InVivo Loss of Function Screening Reveals Carbonic Anhydrase IX as a Key Modulator of Tumor Initiating Potential in Primary Pancreatic Tumors

Reprogramming of energy metabolism is one of the emerging hallmarks of cancer. Up-regulation of energy metabolism pathways fuels cell growth and division, a key characteristic of neoplastic disease, and can lead to dependency on specific metabolic pathways. Thus, targeting energy metabolism pathways might offer the opportunity for novel therapeutics. Here, we describe the application of a novel in vivo screening approach for the identification of genes involved in cancer metabolism using a patient-derived pancreatic xenograft model. Lentiviruses expressing short hairpin RNAs (shRNAs) targeting 12 different cell surface protein transporters were separately transduced into the primary pancreatic tumor cells. Transduced cells were pooled and implanted into mice. Tumors were harvested at different times, and the frequency of each shRNA was determined as a measure of which ones prevented tumor growth. Several targets including carbonic anhydrase IX (CAIX), monocarboxylate transporter 4, and anionic amino acid transporter light chain, xc- system (xCT) were identified in these studies and shown to be required for tumor initiation and growth. Interestingly, CAIX was overexpressed in the tumor initiating cell population. CAIX expression alone correlated with a highly tumorigenic subpopulation of cells. Furthermore, CAIX expression was essential for tumor initiation because shRNA knockdown eliminated the ability of cells to grow in vivo. To the best of our knowledge, this is the first parallel in vivo assessment of multiple novel oncology target genes using a patient-derived pancreatic tumor model.     

The coagulant-active phospholipid content is a major determinant of in vivo thrombogenicity of prothrombin complex (Factor IX) concentrates in rabbits

In vitro evaluation of prothrombin complex concentrates in a thrombin generation assay, using DAPA and purified components of the prothrombinase complex, demonstrated significant levels of coagulant- active "phospholipid replacing" activity. Quantification of this activity showed a significant correlation (r = 0.8747, p less than 0.01) with thrombogenicity measured in vivo in a stasis model in rabbits. Extracted lipid material retained full phospholipid replacing activity in the vitro assay. Thin-layer chromatographic characterization confirmed the presence of phospholipids with known coagulant activity in vitro. In vivo, the extracted material was nonthrombogenic but augmented the thrombogenicity of purified factor Xa. Substitution of a synthetic coagulant-active phospholipid (phosphatidylcholine-phosphatidylserine lipid vesicles) for the extracted phospholipid produced a similar augmentation of a factor-Xa- induced thrombogenicity in vivo. It is concluded that the coagulant- active phospholipid content of prothrombin complex concentrates is a major determinant of thrombogenicity but requires the presence of activated clotting factors for its expression in vivo.     

Effects of recombinant human granulocyte-macrophage colony-stimulating factor on intracellular pH in mature granulocytes

We studied the effects of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSFrh) on the internal pH of granulocytes using the fluorescent probe BCECF. GM-CSFrh did not directly alter the resting pH of granulocytes isolated from the peripheral blood; however, when the cells were preincubated for 90 minutes with the growth factor and then activated with the chemotactic peptide N-formyl met leu phe (fMLP), they exhibited both an acceleration in the initial rate of acidification and a marked delay in realkalinization. The kinetic changes both in initial acidification and in subsequent realkalinization induced by GM-CSFrh priming were not prevented by protein synthesis inhibitors and were observed in granulocytes harvested from patients with both sex-linked and autosomal recessive chronic granulomatous disease (CGD). By directly quantitating H+ ion secretion, by monitoring the effects of sodium repletion on intracellular pH, and through use of the sodium channel inhibitors amiloride and dimethyl amiloride and the Na+/K+-ATPase inhibitor ouabain, we showed that the altered kinetics of intracellular acidification and alkalinization following fMLP stimulation of GM-CSFrh- primed granulocytes could not be accounted for by changes in transmembrane proton exportation regulated by the Na+/H+ antiport channel. Although the initial acidification following fMLP was abrogated by 2-deoxy-D-glucose in both GM-CSFrh-pretreated and GM-CSFrh- untreated granulocytes, retardation of the subsequent phase of alkalinization was observed in GM-CSFrh-primed cells even after inhibition of both glycolytic and mitochondrial metabolism. Our data indicate that the increased cytosolic acidification following fMLP stimulation in granulocytes "primed" with GM-CSFrh does not result from disordered proton excretion but instead from increased release of intracellular free acid which is only partially coupled to glucose catabolism or to the generation of superoxide anion (O2-).     

Detection of platelet-directed immunoglobin G in sera using the peroxidase-anti-peroxidase (PAP) slide technique

An immunohistochemical procedure for the detection of immunoglobulin G adherent to platelets is described. The peroxidase anti-peroxidase method is used to detect antibody activity directed against platelets from normal donors in the sera from 305 individuals. These subjects were divided into three groups: group 1, patients referred for tissue typing; group 2, healthy normal females; group 3, healthy normal males. In group 1, 28% of the sera were found to be positive; in most of these a history of prior transfusions was obtained. In group 2, 7.4% were found to be positive, most having previous pregnancies. Only 1% were found to be positive in group 3, and no reason for presensitization was found. Results from the indirect immunofluorescence technique served as a control and as a means to compare the sensitivity. Under the conditions chosen, the peroxidase anti-peroxidase test was two to eight times more sensitive than the immunofluorescence technique. Specificity of the peroxidase anti-peroxidase technique was demonstrated using a monospecific anti-PLA1 antiserum. It is concluded that the peroxidase anti-peroxidase slide technique may be a useful tool in the study of platelet-related immunophenomena.     

Transforming growth factor beta inhibition increases mortality and left ventricular dilatation after myocardial infarction

BACKGROUND: Transforming growth factor (TGF)-beta is a locally generated cytokine involved in healing processes and tissue fibrosis, all relevant for cardiac remodeling and the development of heart failure after myocardial infarction (MI). However, data regarding the function of TGF-beta after ischemic injury are inconclusive. METHODS AND RESULTS: We tested the effect of TGF-beta inhibition by application of a blocking antibody in mice with MI. Starting 1 week before or 5 days after coronary artery ligation mice were treated with intraperitoneal injections of an anti-TGF-beta (5 mg/kg bodyweight 1D11, Genzyme) or control antibody. Mortality over 8 weeks was significantly higher in the groups treated with the anti-TGF-beta antibody. Both, pre or post MI treatments were associated with increased left ventricular dilatation after MI as determined by serial echocardiography. In anti-TGF-beta treated mice collagen production decreased and matrix-metalloproteinase expression increased. However, the expression of TGF pro-inflammatory cytokine TNF-alpha was not altered by the treatment. Anti-TGF-beta treatment before or after coronary artery ligation increases mortality and worsens left ventricular remodeling in mice with non-reperfused MI. The detrimental effects of TGF-beta inhibition may be mediated by alterations in extracellular matrix remodeling.     

Bone marrow transplantation for patients with Philadelphia chromosome- positive acute lymphoblastic leukemia

We report the treatment outcome of allogeneic bone marrow transplantation in ten patients with Philadelphia chromosome-positive acute lymphoblastic leukemia. Six patients are alive and well for 6 to 30 months (median 19 months) after transplantation. Four patients died with transplant related complications. In view of the poor prognosis associated with this disease, marrow ablation followed by allogeneic or syngeneic marrow grafting may be the preferred treatment modality if a suitable marrow donor is available.     

Outcomes and Excess Costs among Patients with Cardiovascular Disease

ObjectiveTo report on two-year cardiovascular (CV) event rates and quantify the cost of cardiovascular disease using the Australian Reduction of Atherothrombosis for Continued Health (REACH) registry.MethodsProspective registry of 2873 patients with multiple risk factors (MRF), coronary artery disease (CAD), cerebrovascular disease (CerVD) and peripheral artery disease (PAD), recruited through 273 Australian general practitioners. Government reimbursement data from 2011 was used to calculate direct health care costs (pharmaceuticals, outpatient and hospitalisation costs). The main outcome of interest was two-year rates and associated excess costs of cardiovascular death, myocardial infarction, stroke, and hospitalisation for cardiovascular procedures.ResultsThe two year follow-up data were available for 2856 (99.4%) patients. Incidence of any hospitalisation and cardiovascular death was highest among those with previous history of PAD at baseline 49% (n = 126), and 5.1% (n = 13). Non-fatal cardiovascular events were highest among the PAD and CAD groups (21.8% (n = 56) and 14.1% (n = 297) respectively). Those with previous history of PAD and CerVD at baseline had the highest likelihood of CV death (OR = 2.53 (95% CI: 1.58–4.08) and OR = 1.61 (1.05–2.46) respectively) in comparison to other groups. Patients with PAD had the highest likelihood of vascular interventions OR = 3.11 (95% CI: 2.09–4.63) at two years. Overall, the mean (SD) direct expenditure over two years of follow-up per person was A$7544 (A$10,758). In the adjusted model, patients with CAD and PAD incurred A$1093 (95% CI A$24 – A$2072) and A$4890 (95% CI A$3105 – A$6869) more in mean total costs compared to patients with MRF.ConclusionsPatients with PAD had the highest likelihood of vascular interventions and CV death, and incurred high excess costs in comparison to other groups.     

Fibrosing alveolitis: CT-pathologic correlation

Computed tomography (CT) was performed within 10 days of open lung biopsy in nine patients with fibrosing alveolitis. One-centimeter collimation contiguous scans through the chest were obtained in all patients. Additional 1.5-mm collimation scans were obtained in the area in which lung biopsy was later performed in six patients. In seven patients, CT demonstrated patchy involvement of the lung parenchyma, areas with a reticular pattern being intermingled with areas of normal lung. The reticular pattern was associated with cystic spaces 2-4 mm in diameter and was more severe in the lung periphery. Histologically, the reticular pattern corresponded to areas of irregular fibrosis. One patient had diffuse honeycombing (2-20-mm cysts), and one had honeycombing only in the lung periphery. In all patients, CT clearly defined the architectural changes seen on open lung biopsy. These changes were much better seen on the 1.5-mm than on the 10-mm collimation scans. CT may be helpful in determining the pattern and distribution of lung involvement in patients with fibrosing alveolitis and in guiding the surgeon to the most appropriate area(s) for biopsy.