Dunn's Multiple Comparison Research Articles

Abstract 4526: Age dependent disposition of cyclophosphamide (CTX) and metabolites in infants ≤ 1 year old with brain tumors

Abstract An ongoing trial of risk-adapted therapy for infants and young children < 3 yrs old diagnosed with brain tumors includes IV CTX (NCT00602667). One aim is to describe the pharmacokinetics (PK) of CTX, and 2 of its metabolites, the inactive carboxyethylphosphoramide mustard (CEPM) and the active 4-hydroxycyclophosphamide (4-OH CTX) in children < 3 yrs old. This report describes the disposition of CTX and metabolites in infants ≤ 1 yr old. Induction therapy includes 1.5 gm/m2 CTX IV over 1 hr on day 9 of a 28 day course. PK studies for CTX were performed in consenting patients and samples were collected prior to drug infusion, at the end of the infusion (EOI), and 3, 6, and 24 h after EOI. Blood was processed to plasma for determination of CTX and CEPM using a LC-MS/MS method. For 4-OH CTX, samples were immediately derivatized using phenylhydrazine to ensure 4-OH CTX stability until analysis using a LC-MS/MS method. A model with one compartment for CTX and each metabolite was fit to the data using nonlinear mixed effects modeling. In 27 infants ≤ 1 yr, 133 samples were obtained and analyzed for CTX, CEPM, and 4-OH CTX. The median (range) age, BSA, and weight were 6.9 months (0.2 - 12 months), 0.4 m2 (0.2 - 0.6 m2), and 8.1 kg (3.6 - 14.1 kg), respectively. As depicted in the Table below, CTX and 4-OH CTX AUC were 50% and 30% higher in the youngest age group, respectively. Our preliminary analysis showed inclusion of age as a covariate on CTX clearance to 4-OH CTX improved model fits, decreasing the variability observed in 4-OH CTX AUC. Since high 4-OH CTX exposure is associated with increased toxicities, our results indicate the need for better models to accurately simulate 4-OH CTX exposures in infants with the goal of developing rational dosing approaches for CTX in this population. This is the first analysis of CTX and metabolite PK in infants ≤ 1 yr old with brain tumors. Future studies will include a full covariate analysis and an evaluation of exposure-toxicity relationships associated with CTX and metabolites. Age group (months)# ptsCTX AUC0-∞ (μM*hr)4-OH CTX AUC0-∞ (μM*hr)0-363556 (2423-6170)†227 (158-263)†3-652854 (2174-3312)174 (170-237)6-972454 (2356-3281)179 (138-220)9-1292461 (1793-2735)175 (136-210)†Kruskal-Wallis, p<0.05; Dunn's Multiple Comparison, p<0.05†Kruskal-Wallis, p = 0.06 Citation Format: Vinay M. Daryani, Thandranese S. Owens, K. Elaine Harstead, Yogesh T. Patel, David C. Turner, Stacy L. Throm, John C. Panetta, Amar Gajjar, Clinton F. Stewart. Age dependent disposition of cyclophosphamide (CTX) and metabolites in infants ≤ 1 year old with brain tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4526. doi:10.1158/1538-7445.AM2015-4526

Time-dependent effect of clonidine on microvascular permeability during endotoxemia

BackgroundEndothelial leakage with accompanying tissue edema and increased leukocyte adhesion are characteristics of the vascular inflammatory response. Tissue edema formation is a key mechanism in sepsis pathophysiology contributing to impaired tissue oxygenation and the development of shock. Sepsis mortality is directly associated with the severity of these microcirculatory alterations. Dysfunction of the sympathetic nervous system can have deleterious effects in generalized inflammation. This study evaluated the effect of the adrenergic alpha 2 agonist clonidine on microvascular permeability and leukocyte adhesion during endotoxemia. MethodsMacromolecular leakage, leukocyte adhesion, and venular wall shear rate were examined in mesenteric postcapillary venules of rats by using intravital microscopy (IVM). Lipopolysaccharide (LPS) (4mg/kg/h) or equivalent volumes of saline were continuously infused following baseline IVM at 0min. IVM was repeated after 60 and 120min in endotoxemic and nonendotoxemic animals. Clonidine (10μg/kg) was applied as an i.v. bolus. Animals received either (i) saline alone, (ii) clonidine alone, (iii) clonidine 45min prior to LPS, (iv) clonidine 10min prior to LPS, (v) clonidine 30min after LPS, or (vi) LPS alone. Due to nonparametric data distribution, Wilcoxon test and Dunn's multiple comparisons test were used for data analysis. Data were considered statistically significant at p<0.05. ResultsLPS significantly increased microvascular permeability and leukocyte adhesion and decreased venular wall shear rate. Clonidine significantly reduced microvascular permeability when applied 45min before or 30min after LPS administration. Leukocyte adhesion and venular wall shear rate were not affected by clonidine during endotoxemia. ConclusionClonidine reduces microvascular permeability in endotoxemic animals in a time-dependent manner. Adrenergic alpha 2 agonists might prove beneficial in stabilizing capillary leakage during inflammation.

364. Impact of Injection Volume on Hydrodynamic Delivery To the Liver in Mice

Hydrodynamic gene delivery is a widely prevailed method for gene delivery to the liver especially in rodents, because of its efficiency and simplicity. A transient enlargement of the liver derived from physical force, which is generated by a rapid injection of a large amount of solution through the tail vein of a mouse, plays an important role for the gene transfer.We previously demonstrated that the expansion speed of the liver is a primary determinant for gene transfer efficiency, because the liver volume at the end of the injection (final volume) was not significantly different between hydrodynamic (5 sec) and slow (60 sec) injections, as long as the same amount of volume of 9% of body weight (BW) was injected (kanefuji, et al. Mol Ther Methods Clin Deve 1;14029). However, the relationship between the injection volume and final volume has not been clarified.The present study aimed to evaluate volume-dependent physical impacts on the liver in hydrodynamic injection. Physical impacts to the liver were quantified in mice by measuring the final volume using a cone beam computed tomography (CBCT) and serum concentration of alanine aminotransferase (ALT). Hydrodynamic (9% of BW/5 sec), half-hydrodynamic (5% of BW/5 sec), and half-slow (5% of BW/60 sec) injections were performed with contrast medium including 300 mg/ml of iodine through the tail vein of mice. Just after the injections, CBCT studies were performed without any surgical intervention to collect volume data of the liver, and the final volumes were shown as the relative volume of that of control mice. Blood samples were collected for the assay of serum concentration of ALT at the time points of 1, 4, 24, 48, and 168 hours after the injections.The average final volumes were 125.8±11.5 and 119.7±4.8% in half-hydrodynamic and half-slow injections, and were not significantly different from each other, while they were significantly lower than that of 173.1±10.4% in hydrodynamic injection (p>0.99 and <0.05, respectively, Kruskal-Wallis followed by Dunn's Multiple Comparison test). The average levels of ALT 4 hours after the injections were 82.6±72.4 U/L and 1582±701.5 U/L in half-hydrodynamic and hydrodynamic injections, which were significantly different from each other (p<0.01, Mann-Whitney test), and returned to the normal level within 48 hours after the injections.These results clearly indicate that the final volume evidently depends on the injection volume but not speed in both hydrodynamic and half-hydrodynamic injections. Furthermore, the reduction of the injection volume markedly suppressed the elevation of liver enzyme in serum after the injection.From a safety viewpoint, there is no doubt that an injection with less volume is advisable as far as sufficient gene delivery is guaranteed. A further study is on going to make the efficacy of half-hydrodynamic injection comparable with that of hydrodynamic injection.

Serological evidence of vector and parasite exposure in Southern Ghana: the dynamics of malaria transmission intensity.

BackgroundSeroepidemiology provides robust estimates for tracking malaria transmission when intensity is low and useful when there is no baseline entomological data. Serological evidence of exposure to malaria vectors and parasite contribute to our understanding of the risk of pathogen transmission, and facilitates implementation of targeted interventions. Ab to Anopheles gambiae salivary peptide (gSG6-P1) and merozoite surface protein one (MSP-119) reflect human exposure to malaria vectors and parasites. This study estimated malaria transmission dynamics using serological evidence of vector and parasite exposure in southern Ghana.MethodsTotal IgG responses to both antigens in an age stratified cohort (<5, 5–14, >14) were measured from South-eastern Ghana. 295 randomly selected sera were analyzed from archived samples belonging to a cohort study that were followed at 3 consecutive survey months (n = 885); February, May and August 2009. Temporal variations in seroprevalence of both antigens as well as differences between the age-stratified cohorts were determined by χ2 test with p < 0.05 statistically significant. Non-parametric repeated ANOVA – Friedman’s test was used to test differences in antibody levels. Seroprevalence data were fitted to reversible catalytic model to estimate sero-conversion rates.ResultsWhereas parasite prevalence was generally low 2.4%, 2.7% and 2.4% with no apparent trends with season, seroprevalence to both gSG6-P1 and MSP119 were high (59%, 50.9%, 52.2%) and 57.6%, 52.3% and 43.6% in respective order from Feb. to August. Repeated measures ANOVA showed differences in median antibody levels across surveys with specific significant differences between February and May but not August by post hoc Dunn’s multiple comparison tests for gSG6-P1. For MSP119, no differences were observed in antibody levels between February and May but a significant decline was observed from May to August. Seroconversion rates for gSG6-P1 increased by 1.5 folds from February to August and 3 folds for MSP119.ConclusionData suggests exposure to infectious bites may be declining whereas mosquito bites remains high. Sustained malaria control efforts and surveillance are needed to drive malaria further down and to prevent catastrophic rebound. Operational factors for scaling up have been discussed.

Inflammatory responses and intestinal injury development during acute Trypanosoma cruzi infection are associated with the parasite load.

BackgroundChagas disease is caused by the protozoan Trypanosoma cruzi and is characterized by cardiac, gastrointestinal, and nervous system disorders. Although much about the pathophysiological process of Chagas disease is already known, the influence of the parasite burden on the inflammatory process and disease progression remains uncertain.MethodsWe used an acute experimental disease model to evaluate the effect of T. cruzi on intestinal lesions and assessed correlations between parasite load and inflammation and intestinal injury at 7 and 14 days post-infection. Low (3 × 102), medium (3 × 103), and high (3 × 104) parasite loads were generated by infecting C57BL/6 mice with “Y”-strain trypomastigotes. Statistical analysis was performed using analysis of variance with Tukey’s multiple comparison post-test, Kruskal–Wallis test with Dunn’s multiple comparison, χ2 test and Spearman correlation.ResultsHigh parasite load-bearing mice more rapidly and strongly developed parasitemia. Increased colon width, inflammatory infiltration, myositis, periganglionitis, ganglionitis, pro-inflammatory cytokines (e.g., TNF-α, INF-γ, IL-2, IL-17, IL-6), and intestinal amastigote nests were more pronounced in high parasite load-bearing animals. These results were remarkable because a positive correlation was observed between parasite load, inflammatory infiltrate, amastigote nests, and investigated cytokines.ConclusionsThese experimental data support the idea that the parasite load considerably influences the T. cruzi-induced intestinal inflammatory response and contributes to the development of the digestive form of the disease.

Identifying Dietary Patterns Associated with Dairy Consumption in British Children

Milk and dairy products are important components of the diet. However, consumption in children remains relatively low. The aim of this study was to identify dietary patterns associated with variations in dairy consumption in British children.&#x0D; Data were derived from the UK National Diet and Nutrition Survey collected in 2008/09‐2011/12 and included a total of 1687 children (828 girls and 859 boys) aged between 4 and 18 years old. Diet was assessed by a four‐day estimated food diary. Fifty food groups were created and were expressed as g/d. Dietary data was organised in quartiles of dairy intake (included milk, yogurt, ice cream, cheese, other milk and cream, and dairy desserts) and dietary patterns were identified using Kruskal‐Wallis with Dunn's multiple comparisons test, to determine significant differences between dairy quartiles and food groups.&#x0D; The lowest dairy quartile (Q1: 0‐93 g dairy/d) was associated with significantly higher intake of soft drinks (P=0.001), chips (P=0.001), coated chicken (P&lt;0.05), chicken and turkey (P=0.05), and significantly lower intake of wholemeal bread (P=0.01), white fish (P=0.05), tea, coffee and water (P=0.05), biscuits (P=0.001), fruit (P=0.001), fibre (P=0.001) puddings (P=0.01) and sugar and preserves (P=0.05) compared with the highest dairy quartile (Q4: 306‐2022 g dairy/d).&#x0D; Overall, British children consuming a diet high in dairy foods had a dietary pattern that would be considered ‘healthy' compared with those consuming a diet low in dairy foods.&#x0D; This work was funded by DairyCo and The Dairy Council UK.

Parasite accumulation in placenta of non-immune baboons during Plasmodium knowlesi infection.

BackgroundPlacental malaria (PM) causes adverse pregnancy outcomes in the mother and her foetus. It is difficult to study PM directly in humans due to ethical challenges. This study set out to bridge this gap by determining the outcome of PM in non-immune baboons in order to develop a non-human primate model for the disease.MethodsTen pregnant baboons were acquired late in their third trimester (day 150) and randomly grouped as seven infected and three non-infected. Another group of four nulligravidae (non-pregnant) infected was also included in the analysis of clinical outcome. Malaria infection was intravenously initiated by Plasmodium knowlesi blood-stage parasites through the femoral vein on 160th day of gestation (for pregnant baboons). Peripheral smear, placental smear, haematological samples, and histological samples were collected during the study period. Median values of clinical and haematological changes were analysed using Kruskal-Wallis and Dunn’s Multiple Comparison Test. Parasitaemia profiles were analysed using Mann Whitney U test. A Spearman’s rank correlation was run to determine the relationship between the different variables of severity scores. Probability values of P <0.05 were considered significant.ResultsLevels of white blood cells increased significantly in pregnant infected (34%) than in nulligravidae infected baboons (8%). Placental parasitaemia levels was on average 19-fold higher than peripheral parasitaemia in the same animal. Infiltration of parasitized erythrocytes and inflammatory cells were also observed in baboon placenta. Malaria parasite score increased with increase in total placental damage score (rs = 0.7650, P <0.05) and inflammatory score (rs = 0.8590, P <0.05). Although the sample size was small, absence of parasitized erythrocytes in cord blood and foetal placental region suggested lack of congenital malaria in non-immune baboons.ConclusionThis study has demonstrated accumulation of parasitized red blood cells and infiltration of inflammatory cells in the placental intravillous space (IVS) of baboons that are non-immune to malaria. This is a key feature of placental falciparum malaria in humans. This presents the baboon as a new model for the characterization of malaria during pregnancy.

A Quantitative Analysis of a Probiotic Storage Media for Avulsed Teeth.

The aim of the present in vitro study was to investigate the potential of a storage medium, probiotic yogurt (Bifidibacterium animalis DN 173010) in comparison with Hank's balanced salt solution (HBSS), saline and milk in maintaining viable periodontal ligament (PDL) cells on simulated avulsed teeth. Thirty-six freshly extracted single-rooted human teeth with closed apices were divided into six experimental groups (N=6). The teeth were extracted as atraumatically as possible and washed in sterile saline solution to eliminate residual blood. Following extractions, the coronal 3 mm of PDL tissues were scraped with a #15 scalpel to remove cells that may have been damaged. The positive and negative controls corresponded to 0 minutes and an 8-hour dry time, respectively. After extraction, the positive control teeth were immediately treated with dispase and collagenase. The negative control teeth were bench-dried for 8 h, with no follow-up storage solution time, and then placed in the dispase and collagenase. The number of viable protective least significant difference PDL cells were counted under a light microscope with a hemocytometer at 20× magnification and analyzed. Statistical analysis of the data was accomplished using Nonparametric ANOVA complemented by Kruskal-Wallis Test and Dunn's Multiple Comparisons Test. Positive control was found to be significantly better than the others, there were statistically significant differences between positive control and other test groups (p=0.000). The teeth stored in positive control demonstrated the highest number of viable PDL cells followed in order by probiotic yogurt, HBSS, saline and milk. Bifidibacterium animalis DN 173010 seems to be an alternative for the temporary storage of avulsed teeth, due to high number of viable PDL cells. Probiotics may be suitable transport media for avulsed teeth, but further research is warranted using the commercially available products.

Development of novel formulations containing Lysozyme and Lactoferrin and evaluation of antibacterial effects on Mutans Streptococci and Lactobacilli

ObjectiveThe aim of this study was to determine the antibacterial effect of different formulations containing Lysozyme and Lactoferrin and drug delivery system as well as poloxamer 407 with the trade name of Pluronic F-127 and/or freeze dried liposome containing DOTAP [freeze dried Liposomal DOTAP] on Streptococcus sobrinus, Streptococcus mutans and Lactobacillus acidophilus in comparison with 0.2% chlorhexidine. Materials and methodsThe antibacterial effect was assessed by determining the minimum inhibitory concentrations (MIC) for the study and control groups on Streptococcus sobrinus, Streptococcus mutans and Lactobacillus acidophilus. The amounts of biofilm formation accumulation of Mutans Streptococci for 24h on sterile hydroxyapatite discs after application of different formulations were evaluated. The different formulations studied were: (1) Sorensen's Buffer Solution, (2) a gel formulation containing only poloxamer 407, (3) Lysozyme and Lactoferrin dissolved in Sorensen's Buffer Solution, (4) poloxamer 407 combined with the third formulation, (5) Freeze dried Liposomal DOTAP dissolved in Sorensen's Buffer Solution, (6) Freeze dried Liposomal DOTAP combined with poloxamer 407 dispersed in Sorensen's Buffer Solution, (7) Freeze dried Liposomal DOTAP combined with the third formulation, and (8) Lysozyme and Lactoferrin dissolved in Sorensen's Buffer Solution, which was then incorporated into poloxamer 407 and combined with Freeze dried Liposomal DOTAP. The positive and negative control groups were 0.2% chlorhexidine gel and empty hydroxyapatite discs, respectively. Statistical evaluation was carried out with Kruskal–Wallis and Dunn's multiple comparison tests. ResultsIt was observed that the first, third and fifth groups did not have any antibacterial effects on the tested bacteria. The groups that contained poloxamer 407 had nearly identical antibacterial effects on Mutans Streptococci and L. acidophilus. These formulations also inhibited biofilm formation of the bacteria (p<0.05) more effectively. In the positive control group, there was no biofilm formation. ConclusionsAmong the formulations containing poloxamer 407, the one containing Lysozyme and Lactoferrin exhibited the highest inhibitory effect on the tested bacteria. This novel formulation can be beneficial as an antibacterial agent for the prevention of dental caries and biofilm formation.

The characteristics of serum high-density lipoprotein cholesterol levels in urban population from Tangshan City

Objective To understand the characteristics of High-density lipoprotein cholesterol(HDL-C) distribution through analyzing serum HDL-C levels in healthy checkup subjects among local urbanese. Methods The checkup results of 36 454 cases were collected from 2009 to 2013 in Tangshan Gongren hospital(male: n=20 343, female: n=16 111). The cases with liver injury, abnormal blood glucoses , kidney injury and defined cerebrovascular diseases and metabolic disease were excluded. 25 197 cases were analyzed as normal subjects including 11 114 males and 14 083 females. Kolmogorov-Smimov test, kruskal-wallis test, and Dunn multiple comparison test was performed using Bioconductor software 3.0.2 for testing normality distribution and comparing the difference of two or multiple groups, respectively. Results were analyzed statistically with R 3.0.2. Results The results showed that the average level (1.22 ± 0.31) mmol/L of HDL-C among the overall population is lower than that of national average level(1.30 mmol/L). The median serum HDL-C level in female is higher than in male (1.27 and 1.08, χ2=2 606.34, P<0.01). HDL-C levels in male continuously increase from 1.06 mmol/L to 1.11 mmol/L with aging, especially in groups of over 50 years old than in groups of below 50 years old (χ2=75.19, P<0.01). Conclusions Based on 2007 guidance on prevention and treatment by national health bureau, this study showed that there are 29.69% of the apparent healthy subjects, especially about 42.94% of the male, representing low HDL-C level under the low limit of 1.04 mmol/L. These results showed that serum HDL-C level in Tangshan urbanese is lower than that of national average level, and HDL-C level in male is tended to increase with aging. Key words: Cholesterol, HDL; Physical examination; Retrospective studies

The Alpha-defensin Test for Periprosthetic Joint Infection Responds to a Wide Spectrum of Organisms.

BackgroundThe alpha-defensin test has been previously demonstrated to be highly accurate in the diagnosis of prosthetic joint infection (PJI), nearly matching the Musculoskeletal Infection Society definition for PJI. However, the relationship between alpha-defensin levels and differing infecting organism has not yet been investigated.Questions/purposesThe purpose of this study is to describe the breadth of organisms that can trigger a positive synovial fluid alpha-defensin test result in the setting of PJI and also to assess the magnitude of the alpha-defensin result in terms of various pathogen characteristics.MethodsBetween December 2012 and March 2014, one laboratory processed 2319 synovial fluid samples for alpha-defensin testing. The present study reviewed the results of the 1937 samples that simultaneously had a synovial fluid culture performed; these came from 418 surgeons in 42 states. The overall culture-positive rate was 49% (244 of 498) among alpha-defensin-positive synovial fluids and 1% (19 of 1439) among alpha-defensin-negative synovial fluids. The organisms recovered from 244 alpha-defensin-positive, culture-positive fluids were recorded and grouped based on various characteristics, including Gram type, species, virulence, oral pathogenicity, and source joint. Alpha-defensin-negative samples served as uninfected controls. Median alpha-defensin levels were calculated for each group, and Dunn’s multiple comparison test for nonparametric data was used to identify any statistically significant (p < 0.05) organism-specific differences in the alpha-defensin level.ResultsThe alpha-defensin test for PJI was positive in the setting of a wide spectrum of organisms typically causing PJI. The median alpha-defensin level for all 244 alpha-defensin-positive, culture-positive samples (4.7 [interquartile range {IQR}, 3.7–5.3]) was higher than negative controls (0.26 [IQR, 0.22–0.33]) with a median difference of 4.4 (p < 0.001). There were no differences in the median alpha-defensin levels when performing a multiple comparison test among Gram-positive organisms (4.7 [IQR, 3.6–5.3]), Gram-negative organisms (4.8 [IQR, 4.2–5.3]), yeast (4.1 [IQR, 2.2–5.1]), virulent organisms (4.7 [IQR, 3.8–5.2]), less virulent organisms (4.8 [IQR, 3.6–5.4]), oral pathogens (4.5 [IQR, 3.2–5.2]), knees (4.7 [IQR, 3.7–5.3]), hips (4.9 [IQR, 4.1–5.8]), or shoulders (5.3 [IQR, 4.0–10.7]) with all comparisons having a p > 0.999.ConclusionsThe alpha-defensin test provides consistent results regardless of the organism type, Gram type, species, or virulence of the organism and should be seriously considered to be a standard diagnostic tool in the evaluation for PJI. Future research should focus on the performance of this test in specific clinical scenarios such as the immediate postoperative period in the setting of severe immunocompromise and in the setting of a native joint.Level of EvidenceLevel III, diagnostic study.

National survey of back & neck pain amongst consultant ophthalmologists in the United Kingdom.

Repetitive tasks, awkward or prolonged working postures, and high cognitive load are risk factors for occupational musculoskeletal disorders. Ophthalmologists may be vulnerable given that they are exposed to a combination of these factors. This national study assesses the prevalence, severity and associations of back and neck pain amongst UK consultant ophthalmologists. A postal survey was conducted using addresses supplied by the Royal College of Ophthalmologists. Statistical analysis was performed using Pearson correlation coefficient, two-tailed probability testing, analysis of variance (ANOVA) and Dunn's multiple comparison test. 518 responses were received (50.3 % response rate). Back and neck pain were reported by 50.6 % (262/518) and 31.8 % (165/518) of respondents, respectively, with 62.4 % (323/518) reporting one or both. 33.6 % (174/518) reported pain whilst operating, of whom 78.7 % (137/174) found operating exacerbated their pain. 31.7 % (164/518) reported pain when using the slit lamp, of whom 71.3 % (117/164) found it exacerbated their pain. Individual subspecialties showed a significant relative risk of back or neck pain in some circumstances, when compared to ophthalmologists as a whole. Occupational back and neck pain remains a problem amongst ophthalmologists. Recommendations are made for modifications to the working environment, and consideration should be given to improving education for trainees.

Sleep-wake time perception varies by direct or indirect query.

The diagnosis of insomnia rests on self-report of difficulty initiating or maintaining sleep. However, subjective reports may be unreliable, and possibly may vary by the method of inquiry. We investigated this possibility by comparing within-individual response to direct versus indirect time queries after overnight polysomnography. We obtained self-reported sleep-wake times via morning questionnaires in 879 consecutive adult diagnostic polysomnograms. Responses were compared within subjects (direct versus indirect query) and across groups defined by apnea-hypopnea index and by self-reported insomnia symptoms in pre-sleep questionnaires. Direct queries required a time duration response, while indirect queries required clock times from which we calculated time durations. Direct and indirect queries of sleep latency were the same in only 41% of cases, and total sleep time queries matched in only 5.4%. For both latency and total sleep, the most common discrepancy involved the indirect value being larger than the direct response. The discrepancy between direct and indirect queries was not related to objective sleep metrics. The degree of discrepancy was not related to the presence of insomnia symptoms, although patients reporting insomnia symptoms showed underestimation of total sleep duration by direct response. Self-reported sleep latency and total sleep time are often internally inconsistent when comparing direct and indirect survey queries of each measure. These discrepancies represent substantive challenges to effective clinical practice, particularly when diagnosis and management depends on self-reported sleep patterns, as with insomnia. Although self-reported sleep-wake times remains fundamental to clinical practice, objective measures provide clinically relevant adjunctive information.

Inflammatory markers in gingival crevicular fluid of periodontitis patients with type 2 diabetes mellitus according to glycemic control: A pilot study.

Type 2 diabetes mellitus (T2DM) and periodontitis are inflammatory conditions with a bidirectional association. This pilot study aimed to evaluate whether T2DM and glycemic control interfere in inflammatory markers profiles in gingival crevicular fluid (GCF) in periodontitis patients. Fourteen diabetic periodontitis patients were enrolled in this study, seven with adequate glycemic control (glycated hemoglobin [HbA1c] <8.0%) (DMA + P) and seven with inadequate control (HbA1c ≥8.0%) (DMI + P). Seven chronic periodontitis patients without diabetes formed the control group (P). GCF was obtained from diseased sites (probing depth >6 mm) of an entirely hemiarch, pooled and cytokines levels determined using multiplex beads immunoassay. Clinical periodontal parameters were analyzed by Mann-Whitney test and levels of cytokines by Kruskal-Wallis and Dunn's multiple comparison tests with confidence level of 95% (P < 0.05). Cytokines profile of GCF obtained from deep periodontal pockets presented high levels of inflammatory cytokines, and there were no statistical differences between levels of interleukin-6 (IL-6), IL-8 and tumor necrosis factor-α according to presence of diabetes or percentage of HbA1c among the groups, despite groups with T2DM and periodontitis exhibit higher levels of PD. Within the limitations of this study, inflammatory mediators in GCF are dependent to the local response and do not correlate with the diabetic status.

Honey as an alternative fixative for oral tissue: An evaluation of processed and unprocessed honey.

Background:Fixation is the most imperative step in the practice of diagnostic histopathology, which is intimately linked to 10% formalin. As a result of increasing concerns about the potential carcinogenicity of the formaldehyde, attempt to find safer alternatives is necessary. Honey has been shown to possess antimicrobial, antiviral and antimutagenic properties. Many studies have reported that honey possesses dehydrating and preserving effects also.Aims and Objectives:To study the fixative properties of processed and unprocessed honey in oral tissues followed by comparision with formalin.Materials and Methods:The study group comprised 12 different tissues. Each tissue was cut into 3 segments and were immediately fixed in bottles containing 10% unprocessed honey, 10% processed honey and 10% formalin, respectively, for 24 h at room temperature. After fixation, tissues were processed using the routine standard processing protocol followed by hematoxylin and eosin staining. Data were statistically analyzed using descriptive statistics such as mean, standard deviation and percentage. Kruskal–Wallis test followed by post hoc Dunn's multiple comparisons test and Chi-square test or Fisher's exact test for small sample size. A P < 0.05 was considered as significant. Data analysis was done by using software Minitab v14.0.Results:When all the stained sections were assessed for the parameters, there was no statistically significant difference between tissues fixed in processed and unprocessed honey compared to formalin (P = 0.004). The tissue morphology and staining adequacy for diagnosis in honey fixed tissue was at par with formalin fixed tissue. Hence, our results suggest that both processed honey and unprocessed honey can be used as a safe alternative for formalin.

The effect of different drinks on the color stability of different restorative materials after one month

ObjectivesThe aim of this study was to evaluate the effect of three different drinks on the color parameters of four different restorative materials.Materials and MethodsThree different composites (Filtek Ultimate Universal Restorative, Filtek Ultimate Flowable, and Filtek Silorane, 3M ESPE) and a polyacid-modified composite resin material (Dyract XP, Dentsply DeTrey GmbH) were evaluated. Eighty-four disc-shaped specimens of 8 mm in diameter and 2 mm in thickness were prepared (n = 21 each). Color coordinates (L*a*b*, ΔL*, Δa*, Δb*, and ΔE*) were measured using a VİTA Easyshade Compact (VİTA Zahnfabrik) after 24 hr of storage (baseline) and after 30 day of storage in three different beverages of black tea, Coca cola, or water (control) (n = 7). In each beverage, the specimens were stored three times a day, one hr each, for 30 day. The color changes (ΔE) were calculated and were analyzed by Kruskal-Wallis and Dunn multiple comparison test.ResultsThe color difference (ΔE*) of the resin materials ranged between 1.31 and 15.28 after 30 day of immersion in the staining solutions. Dyract XP in Coca cola (15.28 ± 2.61) and black tea (12.22 ± 2.73) showed the highest mean ΔE* value after 30 day, followed by Filtek Ultimate Universal Restorative (5.99 ± 1.25) and Filtek Ultimate Flowable (4.71 ± 1.40) in black tea (p < 0.05).ConclusionsThe compomers displayed unacceptable color changes at the end of 30 day in all beverages. Among resin composites, the silorane based composite exhibited relatively good color stability than the others. Filtek Ultimate Universal Restorative and Filtek Flowable showed similar color changes in all beverages.

33 TELOMERASE ACTIVITIES IN CLONED BEAGLE DOGS

Telomerase is important ribonucleoprotein for restoring telomere length from its own RNA template. Regarding cloned animals derived from somatic cell nuclear transfer (SCNT), interesting questions have been raised about whether the cloning process restores cellular telomerase activity undergone by their donor cells. The present study was conducted to determine the effects of cloning on telomerase activity in the dog and normality of telomerase activity in cloned dogs. Focusing our attention on differences in telomerase activity depending on the age, we analysed telomerase activity in dogs produced by natural breeding of various ages. Comparison of the telomerase activities of cloned dogs and those of dogs produced by natural breeding was also performed. For SCNT, 2 cell donors, 7- and 9-year-old beagles, were used and donor cells were isolated from ear skin. After establishing donor cell lines, the enucleated canine in vivo-matured oocytes and the cells were injected and fused by electrofusion. After 30 days from embryo transfer, pregnancy diagnosis was performed and 7 cloned dogs were produced on the due date. For standardization of telomerase activity in beagles produced by natural breeding, blood of total 14 dogs at each age (10 months, 20 months, 5, 7, and 8 years old) were collected and telomerase activity was measured by the telomeric repeat amplification protocol (TRAP) assay. Telomerase activity measurements of at least 6 replications in each dog were performed. For statistical analysis, one-way ANOVA with Dunn's Multiple Comparison Test was used. Significant differences in telomerase activity were observed between the blood of cloned and donor dogs. It was shown that mean telomerase activities were decreased according to biological aging with significances. Mean telomerase activities in 10 cloned dogs were higher than those of a donor dog. Cloned dogs also showed similar levels of telomerase activities as their age-matched natural bred dogs, suggesting that they are within the variation in normal dogs. These observations indicate that the cloning process restores the telomerase activity in the dog. Thus, complex regulation of telomerase activity during nuclear reprogramming may regulate and be involved in telomerase activity in cloned dogs. It remains to be determined whether telomere length is correlated with telomerase activity and if it accurately reflects the physiological age of cloned dogs.This study was supported by IPET (#311062–04–2-SB010), RDA (PJ008975022013), Research Institute for Veterinary Science, the BK21 program, Nestle Purina Korea, and TS Corporation.

311 TEMPERATURE DURING OVERNIGHT HOLDING IN MEIOSIS INHIBITOR-FREE MEDIUM AFFECTS CHROMATIN CONFIGURATION AND MEIOTIC RESUMPTION IN EQUINE OOCYTES

Immature equine oocytes may be held overnight in an Earle's/Hanks' M199-based medium in the absence of meiotic inhibitors (EH medium) to schedule the onset of in vitro maturation. Holding in EH has been shown not to affect meiotic or developmental competence of equine oocytes (Choi et al. 2006 Theriogenology 66, 955–963). However, no studies have been performed to identify the mode by which this medium suppresses meiosis. We hypothesised that holding temperature may affect oocyte meiotic arrest. The effect of 3 holding temperatures (25, 30, 38°C) on chromatin status was investigated after Hoechst 33258 staining (Hinrichs et al. 2005 Biol. Reprod. 72, 1142–1150). Oocytes were recovered by scraping of follicles from slaughterhouse-derived ovaries. Data were analysed by Chi-squared test and one-way ANOVA followed by Dunn's or Holm-Sidak Multiple Comparison methods. A level of P &lt; 0.05 was considered significant. There were no significant differences in chromatin configuration between oocytes held overnight at 25°C (25°C-held) and controls (immediately-fixed oocytes); the proportion of oocytes showing meiotic resumption was 1/27, 4% and 0/26, 0%, respectively (not significant, NS). In contrast, holding at higher temperature significantly increased meiosis resumption (14/38, 37% and 14/28, 50%, at 30 and 38°C, respectively; P &lt; 0.01) and reduced the proportion of oocytes showing the most meiotically-competent germinal-vesicle (GV) configuration (condensed chromatin, CC; 24 to 29% v. 65 to 70% for control and 25°C-held, respectively; P &lt; 0.05). Based on these results, a subsequent experiment was performed in which oocyte meiotic stage and mitochondrial (mt) potential of 25°C-held (n = 29) and control (n = 36) oocytes was evaluated. Nuclear chromatin, mt activity (MitoTracker orange), intracellular reactive oxygen species (ROS) levels (2′,7′-dichlorodihydrofluorescein diacetate, DCDHFDA), and mt/ROS colocalization (Pearson's coefficient) were analysed by epifluoscence and confocal microscopy (Martino et al. 2012 Fertil. Steril. 97, 720–728). Meiotic arrest after EH treatment at 25°C was confirmed (0/29, 0% v. 5/36, 14% for meiotic resumption in 25°C-held and controls, respectively; NS). At any GV stage, 25°C-held treatment had no effect on mt activity, ROS levels, or mt/ROS colocalization. For example, in CC oocytes, values for control and 25°C-held, respectively, were: MitoTracker, 547.8 ± 499.5 v. 722.9 ± 390.3; DCF fluorescence intensity, 278.5 ± 179.3 v. 378 ± 185, and mt/ROS colocalization, 0.5 ± 0.1 v. 0.5 ± 0.2; these were not significantly different (NS). In conclusion, EH holding at 25°C maintains meiotic arrest, viability, and mt potential of equine oocytes.

Vitamin D enhances production of soluble ST2, inhibiting the action of IL-33

Vitamin D enhances production of soluble ST2, inhibiting the action of IL-33

Polymorphisms of CYP1B1, CYP3A4, CYP3A5, GSTM1, GSTTT1 and SULT1A1 Associated with Early Age Acute Leukemia

The acute leukemia causation pathway has been postulated to be a multistep process with interaction between environmental and genetic susceptibility factors. Based on previous observation, early age leukemia (EAL) was associated with maternal hormone exposures.The aim of the study was to explore whether polymorphisms in genes of the estrogen-metabolism pathway could be related to EAL. The study included 754 children, being 350 EAL and 404 age-matched controls and 134 mothers of EAL and controls. Polymorphisms of CYP1B1 (rs1056836), CYP3A4 (rs2740574), and CYP3A5 (rs776746) were identified by RFLP; GSTM1 and GSTT1 deletions were detected by multiplex PCR, whilst an internal control (CYP1A1) was co-amplified; SULT1A1 rs9282861 and rs1801030 were genotyped on PyroMark Q24. Gene expression was performed by Real-time PCR using Platinum® SYBR® Green qPCR SuperMix. Logistic regression was used to evaluate the associations adjusted on skin color. The risk was determined by calculating odds ratios (ORs) with 95% confidence interval (CI). CYP1B1 and CYP3A5 expression analyzed using Kruskal-Wallis and Dunn's Multiple Comparison tests. GSTT1*0, CYP3A4*1B, and CYP3A5*1 were associated with an increased risk of acute myeloid leukemia (AML) after adjusting for multiple comparisons regarding non-whites and male children (OR 1.91, 95% CI: 1.12, 3.28; OR 2.44, 95% CI: 1.10, 5.43; OR 1.98, 95% CI: 0.96, 4.10, respectively). CYP1B1 was not expressed in 57.1% of EAL cases; expression varied by genotype, gender, and leukemia subtype. AML was associated with distinct modulations: CYP1B1*3/SULT1A1*3 decreased and CYP3A4*1B/SULT1A1*2 increased EAL risk (OR 0.24, 95% CI: 0.10, 0.55; OR 2.06, 95% CI: 0.96, 4.42, respectively). Maternal-fetal genotypes were associated with EAL based on CYP3A4*1B and GSTT1*0. Therefore, genetic polymorphisms involved in estrogen metabolism confer different susceptibilities for EAL, modulating leukemia subtype in males.