The differences in growth of M. avium subsp. paratuberculosis that have historically been reported in diverse settings have been strongly influenced by the type of culture medium used. When an optimal culture medium, such as modified Middlebrook 7H10 agar, is used, very little difference between the growth phenotypes of diverse strains of M. avium subsp. paratuberculosis was observed. This optimal medium is recommended to remove bias in the isolation and cultivation of M. avium subsp. paratuberculosis.

ßarrestin-1 regulates DNA repair by acting as an E3-ubiquitin ligase adaptor for 53BP1.

Cellular DNA is constantly under threat from internal and external insults, consequently multiple pathways have evolved to maintain chromosomal fidelity. Our previous studies revealed that chronic stress, mediated by continuous stimulation of the ß2-adrenergic-ßarrestin-1 signaling axis suppresses activity of the tumor suppressor p53 and impairs genomic integrity. In this pathway, ßarrestin-1 (ßarr1) acts as a molecular scaffold to promote the binding and degradation of p53 by the E3-ubiquitin ligase, MDM2. We sought to determine whether ßarr1 plays additional roles in the repair of DNA damage. Here we demonstrate that in mice ßarr1 interacts with p53-binding protein 1 (53BP1) with major consequences for the repair of DNA double-strand breaks. 53BP1 is a principle component of the DNA damage response, and when recruited to the site of double-strand breaks in DNA, 53BP1 plays an important role coordinating repair of these toxic lesions. Here, we report that ßarr1 directs 53BP1 degradation by acting as a scaffold for the E3-ubiquitin ligase Rad18. Consequently, knockdown of ßarr1 stabilizes 53BP1 augmenting the number of 53BP1 DNA damage repair foci following exposure to ionizing radiation. Accordingly, ßarr1 loss leads to a marked increase in irradiation resistance both in cells and in vivo. Thus, ßarr1 is an important regulator of double strand break repair, and disruption of the ßarr1/53BP1 interaction offers an attractive strategy to protect cells against high levels of exposure to ionizing radiation.

Review of patients with peritoneal malignancy treated with peritonectomy and heated intraperitoneal chemotherapy.

BACKGROUND: Peritoneal dissemination of malignancy is usually considered incurable. The purpose of the present study was to evaluate the efficacy of intraperitoneal chemohyperthermia and cytoreductive surgery. METHODS: The present article is a retrospective review of prospectively recorded data in 60 patients who underwent 71 peritonectomy procedures between January 1996 and May 2004. Hospital records, a database and department notes were studied. Conditions treated were pseudomyxoma peritoneii (PMP) and appendiceal cancer (23), mesothelioma (7), colorectal cancer (CRC, 15), ovarian cancer (6) and other forms of malignancy (9). Following cytoreductive surgery, early postoperative intraperitoneal chemotherapy (EPIC) was given in 47 procedures, five with added i.v. mitomycin C. In 34 procedures, heated intraperitoneal chemotherapy (HIPEC) was administered. A policy change was made from intravenous to intraperitoneal mitomycin C chemotherapy in December 2001. Peritoneal cancer index (PCI) was calculated for all procedures. RESULTS: Of the procedures, 23 had PCI < or = 10, 37 had PCI of 11-20, and 11 had PCI > 20. The median operation time was 9 h. Blood units transfused and length of hospital stay have declined. Mortality was 4/60 patients (6.7%), caused by pancytopenia and sepsis. Morbidity occurred in 28/71 procedures. The 3-year survival rate for the HIPEC group was 71% compared with 28% for the no HIPEC group. In the complete excision group, the 3-year survival rate was 52% compared with 13% for the incomplete excision group. The 3-year survival rate for PMP and appendiceal cancer was 74%. The 2-year survival rate for ovarian cancer was 67%, mesothelioma 57%, and CRC 50%, respectively. CONCLUSIONS: Morbidity is significantly associated with duration of surgery and units of blood transfused. Our findings are consistent with the international experience in patients treated with combined peritonectomy and HIPEC.

Peritoneal carcinomatosis from colorectal cancer and small bowel cancer treated with peritonectomy.

BACKGROUND: This study aims to assess the survival of patients who underwent peritonectomy, to assess the morbidity and mortality associated with the procedure and to review the published reports on the survival of patients with peritoneal spread of colorectal cancer (CRC). METHODS: Peritonectomy involves resection of all visible peritoneal tumour and is followed by heated intraperitoneal chemotherapy. Peritonectomy with heated intraperitoneal chemotherapy is associated with a 3-year survival of 30-50% in patients with low peritoneal cancer index (PCI) with peritoneal carcinomatosis from CRC. There are approximately 1000 patients in phase 2 studies and a large survival advantage was shown in a randomized control trial. We have carried out over 100 peritonectomy procedures. This study describes 22 patients with peritoneal spread of gastrointestinal cancer treated with peritonectomy between 1996 and March 2005. Twenty of these patients had primary colorectal cancer and two patients had primary small bowel cancer. RESULTS: Of the 22 patients who underwent peritonectomy, 8 patients are now deceased. The median follow up is now 16.1 months. At 12 months, the survival was 61.5% and at 24 months the survival was 46.1%, which are creditable results comparable with the world published reports. We found that those patients with all macroscopic residual tumour removed at the end of the procedure (completeness of cancer resection, CCR O) had improved 24-month survival compared with patients in whom there was incomplete tumour resection (53.3% survival vs 22.2%, respectively, P = 0.024). Patients with a PCI score less than 13 had better survival (P = 0.0003). CONCLUSIONS: Peritonectomy for peritoneal carcinomatosis from CRC offers patients improved survival. Our results are consistent with the published data with respect to improved survival in patients with low PCI and complete cytoreduction.

Particulate matter-induced airway hyperresponsiveness is lymphocyte dependent.

BACKGROUND: Exposure to airborne particulate matter (PM), a major component of air pollution, has been associated with increases in both exacerbations of and hospitalizations for asthma. We have previously shown that exposure to ambient PM collected in urban Baltimore (AUB) induces airway hyperresponsiveness (AHR), eosinophilic and neutrophilic inflammation, and the recruitment of T cells. However, the mechanism(s) by which it induces these features of asthma remains unknown. OBJECTIVE: We investigated whether T lymphocytes play a role in AUB-induced AHR. METHODS: We compared the effects of AUB exposure on the allergic phenotype in wild-type (WT) BALB/c mice and in mice deficient in recombinase-activating gene-1 (Rag1-/-) that lack mature lymphocytes. RESULTS: We found that exposure of WT mice to AUB induced AHR concomitant with increases in the numbers of bronchoalveolar lavage (BAL) fluid lymphocytes, eosinophils, neutrophils, and mucus-containing cells in the lungs of WT mice. Interestingly, we show for the first time that these effects were associated with significant elevations in interleukin (IL)-17A, IL-17F, and T-helper 2 cell (TH2) (IL-13, IL-5) cytokine levels in lung cells, as well as reductions in the levels of the suppressive cytokine IL-10. Interestingly, Rag1-/- mice failed to develop AUB-induced AHR; however, AUB-induced BAL fluid cellularity, and mucus cell changes were only partially inhibited in Rag1-/- mice. CONCLUSIONS: Taken together, our results suggest that AUB exposure increases the pathophysiological features of asthma via activation of lymphocyte-dependent pathways. These results provide a plausible biological mechanism for the strong association between PM exposure and the increased severity of asthma.

Learning curve for cytoreductive surgery and perioperative intraperitoneal chemotherapy for peritoneal surface malignancy--a journey to becoming a Nationally Funded Peritonectomy Center.

BACKGROUND: Cytoreductive surgery (CRS) combined with perioperative intraperitoneal chemotherapy (PIC) for peritoneal surface malignancy is associated with a morbidity rate of 30-50% and a mortality rate of 1-10%. Recently, the St George Hospital in Sydney has been commissioned as the Nationally Funded Center for treatment of peritoneal surface malignancy in Australia. METHODS: The clinical and treatment-related data regarding 140 consecutive patients were prospectively collected. A comparison between the initial 70 patients (Group I) and the subsequent 70 patients (Group II) was performed. Univariate and multivariate analyses were conducted to identify the significant risk factors for moderate to severe morbidity. RESULTS: The hospital mortality was 4%. Sixty-one patients (44%) had moderate morbidity. Twenty-eight patients (20%) experienced severe morbidity. The mean hospital stay was 30 days. Twenty-seven patients (19%) were readmitted after initial discharge for management of delayed complications. The severe morbidity rate reduced from 30% to 10%, and the delayed morbidity rate reduced from 29% to 10%, when comparing Groups I and II. There were also reduced transfusion requirement, duration of operation, and intensive care unit stay. In the multivariate analysis, Group I (vs Group II; P = .005), performing small bowel resection (P = .005), and >4 peritonectomy procedures (vs