Cytokine synthesis by intestinal intraepithelial lymphocytes. Both gamma/delta T cell receptor-positive and alpha/beta T cell receptor-positive T cells in the G1 phase of cell cycle produce IFN-gamma and IL-5

Murine intestinal intraepithelial lymphocytes (IEL) have been shown to contain subsets of alpha/beta TCR+ and gamma/delta TCR+ T cells that spontaneously produce cytokines such as IFN-gamma and IL-5. We have now determined the nature and cell cycle stage of these cytokine-producing T lymphocytes in EIL by using IFN-gamma- and IL-5-specific ELISPOT assay, cytokine-specific mRNA-cDNA dot-blot hybridization and polymerase chain reaction, and flow cytometry (FACS) for DNA analysis. When CD3+ T cells from IEL of normal C3H/HeN mice were separated into low and high density fractions by discontinuous Percoll gradients, IFN-gamma and IL-5 spot-forming cells were only found in the former population. Analysis of mRNA for these cytokines by both IFN-gamma- and IL-5-specific dot-blot hybridization and polymerase chain reaction revealed that higher levels of message for IFN-gamma and IL-5 were also seen in the low density fraction. However, cell cycle analysis of these two fractions by FACS using propidium iodide showed a similar pattern of cell cycle stages in both low and high density populations (G0 + G1 approximately 96 to 98% and S/G2 + M approximately 2 to 4%). Finally, mRNA from gamma/delta TCR+ and alpha/beta TCR+ T cells in both low and high density fractions of IEL were analyzed for IFN-gamma and IL-5 message by polymerase chain reaction. After 35 cycles of amplification, both gamma/delta TCR+ and alpha/beta TCR+ T cells in the low density fraction expressed higher levels of message for these two cytokines when compared with the high density population. These results have now shown that both gamma/delta and alpha/beta TCR+ IEL can be separated into low and high density subsets and both fractions possess a similar stage of cell cycle. However, only the low density cells (in G1 phase) of both gamma/delta and alpha/beta TCR types possess increased cytokine-specific mRNA and produce the cytokines IFN-gamma and IL-5. Our results suggest that alpha/beta TCR+ and gamma/delta TCR+ IEL can produce cytokines without cell proliferation.

Planar difference functions

In 1980 Alltop produced a family of cubic phase sequences that nearly meet the Welch bound for maximum non-peak correlation magnitude. This family of sequences were shown by Wooters and Fields to be useful for quantum state tomography. Alltop’s construction used a function that is not planar, but whose difference function is planar. In this paper we show that Alltop type functions cannot exist in fields of characteristic 3 and that for a known class of planar functions, x^3 is the only Alltop type function.

Results of a phase IIa clinical trial of an anti-inflammatory molecule, chaperonin 10, in multiple sclerosis

Background Chaperonin 10 (Cpn10) is a mitochondrial molecule involved in protein folding. The aim of this study was to determine the safety profile of Cpn10 in patients with multiple sclerosis (MS). Methods A total of 50 patients with relapse-remitting or secondary progressive MS were intravenously administered 5 mg or 10 mg of Cpn10 weekly for 12 weeks in a double-blind, randomized, placebo controlled, phase II trial. Clinical reviews, including Expanded Disability Status Scale and magnetic resonance imaging (MRI) with Gadolinium, were undertaken every 4 weeks. Stimulation of patient peripheral blood mononuclear cells with lipopolysaccharide ex vivo was used to measure the in vivo activity of Cpn10. Results No significant differences in the frequency of adverse events were seen between treatment and placebo arms. Leukocytes from both groups of Cpn10-treated patients produced significantly lower levels of critical proinflammatory cytokines. A trend toward improvement in new Gadolinium enhancing lesions on MRI was observed, but this difference was not statistically significant. No differences in clinical outcome measures were seen. Conclusions Cpn10 is safe and well tolerated when administered to patients with MS for 3 months, however, a further extended phase II study primarily focused on efficacy is warranted.

Locked nucleic acid (LNA) single nucleotide polymorphism (SNP) genotype analysis and validation using real-time PCR

With an increased emphasis on genotyping of single nucleotide polymorphisms (SNPs) in disease association studies, the genotyping platform of choice is constantly evolving. In addition, the development of more specific SNP assays and appropriate genotype validation applications is becoming increasingly critical to elucidate ambiguous genotypes. In this study, we have used SNP specific Locked Nucleic Acid (LNA) hybridization probes on a real-time PCR platform to genotype an association cohort and propose three criteria to address ambiguous genotypes. Based on the kinetic properties of PCR amplification, the three criteria address PCR amplification efficiency, the net fluorescent difference between maximal and minimal fluorescent signals and the beginning of the exponential growth phase of the reaction. Initially observed SNP allelic discrimination curves were confirmed by DNA sequencing (n = 50) and application of our three genotype criteria corroborated both sequencing and observed real-time PCR results. In addition, the tested Caucasian association cohort was in Hardy-Weinberg equilibrium and observed allele frequencies were very similar to two independently tested Caucasian association cohorts for the same tested SNP. We present here a novel approach to effectively determine ambiguous genotypes generated from a real-time PCR platform. Application of our three novel criteria provides an easy to use semi-automated genotype confirmation protocol.

Phase III study of matrix metalloproteinase inhibitor prinomastat in non-small-cell lung cancer

Purpose: Matrix metalloproteinases (MMPs) degrade extracellular proteins and facilitate tumor growth, invasion, metastasis, and angiogenesis. This trial was undertaken to determine the effect of prinomastat, an inhibitor of selected MMPs, on the survival of patients with advanced non-small-cell lung cancer (NSCLC), when given in combination with gemcitabine-cisplatin chemotherapy. Patients and Methods: Chemotherapy-naive patients were randomly assigned to receive prinomastat 15 mg or placebo twice daily orally continuously, in combination with gemcitabine 1,250 mg/m2 days 1 and 8 plus cisplatin 75 mg/m2 day 1, every 21 days for up to six cycles. The planned sample size was 420 patients. Results: Study results at an interim analysis and lack of efficacy in another phase III trial prompted early closure of this study. There were 362 patients randomized (181 on prinomastat and 181 on placebo). One hundred thirty-four patients had stage IIIB disease with T4 primary tumor, 193 had stage IV disease, and 34 had recurrent disease (one enrolled patient was ineligible with stage IIIA disease). Overall response rates for the two treatment arms were similar (27% for prinomastat v 26% for placebo; P = .81). There was no difference in overall survival or time to progression; for prinomastat versus placebo patients, the median overall survival times were 11.5 versus 10.8 months (P = .82), 1-year survival rates were 43% v 38% (P = .45), and progression-free survival times were 6.1 v 5.5 months (P = .11), respectively. The toxicities of prinomastat were arthralgia, stiffness, and joint swelling. Treatment interruption was required in 38% of prinomastat patients and 12% of placebo patients. Conclusion: Prinomastat does not improve the outcome of chemotherapy in advanced NSCLC. © 2005 by American Society of Clinical Oncology.

Phase III trial of gemcitabine and carboplatin versus mitomycin, ifosfamide, and cisplatin or mitomycin, vinblastine, and cisplatin in patients with advanced nonsmall cell lung carcinoma

BACKGROUND. The authors compared gemcitabine and carboplatin (GC) with mitomycin, ifosfamide, and cisplatin (MIC) or mitomycin, vinblastine, and cisplatin (MVP) in patients with advanced nonsmall cell lung carcinoma (NSCLC). The primary objective was survival. Secondary objectives were time to disease progression, response rates, evaluation of toxicity, disease-related symptoms, World Health Organization performance status (PS), and quality of life (QoL). METHODS. Three hundred seventy-two chemotherapy-naïve patients with International Staging System Stage III/IV NSCLC who were ineligible for curative radiotherapy or surgery were randomized to receive either 4 cycles of gemcitabine (1000 mg/m2 on Days 1, 8, and 15) plus carboplatin (area under the serum concentration-time curve, 5; given on Day 1) every 4 weeks (the GC arm) or MIC/MVP every 3 weeks (the MIC/MVP arm). RESULTS. There was no significant difference in median survival (248 days in the MIC/MVP arm vs. 236 days in the GC arm) or time to progression (225 days in the MIC/MVP arm vs. 218 days in the GC arm) between the 2 treatment arms. The 2-year survival rate was 11.8% in the MIC/MVP arm and 6.9% in the GC arm. The 1-year survival rate was 32.5% in the MIC/MVP arm and 33.2% in the GC arm. In the MIC/MVP arm, 33% of patients responded (4 complete responses [CRs] and 57 partial responses [PRs]) whereas in the GC arm, 30% of patients responded (3 CRs and 54 PRs). Nonhematologic toxicity was comparable for patients with Grade 3-4 symptoms, except there was more alopecia among patients in the MIC/MVP arm. GC appeared to produce more hematologic toxicity and necessitated more transfusions. There was no difference in performance status, disease-related symptoms, of QoL between patients in the two treatment arms. Fewer inpatient stays for complications were required with GC. CONCLUSIONS. The results of the current study failed to demonstrate any difference in efficacy between the newer regimen of GC and the older regimens of MIC and MVP. © 2003 American Cancer Society.

Phase III trial comparing paclitaxel poliglumex (CT-2103, PPX) in combination with carboplatin versus standard paclitaxel and carboplatin in the treatment of PS 2 patients with chemotherapy-naïve advanced non-small cell lung cancer

INTRODUCTION: Performance status (PS) 2 patients with non-small cell lung cancer (NSCLC) experience more toxicity, lower response rates, and shorter survival times than healthier patients treated with standard chemotherapy. Paclitaxel poliglumex (PPX), a macromolecule drug conjugate of paclitaxel and polyglutamic acid, reduces systemic exposure to peak concentrations of free paclitaxel and may lead to increased concentrations in tumors due to enhanced vascular permeability. METHODS: Chemotherapy-naive PS 2 patients with advanced NSCLC were randomized to receive carboplatin (area under the curve = 6) and either PPX (210 mg/m/10 min without routine steroid premedication) or paclitaxel (225 mg/m/3 h with standard premedication) every 3 weeks. The primary end point was overall survival. RESULTS: A total of 400 patients were enrolled. Alopecia, arthralgias/myalgias, and cardiac events were significantly less frequent with PPX/carboplatin, whereas grade ≥3 neutropenia and grade 3 neuropathy showed a trend of worsening. There was no significant difference in the incidence of hypersensitivity reactions despite the absence of routine premedication in the PPX arm. Overall survival was similar between treatment arms (hazard ratio, 0.97; log rank p = 0.769). Median and 1-year survival rates were 7.9 months and 31%, for PPX versus 8 months and 31% for paclitaxel. Disease control rates were 64% and 69% for PPX and paclitaxel, respectively. Time to progression was similar: 3.9 months for PPX/carboplatin versus 4.6 months for paclitaxel/carboplatin (p = 0.210). CONCLUSION: PPX/carboplatin failed to provide superior survival compared with paclitaxel/carboplatin in the first-line treatment of PS 2 patients with NSCLC, but the results with respect to progression-free survival and overall survival were comparable and the PPX regimen was more convenient. © 2008International Association for the Study of Lung Cancer.

Randomized phase II trial of the efficacy and safety of trastuzumab combined with docetaxel in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer administered as first-line treatment : the M77001 study group

Purpose: This randomized, multicenter trial compared first-line trastuzumab plus docetaxel versus docetaxel alone in patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer (MBC). Patients and Methods: Patients were randomly assigned to six cycles of docetaxel 100 mg/m 2 every 3 weeks, with or without trastuzumab 4 mg/kg loading dose followed by 2 mg/kg weekly until disease progression. Results: A total of 186 patients received at least one dose of the study drug. Trastuzumab plus docetaxel was significantly superior to docetaxel alone in terms of overall response rate (61% v 34%; P = .0002), overall survival (median, 31.2 v 22.7 months; P = .0325), time to disease progression (median, 11.7 v 6.1 months; P = .0001), time to treatment failure (median, 9.8 v 5.3 months; P = .0001), and duration of response (median, 11.7 v 5.7 months; P = .009). There was little difference in the number and severity of adverse events between the arms. Grade 3 to 4 neutropenia was seen more commonly with the combination (32%) than with docetaxel alone (22%), and there was a slightly higher incidence of febrile neutropenia in the combination arm (23% v 17%). One patient in the combination arm experienced symptomatic heart failure (1%). Another patient experienced symptomatic heart failure 5 months after discontinuation of trastuzumab because of disease progression, while being treated with an investigational anthracycline for 4 months. Conclusion: Trastuzumab combined with docetaxel is superior to docetaxel alone as first-line treatment of patients with HER2-positive MBC in terms of overall survival, response rate, response duration, time to progression, and time to treatment failure, with little additional toxicity. © 2005 by American Society of Clinical Oncology.

Dynamic contrast-enhanced magnetic resonance imaging as a biomarker for the pharmacological response of PTK787/ZK 222584, an inhibitor of the vascular endothelial growth factor receptor tyrosine kinases, in patients with advanced colorectal cancer and liver metastases: Results from two phase I studies

Purpose: PTK787/ZK 222584 (PTK/ZK), an orally active inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases, inhibits VEGF-mediated angiogenesis. The pharmacodynamic effects of PTK/ZK were evaluated by assessing changes in contrast-enhancement parameters of metastatic liver lesions using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in patients with advanced colorectal cancer treated in two ongoing, dose-escalating phase I studies. Patients and Methods: Twenty-six patients had DCE-MRI performed at baseline, day 2, and at the end of each 28-day cycle. Doses of oral PTK/ZK ranged from 50 to 2000 mg once daily. Tumor permeability and vascularity were assessed by calculating the bidirectional transfer constant (Ki). The percentage of baseline Ki (% of baseline Ki) at each time point was compared with pharmacokinetic and clinical end points. Results: A significant negative correlation exists between the % of baseline Ki and increase in PTK/ZK oral dose and plasma levels (P = .01 for oral dose; P = .0001 for area under the plasma concentration curve at day 2). Patients with a best response of stable disease had a significantly greater reduction in Ki at both day 2 and at the end of cycle 1 compared with progressors (mean difference in % of baseline Ki, 47%, P = .004%; and 51%, P = .006; respectively). The difference in % of baseline Ki remained statistically significant after adjusting for baseline WHO performance status. Conclusion: These findings should help to define a biologically active dose of PTK/ZK. These results suggest that DCE-MRI may be a useful biomarker for defining the pharmacological response and dose of angiogenesis inhibitiors, such as PTK/ZK, for further clinical development. © 2003 by American Society of Clinical Oncology.

SRL 172 (killed Mycobacterium vaccae) in addition to standard chemotherapy improves quality of life without affecting survival, in patients with advanced non-small-cell lung cancer : phase III results

Background: This open-label, randomised phase III study was designed to further investigate the clinical activity and safety of SRL172 (killed Mycobacterium vaccae suspension) with chemotherapy in the treatment of non-small-cell lung cancer (NSCLC). Patients and methods: Patients were randomised to receive platinum-based chemotherapy, consisting of up to six cycles of MVP (mitomycin, vinblastine and cisplatin or carboplatin) with (210 patients) or without (209 patients) monthly SRL172. Results: There was no statistical difference between the two groups in overall survival (primary efficacy end point) over the course of the study (median overall survival of 223 days versus 225 days; P = 0.65). However, a higher proportion of patients were alive at the end of the 15-week treatment phase in the chemotherapy plus SRL172 group (90%), than in the chemotherapy alone group (83%) (P = 0.061). At the end of the treatment phase, the response rate was 37% in the combined group and 33% in the chemotherapy alone group. Patients in the chemotherapy alone group had greater deterioration in their Global Health Status score (-14.3) than patients in the chemotherapy plus SRL172 group (-6.6) (P = 0.02). Conclusion: In this non-placebo controlled trial, SRL172 when added to standard cancer chemotherapy significantly improved patient quality of life without affecting overall survival times. © 2004 European Society for Medical Oncology.

Prognostic factors in patients with advanced non-small cell lung cancer : data from the phase III FLEX study

The FLEX study demonstrated that the addition of cetuximab to chemotherapy significantly improved overall survival in the first-line treatment of patients with advanced non-small cell lung cancer (NSCLC). In the FLEX intention to treat (ITT) population, we investigated the prognostic significance of particular baseline characteristics. Individual patient data from the treatment arms of the ITT population of the FLEX study were combined. Univariable and multivariable Cox regression models were used to investigate variables with potential prognostic value. The ITT population comprised 1125 patients. In the univariable analysis, longer median survival times were apparent for females compared with males (12.7 vs 9.3 months); patients with an Eastern Cooperative Oncology Group performance status (ECOG PS) of 0 compared with 1 compared with 2 (13.5 vs 10.6 vs 5.9 months); never smokers compared with former smokers compared with current smokers (14.6 vs 11.1 vs 9.0); Asians compared with Caucasians (19.5 vs 9.6 months); patients with adenocarcinoma compared with squamous cell carcinoma (12.4 vs 9.3 months) and those with metastases to one site compared with two sites compared with three or more sites (12.4 months vs 9.8 months vs 6.4 months). Age (<65 vs ≥65 years), tumor stage (IIIB with pleural effusion vs IV) and percentage of tumor cells expressing EGFR (<40% vs ≥40%) were not identified as possible prognostic factors in relation to survival time. In multivariable analysis, a stepwise selection procedure identified age (<65 vs ≥65 years), gender, ECOG PS, smoking status, region, tumor histology, and number of organs involved as independent factors of prognostic value. In summary, in patients with advanced NSCLC enrolled in the FLEX study, and consistent with previous analyses, particular patient and disease characteristics at baseline were shown to be independent factors of prognostic value. The FLEX study is registered with ClinicalTrials.gov, number NCT00148798. © 2012 Elsevier Ireland Ltd.

EGFR expression as a predictor of survival for first-line chemotherapy plus cetuximab in patients with advanced non-small-cell lung cancer : analysis of data from the phase 3 FLEX study

Background: Findings from the phase 3 First-Line ErbituX in lung cancer (FLEX) study showed that the addition of cetuximab to first-line chemotherapy significantly improved overall survival compared with chemotherapy alone (hazard ratio [HR] 0·871, 95% CI 0·762-0·996; p=0·044) in patients with advanced non-small-cell lung cancer (NSCLC). To define patients benefiting most from cetuximab, we studied the association of tumour EGFR expression level with clinical outcome in FLEX study patients. Methods: We used prospectively collected tumour EGFR expression data to generate an immunohistochemistry score for FLEX study patients on a continuous scale of 0-300. We used response data to select an outcome-based discriminatory threshold immunohistochemistry score for EGFR expression of 200. Treatment outcome was analysed in patients with low (immunohistochemistry score <200) and high (≥200) tumour EGFR expression. The primary endpoint in the FLEX study was overall survival. We analysed patients from the FLEX intention-to-treat (ITT) population. The FLEX study is registered with ClinicalTrials.gov, number NCT00148798. Findings: Tumour EGFR immunohistochemistry data were available for 1121 of 1125 (99·6%) patients from the FLEX study ITT population. High EGFR expression was scored for 345 (31%) evaluable patients and low for 776 (69%) patients. For patients in the high EGFR expression group, overall survival was longer in the chemotherapy plus cetuximab group than in the chemotherapy alone group (median 12·0 months [95% CI 10·2-15·2] vs 9·6 months [7·6-10·6]; HR 0·73, 0·58-0·93; p=0·011), with no meaningful increase in side-effects. We recorded no corresponding survival benefit for patients in the low EGFR expression group (median 9·8 months [8·9-12·2] vs 10·3 months [9·2-11·5]; HR 0·99, 0·84-1·16; p=0·88). A treatment interaction test assessing the difference in the HRs for overall survival between the EGFR expression groups suggested a predictive value for EGFR expression (p=0·044). Interpretation: High EGFR expression is a tumour biomarker that can predict survival benefit from the addition of cetuximab to first-line chemotherapy in patients with advanced NSCLC. Assessment of EGFR expression could offer a personalised treatment approach in this setting. Funding: Merck KGaA. © 2012 Elsevier Ltd.

Reactions of the hydroperoxide anion with dimethyl methylphosphonate in an ion trap mass spectrometer : evidence for a gas phase alpha-effect

The gas phase degradation reactions of the chemical warfare agent (CWA) simulant, dimethyl methylphosphonate (DMMP), with the hydroperoxide anion (HOO(-)) were investigated using a modified quadrupole ion trap mass spectrometer. The HOO(-) anion reacts readily with neutral DMMP forming two significant product ions at m/z 109 and m/z 123. The major reaction pathways correspond to (i) the nucleophilic substitution at carbon to form \[CH(3)P(O)(OCH(3))O](-) (m/z 109) in a highly exothermic process and (ii) exothermic proton transfer. The branching ratios of the two reaction pathways, 89% and 11% respectively, indicate that the former reaction is significantly faster than the latter. This is in contrast to the trend for the methoxide anion with DMMP, where proton transfer dominates. The difference in the observed reactivities of the HOO(-) and CH(3)O(-) anions can be considered as evidence for an a-effect in the gas phase and is supported by electronic structure calculations at the B3LYP/aug-cc-pVTZ//B3LYP/6-31+G(d) level of theory that indicate the S(N)2(carbon) process has an activation energy 7.8 kJ mol(-1) lower for HOO(-) as compared to CH(3)O(-). A similar alpha-effect was calculated for nucleophilic addition-elimination at phosphorus, but this process an important step in the perhydrolysis degradation of CWAs in solution - was not observed to occur with DMMP in the gas phase. A theoretical investigation revealed that all processes are energetically accessible with negative activation energies. However, comparison of the relative Arrhenius pre-exponential factors indicate that substitution at phosphorus is not kinetically competitive with respect to the S(N)2(carbon) and deprotonation processes.

Discrete phase-locked loop systems and spreadsheets

This paper demonstrates the use of a spreadsheet in exploring non-linear difference equations that describe digital control systems used in radio engineering, communication and computer architecture. These systems, being the focus of intensive studies of mathematicians and engineers over the last 40 years, may exhibit extremely complicated behaviour interpreted in contemporary terms as transition from global asymptotic stability to chaos through period-doubling bifurcations. The authors argue that embedding advanced mathematical ideas in the technological tool enables one to introduce fundamentals of discrete control systems in tertiary curricula without learners having to deal with complex machinery that rigorous mathematical methods of investigation require. In particular, in the appropriately designed spreadsheet environment, one can effectively visualize a qualitative difference in the behviour of systems with different types of non-linear characteristic.

Electrochemical current rectification – a novel signal amplification strategy for highly sensitive and selective aptamer-based biosensor

Electrochemical aptamer-based (E-AB) sensors represent an emerging class of recently developed sensors. However, numerous of these sensors are limited by a low surface density of electrode-bound redox-oligonucleotides which are used as probe. Here we propose to use the concept of electrochemical current rectification (ECR) for the enhancement of the redox signal of E-AB sensors. Commonly, the probe-DNA performs a change in conformation during target binding and enables a nonrecurring charge transfer between redox-tag and electrode. In our system, the redox-tag of the probe-DNA is continuously replenished by solution-phase redox molecules. A unidirectional electron transfer from electrode via surface-linked redox-tag to the solution-phase redox molecules arises that efficiently amplifies the current response. Using this robust and straight-forward strategy, the developed sensor showed a substantial signal amplification and consequently improved sensitivity with a calculated detection limit of 114 nM for ATP, which was improved by one order of magnitude compared with the amplification-free detection and superior to other previous detection results using enzymes or nanomaterials-based signal amplification. To the best of our knowledge, this is the first demonstration of an aptamer-based electrochemical biosensor involving electrochemical rectification, which can be presumably transferred to other biomedical sensor systems.