Comparison across vehicles of passenger head kinematics in abrupt vehicle maneuvers.

Objective: Studies of vehicle occupant motions in response to abrupt vehicle maneuvers have demonstrated movements that may result in changes in the level of protection for the occupant if a crash subsequently occurs. The previous studies have typically used a single vehicle. The current study assesses whether the patterns of occupant head movement are different across passenger vehicle types.Method: Data collection was conducted on a closed test track with the same driver for all trials. A passenger sedan, a minivan, and a pickup truck were equipped with inertial measurement units to quantify vehicle dynamics. Head location was tracked using Microsoft Kinect v2 sensor and a novel methodology that fits 3 D head scan data to the depth data acquired in the vehicle. Twelve men and women with a wide range of body size and age were recruited. The primary purpose of the study was obfuscated by telling the participants that the focus was on vehicle ride motion. Participants sat in the right front seat and wore the vehicle belt. The first event during the test track route was a hard brake (approximately 1 g) to a stop from 35 mph (56 kph). Within the space of approximately 5 min the participants also experienced two aggressive, right-going lane changes, a sharp right turn with simultaneous hard braking, and a second hard braking event. The vehicles were presented in random order for each participant. This paper presents comparison across vehicles of head motions in the braking and lane-change maneuvers.Results: Accelerations were similar across the vehicles for both braking and lane-change events. The means (standard deviations) of forward head-CG excursion in the first braking event were 162 (54), 112 (39), and 176 (46) mm for the minivan, passenger car, and truck, respectively. The forward head excursion in the passenger car was found to be significantly smaller than in the other two vehicles using a paired t-test (p < 0.01). Across vehicles, the mean excursion in the second braking exposure was smaller than in the first (p < 0.01). In the first lane change event, the mean (SD) inboard head excursions were 126 (51), 110 (49), and 140 (68) mm; the values were not significantly different across vehicles or in the second lane-change event. A detailed investigation did not reveal an explanation for the smaller head excursions in the passenger car.Discussion: This is the first quantitative occupant kinematics study to compare responses across vehicles. Although a significant difference was found between vehicles, the overall responses are similar to those observed in a previous study.Conclusions: The results confirm previous studies showing large variance in excursions across occupants. Further study is needed to understand the factors that affect responses across vehicles.

Passenger head kinematics in abrupt braking and lane change events.

OBJECTIVE: A test track study was conducted to quantify patterns of adult front seat passenger head motion during abrupt vehicle maneuvers. METHOD: Eighty-seven men and women with a wide range of body sizes and ages participated in data collection on a closed test track in a passenger sedan under manual control by a test driver. Because a primary goal of the study was to gather "unaware" data, the participants were instructed that the study was concerned with vehicle dynamics and they were required to read from a questionnaire taped to the top of their thighs as the drive began. The first event was a hard brake (approximately 1 g) to a stop from 35 mph (56 kph). Within the space of approximately 5 min the participants also experienced an aggressive lane change, a sharp right turn with simultaneous hard braking, and a second hard braking event. A Microsoft Kinect v2 sensor was positioned to view the area around the front passenger seat. Head location was tracked using the Kinect data with a novel methodology that fit 3D head scan data to the depth data acquired in the vehicle. RESULT: The mean (standard deviation) forward excursion of the estimated head center of gravity (CG) location in the first braking event was 135 (62) mm. The forward head CG excursion in the second braking event of 115 (51) mm was significantly less than that in the first, but the difference was small relative to the within-condition variance. Head excursion on the second braking trial was less than that on the first trial for 69% of participants. The mean maximum inboard head excursion in lane-change maneuvers was 118 (40) mm. Forward head excursions in braking were significantly smaller for older passengers and those with higher body mass index, but the combined factors accounted for less than 25% of the variance. Inboard head excursion in the lane-change event was significantly related to stature, but only about 7% of variance was related to body size. Head excursions for men and women did not differ significantly after accounting for body size. DISCUSSION: This is the first quantitative occupant dynamics study to use a large, diverse sample of passengers, enabling the exploration of the effects of covariates such as age and body size. CONCLUSIONS: The data demonstrate that a relatively large range of head positions can be expected to result from abrupt vehicle maneuvers. The data do not support simple scaling of excursions based on body size.

Proteomic profiling of rectal cancer reveals acid ceramidase is implicated in radiation response.

BACKGROUND: Neoadjuvant chemoradiotherapy (CRT) is used in locally advanced rectal cancer when tumours threaten the circumferential resection margin, with varying response to treatment. This experimental study aimed to identify significantly differentially expressed proteins between patients responding and not responding to CRT, and to validate any proteins of interest. METHODS: Mass spectrometry (with isobaric tagging for relative quantification) analysis of rectal cancers pre- and post-CRT, and at resection. Validation of proteins of interest was performed by assessing tissue microarray (TMA) immunohistochemistry expression in a further 111 patients with rectal cancer. RESULTS: Proteomic data are available via ProteomeXchange with identifier PXD008436. Reduced abundance of contributing peptide ions for acid ceramidase (AC) (log fold change -1.526, p = 1.17E-02) was observed in CRT responders. Differential expression of AC was confirmed upon analysis of the TMAs. Cancer site expression of AC in stromal cells from post-CRT resection specimens was observed to be relatively low in pathological complete response (p = 0.003), and relatively high with no response to CRT (p = 0.017). CONCLUSION: AC may be implicated in the response of rectal cancer to CRT. We propose its further assessment as a novel potential biomarker and therapeutic target. SIGNIFICANCE: There is a need for biomarkers to guide the use of chemoradiotherapy in rectal cancer, as none are in routine clinical use. We have determined acid ceramidase may have a role in radiation response, based on novel proteomic profiling and validation in a wider dataset using tissue microarrays. The ability to predict or improve response would positively select those patients who will derive benefit, prevent delays in the local and systemic management of disease in non-responders, and reduce morbidity associated with chemoradiotherapy.

Implications of HLA-allele associations for the study of type IV drug hypersensitivity reactions.

INTRODUCTION: Type IV drug hypersensitivity remains an important clinical problem and an obstacle to the development of new drugs. Several forms of drug hypersensitivity are associated with expression of specific HLA alleles. Furthermore, drug-specific T-lymphocytes have been isolated from patients with reactions. Despite this, controversy remains as to how drugs interact with immune receptors to stimulate a T-cell response. Areas covered: This article reviews the pathways of T-cell activation by drugs and how the ever increasing number of associations between expression of HLA alleles and susceptibility to hypersensitivity is impacting on our research effort to understanding this form of iatrogenic disease. Expert opinion: For a drug to activate a T-cell, a complex is formed between HLA molecules, an HLA binding peptide, the drug and the T-cell receptor. T-cell responses can involve drugs and stable or reactive metabolites bound covalently or non-covalently to any component of this complex. Recent research has linked the HLA associations to the disease through the characterization of drug-specific T-cell responses restricted to specific alleles. However, there is now a need to identify the additional genetic or environment factors that determine susceptibility and use our increased knowledge to develop predictive immunogenicity tests that offer benefit to Pharma developing new drugs.

Exome sequencing of synchronously resected primary colorectal tumours and colorectal liver metastases to inform oncosurgical management.

BACKGROUND: Next generation sequencing technology has facilitated mapping of the colorectal cancer genotype and furthered our understanding of metastogenesis. The aim of this study was to investigate for conserved and different mutations in the exomes of synchronously resected primary colorectal tumour and liver metastases. This information could potentially be utilised to guide the treatment of advanced disease with the help of biological information from the primary tumour. METHODS: We performed exome sequencing of synchronously resected primary colorectal cancer and colorectal liver metastases as well as normal colonic mucosa and liver parenchyma, from four patients who had received neo-adjuvant chemotherapy, at a depth of 50X using the Ion Proton platform. Raw data was mapped to the reference genome prior to variant calling, annotation and downstream analysis. RESULTS: Exome sequencing identified 585 non-synonymous missense single nucleotide variants (SNVs), of which 215 (36.8%) were unique to the primary tumour, 226 (38.6%) unique to the metastasis and 81 (13.8%) present in patient matched pairs. SNVs identified in the ErbB pathway appear to be concordant between primary and metastatic tumours. CONCLUSION: Only 13.8% of the metastatic exome can be predicted by the genotype of the primary tumour. We have demonstrated concordance of a number of SNVs in the ErbB pathway, which may inform selection of therapeutic agents in advanced colorectal cancer.

shapecoder: a new method for visual quantification of body mass index in young children.

BACKGROUND: Few tools exist to quantify body mass index visually. OBJECTIVE: To examine the inter-rater reliability and validity (sensitivity and specificity for overweight/obesity and obesity) of a three-dimensional visual rating system to quantify body mass index (BMI) in young children. METHODS: Children (n = 242, mean age 5.9 years, 50.0% male; 40.5% overweight/ obese) participated in a videotaped protocol and weight and height were measured. Research staff applied a novel three-dimensional computer-based figure rating system (shapecoder) to the child's videotaped image. Inter-rater reliability was calculated, as well as correlation with measured body mass index (BMI) and sensitivity, specificity, positive predictive value and negative predictive value for overweight/obesity and obesity. RESULTS: Inter-rater reliability was excellent (intraclass correlation coefficient = 0.98). The correlation of shapecoder-generated BMI with measured BMI was 0.89. For overweight/obesity, the sensitivity, specificity, positive predictive value and negative predictive value were 62%, 97%, 94% and 79% respectively. For obesity, these values were 65%, 99%, 97% and 92% respectively. CONCLUSION: shapecoder provides a method to quantify child BMI from video images with high inter-rater reliability, fair sensitivity and good specificity for overweight/obesity and obesity. The approach offers an improvement over existing two-dimensional rating scales for BMI.

Inhibition of Porcine Endogenous Retrovirus by Multi-Targeting Micro RNA Against Long Terminal Region.

BACKGROUND: There might be much benefit in xenotransplantation, however, the risk of infections across species barriers remains, especially porcine endogenous retrovirus (PERV). To date, many attempts have been made to knock down active PERVs by inhibitory RNA (RNAi) and micro RNA (miRNA), which target different genes of PERV. There are a few studies that have explored whether targeting promoter regions of PERV could exert an inhibition effect. METHODS: miRNAs were automatically selected based on an online program BLOCK-iT RNAi Designer. The inhibition efficiency between miRNAs was compared based on their inhibition of different PERV genes: long terminal repeats (LTR), gag, and pol. Both relative quantitative real-time polymerase chain reaction (PCR) and C-type reverse transcriptase activity were performed. RESULTS: The results demonstrated that miRNA targeting the LTR region degraded the target sequence, and simultaneously inhibited the mRNA expression of both gag and pol genes of PERV. The LTR1, LTR2, and dual LTR1 + LTR2 miRNA inhibited 76.2%, 22%, and 76.8% of gag gene expression, respectively. Similarly, the miRNA was found to knock down the pol gene expression of 69.8%, 25.5%, and 77.7% for single targeting miRNA (LTR1 and LTR2) and multi-targeting miRNA (LTR1 + LTR2), respectively. A stable PK15 clone constitutively expressed dual LTR1 + LTR2 miRNA and exhibited higher inhibitory up to 82.8% and 92.7% of the expressions of the gag and pol genes, respectively. Also, the result of co-cultivation of dual LTR1 + LTR2 miRNA transfected PK15 cell with a human cell line inhibited expression of LTR, gag, and pol genes of PERV. CONCLUSIONS: In conclusion, this study suggested that the LTR might be an alternative target for gene silencing of PERV, and that multi-targeting miRNA had better inhibitory effect than single- targeting miRNA. In an in vitro model, the presence of miRNA was able to reduce PERV infectivity in a human cell line.

Optimising the use of medicines to reduce acute kidney injury in children and babies.

The majority of medications in children are administered in an unlicensed or off-label manner. Paediatricians are obliged to prescribe using the limited evidence available. The 2007 EU regulation on the use of paediatric drugs means pharmaceutical companies are now obliged to (and receive incentives for) contributing to paediatric drug data and carrying out paediatric clinical trials. This is important, as the efficacy and adverse effect profiles of medicines vary across childhood. Additionally, there are significant age-related changes in the pharmacodynamic and pharmacokinetic activity of many drugs. This may be related to physiological (differential expressions of cytochrome P450 enzymes or variable glomerular filtration rates at different ages for example) and psychological (increasing autonomy and risk perception in teenage years) changes. Increasing numbers of children are surviving life-threatening childhood conditions due to medical advances. This means there is an increasing population who are at risk of the consequences of the long-term, early exposure to nephrotoxic agents. The kidney is an organ that is particularly vulnerable to damage as a consequence of drugs. Drug-induced acute kidney injury (AKI) episodes in children and babies are principally due to non-steroidal anti-inflammatory drugs, antibiotics or chemotherapeutic agents. The renal tubules are vulnerable to injury because of their concentrating ability and high-energy hypoxic environment. This review focuses on drug-induced AKI and the methods to minimise its effect, including general management plus the role of child-specific pharmacokinetic data, the use of pharmacogenomics and early detection of AKI using urinary biomarkers and electronic triggers.

First report of Fusarium oxysporum species complex infection in zebrafish culturing system.

Fusarium oxysporum species complex (FOSC) is a highly diverse fungus. Recently, F. oxysporum infection was identified from zebrafish (Danio rerio) culturing system in Korea. Initially, a rapid whitish smudge was appeared in the water with the fungal blooming on walls of fish tanks. Microscopic studies were conducted on fungal hyphae, colony pigmentation and chlamydospore formation and the presence of macro- and microspores confirmed that the isolated fungus as F. oxysporum. Furthermore, isolated F. oxysporum was confirmed by internal transcribed spacer sequencing which matched (100%) to nine F. oxysporum sequences available in GenBank. Experimental hypodermic injection of F. oxysporum into adult zebrafish showed the development of fungal mycelium and pathogenicity similar to signs observed. Histopathologic results revealed a presence of F. oxysporum hyphae in zebrafish muscle. Fusarium oxysporum growth was increased with sea salt in a concentration-dependent manner. Antifungal susceptibility results revealed that F. oxysporum is resistant to copper sulphate (up to 200 µg mL-1 ) and sensitive to nystatin (up to 40 µg mL-1 ). This is the first report of FOSC from zebrafish culture system, suggesting it appears as an emerging pathogen, thus posing a significant risk on zebrafish facilities in the world.

Theileria buffeli infections in grazing cattle in the Republic of Korea.

Theileria infections are encountered worldwide, occasionally resulting in serious economic losses for the livestock industry. This study is an epidemiological survey of Theileria infections in Korean indigenous cattle populations in the Republic of Korea (ROK). Blood samples were collected from 100 cattle in April (n=50) (prior to pastureland grazing), and again four months later, in August (n=50) (half of the cattle put out for grazing and the other half kept in housing). All samples were tested for the presence of Theileria infection based on PCR amplification of the small subunit of ribosomal RNA gene. Twenty-two samples across the whole study were verified as positive for Theileria infection by PCR methods. In August, Theileria infection was markedly increased in grazing cattle (16/25 animals, 64%) compared with indoor cattle (4/25 animals, 16%); affected animals exhibited no clinical signs of infection. The red blood cell, hematocrit, and hemoglobin values were significantly lower in Theileriapositive cattle than in Theileria-negative cattle. Phylogenetic analysis demonstrated that the isolates from this study belonged to the T. buffeli species, and were significantly related to Types A, B, C, and E, and were distinct from T. buffeli Type D, which is known to be more pathogenic. These findings indicate that T. buffeli identified in Korean indigenous cattle have a low-to-mild pathogenicity. These results suggest that the T. buffeli infection is relatively higher in the ROK, and the infection rate may increase following grazing. Taken together, T. buffeli infection may not only be seasonally correlated, but also may be affected by management practices such as pastureland grazing.

Detection of drug-responsive B lymphocytes and antidrug IgG in patients with ß-lactam hypersensitivity.

BACKGROUND: Delayed-type ß-lactam hypersensitivity develops in subset of patients. The cellular immunological processes that underlie the drug-specific response have been described; however, little is known about involvement of the humoral immune system. Thus, the aim of this study was to utilize piperacillin hypersensitivity as an exemplar to (i) develop cell culture methods for the detection of drug-specific B-cell responses, (ii) characterize drug-specific IgG subtypes and (iii) assess reactivity of IgG antibodies against proteins modified to different levels with piperacillin haptens. METHODS: IgG secretion and CD19+ CD27+ expression on B cells were measured using ELISPOT and flow cytometry, respectively. A piperacillin-BSA adduct was used as an antigen in ELISA antibody binding studies. Adducts generated using different ratios of drug to protein were used to determine the degree of conjugation required to detect IgG binding. RESULTS: B cells from hypersensitive patients, but not controls, were stimulated to secrete IgG and increase CD27 expression when cultured with soluble piperacillin. A piperacillin-BSA adduct with cyclized and hydrolysed forms of the hapten bound to eight lysine residues was used to detect hapten-specific IgG 1-4 subclasses in patient plasma. Hapten inhibition and the use of structurally unrelated hapten-BSA adducts confirmed antigen specificity. Antibody binding was detected with antigens generated at piperacillin/BSA ratios of 10:1 and above, which corresponded to a minimum epitope density of 1 for antibody binding. CONCLUSION: These data show that antigen-specific B lymphocytes and T lymphocytes are activated in piperacillin-hypersensitive patients. Further work is needed to define the role different IgG subtypes play in regulating the iatrogenic disease.

Detection and Phylogenetic Analysis of Porcine Deltacoronavirus in Korean Swine Farms, 2015.

This study applied molecular-based method to investigate the presence of porcine deltacoronavirus (PDCoV) in 59 commercial pig farms in South Korea. The results of RT-PCR screening on a relatively large collection of faeces samples (n = 681) from January 2013 to March 2015 did not reveal the presence of PDCoV until the end of 2014. However, on March 2015, PDCoV-positive samples (SL2, SL5) were detected from SL swine farm in Gyeongbuk province. The phylogenetic trees based on the complete spike- and nucleocapsid protein-coding genes showed that SL2 and SL5 closely related to the US PDCoV strains rather than those in China. Thought Korean strains of PDCoV isolated in 2014 (KNU14.04) and in 2015 (SL2 and SL5) grouped within US PDCoV cluster, the reconstruction of ancestral amino acid changes suggested that they are different.

Adaptation to acetaminophen exposure elicits major changes in expression and distribution of the hepatic proteome.

Acetaminophen overdose is the leading cause of acute liver failure. One dose of 10-15 g causes severe liver damage in humans, whereas repeated exposure to acetaminophen in humans and animal models results in autoprotection. Insight of this process is limited to select proteins implicated in acetaminophen toxicity and cellular defence. Here we investigate hepatic adaptation to acetaminophen toxicity from a whole proteome perspective, using quantitative mass spectrometry. In a rat model, we show the response to acetaminophen involves the expression of 30% of all proteins detected in the liver. Genetic ablation of a master regulator of cellular defence, NFE2L2, has little effect, suggesting redundancy in the regulation of adaptation. We show that adaptation to acetaminophen has a spatial component, involving a shift in regionalisation of CYP2E1, which may prevent toxicity thresholds being reached. These data reveal unexpected complexity and dynamic behaviour in the biological response to drug-induced liver injury.

The chemical, genetic and immunological basis of idiosyncratic drug-induced liver injury.

Idiosyncratic drug reactions can be extremely severe and are not accounted for by the regular pharmacology of a drug. Thus, the mechanism of idiosyncratic drug-induced liver injury (iDILI), a phenomenon that occurs with many drugs including ß-lactams, anti-tuberculosis drugs and non-steroidal anti-inflammatories, has been difficult to determine and remains a pressing issue for patients and drug companies. Evidence has shown that iDILI is multifactorial and multifaceted, which suggests that multiple cellular mechanisms may be involved. However, a common initiating event has been proposed to be the formation of reactive drug metabolites and covalently bound adducts. Although the fate of these metabolites are unclear, recent evidence has shown a possible link between iDILI and the adaptive immune system. This review highlights the role of reactive metabolites, the recent genetic innovations which have provided molecular targets for iDILI, and the current literature which suggests an immunological basis for iDILI.

Comprehensive microRNA profiling in acetaminophen toxicity identifies novel circulating biomarkers for human liver and kidney injury.

Our objective was to identify microRNA (miRNA) biomarkers of drug-induced liver and kidney injury by profiling the circulating miRNome in patients with acetaminophen overdose. Plasma miRNAs were quantified in age- and sex-matched overdose patients with (N = 27) and without (N = 27) organ injury (APAP-TOX and APAP-no TOX, respectively). Classifier miRNAs were tested in a separate cohort (N = 81). miRNA specificity was determined in non-acetaminophen liver injury and murine models. Sensitivity was tested by stratification of patients at hospital presentation (N = 67). From 1809 miRNAs, 75 were 3-fold or more increased and 46 were 3-fold or more decreased with APAP-TOX. A 16 miRNA classifier model accurately diagnosed APAP-TOX in the test cohort. In humans, the miRNAs with the largest increase (miR-122-5p, miR-885-5p, miR-151a-3p) and the highest rank in the classifier model (miR-382-5p) accurately reported non-acetaminophen liver injury and were unaffected by kidney injury. miR-122-5p was more sensitive than ALT for reporting liver injury at hospital presentation, especially combined with miR-483-3p. A miRNA panel was associated with human kidney dysfunction. In mice, miR-122-5p, miR-151a-3p and miR-382-5p specifically reported APAP toxicity - being unaffected by drug-induced kidney injury. Profiling of acetaminophen toxicity identified multiple miRNAs that report acute liver injury and potential biomarkers of drug-induced kidney injury.

Change in body surface temperature as an ancillary measurement to motor evoked potentials.

STUDY DESIGN: Experimental study. OBJECTIVES: To study the role of surface temperature as an adjunct to motor evoked potentials (MEPs) in rabbit spinal cord injury (SCI) model. SETTING: Department of Orthopedics, Korea University Guro Hospital, Seoul, Korea. METHODS: Rabbits (n =18) were divided into Complete (n = 9) and Incomplete (n = 9) SCI groups. Complete SCI was defined as being non-responsive to a wake-up test with loss of MEPs after transection of spinal cord. Incomplete SCI was defined as being responsive to a wake-up test with significant attenuation (⩾ 80%) of MEPs after impaction on spinal cord. Surface temperature of upper and lower extremities, core temperature and MEPs signals were checked before, during and after SCI for 20 min. A wake-up test was conducted and spinal cord was histologicaly evaluated. RESULTS: Experimental conditions between the two groups were statistically similar (P > 0.005 for all values). After SCI, upper extremity temperatures did not change in either group (P > 0.005); however, the surface temperature of the lower extremities in the Complete SCI Group elevated to 1.7 ± 0.5°C in comparison to 0.5 ± 0.1°C in the Incomplete SCI Group (P < 0.001). The scores of wake-up test in the Incomplete SCI Group were significantly different from that of the Complete SCI Group (P < 0.001), while white and gray matter damage was variable on histology. CONCLUSIONS: Monitoring of changes of body surface temperature of the lower extremities can be potentially used to identify the completeness of SCI in a rabbit model.

Assessment of early therapeutic response to sorafenib in renal cell carcinoma xenografts by dynamic contrast-enhanced and diffusion-weighted MR imaging.

OBJECTIVE: To investigate the feasibility of dynamic contrast-enhanced MRI (DCE-MRI) and diffusion-weighted MRI (DWI) in monitoring early therapeutic response to sorafenib in renal cell carcinoma (RCC) xenograft models. METHODS: Sorafenib (40 mg kg(-1)) was administered orally to BALB/c nude mice (n = 9) bearing subcutaneous tumours of human RCC ACHN xenografts. DCE-MRI and DWI were obtained 0, 1, 3 and 7 days after therapy, and DCE-MRI parameters (K(trans) and ve) and apparent diffusion coefficient (ADC) values were calculated. Tumour size and volume changes were correlated with changes in DCE-MRI parameters or ADC values after therapy. RESULTS: Following therapy, K(trans) showed a significant decrease over time (p = 0.005), whereas ve did not demonstrate significant changes between time points (p = 0.97). ADC values showed a progressive increase over time (p = 0.004). Compared with pre-therapy, K(trans) showed a significant decrease after 3 days of therapy (p = 0.039), and ADC values increased significantly after 7 days (p = 0.039). Tumour size and volume did not show significant changes during 7 days. Tumour size and volume changes were not associated with changes in DCE-MRI parameters or ADC values. CONCLUSION: DCE-MRI and DWI may show early physiological changes within 1 week after initiating sorafenib treatment on human RCC xenografts. ADVANCES IN KNOWLEDGE: The quantitative parameters of DCE-MRI and DWI may offer the potential for assessing early therapeutic response to sorafenib in clinical trials.

Pediatric microdose and microtracer studies using 14C in Europe.

Important information gaps remain on the efficacy and safety of drugs in children. Pediatric drug development encounters several ethical, practical, and scientific challenges. One barrier to the evaluation of medicines for children is a lack of innovative methodologies that have been adapted to the needs of children. This article presents our successful experience of pediatric microdose and microtracer studies using (14) C-labeled probes in Europe to illustrate the strengths and limitations of these approaches.

HLA-DQ allele-restricted activation of nitroso sulfamethoxazole-specific CD4-positive T lymphocytes from patients with cystic fibrosis.

BACKGROUND: For certain HLA allele-associated drug hypersensitivity reactions, the parent drug has been shown to associate directly with the risk allele. In other forms of hypersensitivity, HLA risk alleles have not been identified and T cells are activated in an allele unrestricted manner. Chemically reactive drug metabolites bind to multiple proteins; thus, it is assumed that the derived peptide antigens interact with a number of HLA molecules to activate T cells; however, HLA restriction of the drug metabolite-specific T-cell response has not been studied. OBJECTIVE: To utilize T cells from sulfamethoxazole (SMX) hypersensitive patients with cystic fibrosis to examine the HLA molecules that interact with nitroso SMX (SMX-NO)-derived antigens. METHODS: T-cell clones were generated from 4 hypersensitive patients. Drug-specific proliferative responses and cytokine secretion were measured. Anti-human class I and class II antibodies were used to analyse HLA restriction. Antigen-presenting cells expressing different HLA molecules were used to determine the alleles involved in the presentation of SMX-NO-derived antigens to T cells. RESULTS: A total of 976 clones were tested for SMX-NO reactivity. Thirty-nine CD4+ clones were activated with SMX-NO and found to proliferate and secrete cytokines. The SMX-NO-specific response was blocked with an antibody against HLA-DQ. SMX-NO-specific responses were detected with antigen-presenting cells expressing HLA-DQB1*05:01 (patient 1) and HLA-DQB1*02:01 (patient 2), but not other HLA-DQB1 alleles. CONCLUSION AND CLINICAL RELEVANCE: HLA-DQ plays an important role in the activation of SMX-NO-specific CD4+ T cells. Detection of HLA-DQ allele-restricted responses suggests that T cells are activated by a limited repertoire of SMX-NO-modified peptides.

Evaluation of a novel tissue stabilization gel to facilitate clinical sampling for translational research in surgical trials.

BACKGROUND: The aim was to establish the feasibility of using a tissue stabilization gel (Allprotect™) as an alternative to liquid nitrogen to facilitate collection of clinical samples for translational research. METHODS: Tumour samples from patients undergoing surgery for primary or metastatic colorectal cancer were either snap-frozen in liquid nitrogen or stored in Allprotect™ under a number of different conditions. Sample integrity was compared across different storage conditions by assessing biomolecule stability and function. DNA quality was assessed spectrophotometrically and by KRas genotyping by pyrosequencing. Total RNA retrieval was determined by nanodrop indices/RNA integrity numbers, and quality assessed by reverse transcription-PCR for two representative genes (high-mobility group box 1, HMGB1; carboxylesterase 1, CES1) and two microRNAs (miR122 and let7d). Western blot analysis of HMGB1 and CES1 was used to confirm protein expression, and the metabolic conversion of irinotecan to its active metabolite, SN-38, was used to assess function. RESULTS: Under short-term storage conditions (up to 1 week) there was no apparent difference in quality between samples stored in Allprotect™ and those snap-frozen in liquid nitrogen. Some RNA degradation became apparent in tissue archived in Allprotect™ after 1 week, and protein degradation after 2 weeks. CONCLUSION: In hospitals that do not have access to liquid nitrogen and -80°C freezers, Allprotect™ provides a suitable alternative for the acquisition and stabilization of clinical samples. Storage proved satisfactory for up to 1 week, allowing transfer of samples without the need for specialized facilities. Surgical relevance Access to clinical material is a fundamental component of translational research that requires significant infrastructure (research personnel, liquid nitrogen, specialized storage facilities). The aim was to evaluate a new-to-market tissue stabilization gel (Allprotect™), which offers a simple solution to tissue preservation without the need for complex infrastructure. Allprotect™ offers comparable DNA, RNA and protein stabilization to tissue snap-frozen in liquid nitrogen for up to 1 week. Degradation of biomolecules beyond this highlights its role as a short-term tissue preservative. Allprotect™ has the potential to increase surgeon participation in translational research and surgical trials requiring tissue collection.