AM Levels Research Articles

Determination of Atto- to Femtogram Levels of Americium and Curium Isotopes in Large-Volume Urine Samples by Compact Accelerator Mass Spectrometry

Ultralow level analysis of actinides in urine samples may be required for dose assessment in the event of internal exposures to these radionuclides at nuclear facilities and nuclear power plants. A new bioassay method for analysis of sub-femtogram levels of Am and Cm in large-volume urine samples was developed. Americium and curium were co-precipitated with hydrous titanium oxide from the urine matrix and purified by column chromatography separation. After target preparation using mixed titanium/iron oxides, the final sample was measured by compact accelerator mass spectrometry. Urine samples spiked with known quantities of Am and Cm isotopes in the range of attogram to femtogram levels were measured for method evaluation. The results are in good agreement with the expected values, demonstrating the feasibility of compact accelerator mass spectrometry (AMS) for the determination of minor actinides at the levels of attogram/liter in urine samples to meet stringent sensitivity requirements for internal dosimetry assessment.

Effects of Meditation on Stress Levels of Physical Therapist Students

Background and Purpose. Due to the arduous nature of the professional (entrylevel) physical therapist (PT) education program, PT students often experience higher levels of stress than their age and gender-matched peers, which can negatively affect their academic performance and wellbeing. Therefore, finding methods to manage stress is important for students’ wellbeing and academic success. The purpose of this study was to investigate the effect of meditation on stress levels of students in a doctor of physical therapy program. Participants. Twenty-four PT students in their first and second year of an entry-level doctoral degree program participated in this study. Methods. This study examined the stress levels of 24 subjects via blood pressure, morning and evening salivary cortisol levels, and 3 different stress questionnaires (Perceived Stress Scale, General Anxiety Disorder 7, Stress Visual Analog Scale) using a quasi-experimental pretest-posttest design. The intervention was a twice-daily 20-minute beeja mantra-based meditation performed for 8 weeks. Results. All 24 students completed the study with average compliance of 91.6% ± 8.7% (mean ± SD) meditating at least once per day, and 77.3% ± 19.8% meditating twice per day. A paired t test was used to analyze the blood pressure readings, with systolic blood pressure decreasing by 2.9 ± 2.3 (standard error of the mean: SEM) mmHg (P = .022), and diastolic blood pressure decreasing by 4.6 ± 2.9 (SEM) mmHg (P = .005). Four subjects with prehypertensive systolic blood pressure readings prior to the meditation intervention moved into healthy normal range by the conclusion of the study. The Wilcoxon signed rank test was used to evaluate the questionnaires, which showed significantly decreased subjective stress levels (P < .05). No significant changes in mean morning (AM) or night (PM) salivary cortisol levels were found (P > .05). However, 9 of the 14 (64%) subjects who had at least 1 cortisol measurement outside of the normal range (established by ZRT Labs) at the start of the study had both AM and PM cortisol levels in the normal range following the intervention. Discussion and Conclusion. The results of this study show that an 8-week mantrabased meditation program significantly reduces blood pressure, significantly reduces perception of stress based on the subjective questionnaires, and that a 1-day AM/PM cortisol sample may not be sufficient to accurately analyze a subject's true daily cortisol level to show statistical change.

Digital PCR (dPCR) Overcomes the Limitations in Detection and in Quantification of Quantitative PCR (qPCR) and Reveals Different Levels of BCR-ABL1 Copies/µl Among the Chronic Myeloid Leukemia (CML) Patients Achieving Major (MR3.0) or DEEP (MR4.0, MR4.5 and MR5.0) Molecular Response with Tyrosin Kynase Inhibitors (TKIs)

Monitoring BCR-ABL1 transcript by quantitative PCR (qPCR) is essential for the management of CML patients treated with TKIs. Currently, up to 40-50% of CML patients treated with TKIs can achieve a deep molecular response (DMR = BCR-ABL1 minor or equal to0,01% IS), but only 50% of them are reported to maintain a stable Treatment Free Remission (TFR). Since qPCR has some intrinsic limitations in detection and quantification of BCR-ABL1 transcript, this method does not appear optimal in the selection of patients eligible for TKIs cessation. A precise monitoring of BCR-ABL1 transcript levels can help even better the clinicians in managing CML patients treated with TKIs and in selecting the best candidates for discontinuation of TKIs without relapse.Digital PCR (dPCR) can be more advantageous than qPCR. It gives the absolute quantification of target nucleic acidsby partitioning the PCR reaction mix over a large number of wells, each containing a single copy or no copies of the target region. Based on the assumption of Poisson's distribution, the number of template copies originally presenting in the sample can be calculated from the number of partitions in which amplification has successfully occurred. In that way, standard curves cannot be necessary and data are more accurate.In this study we set a dPCR assay to quantify the BCR-ABL1 transcript in a preliminary cohort of CML patients with major (MMR or MR3.0) or deep molecular response (MR4.0, MR4.5 and MR5.0).The analysis by dPCR were based on a TaqMan-MGB probes targeting the BCR-ABL1 transcript. Custom assay was designed and produced with a FAM-label, basing on the sequence of routinely-used probes. The experiments were performed on a QuantStudio 3D Digital PCR System (Life Technologies). A commercial BCR-ABL1 Mbcr standard dilutions (Qiagen) and 10 replicates of blank samples (DNA-free, RNA-free water) were used to set dPCR assay and to determine the Limit of Detection and the Limit of Quantification of our method. The values of absolute quantities of BCR-ABL1 transcript assessed by dPCR were expressed as number of copies/ul. Samples for dPCR testing were obtained from peripheral blood (10 ml in EDTA tubes) of CML patients treated with TKIs, namely imatinib, nilotinib, or dasatinib, on time-checks planned for monitoring MMR or DMR through the conventional qPCR. To the purpose of the study, we evaluated 10 cases with stable MR3.0, 7 cases with stable MR4.0, 6 cases with stable MR4.5, 7 cases with stable MR5.0. It has been considered as stable any MR3.0, MR4.0, MR4.5, or MR5.0 molecular response measured by conventional qPCR and detected in the last three consecutive checks performed during the last 12 months. Blank samples served as negative controls, while 10 peripheral blood samples of healthy donors served as normal controls.Digital PCR (dPCR) revealed different levels of BCR-ABL1 copies/µl among the CML patients achieving the major (MR3.0) or deep (MR4.0, MR4.5 AND MR5.0) molecular response with TKIs. Moving from MR3.0 to MR5.0 molecular response the median of BCR-ABL1 copies/µl assessed by dPCR were progressively decreasing (Figure 1), with blanks and healthy controls approximately to zero. Medians with ranges were 0.957 (0.472-1.692) for MR3.0; 0.319 (0.072-0.906) for MR4.0; 0.231 (0.063-0.651) for MR4.5; 0.219 (0.074-0.399) for MR5.0; 0.076 (0.000-0.118) for healthy controls, and 0.000 (0.000-0.129) for blanks. Moreover, dPCR revealed different BCR-ABL1 copies/µl among the patients of each class of molecular response. Importantly, in patients with MR4.5, MR5.0 and with undetectable levels of BCR-ABL1 % IS as measured with qPCR, discrete variable levels of BCR-ABL1 copies/µl have been detected by dPCR. These data, revealing different levels of BCR-ABL1 copies/µl beyond the limit of detection and quantification of conventional qPCR, may explain why no correlation was observed between BCR-ABL1 % IS levels measured by qPCR and numbers of BCR-ABL1 copies/µl measured by dPCR.We are screening CML patients with MMR and DMR, but these preliminary results show that dPCR appears to be more accurate than qPCR for detection and quantification of BCR-ABL1 transcript and it should be seen as a useful step forward in order to better manage the TKI therapy and to better select the candidates for TFR.Acknowledgments:Department of Clinical and Experimental Sciences, University of Brescia; PRIN2009; European Leukaemia Net; BCC "Pompiano e Franciacorta". [Display omitted] DisclosuresTiribelli:Bristol Myers Squibb: Consultancy, Speakers Bureau; Ariad Pharmaceuticals: Consultancy, Speakers Bureau; Novartis Farma: Consultancy, Speakers Bureau. Rosti:Bristol Myers Squibb: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau. Martinelli:Roche: Consultancy; BMS: Speakers Bureau; MSD: Consultancy; ARIAD: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy.

Simultaneous Quantification of Methylated Cytidine and Adenosine in Cellular and Tissue RNA by Nano-Flow Liquid Chromatography-Tandem Mass Spectrometry Coupled with the Stable Isotope-Dilution Method.

The rising interest in understanding the functions, regulation, and maintenance of the epitranscriptome calls for robust and accurate analytical methods for the identification and quantification of post-transcriptionally modified nucleosides in RNA. Monomethylations of cytidine and adenosine are common post-transcriptional modifications in RNA. Herein, we developed an LC-MS/MS/MS coupled with the stable isotope-dilution method for the sensitive and accurate quantifications of 5-methylcytidine (m(5)C), 2'-O-methylcytidine (Cm), N(6)-methyladenosine (m(6)A), and 2'-O-methyladenosine (Am) in RNA isolated from mammalian cells and tissues. Our results showed that the distributions of m(5)C, Cm and Am are tissue-specific. In addition, the 2'-O-methylated ribonucleosides (Cm and Am) are present at higher levels than the corresponding methylated nucleobase products (m(5)C and m(6)A) in total RNA isolated from mouse brain, pancreas, and spleen but not mouse heart. We also found that the levels of m(5)C, Cm, and Am are significantly lower (by 6.5-43-fold) in mRNA than in total RNA isolated from HEK293T cells, whereas the level of m(6)A was slightly higher (by 1.6-fold) in mRNA than in total RNA. The availability of this analytical method, in combination with genetic manipulation, may facilitate the future discovery of proteins involved in the maintenance and regulation of these RNA modifications.

An Anti-Apoptotic Molecule, Anamorsin, Is Upregulated in Anemia and Essential for Imperative Erytropoiesis

IntroductionAnamorsin (AM) is an anti-apoptotic molecule we originally isolated from hematopoietic cell line, Ba/F3. That is dependent on interleukin-3 (IL-3) stimulation. We showed that AM conferred resistance to apoptosis induced by the lack of IL-3 stimulation. AM has no structural homology to any known anti-apoptotic molecules such as Bcl-2 and IAP family members. AM-deficient mice are embryonic lethal at late gestation due to the defect of definitive hematopoiesis. Its embryos are anemic and very small in size. Thus AM is considered to be crucial in erythropoiesis but the mechanisms how AM works remain unclear. In this study, we tried to clarify the roles of AM in erythropoiesis.Methods and resultsAt first, in order to examine the expression levels of AM in each differential stage of erythroid cells, we sorted those cells from eight-week-old mice bone marrow (n=4) using flow cytometry. The expression levels of AM were evaluated by Immunoblotting. The results showed that the proerythroblast population (CD71high Ter119low) expressed AM higher than any other erythroid cells. The expression of AM in CD71high Ter119 high cells and CD71low Ter119high cells, which correspond to basophilic erythroblasts and orthchromatophilic erythroblasts respectively, were markedly decreased. Next, we analyzed whether AM expression is induced by erythropoietin (EPO). Recombinant human EPO was injected intravenously (0.1mg/kg) with a single treatment (day0) to eight-week-old mice (n=3). The bone marrow and spleen cells were collected seven days after injection and the samples were subjected to immunoblotting. The results showed that EPO treatment led to increased expression of AM in both bone marrow and spleen. At 14 days after EPO injection, the expression of AM returned to the basal. Furthermore, we examined the AM expression in the acute anemia model. Blood of 0.2ml was exsanguinated from each mouse every day until hematocrit reached to the value less than 25 % (day 0).In this phlebotomized mice, the AM expression was strongly induced specially in proerythroblast population on day 1 and returned to the basal after the recovery from anemia. To explore the AM expression under the other stress, 5-FU (150 mg/kg), a chemotherapeutic drug, was administered to mice (n=3) with a single treatment (day 0). The increased expression of AM was also detected when the mice were most severely anemic (day7). To investigate the significance of AM expression induced by anemia, we transplanted 106 14.5 dpc wild-type (AM+/+) and AM-deficient (AM–/–) fetal liver cells into lethally irradiated congenic recipient mice. At 8 weeks after transplantation, we analyzed the response to anemia induced by phlebotomy. The results showed that donor-derived cells from AM+/+ fetal liver cells were detected the smooth recovery on day 3 after phlebotomy. On the other hand, donor-derived cells from AM–/– fetal liver cells showed an approximately 5-fold reduction compared to those from AM+/+fetal liver cells.Conclusion and discussionThe proerythroblast population expressed AM at the highest level among various erythroid lineage populations. The AM expression level was increased by acute anemia induced by phlebotomy and AM-deficiency could show inefficient recovery of erythropoiesis from the induced acute anemia. Previously we reported that AM is essential for Fe/S cluster biogenesis and AM deficiency impaired the iron homeostasis (ASH abs. #610 (2012)). We also reported that the proerythroblast population in AM-deficient fetal liver cell showed more increased labile iron pool and significantly induced apoptosis. From our previous and present data, it is possible that AM may control erythropoiesis by preventing apoptosis when erythrocyte production is needed. DisclosuresNo relevant conflicts of interest to declare.

Graphene/MoS(2) heterostructures for ultrasensitive detection of DNA hybridisation.

The photoluminescence signals of a graphene/MoS2 heterostructural stacking film are sensitive to environmental charges, which allows the single-base sequence-selective detection of DNA hybridization with sensitivity to the level of aM.

Description of Industries sources to enviromental exposure to benceno in chidren from Ecatepec, county in Mexico

Background Ecatepec County in Mexico City Metropolitan Area is highly industrialized and considered as one of the most polluted area; however there are no reports about other air pollutants. Objects To describe the sources of environmental exposure to benzene by industrial emissions for children 7-10 years of age from three primary schools from Ecatepec County. Methods: A cross-sectional study was conducted in 170 school children (7-10 years old) enrolled at three different schools from Ecatepec County; urinary concentrations t,t-muconic acid (t,t-MA) as indicators of benzene exposure was determined by HPLC. We take data of inventory of industries from local government were characterized potential point sources for benzene in Ecatepec. Themselves, the homes children´s and industries were located as through a Geographic Information System (GIS). For each child, we calculated the number of industries and distance to nearest industry and assessed the relationship between estimated emissions of VOCs with the metabolite levels. Results: The average of AM levels was 233.3 mg / g creatinine (SD = 245.13), being higher in the students of the school in San Cristobal (Mean = 318.89, SD = 257.5). At a distance of 500 meters, each school had an average 65 industries with 36.78 tons of VOCs (SD = 204.14) by year in average. Each industry was assigned to 13 children on average (SD = 7.9). The predominant industry in the area was the metal followed by chemistry and food, but the industry emitting more VOCs was for cement for construction followed by chemical and fertilizer. A nonlinear relationship was observed between total VOCs emissions and AM levels in children especially in children whose industries were less than 500 meters. Conclusion: Exposure to benzene in these children can be broad impact in terms of health; exposure characterization in this area shows a population at risk that could be exceeding the permissible limits. It is important to consider strategies to mitigate the damage.

Abstract 052: Epicardial-derived Adrenomedullin Drives Cardiac Hyperplasia During Embryogenesis

The multi-functional peptide adrenomedullin ( Adm = gene, AM = protein) plays important roles in embryonic development and disease. Previous studies demonstrated that Adm knockout mice die at embryonic day 13.5 with small, disorganized hearts and hypoplastic lymphatic vessels, highlighting the importance of this peptide in normal cardiovascular development. Since Adm knockout animals are embryonic lethal, our goal was to generate and characterize a novel model of Adm over-expression to study the role of Adm during development and disease processes. Through gene targeting techniques, we generated a novel mouse model of Adm over-expression, abbreviated as Adm hi/hi . When we assessed gene expression of Adm from 10 different tissues, we found Adm hi/hi mice express 3- to 15-fold more Adm than wildtype littermates. Additionally, peptide levels of AM in lung and kidney, as well as circulating plasma levels of AM were elevated 3-fold over wildtype mice, indicating a functional increase in AM. Our initial analysis revealed that adult Adm hi/hi mice have larger heart weight to body weight ratios than wildtype littermates (4.93±0.23 vs. 5.96±0.29, n = 11-12). We found that compared to wildtype, Adm hi/hi embryos have more proliferating cells during heart development (14.46±1.11 vs. 31.97±2.84, n=4), indicating that hyperplasia drives Adm hi/hi heart enlargement. By crossing the Adm hi/hi line to different tissue-specific Cre lines, we were able to excise the stabilizing bovine growth hormone 3’UTR, thereby returning Adm expression levels back to wildtype in cells with active Cre recombinase. Using this approach, we identified the epicardium as a major source of AM during cardiac development. In conclusion, we found that AM derived primarily from the epicardium drives cardiac hyperplasia during embryonic development resulting in persistent, enlarged hearts of adult Adm hi/hi mice. Since our Adm hi/hi mice recapitulate the 3-fold plasma elevation of AM observed during human disease, this mouse line will be a useful tool for studying the role of elevated AM during disease.

Adrenomedullin in peritoneal effluent expressed by peritoneal mesothelial cells

BackgroundAdrenomedullin (AM) possesses vasodilative and cell-protective properties. Glycine combines with the C-terminal of AM to form mature, physiologically active AM (mAM). AM is reportedly induced by high glucose condition in vascular endothelial or smooth muscle cells; however, little is known on how AM is activated by amidation. To investigate the behavior of AM in patients undergoing peritoneal dialysis (PD), the concentrations of AM, mAM and CA125 were measured. The mAM to AM ratio (mAM/AM ratio) was also evaluated as a marker of amidation activity.MethodsTwenty patients were recruited for this study. The effluent at the time of the peritoneal equilibration test was collected and AM, mAM and CA125 concentrations were measured. The expression of AM in peritoneal mesothelial cells (PMCs) collected from effluent was also examined with an indirect immunofluorescent method.ResultsMean values of AM and mAM in effluent were 18.1 ± 1.6 and 4.1 ± 0.3 fmol/mL, respectively. In plasma, they were 42.6 ± 3.3 and 5.6 ± 0.6 fmol/mL, respectively. AM concentrations in effluent did not correlate with plasma AM level but correlated well with the dialysate-to-plasma ratio of creatinine (D/P ratio of creatinine). Moreover, in 7 of 20 cases, concentrations of the mAM and mAM/AM ratio in effluent were higher than in plasma. In effluent, AM concentration but not the mAM/AM ratio correlated with CA125 concentration. Immunocytological study revealed diffuse, cytoplasmic expression of AM in PMCs which were collected from effluent during PD.ConclusionAM is expressed by PMCs and actively amidated in the abdominal cavity of patients undergoing PD.

A Single Center Transition of Care Model from Pediatric Heart to Adult Services

Purpose This first transition year has shown to have increased instances of rejection, graft dysfunction, and death. There is little data or guidelines to transition young adults to adult centered transplant programs. There are no current ISHLT guidelines to establish a transition program. We have two years of data gathered from 2010 to the present from our program. Methods and Materials Benchmarks that have been identified to measure the success of the transition are the following indicators:1.Number of Rejection events. 2. Stable allomap scores and IS levels. 3.Consistent clinical compliance, continued stable mental health -with minimal life style changes/impairment 4. Work/School. Family goals with engagement in departmental return to work program, participation in pregnancy counseling.5. ED visits/hospitalization/ discharges. Results Year to date 12 patients have transitioned. The mean time post transplant has been 12 years. Median age has been 24 years. We have 2 patient deaths; 1 from humoral rejection due to medical non-compliance and 1 patient died from suicide. 5 patients have successfully entered the work force or are attending college.10 others have had good compliance and stable IS/ Allomap levels. There have been no pregnancies reported in our transition program. All 12 patients have been hospitalized for causes including: infection, rejection, rhythm issues, and other end organ failure. Conclusions Initial outcomes shows complexity of establishing a transition program. It is imperative to build an effective transitional care model/program with evidenced based practice without loss of life or graft function. Lessons learned include establishing better education and pre- planning from a peds oriented program to an adult oriented program, establishing a care coordinator to help this special transition population, administrative and financial support, timing of transition criteria (coincide age related mile stones), confidence in the adult team, and establishing peds to adult oriented quality of life benchmarks.

Resetting the baroreflex during snoring: Role of resistive loading and intra-thoracic pressure

Baroreflex sensitivity (BRS) is reduced during snoring in humans and animal models. We utilised our rabbit model to examine the contribution of increased upper airway resistance to baroreflex resetting during snoring, by comparing BRS and baroreflex operating point (OP) values during IS to those obtained during tracheostomised breathing through an external resistive load (RL) titrated to match IS levels of peak inspiratory pleural pressure (Ppl). During both IS and RL, BRS decreased by 45% and 49%. There was a linear relationship between the change in Ppl and the decrease in BRS, which was similar for IS and RL. During both RL and IS, there was a shift in OP driven by ∼16% increase in HR and no change in arterial pressure. Snoring related depression of BRS is likely mediated via a HR driven change in OP, which itself may be the outcome of negative intra-thoracic pressure mediated effects on right atrial wall stretch reflex control of heart rate.

The Innsbruck Hand Transplant Program: An Update More than a Decade after the First Transplant

Introduction: We describe here the outcome after two bilateral hand, one bilateral forearm and one unilateral hand transplantation at 12, 9, 5.5 and 2.5 years after transplantation. Methods: Four patients received a bilateral hand (n=2), a bilateral forearm (n=1) or a unilateral hand transplant between March 2000 and July 2009. Induction therapy with ATG (n=2) or alemtuzumab (n=2) was followed by tacrolimus, prednisolon MMF (n=3) or tacrolimus and MMF (n=1) maintenance IS. Later, sirolimus/everolimus was added under simultaneous withdrawal (n=2) or dose reduction (n=1) of tacrolimus (n=1) or MMF (n=1). Steroids were avoided in one and withdrawn in two patients. Results: Total active range of motion improved continously with a grip strength of 2-10kg. Hand function correlated well with time after transplant and amputation level. Intrinsic hand muscle function recovery and discriminative sensation were observed in all patients. Complications included CMV infection, fungal infection, hypertension, hyperglycemia, transient creatinine increase and headache and a bullous pemphigoid. Three, six, four, and one rejection episode were successfully treated with steroids, anti-CD25, anti-CD52 antibodies and/or intensified maintenance IS. Skin histology at current shows no or mild perivascular lymphocytic infiltrates without signs of progression. Vessels are patent without signs for luminal narrowing or intimal proliferation. Conclusion: The overall functional outcome and patient satisfaction after bilateral hand, bilateral forearm and unilateral hand transplantation are highly encouraging. All patients are now free of rejection with moderate levels of IS.

PARTIALLY HYDROLYZED GUAR GUM INTAKE AMELIORATES CONSTIPATION, IMPROVES NUTRITIONAL STATUS AND REDUCES INDOXYLSULFURIC ACID IN DIALYSIS PATIENTS.

Dialysis patients often develop constipation and changes in intestinal bacterial flora. Indoxylsulfuric acid (IS) levels rise as glomerular filtration decreases, and patients with renal failure have high IS. Elevated IS is also caused by increased indole due to altered intestinal flora (Takayama et al, Am J Kidney Dis. 2003). We investigated whether administering partially hydrolyzed guar gum (PHGG) (Sunfiber: a product of Taiyokagaku Co., Ltd., Japan) ameliorates constipation and improves nutritional status in dialysis patients, while decreasing IS levels. Thirty-five patients on maintenance dialysis (mean age, 71 ± 9; male/female= 22/13) ingested PHGG (10 g/day) for 6 weeks. Defecation was scored before and after PHGG intake using a modified Constipation Assessment Scale-Long Term (Japanese version). Nutritional status was rated according to the Geriatric Nutritional Risk Index (GNRI) before and after PHGG intake. IS was measured in 8 patients taking PHGG orally for 24 weeks, for comparison with those in 8 patients not on PHGG. Constipation scores decreased from 7.9 to 5.0 (p 〈.01) and GNRI increased from 95.0 ± 5.0 to 95.9 ± 5.7 (p 〈.05), reflecting amelioration of constipation and improved nutritional status. The ratio of IS after to that before PHGG intake was calculated to analyze the magnitude of IS change. The ratio in patients not on PHGG was 1.2 ± 0.3, i.e. IS rose, while that in patients taking PHGG was significantly reduced (0.8 ± 0.3, p 〈.05). Our results indicate PHGG consumption to ameliorate constipation and improve nutritional status, and that continued intake reduces IS, in dialysis patients.

Diurnal variations in the phagocytic activity of zebrafish kidney leukocytes can be influenced by melatonin in a time-of-day dependent manner. (117.13)

Abstract Diurnal variations in immune system function result from rhythmic expression of clock genes. Their relationship with neuroendocrine regulators such as melatonin is not well understood. We explored the influence of melatonin on variations of phagocytic activity of zebrafish leukocytes. The percentage of kidney myeloid cells that phagocytosed pHrodo-labeled E. coli varied substantially throughout the day. The greatest increase in activity was between 6 AM and 10 AM, spanning the daily dark-to-light transition, a time when endogenous melatonin levels are falling to their daytime low. To determine whether extending the perceived night by prolonging the presence of melatonin would reduce the peak participation in phagocytosis observed at 10 AM, fish were exposed to exogenous melatonin for two hours beyond the beginning of the daily light period. Treatment with melatonin resulted in significantly lower participation in phagocytosis, similar to the normal 6 AM (dark phase) levels. The opposite effect was observed when fish received melatonin later in the day, prior to the evening light-to-dark transition. Fish exposed to melatonin prior to assay at 6 PM (late day) displayed a significant increase in phagocytosis over control fish, with participation levels surpassing normal 10 PM (early dark phase) levels. At both times of day, pre-treatment with the melatonin receptor antagonist luzindole prevented the changes in phagocytosis associated with extended melatonin presence.

Caffeine Ingestion Reverses the Circadian Rhythm Effects on Neuromuscular Performance in Highly Resistance-Trained Men

PurposeTo investigate whether caffeine ingestion counteracts the morning reduction in neuromuscular performance associated with the circadian rhythm pattern.MethodsTwelve highly resistance-trained men underwent a battery of neuromuscular tests under three different conditions; i) morning (10:00 a.m.) with caffeine ingestion (i.e., 3 mg kg−1; AMCAFF trial); ii) morning (10:00 a.m.) with placebo ingestion (AMPLAC trial); and iii) afternoon (18:00 p.m.) with placebo ingestion (PMPLAC trial). A randomized, double-blind, crossover, placebo controlled experimental design was used, with all subjects serving as their own controls. The neuromuscular test battery consisted in the measurement of bar displacement velocity during free-weight full-squat (SQ) and bench press (BP) exercises against loads that elicit maximum strength (75% 1RM load) and muscle power adaptations (1 m s−1 load). Isometric maximum voluntary contraction (MVCLEG) and isometric electrically evoked strength of the right knee (EVOKLEG) were measured to identify caffeine's action mechanisms. Steroid hormone levels (serum testosterone, cortisol and growth hormone) were evaluated at the beginning of each trial (PRE). In addition, plasma norepinephrine (NE) and epinephrine were measured PRE and at the end of each trial following a standardized intense (85% 1RM) 6 repetitions bout of SQ (POST).ResultsIn the PMPLAC trial, dynamic muscle strength and power output were significantly enhanced compared with AMPLAC treatment (3.0%–7.5%; p≤0.05). During AMCAFF trial, muscle strength and power output increased above AMPLAC levels (4.6%–5.7%; p≤0.05) except for BP velocity with 1 m s−1 load (p = 0.06). During AMCAFF, EVOKLEG and NE (a surrogate of maximal muscle sympathetic nerve activation) were increased above AMPLAC trial (14.6% and 96.8% respectively; p≤0.05).ConclusionsThese results indicate that caffeine ingestion reverses the morning neuromuscular declines in highly resistance-trained men, raising performance to the levels of the afternoon trial. Our electrical stimulation data, along with the NE values, suggest that caffeine increases neuromuscular performance having a direct effect in the muscle.

Implications of long-term medication of oral steroids and antimalarial drugs in primary Sjögren's syndrome

BackgroundImmunomodulating drugs are commonly used in treating patients with autoimmune diseases but with very different outcomes. We aimed to investigate differences in cytokine and autoantibody levels with regard to patient characteristics in patients with primary Sjögren's syndrome (pSS) receiving oral steroids or antimalarial drugs (AM) after a longer period of time. MethodsSerum samples from 141 patients fulfilling the revised EU–US criteria and 99 healthy controls were analysed for 25 cytokines and 8 autoantibodies. ResultsAM-patients had lowered levels of IL-5, IL-10, IL-13 and IFN-γ, though non-significantly. Use of prednisolone was associated with reduced levels of IL-15, IL-2, IL-4, IL-12p40, TNF-α, MIP-1α and MIP-1β (p<0.05), and a trend towards decreased levels of IL-1RA and IL-1β was observed. No associations were seen between AM and antibody levels. Significantly higher protein levels of anti-Ro-52 and anti-Ro-60 were observed in the patients taking prednisolone (p<0.05). The proportion of patients positive for anti-Ro-52 and anti-La-48 did not differ significantly in the groups taking and not taking prednisolone, but a difference was seen for anti-Ro-60 (p<0.05). ConclusionsPrednisolone is a potent anti-inflammatory and immunosuppressive drug commonly used in autoimmune diseases. Our study shows that oral steroids are associated with reduced levels of several pro-inflammatory cytokines, but increased levels of pSS specific autoantibodies. The association between steroid use and increased antibody levels is not readily explained by known steroid effects, and should therefore be confirmed in further studies. Lower levels of pro-inflammatory cytokines indicate a beneficial effect of oral steroids in this patient group.

Evidence for Extrathyroidal Formation of 3-Iodothyronamine in Humans as Provided by a Novel Monoclonal Antibody-Based Chemiluminescent Serum Immunoassay

Thyronamines are thyronergic metabolites of thyroid hormones. Lack of reliable and sensitive detection methods for endogenous 3-iodothyronamine (3-T(1)AM) has so far hampered progress in understanding their physiological action and role in endocrine homeostasis or pathophysiology of diseases. We characterized newly generated mouse monoclonal 3-T(1)AM antibodies and established a monoclonal antibody-based chemiluminescence immunoassay as a powerful tool for monitoring 3-T(1)AM levels in investigations addressing altered serum profiles and potential sites of origin and action of 3-T(1)AM in humans. Our exploratory study on 3-T(1)AM serum levels in humans measured 3-T(1)AM concentrations in comparison with thyroid hormones. Thirteen adult healthy subjects, 10 patients with pituitary insufficiency, and 105 thyroid cancer patients participated. INTERVENTIONS included l-T(4) withdrawal in patients with pituitary insufficiency as well as TSH-suppressive T(4) substitution in thyroid cancer patients. 3-T(1)AM was reliably quantified in human serum and stable after storage at room temperature and 4 C overnight as well as after four freeze-thaw cycles. The median serum concentration in healthy subjects was 66 ± 26 nm. 3-T(1)AM was also detected in T(4)-substituted thyroid cancer patients. Although free T(4) and T(3) significantly decreased during T(4) withdrawal, 3-T(1)AM levels remained constant for 6 d. Because higher 3-T(1)AM levels are detectable in T(4)-substituted thyroid cancer patients after thyroidectomy/radioiodine treatment compared with healthy controls, we concluded that 3-T(1)AM is mainly produced by extrathyroidal tissues. The serum profile during T(4) withdrawal suggests either a long half-life or persisting 3-T(1)AM release into serum from intracellular thyroid hormone precursors or stores.

Hipopituitarism due to primary empty sella and uncommon muscular symptoms

Muscular abnormalities are uncommon manifestations of secondary adrenal insufficiency [1]. Herein, an elderly woman with an unusual muscular presentation of hypopituitarism due to empty sella and secondary adrenal insufficiency is described. A 75-year-old woman reported severe pain and contracture in both thighs associated with progressive difficulty in walking for the past three weeks. Response to analgesics and NSAI was limited. Her past medical history was unremarkable. Physical examination showed a wellappearing woman with normal vital signs. There was pronounced stiffness and pain in the aductor muscles of both thighs. Movement of lower extremities was painful. The electromyography did not show abnormal findings. Laboratory test results showed sodium 118 mEq/l, potassium 4.4 mEq/l, creatin-kinase 356 U/l (0‐167), LDH 777 U/l (240‐480), and C-reactive protein 2.0 mg/dl (0‐0.5). An endocrine evaluation was performed due to presence of severe hyponatremia: free T4 \ 0.4 ng/dl (0.93‐1.7), TSH 1.31 mIU/ml (0.27‐4.2), FSH 1.7 mIU/l (26.5‐139), LH 0.4 mIU/l (20‐65), GH \ 0.13 pg/l, and prolactin was normal. At 10 AM, serum cortisol and plasma ACTH levels were 3.5 lg/dl (5‐20) and 29.4 pg/ml (0‐52), respectively. Twenty-four-hour urinary cortisol was 6.0 lg (10‐90). A short ACTH stimulation test showed low serum cortisol response. MRI of the brain revealed an empty sella (Fig. 1). Replacement therapy was started with hydrocortisone and levothyroxine. She has remained well since. Primary empty sella, a rare neuroradiological finding in the general population, has been related to a congenital incomplete formation of the sellar diaphragm resulting in herniation of the subarachnoid space within the sella and increasing the intracranial pressure. Most common symptoms reported as presentation of primary empty sella were headache, visual disturbances, rhinorrea, and endocrine dysfunction [2]. This patient had biochemical evidence of secondary adrenal insufficiency, and endocrinological testing demonstrated secondary adrenal insufficiency and hypopituitarism related to empty sella. The clinical syndrome developed slowly, over several weeks. Patients with slow onset adrenal insufficiency (as this) usually complain of constitutional syndrome, abdominal pain, nausea, and diarrhea. Musculoskeletal abnormalities are a rare presentation of this disease.

Clinical guidelines in Hematology.

Clinical guidelines are now a powerful tool in decision making in the complex process of healthcare. There is no absolute definition of its impact on the clinical outcomes and in different patient populations. Nevertheless, its role is unquestionable in the regulation and organization of the healthcare system as a whole.(1-8) Evidence-based clinical guidelines balance the diverse interests involved during the process to ensure that patients receive an adequate standard of healthcare. Through clinical guidelines we can compare our experience with recommendations, which not only teaches us and brings our knowledge up-to-date, but also allows us to reflect on the main issue: what level of uncertainty am I accepting with my current conduct of this patient?(9-15) The central principle of the Associacao Medica Brasileira (AMB)/Conselho Federal de Medicina (CFM) Guidelines Program is to prepare the physician to answer four basic questions: a) what do I do in my clinical practice; b) for whom do I do it; c) how do I do it and d)why do I do it?(16) The development of recommendations can be interpreted as a way to limit medical autonomy, but in fact, it is to make our actions in healthcare in Brazil transparent, clearly stating the strength of scientific evidence that supports each of these conducts by estimating the level of uncertainty involved in decision making.(17-22) We established standards, providing conduct options focused on the patient in relation to what we do: recommendations for diagnosis, prevention, treatment and prognosis; for whom we do it: patients with indications to meet their expectations and individuality, and never forgetting the minorities; how we do it: defining the method by which to develop our detailed and explicit conduct; and why we do it: to support our decisions on the benefits, risks and harm to patients.(23-26) The AMB-CFM Guidelines Program together with the societies of medical specialties, members of the AMB, has already prepared 500 clinical guidelines and today has about 120 in development. In addition, continuing medical education and participation in international networks that develop evidence-based guidelines is included in the Guidelines Programs. In 2011, Brazilian hematology through its society (the Associacao Brasileira de Hematologia e Hemoterapia - ABHH) started an unprecedented process of developing evidence-based protocols within the AMB-CFM Guidelines Program. The association initially chose six major hematological diseases: Sickle cell anemia, chronic myeloid leukemia, acute promyelocytic leukemia, non-promyelocytic leukemia, idiopathic thrombocytopenic purpura and multiple myeloma. Each theme (Guideline) is composed of important clinical questions (on average 15) prepared by experts. These questions are structured using the acronym, PICO (P: patient, I: Intervention C: Comparison, O: Outcome) as a guide to search available evidence by an extensive systematic review of the literature to find evidence to support the recommendations for each clinical question. The recommendations are based on the strongest scientific evidence and aim to help hematologists make their decisions on each individual patient.(3,16,19,21) In mid-2012, the first six issues will be completed initiating a series of feasible guidelines developed using a rigorous methodology written in a clear and objective language. Without doubt, participants at all levels of the healthcare system will benefit, but mostly these benefits will be reflected in the care provided to hematology patients in Brazil. We recognize the difficulties of obtaining and critically analyzing the evidence, the pressure of interest that are not always directed to the care of patients and the difficulties of the National Health System in relation to its structure, diversity and inequality. But we also know the effort and determination of many, who, through the guidelines, will establish a discerning, flexible, ethical, and reflective language, based on evidence that meets the basic needs and expectations of patients.(2,21,22)

Stress Reduction at the Work-Family Interface: Positive Parenting and Self-Efficacy as Mechanisms of Change in Workplace Triple P

Workplace Triple P (WPTP) is a group-based parenting skills training specifically designed to meet the needs of employed parents. Several randomized controlled trials have demonstrated the training's efficacy. This study examined possible mechanisms of change that account for the stress reduction effects of this parenting skills training at the work-family interface. It was hypothesized that reductions in dysfunctional parenting (DP) behavior and increases in parental self-efficacy would mediate the stress-reducing effects of WPTP. The mediator effects were analyzed in a German pre- and postcompleter sample (N = 97 parents; 74 mothers and 23 fathers; treatment condition: n = 42 parents; waitlist-control condition n = 55 parents). The results indicate that individual stress- (IS) level reductions were mediated by decreased levels of DP. In addition, changes in IS levels mediated the effects of the training on work-related stress. The discussion focuses on the importance of understanding mechanisms of change to effectively implement interventions at the work-family interface.