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Background Systemic oxidative stress is known to be associated with the severity and prognosis in patients with cardiovascular diseases (CVD), including chronic heart failure. On the other hand, exercise tolerance is closely related to the prognosis in heart failure patients. However, no report has examined how oxidative stress is involved in each parameter evaluated by cardiopulmonary exercise testing (CPET) in patients with dilated cardiomyopathy (DCM). Purpose To examine the relationship between oxidative stress and CPET parameters such as peak VO2 and VE/VCO2 slope in patients with DCM Methods We recruited 214 patients with CVD who were performed CPET and measured brain natriuretic peptide (BNP) and haemoglobin (Hb). Finally, we enrolled 96 patients with DCM who have dilated dimensions of the left ventricular lumen by echocardiography and are diagnosed by endomyocardial biopsy. All patients underwent CPET using 0W warm-up and 10W ramp protocol on an upright electrical bicycle ergometer. We defined low peak VO2 as peak VO234. The oxidative stress level was evaluated by a d-ROMs test, in which the amount of organic hydroperoxide converted into radicals oxidizing N, N-diethyl-p-phenylenediamine hydroperoxide is measured. The high level of d-ROMs was defined as d-ROMs≥401 U.CARR. Results Mean age was 56±15 years old. Mean ejection fraction, peak VO2 and VE/VCO2 slope were 37±15%, 16.3±5.0 ml/min/kg, and 31.0±11.8, respectively. The percentage of the high level of d-ROMs was 25%. In univariable logistic regression analysis, Hb, log BNP, and the high level of oxidative stress were significantly associated with low peak VO2, whereas, in multivariable logistic regression analysis, age, Hb, and log BNP were significant factors for high VE/VCO2 slope. In multivariable logistic regression analysis, only the high level of d-ROMs was independently associated with low peak VO2 (OR 3.18, CI 1.12–9.04, p=0.030). While, in multivariable logistic regression analysis, Hb (OR 0.51, CI 0.32–0.81, p=0.004) and log BNP (OR 2.77, CI 1.33–5.76, p=0.006) were significantly related to high VE/VCO2 slope. Conclusions In patients with DCM, low peak VO2 was associated with the high level of d-ROMs, and high VE/VCO2 slope was associated with BNP. These results suggested that oxidative stress was only related to peak VO2. Funding Acknowledgement Type of funding sources: None.

Background Heart failure with preserved ejection fraction (EF) remains a poor prognosis as same as heart failure with reduced EF. Peak oxygen uptake (VO2) by cardiopulmonary exercise testing (CPET) is a useful parameter for predicting cardiovascular diseases prognosis. Furthermore, though there are some reports that CPET parameters are associated with indicators of diastolic dysfunction, each of these indicators has some limitations. Recently, recommendations for the evaluation of left ventricular diastolic function by echocardiography were reported from the ASE/EACVI. However, no reports have examined the association between exercise tolerance indices and diastolic dysfunction based on these recommended variables. Purpose To examine the relationship between peak VO2 and diastolic dysfunction using the recommendation from ASE/EACVI in cardiovascular diseases patients with preserved EF Methods We recruited 214 patients who were performed both CPX and echocardiography. EF ≥50% was 99 patients. All patients underwent 0W warm-up and 10W ramp on an upright electrical bicycle ergometer. Diastolic dysfunction was assessed using the recommendations for the evaluation of diastolic function by ASE/EACVI. We used abnormal cutoff values are annular e' velocity: septal e' 14, left atrial volume index >34 ml/m2, and peak tricuspid regurgitation (TR) velocity >2.8 m/s. Diastolic dysfunction is present if more than half of the available parameters meet these cutoff values. Results Mean age was 57±14 years old, the portion of women was 69%. The portion of diastolic dysfunction was 16%. In univariable logistic regression analysis, age, log BNP, septal e' 2.8 m/s, and the presence of diastolic dysfunction were significantly associated with peakVO2 Conclusions In preserved EF, low peak VO2 was significantly associated with diastolic dysfunction assessed by the recommendations from the ASE/EACVI. Funding Acknowledgement Type of funding sources: None.

The first enantioselective carbometalation reaction of azabicycloalkenes has been achieved by iron catalysis to in situ form optically active organozinc intermediates, which are amenable to further synthetic elaborations. The observed chiral induction, along with the DFT and XAS analyses, reveals the direct coordination of the chiral phosphine ligand to the iron centre during the carbon–carbon and carbon–metal bond forming step. This new class of iron-catalysed asymmetric reaction will contribute to the synthesis and production of bioactive molecules.

Machine learning is a powerful tool used in many different areas, from image processing to space navigation and high-energy physics. In this paper we present a configuration of different artificial intelligent tools aimed at the extraction of features from data registered in the SciBar Cosmic Ray Telescope (SciCRT). The SciCRT is an array of plastic scintillator bars that work nearly independently as particle detectors. When a particle crosses inside the telescope, scintillation photons are emitted by the plastics. The intensity of photons is directly proportional to the energy deposited in each bar. Taking advantage of the construction of the telescope, the small transverse area of the scintillator bars, it is possible to do particle tracking and analysis. The main purpose of SciCRT is the detection of solar neutrons originated in the violent phenomena taking place at the surface of the Sun. Nonetheless, the SciCRT is capable of detecting different kinds of secondary particles produced by the interactions of primary cosmic rays with the atmospheric nuclei. For this reason, the task of signal classification is essential. Our final goal will be the classification of detected cosmic ray particles, as well as, the unfolding of the neutron energy spectrum and the estimation of the angular distribution. To achieve this our methodology relies of pattern recognition, artificial neural networks, k-means clustering and k-Nearest Neighbors. In addition, our paper presents a Monte Carlo simulation of the SciCRT for the training and evaluation of the machine learning algorithms.

We measured both the differential cross section ($\sigma_{p,p^\prime}$ $=d^2\sigma/d\Omega dE_{x}$) and the $\gamma$-ray emission probability ($R_\gamma(E_x)$ $=\sigma_{p,p^\prime\gamma}$/$\sigma_{p,p^\prime}$) from the giant resonances excited by $\rm^{12}C$(\textit{p,p}$^\prime$) reaction at 392 MeV and 0$^\circ$, using a magnetic spectrometer and an array of NaI(Tl) counters. The absolute value of $R_\gamma(E_x)$ was calibrated by using the well-known $\gamma$-ray emission probability from $\rm^{12}C^* ( 15.11$ MeV, $ 1^+$, $T=1$) and $\rm^{16}O^* ( 6.9$ MeV, $2^+$, $T=0$) states within 5\% uncertainty. We found that $R_\gamma(E_x)$ starts from zero at $E_x=16$ MeV, increases to a maximum of 53.3$\pm$0.4$\pm$3.9\% at $E_x=27$ MeV and then decreases. We also compared the measured values of $R_\gamma(E_x)$ with statistical model calculation based on the Hauser-Feshbach formalism in the energy region $E_x=$ 16-32 MeV and discussed the features of $\gamma$-ray emission probability quantitatively.

This paper presents a simulation/experimental technique used in the design of optimum performance read-out electronics for a new cosmic ray telescope composed of scintillator bars. This new detector, called SciBar Cosmic Ray Telescope (SciCRT), is installed on top of Sierra Negra volcano in Mexico, operating at an atmospheric depth of 575 g cm−2. The severe atmospheric conditions and high background rate of the place currently limit the performance of the detector, therefore the requirements of our design are low power consumption and high throughput rate. The simulation developed considers the interaction of high energy cosmic ray muons with the scintillator bar, generation and propagation of photons inside a wavelength shifting fiber (WLS) fiber and the detection by a multi-anode photomultiplier (MAPMT). To produce realistic signals coming from the interaction of particles, our method relies on the extraction of different parameters throughout experiment to include them in the simulation. In addition, the paper presents a muon cosmic ray experiment designed to validate the results of the simulation and test the performance of the new electronics.

We perform the first direct mass measurements of neutron-rich calcium isotopes beyond neutron number 34 at the RIKEN Radioactive Isotope Beam Factory by using the time-of-flight magnetic-rigidity technique. The atomic mass excesses of ^{55-57}Ca are determined for the first time to be -18650(160), -13510(250), and -7370(990) keV, respectively. We examine the emergence of neutron magicity at N=34 based on the new atomic masses. The new masses provide experimental evidence for the appearance of a sizable energy gap between the neutron 2p_{1/2} and 1f_{5/2} orbitals in ^{54}Ca, comparable to the gap between the neutron 2p_{3/2} and 2p_{1/2} orbitals in ^{52}Ca. For the ^{56}Ca nucleus, an open-shell property in neutrons is suggested.

The (^{12}N, ^{12}C) charge-exchange reaction at 175 MeV/u was developed as a novel probe for studying the isovector spin giant monopole resonance (IVSMR), whose properties are important for better understanding the bulk properties of nuclei and asymmetric nuclear matter. This probe, now available through the production of ^{12}N as a secondary rare-isotope beam, is exothermic, is strongly absorbed at the surface of the target nucleus, and provides selectivity for spin-transfer excitations. All three properties enhance the excitation of the IVSMR compared to other, primarily light-ion, probes, which have been used to study the IVSMR thus far. The ^{90}Zr(^{12}N,^{12}C) reaction was measured and the excitation energy spectra up to about 70 MeV for both the spin-transfer and non-spin-transfer channels were deduced separately by tagging the decay by γ emission from the ^{12}C ejectile. Besides the well-known Gamow-Teller and isobaric analog transitions, a clear signature of the IVSMR was identified. By comparing with the results from light-ion reactions on the same target nucleus and theoretical predictions, the suitability of this new probe for studying the IVSMR was confirmed.

This work report PVDF micro machining process by RIE without much loss of its piezoelectric constant. L/S (70/130_μm) of fine structure was realized in this work with high etch rate. O 2 mixing feed gases of CF 4 , CHF 3 , SF 6 and 100 % of O 2 were used. At the condition of 100% O 2 gas plasma and at a higher of RF power, the better of surface state and higher of aspect ratio structure could be realized.

The SciBar Cosmic ray telescope (SciCRT) is installed on the top of the Sierra Negra volcano with the main goal of observing solar neutrons to investigate the ion acceleration process during solar flares. Using scintillator bars as a medium to stop energetic particles, the SciCRT is capable of recording both energy deposited on the bars and direction of the incoming particles with high resolution. The original DAQ system was used in neutrino oscillation experiment (low event rate), therefore operation of the electronics on cosmic ray experiment is limited. To improve the SciCRT performance as a solar neutron telescope, development of custom made DAQ electronics is essential. Our first step onto this task was the design and construction of a new fast readout back-end board using SiTCP. The installation of this new system on Sierra Negra and its further improvement on the data acquisition for the detector will be analyzed on separate paper on this conference. The development of new front end electronics is the next stage of the upgrading process. To achieve this goal, we are developing new electronics applying the time over threshold (ToT) technique, using a FPGA to process the signal from one 64 channel multi anode photomutiplier tube (MAPMT). In this paper we will present the details of this new system and several tests performed to guarantee its proper operation to detect solar neutrons.

The SciBar Cosmic Ray Telescope (SciCRT) is aimed to help elucidate the acceleration mechanism of high-energy ions that may produce neutrons at the Sun. It is a fully active scintillator tracker which consists of 14,848 plastic scintillator bars, originally constructed for accelerator neutrino oscillation experiments. The SciCRT; it has a huge detector volume compared with conventional Solar Neutron Telescopes (SNTs), e.g. 15 times larger than Mexico SNT. Furthermore, the SciCRT can measure the energy deposition of each particle as neutron ADC data which have not been registered before. Neutron ADC data provide us with a precise measurement of energies deposited at the detector. The SciCRT was deployed at the summit of Mt. Sierra Negra (4,600 m) and began to acquire data in September 2013. Then we partially upgraded the DAQ system developed originally for an accelerator experiment, as the readout rate of the DAQ system was significantly limited for our experiment. This paper highlights sensitivity numerical studies of solar neutrons that the SciCRT is able to register. At first, we focus in the accuracy to determine the spectrum power-law index, assuming an instantaneous emission of solar neutrons. This is required to determine the power-law index within an error of ±1.0 in order to discuss the efficiency of the acceleration. Then in the case of the fixed power-law index, we discuss the capability of discriminating three different lengths of emission times: 0 min, 5 min, and 8 min. Finally we evaluate whether it is possible to discriminate a different combination of these two parameters simultaneously. Thus, we show that data from the SciCRT will unlock the degeneracy problem amid the emission time and the energy spectrum of solar neutrons.

CRIB (CNS Radioactive Ion Beam separator) is a low-energy RI beam separator at the Center for Nuclear Study (CNS) of the University of Tokyo. An overview of the recent developments and status of CRIB, including a detailed summary of beam parameters, is presented. Studies on proton and α resonant scatterings, direct measurements of ( α , p ) reactions, and other types of measurements ( β -decay lifetimes, etc.) have been performed using RI beams at CRIB, motivated by interests in astrophysical reactions and exotic nuclear structure. Among the studies at CRIB, the measurement of 7Be + α resonant scattering is discussed.

SummaryBackground The Janus kinase (JAK) inhibitor, tofacitinib, has shown efficacy for the treatment of psoriasis in a phase IIb trial (A3921047; NCT00678210). Objectives To report haematology data from the phase IIb trial, given the importance of JAK-dependent signalling in haematopoiesis. Methods Patients with moderate-to-severe chronic plaque psoriasis were randomized to receive tofacitinib 2, 5 or 15 mg, or placebo, twice daily over 12 weeks. Blood samples were collected at screening, baseline, weeks 2, 4, 8 and 12 during treatment, and weeks 14 and 16 during off-treatment follow-up. Results Baseline haematology was similar across patients receiving tofacitinib 2 mg (n = 49), 5 mg (n = 49) or 15 mg (n = 49), or placebo (n = 50). Tofacitinib conferred dose-dependent decreases in haemoglobin, haematocrit and red blood cell counts, while reticulocyte counts initially declined, before recovering by week 8, and exceeding baseline levels after treatment cessation. With regard to white blood cells, tofacitinib had no clear dose-dependent effects on basophils or monocytes, but appeared to be associated with transient or reversible dose-dependent decreases in neutrophil and eosinophil counts and transient increases in lymphocyte counts, which were primarily attributable to increases in B-cell counts. Natural killer cell counts declined with tofacitinib. Conclusions Tofacitinib conferred tolerable, dose-dependent changes in haematological parameters during short-term administration in patients with psoriasis. The effects did not appear to be progressive, and were often transient or reversible.

Evidence suggests that bio-activation of drugs to generate chemically reactive metabolites (RM) that act as haptens to form immunogenic protein conjugates may be an important cause of immune-mediated drug hypersensitivity reactions (IDHR). Although many drugs that form RMs raise concerns about producing IDHR, standard non-clinical testing methods are rarely able to identify compounds with the potential to produce IDHR in humans. The objective of this study was to develop a predictive assay for IDHR that involves: (1) the use of an in vitro drug-metabolizing system to generate the RM that is captured by GSH, (2) conjugating the RM-GSH conjugate to mouse serum albumin (MSA) by using a chemical cross-linker, (3) immunization of mice with RM-GSH-MSA adducts, and (4) ex vivo challenge with RM-GSH-MSA adduct and measurement of lymphocyte proliferation to determine if the RM is immunogenic. The predictivity of the assay was evaluated by using drugs that produce RM and have been strongly, weakly, or not associated with IDHRs in the clinic. While this method requires additional validation with more drugs, the results demonstrate the feasibility of identifying drugs strongly associated with IDHR and the utility of the assay for rank ordering drugs with respect to their potential to cause IDHR.

The ICH S8 immunotoxicity testing guideline for human pharmaceuticals was published in 2006 and was intended to provide guidance for assessing the immunotoxicity potential of low-molecular-weight drugs that are not intended to alter the immune system. For drugs intended to modulate the immune system, immunotoxicity testing strategies are generally developed on a case-by-case approach since the targets, intended patient population, and mechanisms of action of the test compound will determine the type of testing needed. Some of the general principles of ICH S8, however, may be applied to immunotoxicity testing strategies for immunomodulatory drugs. A weight-of-evidence approach using factors discussed in ICH S8 in concert with an assessment of the potential value of additional immunotoxicity testing should be considered. For most situations, immunotoxicity studies with immunomodulatory compounds evaluate off-target effects on the immune system and exaggerated pharmacology. The potential use of data from these studies and considerations such as translatability to humans are discussed.

Epstein-Barr virus (EBV)-associated lymphomas are a known risk for immunosuppressed individuals. Non-clinical methods to determine the potential of new immunomodulatory compounds to produce EBV-associated lymphomas (hazard identification) have not been developed. Since lymphocryptovirus (LCV) in non-human primates (NHP) has similar characteristics to EBV in humans, a Roundtable meeting was held in October 2010 to explore how the potential for EBV-related lymphomas in humans can be assessed by using surrogate biomarkers for lymphoma risk in NHP toxicity studies. Stakeholders from regulatory agencies, academia, and industry came together to determine the research gaps and potential benefits and considerations of such an approach given the current state-of-the-science. Key conclusions from the discussion included considerations raised about the potential usefulness of LCV-related biomarkers from NHP studies since there is significant controversy over the reliability of using EBV viral load or EBV-specific T-lymphocytes to predict for lymphoproliferative disorders in transplant patients. In addition, there are technical challenges that need to be further addressed in order to develop methods to measure LCV viral load and LCV-specific T-lymphocytes from cynomolgus monkeys.

Up to 10% of patients with severe immune-mediated drug hypersensitivity reactions have tendencies to develop multiple drug hypersensitivities (MDH). The reason why certain individuals develop MDH and the underlying pathomechanism are unclear. We investigated different T cell subpopulations in MDH patients and compared them with patients allergic to a single drug and with healthy controls (HC).

The current study examined the prevalence of intestinal parasites and genotypes of Giardia intestinalis in puppies from nine pet shops in east Japan. Fresh fecal samples from 1794 puppies (≦3 months old) were collected on one occasion. Giardia spp. was examined for specific coproantigen using ELISA kit (SNAP®Giardia, IDEXX Laboratories, Inc., USA). Other intestinal parasites were detected microscopically using the formalin-ethyl acetate sedimentation technique. Genotyping was determined for the random 29 stool samples identified as Giardia spp. positive using PCR and direct sequencing of the glutamate dehydrogenase (gdh) gene. Overall prevalence of protozoan Giardia spp. and Cystoisospora spp. revealed 23.4% and 11.3%, respectively. Prevalence of ascarids, Strongyloides spp. and hookworms were recorded 1.8%, 1.1% and 0.1%, respectively. Protozoan Giardia spp. and Cystoisospora spp., thus, represent important pathogens among pet shop puppies. All genotyped G. intestinalis isolates were belonged to assemblage C or D, identified as dog-specific genotypes. Zoonotic assemblage A and B were not demonstrated. The result suggests that the risk of zoonotic transmission of G. intestinalis from pet shops puppies to humans may be quite low in Japan.

In drug hypersensitivity, change of drug treatment and continuation with a new drug may result in reappearance of drug hypersensitivity symptoms. This is not uncommon in patients with chronic infections requiring continued and long-lasting antibiotic treatments. For the clinician, the question arises whether these symptoms are due to cross-reactivity, are due to a new sensitization or are a reflection of a multiple drug hypersensitivity syndrome. Based on the p-i concept (pharmacological interaction with immune receptors), we propose that the efficient stimulation of T cells by a drug is the sum of drug-T-cell receptor affinity and readiness of the T cell to react, and therefore not constant. It heavily depends on the state of underlying immune activation. Consequently, drug hypersensitivity diseases, which go along with massive immune stimulations and often high serum cytokine values, are themselves risk factors for further drug hypersensitivity. The immune stimulation during drug hypersensitivity may, similar to generalized virus infections, lower the threshold of T-cell reactivity to drugs and cause rapid appearance of drug hypersensitivity symptoms to the second drug. We call the second hypersensitivity reaction a "flare-up" reaction; this is clinically important, as in most cases the second drug may be tolerated again, if the cofactors are missing. Moreover, the second treatment is often too short to cause a relevant sensitization.

Preclinical immunotoxicity assessments may be performed during pharmaceutical drug development in order to identify potential cause for concern prior to use in the clinic. The in vivo T-dependent antibody response (TDAR) is widely used in this regard, given its sensitivity to known immunosuppressive compounds, but may be impractical early in drug development where quantities of test article are limited. The goal of the current work is to develop an in vitro human cell-based assay that is sensitive to immunosuppression, uses relatively small quantities of test article, and is simple to perform with moderate to high throughput. Ideally, this assay would require the cooperation of multiple cellular compartments to produce a response, similar to the TDAR. Although the Mishell-Dutton assay (in vitro mouse splenic sheep red blood cell response) has been used for this purpose, it shows considerable inter-laboratory variability, and rodent cells are used which leads to potential difficulty in translation of findings to humans. We have developed an assay that measures an influenza antigen-specific response using frozen-stored human peripheral blood mononuclear cells, which we have termed the human lymphocyte activation (HuLA) assay. The HuLA assay is sensitive to cyclosporine, dexamethasone, rapamycin, mycophenolic acid, and methotrexate at concentrations within their respective therapeutic ranges. Although proliferation is the primary endpoint, we demonstrate that flow cytometry approaches may be used to characterize the proliferating lymphocyte subsets. Flu antigen-specific proliferation in the HuLA assay primarily involves both CD4+ and CD8+ T-lymphocytes and B-lymphocytes, although other lymphocyte subsets also proliferate. In addition, flu-specific antibody-secreting cells can be measured in this assay by ELISPOT, a response that is also sensitive to known immunosuppressive compounds. The HuLA assay represents a relatively straightforward assay with the capability of detecting immune suppression in human cells and can be applied to compound ranking and immunotoxicity assessment.

An evaluation of potential antibody formation to biologic therapeutics during the course of nonclinical safety studies and its impact on the toxicity profile is expected under current regulatory guidance and is accepted standard practice. However, approaches for incorporating this information in the interpretation of nonclinical safety studies are not clearly established. Described here are the immunological basis of anti-drug antibody formation to biopharmaceuticals (immunogenicity) in laboratory animals, and approaches for generating and interpreting immunogenicity data from nonclinical safety studies of biotechnology-derived therapeutics to support their progression to clinical evaluation. We subscribe that immunogenicity testing strategies should be adapted to the specific needs of each therapeutic development program, and data generated from such analyses should be integrated with available clinical and anatomic pathology, pharmacokinetic, and pharmacodynamic data to properly interpret nonclinical studies.

Background and purpose: The adhesion molecule mucosal addressin cell adhesion molecule (MAdCAM) plays an essential role in the recruitment of lymphocytes to specialized high endothelial venules of the gastrointestinal tract and in their excessive tissue extravasation observed in inflammatory conditions, such as Crohn's disease. We have characterized the in vitro pharmacological properties of two monoclonal antibodies blocking MAdCAM, MECA-367 and PF-00547659, and determined their pharmacokinetic/pharmacodynamic profiles in vivo. Experimental approach: Functional adhesion assays and surface plasmon resonance were used to characterize, in vitro, the pharmacological properties of MECA-367 and PF-00547659. The in vivo effects of MECA-367 and PF-00547659 on restriction of β7+ memory T lymphocytes were determined in mice and macaques, respectively, over the pharmacological dose range to confirm pharmacokinetic/pharmacodynamic relationships. Key results: MECA-367 and PF-00547659 bound with high affinity to mouse and human MAdCAM with Kd values of 5.1 and 16.1 pmol·L−1 respectively and blocked the adhesion of α4β7+ leukocytes to MAdCAM with similar potency. MECA-367 and PF-00547659 induced a similar, dose-dependent two- to threefold increase in circulating populations of β7+ memory T-cells in the mouse and macaque; without affecting the β7- populations. Conclusions and implications: PF-00547659 has potential utility in the treatment of inflammatory conditions by blocking tissue homing of activated α4β7+ leukocytes. The characterization of a rodent cross-reacting antibody as a surrogate for PF-00547659 in the search for potential pharmacological biomarkers and the determination of efficacious doses was effective in addressing the restricted orthologous cross-reactivity of PF-00547659 and the challenges this poses with respect to efficacy and safety testing.

The number of anti-inflammatory and immunomodulatory drugs being developed in the pharmaceutical industry has increased considerably in the past decade. This increase in research and development has been paralleled by questions from both regulatory agencies and industry on how best to assess decreased host resistance to infections or adverse immunostimulation caused by immunomodulatory agents such as anti-cytokine antibodies (e.g., the tumor necrosis factor-alpha inhibitors), anti-adhesion molecule antibodies (e.g., anti-alpha-4 integrin inhibitors) and immunostimulatory molecules (e.g., anti-CD28 antibodies). Although several methods have been developed for nonclinical assessment of immunotoxicity, highly publicized adverse events have brought to light significant gaps in the application of nonclinical immunotoxicity testing in assessing potential risk in humans. Confounding this problem is inconsistent application of immunotoxicology methods for risk assessment within the scientific community, limited understanding of appropriate immunotoxicity testing strategy for immunomodulators and inconsistent testing requests by regulatory agencies. To address these concerns, The Immunotoxicology Technical Committee (ITC) of the International Life Science Institute (ILSI) Health and Environmental Sciences Institute (HESI) organized a workshop on Immunomodulators and Clinical Immunotoxicology in May 2007. The Workshop was convened to identify key gaps in nonclinical and clinical immunotoxicity testing of anti-inflammatory and immunomodulatory agents and to begin to develop consistent approaches for immunotoxicity testing and risk assessment. This paper summarizes the outcome of the HESI ITC Immunomodulators and Clinical Immunotoxicology Workshop. Topics not discussed at the Workshop were outside the scope of this report. Although more work is needed to develop consistent approaches for immunotoxicity assessment of immunomodulators, this Workshop provided the foundation for future discussion.

The administration of biological therapeutics can evoke some level of immune response to the drug product in the receiving subjects. An immune response comprised of neutralizing antibodies can lead to loss of efficacy or potentially more serious clinical sequelae. Therefore, it is important to monitor the immunogenicity of biological therapeutics throughout the drug product development cycle. Immunoassays are typically used to screen for the presence and development of anti-drug product antibodies. However, in-vitro cell-based assays prove extremely useful for the characterization of immunoassay-positive samples to determine if the detected antibodies have neutralizing properties. This document provides scientific recommendations based on the experience of the authors for the development of cell-based assays for the detection of neutralizing antibodies in non-clinical and clinical studies.

We have deposited diamond-like carbon (DLC) films by radio-frequency magnetron sputtering, and have annealed the films under various conditions to investigate the effects of annealing on the structural properties by visible Raman spectroscopy, X-ray photoelectron spectroscopy and atomic force microscopy. The structural ordering of hydrogenated DLC films occurs during annealing below 400 °C in a vacuum and a hydrogen gas atmosphere, while unhydrogenated DLC films are not ordered during annealing even at 700 °C. On the other hand, the ordering and the decrease of the sp 3 content are observed for both the films after annealing under an atomic hydrogen exposure. The ordering progresses as the annealing temperature and time are increased. The reduction of the film thickness after annealing is suppressed with increasing annealing temperature. The results suggest that both the preferential etching by atomic hydrogen and the hydrogen evolution encourage the structural changes under an atomic hydrogen exposure.

Toxicological testing of compounds often is conducted at the maximum tolerated dose to identify potential target organs. Toxicities observed at these high doses may result in decreased body weight gain, food consumption and activity. These clinical signs are often associated with a generalized stress response. It has been known that stress may cause increased levels of corticosterone, which causes changes in circulating leukocyte profiles, decreases in thymus and spleen weights and changes in the microscopic structure of lymphoid organs. This makes it difficult to differentiate between stress-related changes and direct toxicity to the immune system in standard non-clinical toxicity testing in rats. In mice, MHC Class II expression was found to be a very sensitive biomarker of stress and maybe useful for the rat. Therefore, the objective of studies presented was to further characterize the effects of corticosterone and stressors on the immune system and identify potential biomarkers of stress in rats. Rats were treated with exogenous corticosterone (20 or 30 mg/kg BID) or ethanol (5 g/kg) for either 1 or 4 days. Restraint stress was also evaluated for a 3-day period. Blood and urine samples were collected during the treatment period for corticosterone measurements. At necropsy, blood samples for leukocyte differentials were collected. Spleen and thymus weights, cellularity, lymphocyte subpopulations and histopathology were also evaluated. Urine corticosterone levels were also investigated as a surrogate to measuring serum corticosterone. The results demonstrate that the pattern of responses to corticosterone or the stressors is different in mice and rats. Although, decreases in MHC Class II were found to be a sensitive indicator of stress in mice, only slight decreases were observed in rats with similar serum corticosterone AUC levels. Decreases in thymus weight were greater than spleen weight with corticosterone or ethanol or restraint stressor. No other single parameter or combination of parameters tested were obvious candidates as sensitive biomarkers of stress in rats. However, the good correlation between urine and serum corticosterone levels suggest that urine corticosterone may be a potential biomarker of stress induced changes to the immune response.

The process of a life skills-based nutrition education program and impact on upper grade students in elementary schools were evaluated with regard to their knowledge, attitude, behavior while eating snacks and breakfast, and self-esteem. The subjects for this study were students from two elementary schools in Osaka prefecture. The students in the intervention school participated in a nutrition education program between the second trimester of the fifth grade and the first trimester of the sixth grade that focused on eating healthier snacks and eating breakfast every day. Questionnaires were completed by the students in the intervention and control schools before and after the program. A total of 160 students (88 in the intervention school and 72 in the control school) participated in the survey. A chi-square test, t-test, McNemar test, and Wilcoxon signed-ranks test were used to examine the effects of the program. The statistical significance level was 0.05.Although the teachers could implement well the program for eating healthier snacks, they pointed out the need to reexamine the process and worksheets of the program for eating breakfast. The overall program had a positive effect on nutritional and behavioral knowledge and on the self-control of students in selecting healthy snacks. However, it did not change to the expected extent the intake of oily snacks, attitude and behavior toward eating breakfast, and self-esteem. These results suggest that the program should be revised to improve life skills including decision-making and goal-setting abilities among elementary school students, and to strengthen the relationship between schools and families.

A candidate resonant tetraneutron state is found in the missing-mass spectrum obtained in the double-charge-exchange reaction ^{4}He(^{8}He,^{8}Be) at 186 MeV/u. The energy of the state is 0.83±0.65(stat)±1.25(syst) MeV above the threshold of four-neutron decay with a significance level of 4.9σ. Utilizing the large positive Q value of the (^{8}He,^{8}Be) reaction, an almost recoilless condition of the four-neutron system was achieved so as to obtain a weakly interacting four-neutron system efficiently.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterised by infiltration of immune cells into the affected synovium, release of inflammatory cytokines and degradative mediators, and subsequent joint damage. Both innate and adaptive arms of the immune response play a role, with activation of immune cells leading to dysregulated expression of inflammatory cytokines. Cytokines work within a complex regulatory network in RA, signalling through different intracellular kinase pathways to modulate recruitment, activation and function of immune cells and other leukocytes. As our understanding of RA has advanced, intracellular signalling pathways such as Janus kinase (JAK) pathways have emerged as key hubs in the cytokine network and, therefore, important as therapeutic targets. Tofacitinib is an oral JAK inhibitor for the treatment of RA. Tofacitinib is a targeted small molecule, and an innovative advance in RA therapy, which modulates cytokines critical to the progression of immune and inflammatory responses. Herein we describe the mechanism of action of tofacitinib and the impact of JAK inhibition on the immune and inflammatory responses in RA.

The human monoclonal antibody CP-870,893 is a CD40 receptor agonist currently being developed for the treatment of cancer. A bioassay to measure neutralizing antibodies (Nab) to CP-870,893 in 5% human serum matrix was developed and validated utilizing the Daudi cell line and flow cytometric detection. Additionally, samples from CP-870,893 treated cynomolgus monkeys were analyzed in the bioassay and compared to results obtained using a competitive receptor-binding (CRB) Nab immunoassay to determine if the CRB assay may be used in place of the bioassay. Treatment of Daudi cells for 2 d with CP-870,893 leads to a concentration-dependent increase in CD54 cell surface expression. The presence of antidrug Nab attenuates CP-870,893 binding to CD40 and the induction of CD54. An anti-idiotype monoclonal antibody (Mab) and a monkey sera pool were identified as positive controls for neutralization of CP-870,893. During development, it was observed that the assay robustness was altered by culture media and FBS substitutions. For validation the following parameters were established: cutpoint factors in the presence (0.779) and absence (1.282) of 50 ng/ml CP-870,893, linear region of the concentration-response (1-100 ng/ml CP-870,893), intra- and inter-assay precision (CV/= 25%), specificity and recovery (+/-25%), sensitivity ( approximately 500 ng anti-idiotype Mab per ml serum), technician to technician ruggedness (CV/= 25%), and stability (positive control, CD54 labeling, and cell line). A concentration dependent increase in CP-870,893 neutralization was observed in a 3-mo toxicity study in monkeys using both the Bioassay and CRB assay (R(2) = 0.94) suggesting the CRB Nab assay may be a suitable alternative to a bioassay. Based on the precision, specificity, sensitivity, and robustness, the validated bioassay is suitable for quasi-quantitative analysis of neutralizing anti-CP-870,893 antibodies in human serum.

Differential cross sections of isoscalar and isovector spin-M1 (0(+)→1(+)) transitions are measured using high-energy-resolution proton inelastic scattering at E(p)=295 MeV on (24)Mg, (28)Si, (32)S, and (36)Ar at 0°-14°. The squared spin-M1 nuclear transition matrix elements are deduced from the measured differential cross sections by applying empirically determined unit cross sections based on the assumption of isospin symmetry. The ratios of the squared nuclear matrix elements accumulated up to E(x)=16 MeV compared to a shell-model prediction are 1.01(9) for isoscalar and 0.61(6) for isovector spin-M1 transitions, respectively. Thus, no quenching is observed for isoscalar spin-M1 transitions, while the matrix elements for isovector spin-M1 transitions are quenched by an amount comparable with the analogous Gamow-Teller transitions on those target nuclei.

The mouse drug allergy model (MDAM) was developed as a tool to predict the potential of systemically administered drugs to produce hypersensitivity reactions (HR). Drugs associated with HR in the clinic produce a marked increase in the cellularity of the draining lymph nodes (DLN) in the MDAM. The objective of this study was to characterize the changes in the DLN following exposure to drugs associated with HR and to investigate whether lymphocyte migration and/or proliferation play a role in the response. These endpoints were also investigated in the local lymph node assay (LLNA) to determine whether responses between the two assays occur via similar mechanisms. Results demonstrated that total numbers of T- and B-cells were proportionally increased in the DLN of mice treated with positive control drugs (i.e. abacavir, amoxicillin, ofloxacin, and sulfamethoxazole) compared to animals administered the vehicle or negative control drugs (metformin and cimetidine). In contrast, a significant increase in the B-cell population of the DLN was observed for 2,4-dinitrofluorobenzene (DNFB) following the LLNA protocol. Down-regulation of CD62L and up-regulation of CCR7 were observed for T-cells from the DLN of the positive control treated mice in the MDAM, but not with DNFB in the LLNA. A mild increase in T-cell proliferation was observed in the MDAM with positive control drugs, while DNFB in the LLNA induced proliferation within the B-cell population only. Anti-CD40L antibody administration inhibited MDAM responses to positive control drugs, but did not affect DNFB-induced increases in total cell number in the LLNA. These results suggest that the increased cellularity of the DLN in the MDAM may be the result of drug-induced alterations in lymphocyte migration and/or effects on lymphocyte proliferation. Moreover, it appears that different mechanisms may be involved in driving the MDAM and LLNA responses.

Radioligand binding assays for total and Ig classes of insulin antibodies (IAB) were developed and validated. For each assay, insulin-extracted serum samples were incubated with radiolabeled insulin in the presence and absence of high levels of unlabeled insulin to determine nonspecific binding and total binding, respectively. To measure total IAB, antibody-bound insulin was precipitated with a polyethylene glycol solution, washed, and counted in a gamma-counter. To measure IgG IAB, samples were treated with protein G-Sepharose beads, centrifuged, washed, and counted. For the measurement of IgA, IgE, and IgM IAB, IgG was removed from the samples and treated with anti-IgA, -IgE, or -IgM conjugated to Sepharose beads, centrifuged, washed, and counted. The acid/charcoal extraction of bound and unbound insulin from serum samples was optimized. Specificity and binding capacity of the protein G and antibody-bound beads were evaluated and optimized. The linear region of the total and IgG IAB assays was determined using serum samples containing high levels of insulin antibodies. The limit of quantitation, limit of detection, and precision for all the assays were also determined.