"Boy, what are we all doing? We are crazy, really crazy": a qualitative study of psychosocial processes around an atypical one-time smoking cessation course.

BACKGROUND: Smoking prevalence is still high, which requires effective interventions that help many people who smoke at once in addition to time-consuming individual interventions. 'I Quit' is a large-scale smoking cessation course in The Netherlands. This qualitative study explored I Quit participants' experiences during and after the course, and perceptions of whether and how the course may have altered their smoking behavior. METHODS: We performed individual semi-structured interviews with course participants (N = 21) who had either quit successfully, attempted to quit but relapsed, or had continued to smoke after 'I Quit'. Shortly after qualitative data collection was completed, Foundation I Quit was accused in the media of a number of misbehaviors. Although unplanned, this provided a unique opportunity to explore participants' views on alleged fraud in a second round of interviews (N = 16). Data were collected from 2016 to 2018. RESULTS: Qualitative findings showed two psychosocial processes that may explain smoking cessation after course attendance. First, the confrontation with a large group of people who smoke, of whom some had already developed smoking-related complaints, triggered identity processes both towards and away from quitting smoking. Unorthodox methods used in the course appeared to trigger identity processes. Second, social support after the course from participants' own social network facilitated maintenance of successful quitting. The study also found that interview participants' opinions on I Quit did not change much after allegations of fraud in the media. CONCLUSIONS: Findings suggest that a one-time course might initiate psychosocial processes that could help certain smokers to gain motivation to quit, requiring a minimum of resources. Identity processes triggered by the course seem tricky as people have different ways of dealing with identity threat, some of which can be counterproductive and even result in more difficulty quitting. More research is needed to examine who can benefit from a one-time course, and who needs more support in order to quit successfully.

Lessons learned during implementation of MR-guided High-Intensity Focused Ultrasound treatment of uterine fibroids.

BACKGROUND: Although promising results have been reported for Magnetic Resonance image-guided High-Intensity Focused Ultrasound (MR-HIFU) treatment of uterine fibroids, this treatment is not yet widely implemented in clinical practice. During the implementation of a new technology, lessons are learned and an institutional learning-curve often has to be completed. The primary aim of our prospective cohort study was to characterize our learning-curve based on our clinical outcomes. Secondary aims included identifying our lessons learned during implementation of MR-HIFU on a technical, patient selection, patient counseling, medical specialists and organizational level. RESULTS: Our first seventy patients showed significant symptom reduction and improvement of quality of life at 3, 6 and 12 months after MR-HIFU treatment compared to baseline. After the first 25 cases, a clear plateau phase was reached in terms of failed treatments. The median non-perfused volume percentage of these first 25 treatments was 44.6% (range: 0-99.7), compared to a median of 74.7% (range: 0-120.6) for the subsequent treatments. CONCLUSIONS: Our findings describe the learning-curve during the implementation of MR-HIFU and include straightforward suggestions to shorten learning-curves for future users. Moreover, the lessons we learned on technique, patient selection, patient counseling, medical specialists and organization, together with the provided supplements, may be of benefit to other institutions aiming to implement MR-HIFU treatment of uterine fibroids. Trial registration ISRCTN14634593. Registered January 12, 2021-Retrospectively registered, https://www.isrctn.com/ISRCTN14634593 .

Halting targeted and collateral damage to red blood cells by the complement system.

The complement system is an important defense mechanism against pathogens; however, in certain pathologies, the system also attacks human cells, such as red blood cells (RBCs). In paroxysmal nocturnal hemoglobinuria (PNH), RBCs lack certain complement regulators which sensitize them to complement-mediated lysis, while in autoimmune hemolytic anemia (AIHA), antibodies against RBCs may initiate complement-mediated hemolysis. In recent years, complement inhibition has improved treatment prospects for these patients, with eculizumab now the standard of care for PNH patients. Current complement inhibitors are however not sufficient for all patients, and they come with high costs, patient burden, and increased infection risk. This review gives an overview of the underlying pathophysiology of complement-mediated hemolysis in PNH and AIHA, the role of therapeutic complement inhibition nowadays, and the high number of complement inhibitors currently under investigation, as for almost every complement protein, an inhibitor is being developed. The focus lies with novel therapeutics that inhibit complement activity specifically in the pathway that causes pathology or those that reduce costs or patient burden through novel administration routes.

Avoiding Unnecessary Major Rectal Cancer Surgery by Implementing Structural Restaging and a Watch-and-Wait Strategy After Neoadjuvant Radiochemotherapy.

BACKGROUND: Pathologic complete response (pCR) after neoadjuvant chemoradiotherapy (nCRT) is found in 15-20% of patients with locally advanced rectal cancer. A watch-and-wait (W&W) strategy has been introduced as an alternative strategy to avoid surgery for selected patients with a clinical complete response at multidisciplinary response evaluation. The primary aim of this study was to evaluate the efficacy of the multidisciplinary response evaluation by comparing the proportion of patients with pCR since the introduction of the structural response evaluation with the period before response evaluation. METHODS: This retrospective cohort study enrolled patients with locally advanced rectal cancer who underwent nCRT between January 2009 and May 2018, categorizing them into cohort A (period 2009-2015) and cohort B (period 2015-2018). The patients in cohort B underwent structural multidisciplinary response evaluation with the option of the W&W strategy. Proportion of pCR (ypT0N0), time-to-event (pCR) analysis, and stoma-free survival were evaluated in both cohorts. RESULTS: Of the 259 patients in the study, 21 (18.4%) in cohort A and in 8 (8.7%) in cohort B had pCR (p = 0.043). Time-to-event analysis demonstrated a significant pCR decline in cohort B (p < 0.001). The stoma-free patient rate was 24% higher in cohort B (p < 0.001). CONCLUSION: Multidisciplinary clinical response evaluation after nCRT for locally advanced rectal cancer led to a significant decrease in unnecessary surgery for the patients with a complete response.

Optimization of pulmonary emphysema quantification on CT scans of COPD patients using hybrid iterative and post processing techniques: correlation with pulmonary function tests.

OBJECTIVES: The aim of this study was to assess the effect of hybrid iterative reconstruction and post processing on emphysema quantification in low-dose CT scans of COPD patients using pulmonary function tests (PFT) as a reference. METHODS: CT scans of 23 COPD patients diagnosed with GOLD I or higher were reconstructed with iDose4 level 1 to 7 in IntelliSpace Portal (ISP) 6 and 7. ISP7 was used with and without specific denoising filter for COPD. The extent of emphysema was measured as percentage of lung voxels with attenuation < - 950 Hounsfield units (%LAA-950). The correlation between %LAA-950 and PFT, age, BMI, pack years, and the Clinical COPD Questionnaire (CCQ) and Medical Research Council dyspnea scale (MRC) was determined. RESULTS: Denoising significantly reduced %LAA-950 as was demonstrated by lower %LAA-950 in ISP7 with denoising filter and a significant reduction in %LAA-950 with higher iDose4 levels. All PFT except forced vital capacity (FVC) were significantly inversely correlated with %LAA-950. There was a trend toward a stronger correlation at higher iDose4 levels. %LAA-950 was also significantly correlated with BMI, GOLD class, and CCQ scores. CONCLUSIONS: Our study showed that hybrid iterative reconstruction and use of post processing denoising can optimize the use of emphysema quantification in CT scans as a complimentary diagnostic tool to stage COPD in addition to PFT.

Clinical validation of a pressure-standardized compression mammography system.

OBJECTIVES: Validation of a pressure-standardized compression mammography (PSCM) system, which aims to reduce discomfort and pain by applying the same pressure to every breast, independent of breast size. METHODS: We retrospectively studied mammograms of 39 patients acquired with a conventional force-standardized compression mammography (FSCM) technique and intra-individually compared them to mammograms acquired on a checkup visit with PSCM technique. Patients received one craniocaudal (CC) and one mediolateral oblique (MLO) compression for both breasts. All images were processed to obtain the contact area between the breast and the compression paddle. The pressure was calculated by dividing the compression force by the contact area. RESULTS: A total of 150 FSCM and 150 PSCM images were analyzed. The mean pressure decreased significantly from 17.1 to 12.8 kPa (p < 0.001), when using PSCM instead of FSCM. The applied pressure hardly depended on the breast contact area with the paddle (-0.014 kPa/cm2), while a clear dependency was observed using FSCM. Furthermore, the relative number of over-compressions reduced from 26% to 2%, benefitting patients with smaller breasts. CONCLUSIONS: Our study suggests that using PSCM can reduce patient discomfort and pain during mammographic compression compared to conventional FSCM as a result of lower average pressure. Moreover, standardized pressure may provide a more constant image quality, which could improve diagnostic performance.

Traditional methods v. new technologies - dilemmas for dietary assessment in large-scale nutrition surveys and studies: a report following an international panel discussion at the 9th International Conference on Diet and Activity Methods (ICDAM9), Brisbane, 3 September 2015.

The aim of the present paper is to summarise current and future applications of dietary assessment technologies in nutrition surveys in developed countries. It includes the discussion of key points and highlights of subsequent developments from a panel discussion to address strengths and weaknesses of traditional dietary assessment methods (food records, FFQ, 24 h recalls, diet history with interviewer-assisted data collection) v. new technology-based dietary assessment methods (web-based and mobile device applications). The panel discussion 'Traditional methods v. new technologies: dilemmas for dietary assessment in population surveys', was held at the 9th International Conference on Diet and Activity Methods (ICDAM9), Brisbane, September 2015. Despite respondent and researcher burden, traditional methods have been most commonly used in nutrition surveys. However, dietary assessment technologies offer potential advantages including faster data processing and better data quality. This is a fast-moving field and there is evidence of increasing demand for the use of new technologies amongst the general public and researchers. There is a need for research and investment to support efforts being made to facilitate the inclusion of new technologies for rapid, accurate and representative data.

The current aetiology of malignant pleural effusion in the Western Cape Province, South Africa.

BACKGROUND: Malignant pleural effusion (MPE) represents a very common cause of pleural exudates, and is one of the most challenging pleural disorders to manage. This could be attributed to the paucity of high-quality experimental evidence, and inconsistent practice worldwide. South Africa (SA) currently has no data regarding the aetiology of MPE. OBJECTIVES: To identify the most common malignancies causing MPE in a population served by a large tertiary hospital in SA, and specifically the relative contribution of mesothelioma. A secondary objective was to evaluate the efficacy of chemical pleurodesis in a subset of patients. METHODS: We retrospectively included all known cases of MPE evaluated at our institution over a 3-year period with a tissue diagnosis of MPE. RESULTS: The most common causes of MPE in a total of 274 patients were lung cancer (n=174, 63.5%), breast cancer (n=32, 11.7%), unknown primary (n=22, 11.7%) and mesothelioma (n=27, 9.9%). Talc pleurodesis was performed in 81 of 194 patients (41.8%) referred to our division, and was radiologically successful in 22 of 25 (88.0%) followed up to 3 months. CONCLUSIONS: The main cause of MPE in our setting was lung cancer, followed by breast cancer, unknown primary and mesothelioma. Chemical pleurodesis was a viable palliative measure for MPE in this population.

Novel Algorithms for Improved Sensitivity in Non-Invasive Prenatal Testing.

Non-invasive prenatal testing (NIPT) of cell-free DNA in maternal plasma, which is a mixture of maternal DNA and a low percentage of fetal DNA, can detect fetal aneuploidies using massively parallel sequencing. Because of the low percentage of fetal DNA, methods with high sensitivity and precision are required. However, sequencing variation lowers sensitivity and hampers detection of trisomy samples. Therefore, we have developed three algorithms to improve sensitivity and specificity: the chi-squared-based variation reduction (χ2VR), the regression-based Z-score (RBZ) and the Match QC score. The χ2VR reduces variability in sequence read counts per chromosome between samples, the RBZ allows for more precise trisomy prediction, and the Match QC score shows if the control group used is representative for a specific sample. We compared the performance of χ2VR to that of existing variation reduction algorithms (peak and GC correction) and that of RBZ to trisomy prediction algorithms (standard Z-score, normalized chromosome value and median-absolute-deviation-based Z-score). χ2VR and the RBZ both reduce variability more than existing methods, and thereby increase the sensitivity of the NIPT analysis. We found the optimal combination of algorithms was to use both GC correction and χ2VR for pre-processing and to use RBZ as the trisomy prediction method.

Consequences of CT colonography in stenosing colorectal cancer.

BACKGROUND: In patients with stenosing colorectal cancer (CRC), visualization of the entire colon prior to surgery is recommended to exclude synchronous tumors. Therefore, most centers combine computed tomographic colonography (CTC) with staging CT. The aims of this study were to evaluate the yield and clinical implications of CTC. METHODS: In this multicenter retrospective study, patients with stenosing CRC that underwent CTC and subsequent surgery between April 2013 and November 2015 were included. Result of the CTC, its influence on the surgical treatment plan, and final histology report were evaluated. RESULTS: One hundred sixty-two patients with stenosing CRC were included. Nine (5.6 %) synchronous cancers proximal to the stenosing tumor were suspected with CTC. In four of nine patients, the CTC did not change the primary surgical plan because the tumors were located in the same surgical segment. In five of nine patients, CTC changed the surgical treatment plan. Three of these five patients underwent an extended resection and the presence of the tumors was confirmed. Two of these three synchronous CRCs were also visible on abdominal staging CT. In the other two patients, the result of CTC was false positive which led to an unnecessary extended resection in one patient. CONCLUSION: The yield of CTC was relatively low. In only three patients (1.9 %), CTC correctly changed the primary surgical plan, but in two of them, the tumor was also visible on abdominal staging CT. Moreover, in two patients, CTC was false positive. The clinical value of CTC in stenosing CRC appears to be limited.

Fire, climate and vegetation linkages in the Bolivian Chiquitano seasonally dry tropical forest.

South American seasonally dry tropical forests (SDTFs) are critically endangered, with only a small proportion of their original distribution remaining. This paper presents a 12 000 year reconstruction of climate change, fire and vegetation dynamics in the Bolivian Chiquitano SDTF, based upon pollen and charcoal analysis, to examine the resilience of this ecosystem to drought and fire. Our analysis demonstrates a complex relationship between climate, fire and floristic composition over multi-millennial time scales, and reveals that moisture variability is the dominant control upon community turnover in this ecosystem. Maximum drought during the Early Holocene, consistent with regional drought reconstructions, correlates with a period of significant fire activity between 8000 and 7000 cal yr BP which resulted in a decrease in SDTF diversity. As fire activity declined but severe regional droughts persisted through the Middle Holocene, SDTFs, including Anadenanthera and Astronium, became firmly established in the Bolivian lowlands. The trend of decreasing fire activity during the last two millennia promotes the idea among forest ecologists that SDTFs are threatened by fire. Our analysis shows that the Chiquitano seasonally dry biome has been more resilient to Holocene changes in climate and fire regime than previously assumed, but raises questions over whether this resilience will continue in the future under increased temperatures and drought coupled with a higher frequency anthropogenic fire regime.This article is part of the themed issue 'The interaction of fire and mankind'.

[A woman with episodes of abdominal pain]. / Een vrouw met buikpijnaanvallen.

A 63-year-old woman was referred to the gastroenterology outpatient clinic with gallstones and a big liver mass on ultrasound. She had episodes of upper abdominal pain. Magnetic resonance imaging showed typical images for a giant liver haemangioma. We performed a surgical resection of the giant haemangioma and the gallbladder.

Biodistribution Study of Intravenously Injected Cetuximab-IRDye700DX in Cynomolgus Macaques.

PURPOSE: The use of receptor-targeted antibodies conjugated to photosensitizers is actively being explored to enhance treatment efficacy. To facilitate clinical testing, we evaluated cetuximab conjugated to IRDye700DX (IR700) in cynomolgus macaques. PROCEDURES: Total IR700 and intact cetuximab-IR700 were measured in 51 tissues at 2 and 14 days after intravenous injection of 40 and 80 mg/kg cetuximab-IR700, respectively, and compared with an unlabeled cetuximab-dosed control group (two each per sex per time point per group). RESULTS: The IR700 retrieved from all tissues at 2 and 14 days after dosing was estimated at 34.9 ± 1.8 and 2.53 ± 0.67% of the total dose, respectively. The tissues with the highest levels of intact cetuximab-IR700 at 2 days after dosing were the blood, lung, and skin. Formalin-fixed paraffin-embedded tissue sections at 2 days after dosing showed the highest IR700 signals in the axillary lymph node, mammary gland, and gall bladder. CONCLUSIONS: Both IR700 and intact cetuximab-IR700 biodistributions were consistent with known epidermal growth factor receptor (EGFR) expression, and changes between 2 and 14 days were consistent with rapid metabolism and excretion of the cetuximab-IR700.

Optical innovations in surgery.

BACKGROUND: In the past decade, there has been a major drive towards clinical translation of optical and, in particular, fluorescence imaging in surgery. In surgical oncology, radical surgery is characterized by the absence of positive resection margins, a critical factor in improving prognosis. Fluorescence imaging provides the surgeon with reliable and real-time intraoperative feedback to identify surgical targets, including positive tumour margins. It also may enable decisions on the possibility of intraoperative adjuvant treatment, such as brachytherapy, chemotherapy or emerging targeted photodynamic therapy (photoimmunotherapy). METHODS: This article reviews the use of optical imaging for intraoperative guidance and decision-making. RESULTS: Image-guided cancer surgery has the potential to be a powerful tool in guiding future surgical care. Photoimmunotherapy is a theranostic concept (simultaneous diagnosis and treatment) on the verge of clinical translation, and is highlighted as an effective combination of image-guided surgery and intraoperative treatment of residual disease. Multispectral optoacoustic tomography, a technique complementary to optical image-guided surgery, is currently being tested in humans and is anticipated to have great potential for perioperative and postoperative application in surgery. CONCLUSION: Significant advances have been achieved in real-time optical imaging strategies for intraoperative tumour detection and margin assessment. Optical imaging holds promise in achieving the highest percentage of negative surgical margins and in early detection of micrometastastic disease over the next decade.

Development of the HELIUS food frequency questionnaires: ethnic-specific questionnaires to assess the diet of a multiethnic population in The Netherlands.

OBJECTIVES: Ethnic minorities are often not included in studies of diet and health because of a lack of validated instruments to assess their habitual diets. Given the increased ethnic diversity in many high-income countries, insight into the diets of ethnic minorities is needed for the development of nutritional policies and interventions. In this paper, we describe the development of ethnic-specific food frequency questionnaires (FFQs) to study the diets of Surinamese (African and South Asian), Turkish, Moroccan and ethnic Dutch residents of The Netherlands. METHODS: An existing Dutch FFQ was adapted and formed the basis for three new FFQs. Information on food intake was obtained from single 24 h recalls. Food items were selected according to their percentage contribution to and variance in absolute nutrient intake of the respective ethnic groups. A nutrient database for each FFQ was constructed, consisting of data from the Dutch Food Composition table; data on ethnic foods were based on new chemical analyses and available international data. RESULTS: We developed four ethnic-specific FFQs using a standardised approach that included ~200 food items each and that covered more than 90% of the intake of the main nutrients of interest. CONCLUSIONS: The developed FFQs will enable standardised and comparable assessment of the diet of five different ethnic groups and provide insight into the role of diet in differences in health between ethnic groups. The methodology described in this paper and the choices made during the development phase may be useful in developing similar FFQs in other settings.

Genetic and metabolic determinants of methotrexate-induced mucositis in pediatric acute lymphoblastic leukemia.

Methotrexate (MTX) is an effective and toxic chemotherapeutic drug in the treatment of pediatric acute lymphoblastic leukemia(ALL). In this prospective study, we aimed to identify metabolic and genetic determinants of MTX toxicity. One hundred and thirty-four Dutch pediatric ALL patients were treated with four high infusions MTX (HD-MTX: 5 g m(-2)) every other week according to the DCOG-ALL-10 protocol. Mucositis (National Cancer Institute grade ⩾ 3) was the most frequent occurring toxicity during the HD-MTX phase (20%) and occurred especially after the first MTX course. Mucositis was not associated with plasma MTX, plasma folate or plasma homocysteine levels. Patients with mucositis had higher erythrocyte folate levels at the start of protocol M than patients without mucositis (median 1.4 vs 1.2 µmol l(-1), P<0.008), this could reflect an increased MTX uptake in mucosal cells of patients with mucositis. From 17 single-nucleotide polymorphisms in the MTX pathway, only patients with the wild-type variant of rs7317112 SNP in the ABCC4 gene had more mucositis (AA (39%) vs AG/GG (15%), P=0.016). We found no evidence that erythrocyte folate levels mediate in the association between the rs7317112 and mucositis.

Platelet function alterations in dengue are associated with plasma leakage.

Severe dengue is characterised by thrombocytopenia, plasma leakage and bleeding. Platelets are important for preservation of endothelial integrity. We hypothesised that platelet activation with secondary platelet dysfunction contribute to plasma leakage. In adult Indonesian patients with acute dengue, we measured platelet activation status and the response to the platelet agonist TRAP using flow cytometer-based assays. Patients were monitored daily for plasma leakage by ultrasonography. Acute dengue was associated with platelet activation with an increased expression of the activated fibrinogen receptor (αIIbß3), the lysosomal marker CD63 and the alpha-granule marker CD62P (P-selectin). Upon maximal platelet activation by TRAP, platelet function defects were observed with a significantly reduced maximal activated αIIbß3 and CD63 expression and reduced platelet-monocyte and platelet-neutrophil complexes. Patients in the lowest tertile of activated αIIbß3 and CD63 expression had an odds ratio for plasma leakage of 5.2 (95% confidence interval [CI] 1.3-22.7) and 3.9 (95% CI 1.1-13.7), respectively, compared to the highest tertile. Platelet-derived serotonin has previously been related to plasma leakage and we found increased intra-platelet serotonin concentrations in our patients. In conclusion, platelet activation with platelet function alterations can be found in patients with acute dengue and this may contribute to dengue-associated plasma leakage.

Value of cone-beam computed tomography in the process of diagnosis and management of disorders of the temporomandibular joint.

The objective of this study was to assess the value of cone-beam computed tomographic (CT) images in the primary diagnosis and management of 128 outpatients with disorders of the temporomandibular joint (TMJ). Before a diagnosis was made and treatment planned, the history was taken, physical examination made, and the orthopantomogram studied. After assessment of the cone-beam CT, the oral and maxillofacial surgeon (specialist or resident) was allowed to revise the provisional primary diagnosis and management. The degree of certainty was rated by the clinician before and after the cone-beam CT had been assessed. The primary diagnosis was changed in 32 patients (25%), additional diagnostic procedures were changed in 57 (45%), and the treatment was changed in 15 (12%) (in 4 the treatment was changed to a (minimally) invasive procedure). A total of 74 patients (58%) had their diagnosis and management changed after the cone-beam CT had been assessed. Changes in diagnosis and management were clinically relevant in 9/32 and 9/61 patients, respectively. The clinician's certainty about the primary diagnosis increased after the cone-beam CT had been assessed in 57 patients. Logistic regression analysis showed that the odds in favour of changes in primary diagnosis and management increased when limited mandibular function was a primary symptom, the patient was taking medication for pain, and the articular eminence could not be assessed on OPT. Assessment of cone-beam CT led to changes in primary diagnosis and management in more than half the patients with disorders of the TMJ.

The effects of necrotic enteritis, aflatoxin B1, and virginiamycin on growth performance, necrotic enteritis lesion scores, and mortality in young broilers.

The effects of increasing aflatoxin B1 concentration (0, 0.75, 1.5 mg/kg) on broilers with or without necrotic enteritis or virginiamycin were determined. In the 23-d study, 22 male Cobb 500 chicks per pen were allotted to 12 treatments (3 × 2 × 2 factorial arrangement) with 8 replications. Intestines of 5 birds per pen were examined for lesions on d 21. Birds were allowed to consume feed and water ad libitum. Aflatoxin was included in the diets from d 0. All birds received a 10× dose of coccidiosis vaccine on d 10. Pens of birds where necrotic enteritis was being induced were on Clostridium perfringens pathogen (CPP) contaminated litter from d 0. Aflatoxin decreased gain and feed intake and resulted in poorer feed:gain, increased mortality, and higher lesion scores. Inducing necrotic enteritis increased lesion scores and decreased feed intake and gain. Adding virginiamycin to the diets improved gain, feed intake, feed conversion, and decreased mortality. There was a 3-way interaction (aflatoxin × virginiamycin × CPP) on gain; increasing aflatoxin decreased gain and the effects of CPP and virginiamycin were dependent on aflatoxin concentration. In the absence of aflatoxin virginiamycin increased gain but was unable to prevent the growth suppression caused by CPP. At 0.75 mg/kg of aflatoxin virginiamycin no longer increased growth in non-CPP challenged birds but was able to increase growth in CPP-challenged birds. At the 1.5 mg/kg of aflatoxin concentration, virginiamycin increased gain in non-CPP-challenged birds but challenging birds with CPP had no effect on gain. Virginiamycin improved overall feed conversion with the greatest improvement at 1.5 mg/kg (aflatoxin × virginiamycin, P < 0.05). Aflatoxin increased lesion scores in unchallenged birds but not in challenged birds (aflatoxin × CPP, P < 0.001). Aflatoxin and necrotic enteritis decrease broiler performance and interact to decrease weight gain, virginiamycin helps improve gain in challenged birds at 0.75 mg/kg of aflatoxin, but not at 1.5 mg/kg of aflatoxin.

Measuring methotrexate polyglutamates in red blood cells: a new LC-MS/MS-based method.

The folate antagonist methotrexate (MTX) is the anchor drug in the treatment of rheumatoid arthritis. The therapeutic effects of MTX are attributed to the intracellular levels of MTX, present in the cell as polyglutamates (MTXPGn). We developed a new liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS)-based assay to separately quantitate MTXPGn in red blood cells using stable-isotope-labelled internal standards. Samples were analyzed by LC-ESI-MS/MS using a Waters Acquity UPLC BEH C18 column with a 5-100% organic gradient of 10 mM ammonium bicarbonate (pH 10) and methanol. The analysis consisted of simple sample preparation and a 6-min run time. Detection was done using a Waters Acquity UPLC coupled to a Waters Quattro Premier XE with electrospray ionization operating in the positive ionization mode. Assay validation was performed following recent Food and Drug Administration guidelines. The method was linear from 1-1,000 nM for all MTXPGn (R(2) > 0.99). The coefficient of variation ranged from 1-4% for intraday precision and 6-15% for interday precision. Samples were stable for at least 1 month at -80 °C. Recovery ranged from 98-100%, and the relative matrix-effect varied from 95-99%. The lower limit of quantitation was 1 nM for each MTXPGn. Fifty patient samples from the tREACH study were analyzed. The MTXPGn concentration and distribution of these samples were comparable with values reported in literature. The developed LC-ESI-MS/MS method for the quantitative measurement of MTXPGn in red blood cells is both sensitive and precise within the clinically relevant range. The method can be easily applied in clinical laboratories due to the combination of simple pre-treatment with robust LC-ESI-MS/MS.