Targeting fibroblast activation protein in tumor stroma with chimeric antigen receptor T cells can inhibit tumor growth and augment host immunity without severe toxicity.

The majority of chimeric antigen receptor (CAR) T-cell research has focused on attacking cancer cells. Here, we show that targeting the tumor-promoting, nontransformed stromal cells using CAR T cells may offer several advantages. We developed a retroviral CAR construct specific for the mouse fibroblast activation protein (FAP), comprising a single-chain Fv FAP [monoclonal antibody (mAb) 73.3] with the CD8α hinge and transmembrane regions, and the human CD3ζ and 4-1BB activation domains. The transduced muFAP-CAR mouse T cells secreted IFN-γ and killed FAP-expressing 3T3 target cells specifically. Adoptively transferred 73.3-FAP-CAR mouse T cells selectively reduced FAP(hi) stromal cells and inhibited the growth of multiple types of subcutaneously transplanted tumors in wild-type, but not FAP-null immune-competent syngeneic mice. The antitumor effects could be augmented by multiple injections of the CAR T cells, by using CAR T cells with a deficiency in diacylglycerol kinase, or by combination with a vaccine. A major mechanism of action of the muFAP-CAR T cells was the augmentation of the endogenous CD8(+) T-cell antitumor responses. Off-tumor toxicity in our models was minimal following muFAP-CAR T-cell therapy. In summary, inhibiting tumor growth by targeting tumor stroma with adoptively transferred CAR T cells directed to FAP can be safe and effective, suggesting that further clinical development of anti-human FAP-CAR is warranted

Chemical dissociation of human awareness: focus on non-competitive NMDA receptor antagonists

Since the mid-1950s the pharmaceutical industry has developed a number of chemicals, including phencyclidine, ketamine and related arylcyclohexylamines (PCE and TCP), dizocilpine (MK-801), N-allylnormetazocine [ NANM, (±)SKF-10,047], etoxadrol, dioxadrol and its enantiomers dexoxadrol and levoxadrol, which produce a constellation of unusual behavioral effects in animals and man. The compounds best studied in humans are phencyclidine and ketamine. They produce a remarkable dose-dependent dissociation of awareness. All of these substances are now known to be non-competitive antagonists of NMDA receptors of glutamic acid. They act in the NMDA receptor ion channel. One can conclude, on the basis of the effects observed with these agents, that glutamic acid and related excitatory amino acids are extremely important in the maintenance of human awareness.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/68872/2/10.1177_026988119200600312.pd

Deployable Laboratory Response to Influenza Pandemic; PCR Assay Field Trials and Comparison with Reference Methods

Background: The influenza A/H1N1/09 pandemic spread quickly during the Southern Hemisphere winter in 2009 and reached epidemic proportions within weeks of the official WHO alert. Vulnerable population groups included indigenous Australians and remote northern population centres visited by international travellers. At the height of the Australian epidemic a large number of troops converged on a training area in northern Australia for an international exercise, raising concerns about their potential exposure to the emerging influenza threat before, during and immediately after their arrival in the area. Influenza A/H1N1/09 became the dominant seasonal variant and returned to Australia during the Southern winter the following year. Methods: A duplex nucleic acid amplification assay was developed within weeks of the first WHO influenza pandemic alert, demonstrated in northwestern Australia shortly afterwards and deployed as part of the pathology support for a field hospital during a military exercise during the initial epidemic surge in June 2009. Results: The nucleic acid amplification assay was twice as sensitive as a point of care influenza immunoassay, as specific but a little less sensitive than the reference laboratory nucleic acid amplification assay. Repetition of the field assay with blinded clinical samples obtained during the 2010 winter influenza season demonstrated a 91.7% congruence with the reference laboratory method. Conclusions: Rapid in-house development of a deployable epidemic influenza assay allowed a flexible laboratory response, effective targeting of limited disease control resources in an austere military environment, and provided the public health laboratory service with a set of verification tools for resource-limited settings. The assay method was suitable for rapid deployment in time for the 2010 Northern winter

A Mouse to Human Search for Plasma Proteome Changes Associated with Pancreatic Tumor Development

Background: The complexity and heterogeneity of the human plasma proteome have presented significant challenges in the identification of protein changes associated with tumor development. Refined genetically engineered mouse (GEM) models of human cancer have been shown to faithfully recapitulate the molecular, biological, and clinical features of human disease. Here, we sought to exploit the merits of a well-characterized GEM model of pancreatic cancer to determine whether proteomics technologies allow identification of protein changes associated with tumor development and whether such changes are relevant to human pancreatic cancer. Methods and Findings: Plasma was sampled from mice at early and advanced stages of tumor development and from matched controls. Using a proteomic approach based on extensive protein fractionation, we confidently identified 1,442 proteins that were distributed across seven orders of magnitude of abundance in plasma. Analysis of proteins chosen on the basis of increased levels in plasma from tumor-bearing mice and corroborating protein or RNA expression in tissue documented concordance in the blood from 30 newly diagnosed patients with pancreatic cancer relative to 30 control specimens. A panel of five proteins selected on the basis of their increased level at an early stage of tumor development in the mouse was tested in a blinded study in 26 humans from the CARET (Carotene and Retinol Efficacy Trial) cohort. The panel discriminated pancreatic cancer cases from matched controls in blood specimens obtained between 7 and 13 mo prior to the development of symptoms and clinical diagnosis of pancreatic cancer. Conclusions: Our findings indicate that GEM models of cancer, in combination with in-depth proteomic analysis, provide a useful strategy to identify candidate markers applicable to human cancer with potential utility for early detection

Proteomic-based identification of haptoglobin-1 precursor as a novel circulating biomarker of ovarian cancer

Screening for specific biomarkers of early-stage detection of ovarian cancer is a major health priority due to the asymptomatic nature and poor survival characteristic of the disease. We utilised two-dimensional gel electrophoresis (2DE) to identify differentially expressed proteins in the serum of ovarian cancer patients that may be useful as biomarkers of this disease. In this study, 38 ovarian cancer patients at different pathological grades (grade 1 (n=6), grade 2 (n=8) and grade 3 (n=24)) were compared to a control group of eight healthy women. Serum samples were treated with a mixture of Affigel-Blue and protein A (5 : 1) for 1 h to remove high abundance protein (e.g. immunoglobulin and albumin) and were displayed using 11 cm, pH 4-7 isoelectric focusing strips for the first dimension and 10% acrylamide gel electrophoresis for the second dimension. Protein spots were visualised by SYPRO-Ruby staining, imaged by FX-imager and compared and analysed by PDQuest software. A total of 24 serum proteins were differentially expressed in grade 1 (P<0.05), 31 in grade 2 (P<0.05) and 25 in grade 3 (P<0.05) ovarian cancer patients. Six of the protein spots that were significantly upregulated in all groups of ovarian cancer patients were identified by nano-electrospray quadrupole quadrupole time-of-flight mass spectrometry (n-ESIQ(q)TOFMS) and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOFMS) as isoforms of haptoglobin-1 precursor (HAP1), a liver glycoprotein present in human serum. Further identification of the spots at different pathological grades was confirmed by Western blotting using monoclonal antibody against a haptoglobin epitope contained within HAP1. Immunohistochemical localisation of HAP1-like activity was present in malignant ovarian epithelium and stroma but strong immunostaining was present in blood vessels, areas with myxomatous stroma and vascular spaces. No tissue localisation of HAP1-like immunoreactivity was observed in normal ovarian surface epithelium. These data highlight the need to assess circulating concentration of HAP1 in the serum of ovarian cancer patients and evaluate its potential as a biomarker in the early diagnosis of ovarian cancer.N Ahmed, G Barker, KT Oliva, P Hoffmann, C Riley, S Reeve, AI Smith, BE Kemp, MA Quinn and GE Ric

Effects of Blood Collection Conditions on Ovarian Cancer Serum Markers

Evaluating diagnostic and early detection biomarkers requires comparing serum protein concentrations among biosamples ascertained from subjects with and without cancer. Efforts are generally made to standardize blood processing and storage conditions for cases and controls, but blood sample collection conditions cannot be completely controlled. For example, blood samples from cases are often obtained from persons aware of their diagnoses, and collected after fasting or in surgery, whereas blood samples from some controls may be obtained in different conditions, such as a clinic visit. By measuring the effects of differences in collection conditions on three different markers, we investigated the potential of these effects to bias validation studies.We analyzed serum concentrations of three previously studied putative ovarian cancer serum biomarkers-CA 125, Prolactin and MIF-in healthy women, women with ovarian cancer undergoing gynecologic surgery, women undergoing surgery for benign ovary pathology, and women undergoing surgery with pathologically normal ovaries. For women undergoing surgery, a blood sample was collected either in the clinic 1 to 39 days prior to surgery, or on the day of surgery after anesthesia was administered but prior to the surgical procedure, or both. We found that one marker, prolactin, was dramatically affected by collection conditions, while CA 125 and MIF were unaffected. Prolactin levels were not different between case and control groups after accounting for the conditions of sample collection, suggesting that sample ascertainment could explain some or all of the previously reported results about its potential as a biomarker for ovarian cancer.Biomarker validation studies should use standardized collection conditions, use multiple control groups, and/or collect samples from cases prior to influence of diagnosis whenever feasible to detect and correct for potential biases associated with sample collection

HE4 and CA125 as a diagnostic test in ovarian cancer: prospective validation of the Risk of Ovarian Malignancy Algorithm

BACKGROUND: Recently, a Risk of Ovarian Malignancy Algorithm (ROMA) utilising human epididymis secretory protein 4 (HE4) and CA125 successfully classified patients as presenting a high or low risk for epithelial ovarian cancer (EOC). We validated this algorithm in an independent prospective study. METHODS: Women with a pelvic mass, who were scheduled to have surgery, were enrolled in a prospective study. Preoperative serum levels of HE4 and CA125 were measured in 389 patients. The performance of each of the markers, as well as that of ROMA, was analysed. RESULTS: When all malignant tumours were included, ROMA (receiver operator characteristic (ROC)-area under curve (AUC) = 0.898) and HE4 (ROC-AUC) = 0.857) did not perform significantly better than CA125 alone (ROC-AUC = 0.877). Using a cutoff for ROMA of 12.5% for pre-menopausal patients, the test had a sensitivity of 67.5% and a specificity of 87.9%. With a cutoff of 14.4% for post-menopausal patients, the test had a sensitivity of 90.8% and a specificity of 66.3%. For EOC vs benign disease, the ROC-AUC of ROMA increased to 0.913 and for invasive EOC vs benign disease to 0.957. CONCLUSION: This independent validation study demonstrated similar performance indices to those recently published. However, in this study, HE4 and ROMA did not increase the detection of malignant disease compared with CA125 alone. Although the initial reports were promising, measurement of HE4 serum levels does not contribute to the diagnosis of ovarian cancer. British Journal of Cancer (2011) 104, 863-870. doi:10.1038/sj.bjc.6606092 www.bjcancer.com Published online 8 February 2011 (C) 2011 Cancer Research U

Methadone, Buprenorphine, and Street Drug Interactions with Antiretroviral Medications

While street drugs appear unlikely to alter the metabolism of antiretroviral (ARV) medications, several ARVs may induce or inhibit metabolism of various street drugs. However, research on these interactions is limited. Case reports have documented life-threatening overdoses of ecstasy and gamma-hydroxybutyrate after starting ritonavir, an ARV that inhibits several metabolic enzymes. For opioid addiction, methadone or buprenorphine are the treatments of choice. Because a number of ARVs decrease or increase methadone levels, patients should be monitored for methadone withdrawal or toxicity when they start or stop ARVs. Most ARVs do not cause buprenorphine withdrawal or toxicity, even if they alter buprenorphine levels, with rare exceptions to date including atazanavir/ritonavir associated with significant increases in buprenorphine and adverse events related to sedation and mental status changes in some cases. There are newer medications yet to be studied with methadone or buprenorphine. Further, there are many frequently used medications in treatment of complications of HIV disease that have not been studied. There is need for continuing research to define these drug interactions and their clinical significance