Approaches and considerations for the assessment of immunotoxicity for environmental chemicals: A workshop summary

As experience is gained with toxicology testing and as new assays and technologies are developed, it is critical for stakeholders to discuss opportunities to advance our overall testing strategies. To facilitate these discussions, a workshop on practices for assessing immunotoxicity for environmental chemicals was held with the goal of sharing perspectives on immunotoxicity testing strategies and experiences, developmental immunotoxicity (DIT), and integrated and alternative approaches to immunotoxicity testing. Experiences across the chemical and pharmaceutical industries suggested that standard toxicity studies, combined with triggered-based testing approaches, represent an effective and efficient approach to evaluate immunotoxic potential. Additionally, discussions on study design, critical windows, and new guideline approaches and experiences identified important factors to consider before initiating DIT evaluations including assay choice and timing and the impact of existing adult data. Participants agreed that integrating endpoints into standard repeat-dose studies should be considered for fulfilling any immunotoxicity testing requirements, while also maximizing information and reducing animal use. Participants also acknowledged that in vitro evaluation of immunosuppression is complex and may require the use of multiple assays that are still being developed. These workshop discussions should contribute to developing an effective but more resource and animal efficient approach for evaluating chemical immunotoxicity.     

DART-MS/MS法直接实时检测保健食品中非法添加的6个PDE5抑制剂的应用研究

目的:建立一种可以快速检测保健食品中非法添加6个磷酸二酯酶5(PDE5)抑制剂西地那非、他达拉非、红地那非、羟基豪莫西地那非、氨基他达拉非、伪伐地那非的方法。方法:采用直接实时分析(DART)离子源结合三重四极杆质谱,DART离子源的样品传输速度为0.2 mm·s-1,载气温度为450℃。Agilent 6410B Triple Quad LC/MS毛细管电压4 kV,干燥气流速为10 L·min-1。扫描模式为子离子全扫描,扫描范围为50～550 amu。待测成分的前体离子为m/z 475.1(西地那非),m/z467.2(红地那非),m/z 505.1(羟基豪莫西地那非),m/z 390.1(他达拉非),m/z 391.2(氨基他达拉非),m/z 460.3(伪伐地那非)。通过对照品与样品的二级质谱图对比进行样品中非法添加成分的定性鉴别。结果:若干批市售保健食品中有4批检出西地那非,1批检出他达拉非。检出限达1μg·g-1,检测时间仅为几分钟。结论:DART-MS可用于固体样品中添加PDE5抑制剂类成分的快速检测,用于药品快速检验中批量样品的初筛更有优势。     

Enhanced normograms and pregnancy outcome analysis in nonhuman primate developmental toxicity studies

The incidence of spontaneous pregnancy/infant losses is highly variable in long-tailed macaques (cynomolgus monkey), making it potentially difficult to ascertain test item-related effects in developmental toxicity studies. Therefore, pregnancy normograms had been developed by Jarvis et al. [1] to aid in the distinction of normal (e.g. test facility background) versus non-normal pregnancy outcomes. These normograms were mostly derived from embryo-fetal development studies and from PPND studies with a postnatal phase limited to seven days. However, the enhanced pre- and postnatal developmental (ePPND) study paradigm has essentially replaced these former study types. This work aims at providing enhanced normograms (e-normograms) in the context of regulatory ePPND studies. Survival functions for the prenatal phase (286 control pregnancies) and the postnatal phase (222 live infants) were estimated using the Kaplan-Meier estimator. Normograms were generated from survival curves and pseudo-study simulations. Data were available from two test facilities with comparable EU-compliant animal husbandry. Pregnancy duration/outcome as well as survival functions did not differ significantly between test facilities indicating that this husbandry system yields comparable developmental observations across different test facilities, at least in this NHP species. These novel e-normograms were developed for pregnant long-tailed macaques and provide an extended postnatal period up to three months, a new concept of separate normograms for the prenatal and the postnatal period, specific information on the perinatal phase events, a prediction of expected number of live infants for group size management, and the option to evaluate effects on pregnancy duration through distinction of live births and infant losses.     

Rapid screening of 35 new psychoactive substances by ion mobility spectrometry (IMS) and direct analysis in real time (DART) coupled to quadrupole time‐of‐flight mass spectrometry (QTOF‐MS)

The recent propagation of new psychoactive substances (NPS) has led to the development of new techniques for the rapid characterization of controlled substances in this category. A commercial bench‐top ion mobility spectrometer (IMS) with a ⁶³Ni ionization source and a direct analysis in real time (DART) coupled to quadrupole time‐of‐flight (QTOF) were used for the rapid characterization of 35 NPS. The advantages of these techniques are fast response, ease of operation, and minimal sample preparation. The characteristic reduced mobilities of each substance are reported as are the mass spectra of the 35 compounds. The acquired product ion scan mass spectra were also compared to a library database constructed by QTOF with a electrospray ionization (ESI) source and showed a consistent relative abundance for each peak over time. A total of four seized drug samples provided by the local forensic laboratory were analyzed in order to demonstrate the utility of this approach. The results of this study suggest that both IMS and DART‐QTOF are promising alternatives for the rapid screening and characterization of these new psychoactive substances. Copyright © 2015 John Wiley & Sons, Ltd.     

DART-MS: A New Research Tool for Herbal Medicine Analysis

Direct analysis in real time(DART) possesses the merits of analyzing sample in its native status with minimal or even no sample pretreatment.In this review, we summarized the recent applications of DART in the field of herbal medicine analysis such as compound detection, species identification, metabolites profiling and initial quantification. DART with the characters of hyper-rapid, easy-hyphenated offers a new research tool for herbal medicines to complete the experimental process in a very simple but still reliable way. It is anticipated that more wide and deep applications of DART in herbal medicine analysis, as rapid quantification, high-throughput active compounds screening, rapid species identification, and fast illegal additives screening will be promising and foreseeable in the near future.     

Getting on target: Development of the novel,prozone-resistant,dual antibody rapid test (DART) for the LABScreen single antigen bead (SAB) assay

A major limitation of the single antigen bead (SAB) assay is the so called prozone effect, whereby the detection of high titer complement fixing HLA antibodies is compromised due to complement split product (from C3 and C4 components) deposition and interference with the reporter anti-IgG-PE antibody binding. Strategies to minimize prozone include serum titration or treatment with heat, dithiotreitol (DTT), or ethylenediaminetetraacetic acid (EDTA). While effective, these treatments may compromise HLA antibody binding and detection. Here we describe the Dual Antibody Rapid Test (DART), a modified version of the rapid optimized SAB (ROB) protocol, in which we use an IgG-PE/C3d-PE antibody cocktail to simultaneously detect bead bound IgG and C3d, which allows for detection of HLA antibodies independent of the prozone effect. Twenty prozone positive sera (10 class I and 10 class II), identified by titration, were tested by the ROB protocol, with or without EDTA pre-treatment, using three reporter antibody cocktails: (1) IgG-PE, (2) C3d-PE, or (3) IgG-PE/C3d-PE (DART). Mean fluorescence intensity (MFI) values were then compared. IgG negative (n = 735) vs IgG positive (n = 1185) reactions were identified using a 1000 MFI IgG EDTA cutoff. IgG positive reactions were classified based on ΔMFI (IgG EDTA – IgG) as follows: (1) prozone negative (ΔMFI < 3000; n = 737), (2) slight prozone (ΔMFI 3001–5000; n = 49), (3) moderate prozone (ΔMFI 5001–10,000; n = 93), and (4) marked prozone (ΔMFI > 10,001; n = 306). No C3d deposition was present on IgG negative beads, and the majority of prozone positive specificities (438/448; 98%) fixed complement and were detected with the C3d-PE reporter. Interestingly, C3d-PE MFI was directly proportional to the degree of prozone (mean C3d-PE MFI = 4419.5 ± 1606.3 for slight, 5991.0 ± 2302.7 for moderate, and 12,417.4 ± 2969.9 for marked prozone specificities). Interestingly, EDTA treatment was found to have a negative impact on MFI of up to 15% of prozone negative specificities. Importantly, the DART protocol detected all prozone positive specificities while MFI for prozone negative specificities correlated well with those seen with the IgG-PE reporter alone (R² = 0.97). In conclusion, the DART protocol accurately detects HLA antibodies independent of the prozone effect. Implementation of DART is an easy way to overcome the prozone effect without compromising HLA antibody detection.     

Testing strategies for embryo-fetal toxicity of human pharmaceuticals. Animal models vs. in vitro approaches : A workshop report

Reproductive toxicity testing is characterized by high animal use. For registration of pharmaceutical compounds, developmental toxicity studies are usually conducted in both rat and rabbits. Efforts have been underway for a long time to design alternatives to animal use. Implementation has lagged, partly because of uncertainties about the applicability domain of the alternatives.     

Nettle extract (Urtica dioica) affects key receptors and enzymes associated with allergic rhinitis

A nettle (Urtica dioica) extract shows in vitro inhibition of several key inflammatory events that cause the symptoms of seasonal allergies. These include the antagonist and negative agonist activity against the Histamine‐1 (H₁) receptor and the inhibition of mast cell tryptase preventing degranulation and release of a host of pro‐inflammatory mediators that cause the symptoms of hay fevers. The nettle extract also inhibits prostaglandin formation through inhibition of Cyclooxygenase‐1 (COX‐1), Cyclooxygenase‐2 (COX‐2), and Hematopoietic Prostaglandin D₂ synthase (HPGDS), central enzymes in pro‐inflammatory pathways. The IC₅₀ value for histamine receptor antagonist activity was 251 (±13) µg mL^?1 and for the histamine receptor negative agonist activity was 193 (±71) µg mL^?1. The IC₅₀ values for inhibition of mast cell tryptase was 172 (±28) µg mL^?1, for COX‐1 was 160 (±47) µg mL^?1, for COX‐2 was 275 (±9) µg mL^?1, and for HPGDS was 295 (±51) µg mL^?1. Through the use of DART TOF‐MS, which yields exact masses and relative abundances of compounds present in complex mixtures, bioactives have been identified in nettle that contribute to the inhibition of pro‐inflammatory pathways related to allergic rhinitis. These results provide for the first time, a mechanistic understanding of the role of nettle extracts in reducing allergic and other inflammatory responses in vitro. Copyright © 2009 John Wiley & Sons, Ltd.     

Combined Modality Treatment for Prostate Cancer With Dynamic Adaptive Radiation Therapy Using Four-Dimensional Image-Guided Intensity-Modulated Radiation Therapy and Brachytherapy

Prostate cancer can be successfully treated using dynamic adaptive external beam radiation techniques along with interstitial brachytherapy to deliver curative therapies with low urinary, rectal and erectile function morbidity. Through the use of sophisticated, state-of-the art radiographic imaging for staging and treatment planning, a precise, individual design for treatment is accomplished. Symptom management and patient education are of paramount importance and are integrated throughout the treatment process.