The effect of 2 different premilking stimulation regimens, with and without a latency period, on teat tissue condition and milking performance in Holstein dairy cows.

The objectives of this study were to assess the effect of 2 different premilking stimulation regimens, with and without a latency period between tactile stimulation and the attachment of the milking unit, on the teat tissue condition and milking performance of dairy cows. In a randomized controlled crossover study, 145 Holstein cows milked 3 times daily were assigned to treatment (TRT) or control (CON) groups. Premilking udder preparation for the TRT group consisted of the application of a latency period resulting in a preparation lag time of 90 s. The only difference in the premilking udder preparation of the CON group was the absence of latency period; the milking unit was attached immediately after completion of the tactile stimulation. The average duration of total tactile stimulation in TRT and CON group was 8 ± 2 and 9 ± 2 s, respectively. The study lasted for 14 d and was split into 2 periods, each consisting of a 2-d adjustment period followed by 5 d of data collection. We assessed machine milking-induced short-term changes to the teat tissue by palpation and visual inspection postmilking. Electronic on-farm milk meters were used to assess milking characteristics (milk yield [kg/milking session], machine-on time [s], 2-min milk yield [kg], and duration of low milk flow rate [s]). Generalized linear mixed models were used to analyze the effect of treatment on the outcome variables. The odds of machine milking-induced short-term changes to the teat tissue were lower for cows that received a 90-s preparation lag time (TRT cows) compared with cows in the CON group (odds ratio [95% confidence interval; 95% CI] = 0.13 [0.08-0.20]). The least squares means (95% CI) values of cows in the TRT and CON groups were 15.4 (14.9-15.9) and 15.3 (14.8-15.8) kg, respectively, for milk yield, and 246 (239-253) and 253 (247-260) s for machine-on time. The 2-min milk yield was higher for the TRT compared with CON group cows at all the parity levels. The 2-min milk yields of animals in lactation 1, 2, and ≥3 were 5.7, 5.7, and 6.5 kg, respectively, in the TRT group and 4.6, 5.0, and 5.9 kg in the CON group. The TRT cows spent less time in low milk flow rate compared with CON cows at all parity levels. The durations of low milk flow rate of cows in lactation 1, 2, and ≥3 in the TRT group were 19, 17 and 13 s, respectively, and those in the CON group were 31, 22, and 15 s. In this study, cows that received a latency period, and thus were subjected to a 90-s preparation lag time had lower odds of exhibiting short-term changes to the teat tissue after machine milking, shorter machine-on time, higher 2-min milk yields, and lower durations of low milk flow rates. We conclude that consideration of latency period leading to a 90-s preparation lag time in the premilking stimulation regimen facilitated cows' milk-ejection reflex. This latency period can alleviate the adverse effects of vacuum-induced forces on teat tissue during machine milking, improve udder health, and promote animal well-being.

Dietary Tyrosine Intake (FFQ) Is Associated with Locus Coeruleus, Attention and Grey Matter Maintenance: An MRI Structural Study on 398 Healthy Individuals of the Berlin Aging Study-II.

BACKGROUND AND OBJECTIVE: It is documented that low protein and amino-acid dietary intake is related to poorer cognitive health and increased risk of dementia. Degradation of the neuromodulatory pathways, (comprising the cholinergic, dopaminergic, serotoninergic and noradrenergic systems) is observed in neurodegenerative diseases and impairs the proper biosynthesis of key neuromodulators from micro-nutrients and amino acids. How these micro-nutrients are linked to neuromodulatory pathways in healthy adults is less studied. The Locus Coeruleus-Noradrenergic System (LC-NA) is the earliest subcortical structure affected in Alzheimer's disease, showing marked neurodegeneration, but is also sensitive for age-related changes. The LC-NA system is critical for supporting attention and cognitive control, functions that are enhanced both by tyrosine administration and chronic tyrosine intake. The purpose of this study was to 1) investigate whether the dietary intake of tyrosine, the key precursor for noradrenaline (NA), is related to LC signal intensity 2) whether LC mediates the reported association between tyrosine intake and higher cognitive performance (measured with Trail Making Test - TMT), and 3) whether LC signal intensity relates to an objective measure of brain maintenance (BrainPAD). METHODS: The analyses included 398 3T MRIs of healthy participants from the Berlin Aging Study II to investigate the relationship between LC signal intensity and habitual dietary tyrosine intake-daily average (HD-Tyr-IDA - measured with Food Frequency Questionnaire - FFQ). As a control procedure, the same analyses were repeated on other main seeds of the neuromodulators' subcortical system (Dorsal and Medial Raphe, Ventral Tegmental Area and Nucleus Basalis of Meynert). In the same way, the relationships between the five nuclei and BrainPAD were tested. RESULTS: Results show that HD-Tyr-IDA is positively associated with LC signal intensity. Similarly, LC disproportionally relates to better brain maintenance (BrainPAD). Mediation analyses reveal that only LC, relative to the other nuclei tested, mediates the relationship between HD-Tyr-IDA I and performance in the TMT and between HD-Tyr-IDA and BrainPAD. CONCLUSIONS: These findings provide the first evidence linking tyrosine intake with LC-NA system signal intensity and its correlation with neuropsychological performance. This study strengthens the role of diet for maintaining brain and cognitive health and supports the noradrenergic theory of cognitive reserve. Within this framework, adequate tyrosine intake might increase the resilience of LC-NA system functioning, by preventing degeneration and supporting noradrenergic metabolism required for LC function and neuropsychological performance.

Medicines postpartum in Sweden and coverage in Janusmed Breastfeeding.

PURPOSE: The purpose of this article is (1) to investigate which medicines are prescribed and dispensed to women the first 6 months postpartum, (2) to identify medicines dispensed postpartum but not recommended during breastfeeding, and (3) to find medicines commonly dispensed postpartum, but not currently included in Janusmed Breastfeeding. METHODS: In this register-based cohort study covering births between January 2017 and August 2019, the Swedish Medical Birth Register (MBR), the Prescribed Drug Register, and Janusmed Breastfeeding were linked to identify medicines dispensed to women during the first 6 months postpartum, and how they are covered and classified in Janusmed Breastfeeding. RESULTS: During the first 6 months postpartum, 66% of women purchased at least one prescription medicine from the pharmacy. The most common medicines were contraceptive agents, analgesics, antibiotics, and glucocorticoids. A third of the 30 most commonly dispensed medicines have no information available about the safety of use in breastfeeding. The most dispensed medicines, where the database advises against use in breastfeeding, included several antitussive agents, a local anaesthetic, and several gestagens. The most commonly dispensed medicines not covered by the Janusmed Breastfeeding were medicines for dry eyes, for assisted reproduction, and HIV. CONCLUSION: Prescribed medicines compatible with breastfeeding are more common during the first 6 months postpartum than medicines not compatible with breastfeeding, but medicines which lack evidence for safety in breastfeeding are still commonly used.

Tools for optimising pharmacotherapy in psychiatry (therapeutic drug monitoring, molecular brain imaging and pharmacogenetic tests): focus on antidepressants.

Objectives: More than 40 drugs are available to treat affective disorders. Individual selection of the optimal drug and dose is required to attain the highest possible efficacy and acceptable tolerability for every patient.Methods: This review, which includes more than 500 articles selected by 30 experts, combines relevant knowledge on studies investigating the pharmacokinetics, pharmacodynamics and pharmacogenetics of 33 antidepressant drugs and of 4 drugs approved for augmentation in cases of insufficient response to antidepressant monotherapy. Such studies typically measure drug concentrations in blood (i.e. therapeutic drug monitoring) and genotype relevant genetic polymorphisms of enzymes, transporters or receptors involved in drug metabolism or mechanism of action. Imaging studies, primarily positron emission tomography that relates drug concentrations in blood and radioligand binding, are considered to quantify target structure occupancy by the antidepressant drugs in vivo. Results: Evidence is given that in vivo imaging, therapeutic drug monitoring and genotyping and/or phenotyping of drug metabolising enzymes should be an integral part in the development of any new antidepressant drug.Conclusions: To guide antidepressant drug therapy in everyday practice, there are multiple indications such as uncertain adherence, polypharmacy, nonresponse and/or adverse reactions under therapeutically recommended doses, where therapeutic drug monitoring and cytochrome P450 genotyping and/or phenotyping should be applied as valid tools of precision medicine.

Attention difficulties and physical dysfunction common in children with complex congenital malformations: a study of preschool children with VACTERL association.

AIM: Knowledge on the neurodevelopmental and physical function in children with vertebral defects, anorectal malformations, cardiac defects, tracheo-oesophageal fistula, renal and limb malformations (VACTERL) is scarce. We evaluated Swedish preschool children with VACTERL and identified whether they would need extra support in school. METHODS: From 2015 to 2017, we recruited children aged 5-7 with VACTERL association from the paediatric surgical centre at the University Children's Hospital at Uppsala. Neurodevelopmental function was assessed by age-appropriate intelligence and visual and auditory attention tests, and the children's behaviour and attention were observed by an experienced psychologist. Physical function was evaluated through parental interviews and examinations. Data on patient characteristics, including any surgery and anaesthesia, were extracted from medical records. RESULTS: Of the 13 eligible families, 10 agreed to participate. Intelligence was within the normal range for all children, but attention difficulties were found in eight of the children, requiring adjustments at school, and two of these were later diagnosed with attention deficit hyperactivity disorder. All children had physical dysfunctions that affected their daily nutrition, bowel or bladder functions. CONCLUSION: Attention difficulties and physical dysfunction were common in Swedish preschool children aged 5-7 with VACTERL and they would need support and adjustments when they started school.

Fluoroquinolone use for uncomplicated urinary tract infections in women: a retrospective cohort study.

OBJECTIVES: The United States Food & Drug Administration released an advisory in 2016 that fluoroquinolones be relegated to second-line agents for uncomplicated urinary tract infections (UTIs) given reports of rare but serious side effects; similar warnings have followed from Health Canada and the European Medicines Agency. The objective was to determine whether alternative non-fluoroquinolone agents are as effective as fluoroquinolones in the treatment of UTIs. METHODS: We conducted a retrospective population-based cohort study using administrative health data from six Canadian provinces. We identified women (n = 1 585 997) receiving antibiotic treatment for episodes of uncomplicated UTIs (n = 2 857 243) between January 1 2005 and December 31 2015. Clinical outcomes within 30 days from the initial antibiotic dispensation were compared among patients treated with a fluoroquinolone versus non-fluoroquinolone agents. High-dimensional propensity score adjustments were used to ensure comparable treatment groups and to minimize residual confounding. RESULTS: Fluoroquinolone use for UTI declined over the study period in five of six Canadian provinces and accounted for 22.3-48.5% of treatments overall. The pooled effect across the provinces indicated that fluoroquinolones were associated with fewer return outpatient visits (OR 0.89, 95%CI 0.87-0.92), emergency department visits (OR 0.74, 95%CI 0.61-0.89), hospitalizations (OR 0.83, 95%CI 0.77-0.88), and repeat antibiotic dispensations (OR 0.77, 95%CI 0.75-0.80) within 30 days. CONCLUSIONS: Fluoroquinolones are associated with improved clinical outcomes among women with uncomplicated UTIs. This benefit must be weighed against the risk of fluoroquinolone resistance and rare but serious fluoroquinolone side effects when selecting first-line treatment for these patients.

Use of point-of-care HbA1c measurement to estimate the level of undiagnosed diabetes mellitus among 67-year-old participants in a cardiovascular screening programme in the municipality of Viborg, Denmark.

AIMS: To determine the prevalence of unidentified diabetes mellitus among 67-year-olds in Denmark participating in a screening programme focusing on cardiovascular disease and diabetes, and to describe glycaemic levels in individuals according to point-of-care HbA1c combined with self-reported diabetes status. METHODS: In this cross-sectional, retrospective, population-based study, all people aged 67 years living in the Viborg municipality were invited to take part in the Viborg Inter-sectorial Screening Programme (VISP), which focuses on cardiovascular disease and diabetes. The VISP study was initiated in August 2014 and is ongoing. During the first 2 years of the programme, we stratified participants into groups based on their self-reported diabetes status and a single HbA1c measurement. RESULTS: A total of 1802 individuals were invited to participate, and 1501 consented, seven of whom were excluded because of missing data (HbA1c or diabetes status), resulting in an 82.9% participation rate (n=1494). Among those reporting not to have diabetes, 3.3% (n=45) had an HbA1c level ≥48 mmol/mol (6.5%). In the same group, 16.7% (n=226) had an HbA1c level of 41-48 mmol/mol (5.9-6.5%). Among those self-reporting the presence of diabetes, 30.1% (n=43) had an HbA1c level ≥58 mmol/mol (7.5%). CONCLUSIONS: The prevalence of unidentified diabetes was 3.3% based on a single HbA1c measurement. Furthermore, 16.7% of those reporting not to have diabetes had an HbA1c level of 41-48 mmol/mol (5.9-6.5%), representing a subgroup with an increased risk of developing diabetes. Among those with self-reported diabetes, 30.1% had an HbA1c level ≥58 mmol/mol (7.5%) and 6.3% had a level >74 mmol/mol (8.9%).

Physiological response of broiler embryos to different incubator temperature profiles and maternal flock age during incubation. 1. Embryonic metabolism and day-old chick quality.

Broiler strain, maternal age, and incubation temperature influence embryo metabolism. Hatching eggs were obtained from young (Y; 28 to 34 wk, $\bar{\rm x}$ = 31.2 wk), mid (M; 36 to 45 wk, $\bar{\rm x}$ = 40.5 wk) and old (O; 49 to 54 wk, $\bar{\rm x}$ = 51.4 wk) Ross 708 (n = 88; Experiment 1) and Ross 308 [(n = 45; Experiment 2: (Y; 25 to 34 wk, $\bar{\rm x}$ = 30.5 wk), (M; 35 to 44 wk, $\bar{\rm x}$ = 40.2 wk), and (O; 49 to 54 wk, $\bar{\rm x}$ = 51.6 wk)] breeders. Eggs were stored for 2 to 4 d (18°C, 73% RH), and incubated for 14 d at 37.5°C and 56% RH. At 15 d (E15), 8 fertile eggs per flock age were incubated in individual metabolic chambers at 36.0, 36.5, 37.0, or 37.5°C until E21.5. Each temperature was repeated one additional time. O2 consumption and CO2 production were used to calculate embryonic heat production (EHP). Embryo temperature was measured as eggshell temperature (EST). Initial egg weight was used as a covariate; significance was assessed at P < 0.05. In Ross 708, daily EHP tended to be higher in M and O than Y treatments at E16; EHP of M was higher than Y and O eggs at E18; M and O were higher than O eggs at E19. Incubation at 37.0°C resulted in the highest EHP from E15 to E21, except at E17. Embryos at 37.5°C had reduced EHP beyond E17. Daily EST from E15 to E21 was higher at 37.5 and 37.0°C than at 36.0 and 36.5°C. In Ross 308, daily EST was highest at 37.5°C except at E20. Incubation temperature and EST were highly correlated (R2 = 0.90 to 0.89; P < 0.001). Ross 708 chicks were longer and hatched earlier at 37.0°C than at 36.0 and 37.5°C. EST and EHP increased with incubation temperature in Ross 708. In Ross 308, maternal flock age and incubation temperature did not impact EHP. However, EST was highest at 37.5°C except at E20. Ross 708 was more sensitive to incubation temperature than Ross 308.

Prediction of bleeding and thrombosis by standard biochemical coagulation variables in haematological intensive care patients.

PURPOSE: We assessed the value of standard biochemical coagulation parameters in predicting bleeding, thrombosis and mortality in adult Intensive Care Unit (ICU) patients with haematological malignancies. METHODS: We screened all patients with acute leukaemia and myelodysplastic syndrome admitted to a university hospital ICU during 2008-2012. Data were obtained from the clinical chemistry laboratory database and patient files. We graded bleeding according to the World Health Organisation (WHO)-system within 24-h, within 5-days and during the whole ICU stay. We analysed the predictive values of laboratory parameters using multiple logistic regression and receiver operator characteristics (ROC) curves. As we previously have established that platelet count at admission was associated with bleeding, we focused on International Normalised Ratio (INR), activated pro-thrombin time (APTT), anti-thrombin, D-dimer and fibrinogen, and markers of infection (C-reactive protein, pro-calcitonin), kidney function (creatinine) and tissue damage (lactate dehydrogenase (LDH)). RESULTS: We included 116 patients; 66 (57%) had at least one bleeding episode and 11 (9%) patients had at least one thrombotic event. The differences in coagulation values when bleeding compared to baseline values were minor. INR was the only variable we found associated with subsequent bleeding within 24 h from admission to ICU (odds ratio 2.91, 95% CI: 1.01-8.43, P = 0.048). ROC analyses did not show predictive value of any of the other variables with regards to bleeding and none of the variables were associated with thrombosis in adjusted analyses. Increased levels of LDH at admission were associated with increased 7-day and 30-day mortality. CONCLUSIONS: Increased INR at admission was associated with a higher rate of bleeding in ICU patients with haematological malignancies. No other biochemical coagulation or other parameter had any association with bleeding, thrombosis or mortality except increased LDH, which at ICU admission was associated with increased 30-day mortality.

Identification of the flotillin-1/2 heterocomplex as a target of autoantibodies in bona fide multiple sclerosis.

BACKGROUND: Autoantibodies, in particular those against aquaporin-4 and myelin-oligodendrocyte glycoprotein (MOG), aid as biomarkers in the differential diagnosis of demyelination. Here, we report on discovery of autoantibodies against flotillin in patients with multiple sclerosis (MS). METHODS: The target antigen was identified by histo-immunoprecipitation using the patients' sera and cryosections of rat or pig cerebellum combined with mass spectrometrical analysis. Correct identification was ascertained by indirect immunofluorescence and neutralization tests using the target antigens recombinantly expressed in HEK293 cells. RESULTS: Serum and CSF of the index patient produced a fine-granular IgG indirect immunofluorescence staining of the hippocampal and cerebellar molecular layers. Flotillin-1 and flotillin-2 were identified as target autoantigens. They also reacted with recombinant human flotillin-1/2 co-expressed in HEK293 cells, but not with the individual flotillins in fixed- and live-cell assays. Moreover, neutralization using flotillin-1/2, but not the single flotillins, abolished the tissue reactivity of patient serum. Screening of 521 patients, for whom anti-aquaporin-4 testing was requested and negative, revealed 8 additional patients with anti-flotillin-1/2 autoantibodies. All eight were negative for anti-MOG. Six patients ex post fulfilled the revised McDonald criteria for MS. Vice versa, screening of 538 MS sera revealed anti-flotillin-1/2 autoantibodies in eight patients. The autoantibodies were not found in a cohort of 67 patients with other neural autoantibody-associated syndromes and in 444 healthy blood donors. CONCLUSIONS: Autoantibodies against the flotillin-1/2 heterocomplex, a peripheral membrane protein that is involved in axon outgrowth and regeneration of the optic nerve, are present in 1-2% of patients with bona fide MS.

The effect of maternal canthaxanthin supplementation and hen age on breeder performance, early chick traits, and indices of innate immune function.

Hen age and nutrition influence chick innate immunity. The immunomodulatory antioxidant carotenoid canthaxanthin is transferred from the hen diet to the egg. Antioxidants could protect the chick from bactericidal oxidative species produced by the immune system. Broiler breeder hen diets were supplemented with 0 (Control), 6 (Low), or 12 (High) mg/kg canthaxanthin. Chick early growth and ex vivo innate immunity were measured at 31 to 33 (Early), 45 to 47 (Mid), and 57 to 59 (Late) wk of hen age. Escherichia coli (E. coli) bactericidal capacity, phagocyte activation (number of phagocytes containing at least one E. coli), phagocytic capacity (number of phagocytes containing one or more E. coli), and oxidative burst at 1 and 4 d of age were determined. Egg and chick liver canthaxanthin and chick plasma total antioxidant capacity were measured. Differences were considered significant at P ≤ 0.05. Breeder productivity was greatest for the Low hens; diet did not affect egg yolk, albumen, or shell proportions. Egg canthaxanthin increased with maternal supplementation and plateaued after 28 days, but was not affected by hen age. Chick liver canthaxanthin increased with maternal supplementation, but decreased as hens aged. Hen diet did not affect broiler chick performance to 21 days of age. Maternal canthaxanthin at 6 mg/kg increased chick E. coli bactericidal capacity and d 1 oxidative burst; phagocytosis was unaffected. E. coli bactericidal capacity decreased as hens aged, but increased from 1 to 4 d, indicating maturation of chick innate immunity. Plasma total antioxidant capacity at d 1 increased with maternal canthaxanthin in chicks from Mid and Late hens. Canthaxanthin possesses immuno-modulatory and antioxidant properties, and hen age affected chick innate immune development. Single-comb White Leghorn hens were fed the same levels of canthaxanthin to determine the rate of incorporation into eggs. Egg canthaxanthin reached a plateau after 7 d. Canthaxanthin in the hen diet at 6 mg/kg resulted in the greatest positive effect on hen performance, with little effect on the chick.

Next-generation sequencing of 100 candidate genes in young victims of suspected sudden cardiac death with structural abnormalities of the heart.

BACKGROUND: In sudden, unexpected, non-traumatic death in young individuals, structural abnormalities of the heart are frequently identified at autopsy. However, the findings may be unspecific and cause of death may remain unclear. A significant proportion of these cases are most likely caused by inherited cardiac diseases, and the cases are categorized as sudden cardiac death (SCD). The purpose of this study was to explore the added diagnostic value of genetic testing by next-generation sequencing (NGS) of a broad gene panel, as a supplement to the traditional forensic investigation in cases with non-diagnostic structural abnormalities of the heart. METHODS AND RESULTS: We screened 72 suspected SCD cases (<50 years) using the HaloPlex Target Enrichment System (Agilent) and NGS (Illumina MiSeq) for 100 genes previously associated with inherited cardiomyopathies and channelopathies. Fifty-two cases had non-diagnostic structural cardiac abnormalities and 20 cases, diagnosed with a cardiomyopathy post-mortem (ARVC = 14, HCM = 6), served as comparators. Fifteen (29%) of the deceased individuals with non-diagnostic findings had variants with likely functional effects based on conservation, computational prediction, allele-frequency and supportive literature. The corresponding frequency in deceased individuals with cardiomyopathies was 35% (p = 0.8). CONCLUSION: The broad genetic screening revealed variants with likely functional effects at similar high rates, i.e. in 29 and 35% of the suspected SCD cases with non-diagnostic and diagnostic cardiac abnormalities, respectively. Although the interpretation of broad NGS screening is challenging, it can support the forensic investigation and help the cardiologist's decision to offer counselling and clinical evaluation to relatives of young SCD victims.

Next-generation sequencing of 34 genes in sudden unexplained death victims in forensics and in patients with channelopathic cardiac diseases.

Sudden cardiac death (SCD) is responsible for a large proportion of sudden deaths in young individuals. In forensic medicine, many cases remain unexplained after routine postmortem autopsy and conventional investigations. These cases are called sudden unexplained deaths (SUD). Genetic testing has been suggested useful in forensic medicine, although in general with a significantly lower success rate compared to the clinical setting. The purpose of the study was to estimate the frequency of pathogenic variants in the genes most frequently associated with SCD in SUD cases and compare the frequency to that in patients with inherited cardiac channelopathies. Fifteen forensic SUD cases and 29 patients with channelopathies were investigated. DNA from 34 of the genes most frequently associated with SCD were captured using NimbleGen SeqCap EZ library build and were sequenced with next-generation sequencing (NGS) on an Illumina MiSeq. Likely pathogenic variants were identified in three out of 15 (20%) forensic SUD cases compared to 12 out of 29 (41%) patients with channelopathies. The difference was not statistically significant (p = 0.1). Additionally, two larger deletions of entire exons were identified in two of the patients (7%). The frequency of likely pathogenic variants was >2-fold higher in the clinical setting as compared to SUD cases. However, the demonstration of likely pathogenic variants in three out of 15 forensic SUD cases indicates that NGS investigations will contribute to the clinical investigations. Hence, this has the potential to increase the diagnostic rate significantly in the forensic as well as in the clinical setting.

An in situ tensile test apparatus for polymers in high pressure hydrogen.

Degradation of material properties by high-pressure hydrogen is an important factor in determining the safety and reliability of materials used in high-pressure hydrogen storage and delivery. Hydrogen damage mechanisms have a time dependence that is linked to hydrogen outgassing after exposure to the hydrogen atmosphere that makes ex situ measurements of mechanical properties problematic. Designing in situ measurement instruments for high-pressure hydrogen is challenging due to known hydrogen incompatibility with many metals and standard high-power motor materials such as Nd. Here we detail the design and operation of a solenoid based in situ tensile tester under high-pressure hydrogen environments up to 42 MPa (6000 psi). Modulus data from high-density polyethylene samples tested under high-pressure hydrogen at 35 MPa (5000 psi) are also reported as compared to baseline measurements taken in air.

Evaluation of a competitive enzyme-linked immunosorbent assay for measurements of soluble HLA-G protein.

The human leukocyte antigen (HLA) class Ib molecule, HLA-G, has gained increased attention because of its assumed important role in immune regulation. The HLA-G protein exists in several soluble isoforms. Most important are the actively secreted HLA-G5 full-length isoform generated by alternative splicing retaining intron 4 with a premature stop codon, and the cleavage of full-length membrane-bound HLA-G1 from the cell surface, so-called soluble HLA-G1 (sHLA-G1). A specific and sensitive immunoassay for measurements of soluble HLA-G is mandatory for conceivable routine testing and research projects. We report a novel method, a competitive immunoassay, for measuring HLA-G5/sHLA-G1 in biological fluids. The sHLA-G immunoassay is based upon a competitive enzyme-linked immunosorbent assay (ELISA) principle. It includes a recombinant sHLA-G1 protein in complex with ß2-microglobulin and a peptide as a standard, biotinylated recombinant sHLA-G1 as an indicator, and the MEM-G/9 anti-HLA-G monoclonal antibody (mAb) as the capture antibody. The specificity and sensitivity of the assay were evaluated. Testing with different recombinant HLA class I proteins and different anti-HLA class I mAbs showed that the sHLA-G immunoassay was highly specific. Optimal combinations of competitor sHLA-G1 and capture mAb concentrations were determined. Two versions of the assay were tested. One with a relatively wide dynamic range from 3.1 to 100.0 ng/ml, and another more sensitive version ranging from 1.6 to 12.5 ng/ml. An intra-assay coefficient of variation (CV) of 15.5% at 88 ng/ml and an inter-assay CV of 23.1% at 39 ng/ml were determined. An assay based on the competitive sHLA-G ELISA may be important for measurements of sHLA-G proteins in several conditions: assisted reproduction, organ transplantation, cancer, and certain pregnancy complications, both in research studies and possibly in the future also for clinical routine use.

Glucocorticoid-induced hyperglycemia is prevalent and unpredictable for patients undergoing cancer therapy: an observational cohort study.

BACKGROUND: Patients with cancer are often treated with glucocorticoids (gcs) as part of therapy, which may cause hyperglycemia. We sought to define the prevalence of, and risk factors for, hyperglycemia in this setting. METHODS: Adult patients taking gc as part of therapy protocols for primary brain tumour or metastasis, for lymphoma, or for bone marrow transplant (bmt) were screened with random glucometer measurements taken at least 3 hours after the last dose gcs. RESULTS: We screened 90 patients [44.4% women, 55.6% men; mean age: 59.6 years (range: 25-82 years); mean body mass index (bmi): 26.4 kg/m(2) (range: 15.8-45.3 kg/m(2))] receiving gc as part of cancer treatment. Mean total daily gc dose in the group was 238.5 mg (range: 30-1067 mg) hydrocortisone equivalents. Hyperglycemia (glucose ≥ 8.0 mmol/L) was found in 58.9% (53 of 90), and diabetes mellitus (dm)-range hyperglycemia (glucose ≥ 11.1 mmol/L) in 18.9% (17 of 90). The mean time from gc ingestion to glucometer testing was 5.5 hours (range: 3-20 hours). Presence of hyperglycemia did not correlate with traditional dm risk factors such as age, sex, bmi, and personal or family history of dm. A longer interval from gc dose to testing (p < 0.05), a higher gc dose (p = 0.04), and a shorter interval between the preceding meal and testing (p = 0.02) were risk factors for hyperglycemia in some patient groups. CONCLUSIONS: Glucocorticoid-induced hyperglycemia is common in patients undergoing cancer treatment and cannot be predicted by traditional risk factors for dm. We recommend that all cancer patients receiving gc be screened for hyperglycemia at least 4-6 hours after gc administration.

Role of histone deacetylases in regulation of phenotype of ovine newborn pulmonary arterial smooth muscle cells.

OBJECTIVE: Pulmonary arterial hypertension, characterized by pulmonary vascular remodelling and vasoconstriction, is associated with excessive proliferative changes in pulmonary vascular walls. However, the role of HDACs in the phenotypic alteration of pulmonary arterial smooth muscle cells (PASMC) is largely unknown. MATERIAL AND METHODS: Pulmonary arterial smooth muscle cells were isolated from newborn sheep. Cell cycle analysis was performed by flow cytometry. mRNA and protein expression were measured by real-time PCR and Western blot analysis. Wound-healing scratch assay was used to measure cell migration. Contractility of newborn PASMCs was determined by gel contraction assay. Chromatin immunoprecipitation was used to examine histone modifications along the p21 promoter region. Global DNA methylation was measured by liquid chromatography-mass spectroscopy. RESULTS: Inhibition of class I and class II HDACs by apicidin and HDACi VIII suppressed proliferation of newborn PASMC and induced cell cycle arrest in G1 phase. Acetyl H3 levels were higher in newborn PASMC treated with apicidin and HDACi VIII. This was accompanied by increased expression of p21 and reduced expression of CCND1 but not p53. HDAC inhibition altered histone codes around the p21 promoter region in NPASMC. Apicidin inhibited serum-induced cell migration, and modulated profiling of expression of genes encoding pro-oxidant and antioxidant enzymes. Contractility and global DNA methylation levels of newborn PASMCs were also markedly modulated by apicidin. CONCLUSION: Our results demonstrate that class I HDACs are clearly involved in phenotypic alteration of newborn PASMC.

Influence of FMO1 and 3 polymorphisms on serum olanzapine and its N-oxide metabolite in psychiatric patients.

The widely used antipsychotic drug, olanzapine (OLA) shows large interindividual variability in metabolic clearance. Although the role of the enzymes CYP1A2, CYP2D6 and UGT1A4 has been extensively explored, little is known about the in vivo role of flavin-containing monooxygenases (FMOs) catalyzing the N-oxidation of OLA in vitro. We investigated the influence of FMO1 and 3 polymorphisms on the steady state serum concentrations of OLA and its N-oxide metabolite in 379 patients. The upstream FMO1*6 was associated with increased dose-adjusted serum OLA concentrations (C/Ds; P=0.008), an effect further enhanced by FMO1rs7877C>T in smokers. The influence of FMO3 polymorphisms was limited to variability in OLA N-oxide. Homozygous carriers of FMO3rs2266780A>G (p.E308G) displayed 50% lower C/D of OLA N-oxide compared with subjects homo- or heterozygous for the A-variant (P<0.003). Our data support the role of FMO3 in the N-oxidation of OLA and implicate for the first time the contribution of FMO1 and its functional *6 variant in OLA disposition.

ß2-adrenergic receptor polymorphisms, asthma and COPD: two large population-based studies.

The ß(2)-adrenergic receptor (ADRB2) is an important regulator of airway smooth muscle tone. We tested the hypothesis that three functional polymorphisms in the ADRB2 gene (Thr164Ile, Gly16Arg and Gln27Glu) are associated with reduced lung function, asthma or chronic obstructive pulmonary disease (COPD). We first genotyped 8,971 individuals from the Copenhagen City Heart Study for all three polymorphisms. To validate our findings, we genotyped an additional 53,777 individuals from the Copenhagen General Population Study for the Thr164Ile polymorphism. We identified 60,910 Thr164Ile noncarriers, 1,822 heterozygotes and 16 homozygotes. In the Copenhagen City Heart Study, the Thr164Ile genotype was associated with reduced forced expiratory volume in 1 s (FEV(1)) % predicted (trend p = 0.01) and FEV(1)/forced vital capacity (FVC) (p = 0.001): Thr164Ile heterozygotes had 3% and 2% reduced FEV(1) % pred and FEV(1)/FVC, respectively, compared with noncarriers. The odds ratio for COPD in Thr164Ile heterozygotes was 1.46 (95% CI 1.05-2.02). In the Copenhagen General Population Study, the Thr164 genotype associated with reduced FEV(1) % pred (p = 0.04) and FEV(1)/FVC (p < 0.001): Thr164Ile homozygotes and heterozygotes had 7% and 1% reduced FEV(1) % pred and 6% and 1% reduced FEV(1)/FVC, respectively, compared with noncarriers. The odds ratios for COPD in Thr164Ile homozygotes and heterozygotes were 4.53 (95% CI 1.54-13.3) and 1.07 (95% CI 0.92-1.25), respectively. Our results suggest that ADRB2 Thr164Ile is associated with reduced lung function and increased risk of COPD in the general population.