Proportion Of White Blood Cells Research Articles

Clinical analysis on the therapeutic effect of reduning injection in acute upper respiratory infection

Objective: This study aims to investigate the clinical value of reduning injection in treating acute upper respiratory infection. Methods: A total of 72 patients with acute upper respiratory infection in our hospital from March 2015 to February 2017 were selected and divided into the control and observation groups randomly by the odd-even method. The control group (36 cases) was treated by ribavirin injection, whereas the observation group (36 cases) was treated by reduning injection. The effect of clinical therapies, improvement of symptoms, normal proportions of White Blood Cells (WBCs) and neutrophils, and statistical results of occurrence rate of untoward effect between the two groups were compared. Results: The observation group presents significantly higher therapeutic effect (97.22%) than that in the control group (58.33%) (P<0.05). The improvement time of symptoms in the observation group is shortened significantly compared with that in the control group (P<0.05). The observation group shows dramatically higher proportions of WBCs and neutrophils than those in the control group (P<0.05). The occurrence rate of untoward effect in the observation group is 2.78%, which is reduced dramatically compared with that in the control group (22.22%) (P<0.05). Conclusions: Reasonable reduning injection in clinical treatment of acute upper respiratory infection can guarantee improvement in therapeutic effect, shortening in improvement time of symptoms, growth in proportions of WBCs and neutrophils, and reduction in untoward effect. This approach can optimize prognosis of patients with acute upper respiratory infection.

Serum lipid, enzyme, and hematological responses of broilers fed Acacia angustissima leaf meal-based diets.

The objective of the study was to determine serum lipid, enzyme, and hematological responses of broilers to Acacia angustissima leaf meal. Acacia angustissima leaves were harvested at mid maturity, ground into a leaf meal, and included in broiler diets at 0, 5, and 10%. One hundred and fifty-day-old chicks of 41.7 ± 1.560g were randomly allocated to 15 groups of the three diets. Feed and water were provided ad libitum throughout the trial. At days 14, 28, and 42, 4ml of blood were collected from the jugular vein in plain vacutainer tubes for alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and total cholesterol (TC) analyses. At the end of the experiment, 4ml of blood were collected from two randomly selected birds of each replicate in sterilized bottles containing ethylene diamine tetra acetic acid (EDTA) for full blood count and packed cell volume assessment. Data was tested for normality and analyzed using the General Linear Model Procedure of SAS version 9.3 (SAS 2010). Comparison of means was done using Tukey's test. A. angustissima had no effect on serum ALP, AST, LDH, TC, HDL-C, LDL-C, TG, full blood count, and packed cell volume. Increasing levels of A. angustissima leaf meal increased the ALT in the serum (p <0.05) at 4 and 6weeks. However, serum ALT indices for birds on the control and 5% diet were not different. It was concluded that 5% A. angustissima leaf meal can be included in broiler diets without a negative effect on serum lipid and enzyme profile, production and proportions of white blood cells and packed cell volume.

Influence of cell distribution and diabetes status on the association between mitochondrial DNA copy number and aging phenotypes in the InCHIANTI study

SummaryMitochondrial DNA copy number (mtDNA‐CN) estimated in whole blood is a novel marker of mitochondrial mass and function that can be used in large population‐based studies. Analyses that attempt to relate mtDNA‐CN to specific aging phenotypes may be confounded by differences in the distribution of blood cell types across samples. Also, low or high mtDNA‐CN may have a different meaning given the presence of diseases associated with mitochondrial damage. We evaluated the impact of blood cell type distribution and diabetes status on the association between mtDNA‐CN and aging phenotypes, namely chronologic age, interleukin‐6, hemoglobin, and all‐cause mortality, among 672 participants of the InCHIANTI study. After accounting for white blood cell count, platelet count, and white blood cell proportions in multivariate models, associations of mtDNA‐CN with age and interleukin‐6 were no longer statistically significant. Evaluation of a statistical interaction by diabetes status suggested heterogeneity of effects in the analysis of mortality (P < 0.01). The magnitude and direction of associations between mtDNA‐CN estimated from blood samples and aging phenotypes are influenced by the sample cell type distribution and disease status. Therefore, accounting for these factors may aid understanding of the relevance of mitochondrial DNA copy number to health and aging.

Nanotechnology-based selection of boar spermatozoa: growth development and health assessments of produced offspring

Abstract The heterogeneous population of spermatozoa within the semen ejaculate influences the sire fertility. The current design of magnetic nanoparticle conjugates allows for selective targeting and removal of non-viable spermatozoa within the semen ejaculate. However, the safe application of this process, termed as nanoselection or nanopurification in previous studies, in food animal production and toxicity concerns has yet to be explored. Here, we assessed the fertility potential of nanoselected boar spermatozoa and the subsequent post-natal growth and health characteristics of resulting offspring. Semen doses were harvested from three fertile boars (n=4 doses per boar) and split in two groups (2 doses/boar). Six semen doses (2 per boar)) were subjected to the targeted depletion of acrosome membrane damaged and apoptotic spermatozoa (nanoselected). Meanwhile, the remaining semen doses (n=6) were maintained in the shipping Styrofoam box (control). The motility characteristics of both control and nanoselected spermatozoa were evaluated before and after nanoselection, followed by their use for double inseminations of six estrus synchronized gilts (2 doses/boar/gilt; 3 gilts /control or nanoselected). In comparison to the controls, the computer-assisted sperm analyzer (Hamilton-Thorne) revealed greater motion characteristics of nanoselected spermatozoa, with a significantly higher proportion of progressive spermatozoa and straightness (P 0.05). Various developmental and health parameters of produced offspring such as hepatic cytochrome P450 enzyme activities, blood glucose and immunoglobulin G concentrations, hematocrit, and white blood cell proportions were similar between and across all pigs (control and nanoselected). In addition, reproductive tracts of females born from nanoselected spermatozoa showed no indication of impaired fertility potential, although a significant shortened uterine horn length was measured (56.3±2.6 cm vs. 64.4±2.2 cm in the control group, P=0.04). In conclusion, findings revealed no obvious perturbations of sperm function following nanoselection, while post-natal growth, development, or health data of derived offspring suggest absence of inflicted sub-lethal toxicities attributable to sperm nanoselection. This study supports the safe use of the proposed nanotechnology-based selection for effective semen handling in terminal line swine production systems.

CRP polymorphisms and DNA methylation of the AIM2 gene influence associations between trauma exposure, PTSD, and C-reactive protein

BackgroundRecent studies have implicated inflammatory processes in the pathophysiology of posttraumatic stress disorder (PTSD). C-reactive protein (CRP) is a widely-used measure of peripheral inflammation, but little is known about the genetic and epigenetic factors that influence blood levels of C-reactive protein (CRP) in individuals with PTSD. MethodsParticipants were 286 U.S. military veterans of post-9/11 conflicts (57% with current PTSD). Analyses focused on single nucleotide polymorphisms (SNPs) in the CRP gene and DNA methylation at cg10636246 in AIM2—a locus recently linked to CRP levels through results from a large-scale epigenome-wide association study. ResultsPTSD was positively correlated with serum CRP levels with PTSD cases more likely to have CRP levels in the clinically-elevated range compared to those without a PTSD diagnosis. Multivariate analyses that controlled for white blood cell proportions, genetic principal components, age and sex, showed this association to be mediated by methylation at the AIM2 locus. rs3091244, a functional SNP in the CRP promoter region, moderated the association between lifetime trauma exposure and current PTSD severity. Analyses also revealed that the top SNPs from the largest genome-wide association study of CRP conducted to date (rs1205 and rs2794520) significantly interacted with PTSD to influence CRP levels. ConclusionsThese findings provide new insights into genetic and epigenetic mechanisms of inflammatory processes in the pathophysiology of PTSD and point to new directions for biomarker identification and treatment development for patients with PTSD.

Cadmium exposure and age-associated DNA methylation changes in non-smoking women from northern Thailand.

DNA methylation changes with age, and may serve as a biomarker of aging. Cadmium (Cd) modifies cellular processes that promote aging and disrupts methylation globally. Whether Cd modifies aging processes by influencing establishment of age-associated methylation marks is currently unknown. In this pilot study, we characterized methylation profiles in > 450 000 CpG sites in 40 non-smoking women (age 40–80) differentially exposed to environmental Cd from Thailand. Based on specific gravity adjusted urinary Cd, we classified them as high (HE) and low (LE) exposed and age-matched within 5 years. Urinary Cd was defined as below 2 µg/l in the LE group. We predicted epigenetic age (DNAm-age) using two published methods by Horvath and Hannum and examined the difference between epigenetic age and chronologic age (Δage). We assessed differences by Cd exposure using linear mixed models adjusted for estimated white blood cell proportions, BMI, and urinary creatinine. We identified 213 age-associated CpG sites in our population (P < 10−4). Counterintuitively, the mean Δage was smaller in HE vs. LE (Hannum: 3.6 vs. 7.6 years, P = 0.0093; Horvath: 2.4 vs. 4.5 years, P = 0.1308). The Cd exposed group was associated with changes in methylation (P < 0.05) at 12, 8, and 20 age-associated sites identified in our population, Hannum, and Horvath. From the results of this pilot study, elevated Cd exposure is associated with methylation changes at age-associated sites and smaller differences between DNAm-age and chronologic age, in contrast to expected age-accelerating effects. Cd may modify epigenetic aging, and biomarkers of aging warrant further investigation when examining Cd and its relationship with chronic disease and mortality.

Epigenome-wide Association of DNA Methylation in Whole Blood With Bone Mineral Density.

ABSTRACTGenetic and environmental determinants of skeletal phenotypes such as bone mineral density (BMD) may converge through the epigenome, providing a tool to better understand osteoporosis pathophysiology. Because the epigenetics of BMD have been largely unexplored in humans, we performed an epigenome‐wide association study (EWAS) of BMD. We undertook a large‐scale BMD EWAS using the Infinium HumanMethylation450 array to measure site‐specific DNA methylation in up to 5515 European‐descent individuals (NDiscovery = 4614, NValidation = 901). We associated methylation at multiple cytosine‐phosphate‐guanine (CpG) sites with dual‐energy X‐ray absorptiometry (DXA)‐derived femoral neck and lumbar spine BMD. We performed sex‐combined and stratified analyses, controlling for age, weight, smoking status, estimated white blood cell proportions, and random effects for relatedness and batch effects. A 5% false‐discovery rate was used to identify CpGs associated with BMD. We identified one CpG site, cg23196985, significantly associated with femoral neck BMD in 3232 females (p = 7.9 × 10−11) and 4614 females and males (p = 3.0 × 10−8). cg23196985 was not associated with femoral neck BMD in an additional sample of 474 females (p = 0.64) and 901 males and females (p = 0.60). Lack of strong consistent association signal indicates that among the tested probes, no large‐effect epigenetic changes in whole blood associated with BMD, suggesting future epigenomic studies of musculoskeletal traits measure DNA methylation in a different tissue with extended genome coverage. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

Epigenome-wide association study (EWAS) on lipids: the Rotterdam Study

BackgroundDNA methylation is a key epigenetic mechanism that is suggested to be associated with blood lipid levels. We aimed to identify CpG sites at which DNA methylation levels are associated with blood levels of triglycerides, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and total cholesterol in 725 participants of the Rotterdam Study, a population-based cohort study. Subsequently, we sought replication in a non-overlapping set of 760 participants.ResultsGenome-wide methylation levels were measured in whole blood using the Illumina Methylation 450 array. Associations between lipid levels and DNA methylation beta values were examined using linear mixed-effect models. All models were adjusted for sex, age, smoking, white blood cell proportions, array number, and position on array. A Bonferroni-corrected p value lower than 1.08 × 10−7 was considered statistically significant. Five CpG sites annotated to genes including DHCR24, CPT1A, ABCG1, and SREBF1 were identified and replicated. Four CpG sites were associated with triglycerides, including CpG sites annotated to CPT1A (cg00574958 and cg17058475), ABCG1 (cg06500161), and SREBF1 (cg11024682). Two CpG sites were associated with HDL-C, including ABCG1 (cg06500161) and DHCR24 (cg17901584). No significant associations were observed with LDL-C or total cholesterol.ConclusionsWe report an association of HDL-C levels with methylation of a CpG site near DHCR24, a protein-coding gene involved in cholesterol biosynthesis, which has previously been reported to be associated with other metabolic traits. Furthermore, we confirmed previously reported associations of methylation of CpG sites within CPT1A, ABCG1, and SREBF1 and lipids. These results provide insight in the mechanisms that are involved in lipid metabolism.

Validated flow cytometry allows rapid quantitative assessment of immune responses in amphibians

Assessments of amphibian immune state have been commonly made through indirect methods like phytohemagglutinin (PHA) injections, or by direct methods like cell counts from blood smears. Here we validate a simple method to assess immune responses in amphibians by means of flow cytometry with a fluorescent lipophilic dye (3,3′ Dipentyloxacarbocyanine), which removes the need for specific antibodies. We experimentally altered the immunological state ofPelobates cultripestadpoles by exposing some to exogenous corticosterone. We then determined the immune state of each tadpole through both blood smears and flow cytometry. We found that both techniques showed similar patterns of the proportion of white blood cells. Once validated, flow cytometry also allowed quantitation of changes in absolute number of white cells. We discuss the suitability of both techniques attending to the accuracy of each technique, body size requirements, or the tractability in field studies.

Leukemic Cell Expressed CTLA-4 Suppresses T Cells Via Down-Modulation of CD80 By Trans-Endocytosis

The function of CTLA-4 on non-T cells is largely ignored and currently ill defined despite rapidly growing interest in targeting this immune checkpoint protein in several cancers. While anti-CTLA-4 therapy is proposed to work through inhibition of the immunosuppressive effect of CTLA-4 on T cells, multiple examples of non-T cell expressed CTLA-4 have been reported. These cells include tumor cells of hematological and non-hematological origin and normal B cells. In this study, we have defined a novel immune suppressive role for non-T cell, tumor expressed CTLA-4 in Chronic Lymphocytic Leukemia (CLL). We have detected by microarray that CTLA-4 is in the top 5 most differentially expressed genes between pooled samples of healthy donor normal B cells (N=6) and pooled CLL leukemic B cells (N=5). Upregulation of CTLA-4 by CLL B cells compared to normal B cells was validated by RT-qPCR and flow cytometry. CTLA-4 was predominantly intracellular (42/46 CTLA-4+) and not on the cell surface (2/48 CTLA-4+) in primary CLL samples. B cell activating factors (CD40L, PMA/Ionomycin, LPS, IL4, LPS+IL4, CD40L+IL4, CpG, and anti-IgM) could not induce surface expression of CTLA-4; however, co-culture with anti-CD3/anti-CD28 or ConA activated T cells (autologous or allogeneic) resulted in detectable CTLA-4 on the cell surface of leukemic B cells. This induction did not occur with resting T cells. This finding suggests a role for CTLA-4+ tumor cells in sites of T cell activation, such as the lymph node, a site of leukemic cell proliferation in CLL. To mechanistically study leukemic B cell expressed CTLA-4, we generated CLL-derived Mec1 and OSU-CLL that inducibly express CTLA-4 upon doxycycline (dox) treatment. Mec1 and OSU-CLL cells highly express the ligands for CTLA-4, CD80 and CD86. Dox-induction of CTLA-4 resulted in decreased expression of Mec1 and OSU-CLL expressed CD80, a critical T cell co-stimulatory protein (N=3). Blockade of CTLA-4 using the anti-CTLA-4 therapeutic antibody, Ipilimumab, could restore CD80 on Mec1 and OSU-CLL cells (N=3). Because T cell-expressed CTLA-4 has been previously shown by others to down-modulate CD80 via trans-endocytosis, we co-cultured CTLA-4+ Mec1 and CTLA-4+ primary CLL cells with stably transfected CD80-GFP or CD86-GFP Hek293 cell lines to assess uptake of CD80/CD86 into CTLA-4 expressing tumor cells as the mechanism of CD80 down-modulation. Transfer of CD80-GFP and CD86-GFP was detected by flow cytometry in primary CLL cells and the Mec1 cell line, consistent with the ability of T cell expressed CTLA-4 to trans-endocytose CD80 and CD86. Furthermore, uptake of CD80-GFP or CD86-GFP by primary tumor cells was CTLA-4 dependent, demonstrated by inhibition of GFP uptake in the presence of Ipilimumab. Following determination of decreased CD80, we found that co-culture of primary T cells with Mec1 CTLA-4+ cells resulted in decreased IL2 production measured by Cytokine Bead Array. The loss of IL2 signified decreased co-stimulation as a result of tumor expressed CTLA-4. Studies are ongoing regarding dependence on CD80 or CD86. A minor subset of T cells, Tregs, are known to exert profound immunosuppressive effects through their expression of CTLA-4. Due to our results, tumor expressed CTLA-4 has an overlapping function with Treg CTLA-4, and it is imperative that we define the immunosuppressive effects as, in patients, the leukemic cells may comprise a much larger proportion of white blood cells than T cells. Efforts are now underway to address the effect of tumor expressed CTLA-4 in suppressing anti-tumor immunity in vivo utilizing a novel mouse model. Suppression of T cells by tumor expressed CTLA-4 is a novel finding that is broadly applicable to fields within and outside of cancer research as the pathway and mechanism described here are potentially applicable to CTLA-4 in diverse disease contexts and to the general biology of CTLA-4.[Funding: This work was supported by P01 CA95426. PD received the Pelotonia Graduate Fellowship. Any opinions, findings, and conclusions expressed in this material are those of the author(s) and do not necessarily reflect those of the Pelotonia Fellowship Program] DisclosuresJones:AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract A17: Physical activity and epigenetic age among normative aging study participants

Abstract Low levels of physical activity are associated with increased risk and mortality of common cancers including breast and colon, with strong evidence for increasing risk with age. Epigenetic age is a recently developed concept using DNA methylation measurements to describe biological age at the level of human tissues, cells, and organs. Epigenetic age represents deviation from expected DNA methylation patterns associated with chronological age (defined as Δ_age), and is suggested as a possible biological mechanism driving observed associations between physical activity and cancer risk. However, no studies have evaluated associations between physical activity level and Δ_age. The Normative Aging Study (NAS) is a cohort study of healthy aging conducted by the Veterans Affair Administration. Among NAS participants (n=656), we conducted multivariate linear regression to prospectively evaluate associations between physical activity (MET-hours per week) and Δ_age across two follow-up visits. The visits were on average 3.6 years apart, with a range of 2-8 years. Δ_age was estimated using Horvath’s DNA methylation online calculator and based upon DNA methylation patterns at the 71 cytosine-phosphate-guanine (CpGs) loci used in Hannum’s model. The calculator accounts for cell proportion of white blood cells. We adjusted models for chronological age, body mass index, education, smoking, alcohol consumption, and prevalent cancer status. Overall, we observed statistically significant associations between high physical activity levels and lower Δ_age, or younger epigenetic age. Among individuals reporting the highest level of physical activity (≥ 30 MET-hours per week), Δ_age was 1.44 (95% CI: 0.43 to 2.45) and 2.20 (95% CI: 1.04 to 3.36) years lower compared to those reporting low activity (&amp;lt;12 MET-hours per week) at the first and second visit, respectively. Furthermore, compared to low physical activity, high activity at the first visit was associated with 2.46 (95% CI: 1.25 to 3.67) years lower Δ_age at the second visit, and 3.72 (95% CI: 1.08 to 6.36) months/calendar year lower Δ_age rate of change, or decelerated epigenetic aging over time. In summary, the results of the current study demonstrate that physical activity influences DNA methylation patterns at the CpG loci associated with epigenetic age. Future studies are necessary to characterize further the relationship between physical activity and epigenetic age, especially as a potential biological mechanism of action in carcinogenesis. However, epigenetic age represents a unique set of markers with the potential to identify high-risk populations and, in turn, develop interventions to mitigate risk of cancer. Citation Format: Elizabeth A. Hibler, Yinan Zheng, Lei Liu, Wei Zhang, Frank J. Penedo, Siobhan M. Phillips, David E. Conroy, Joel Schwartz, Pantel Vokonas, Andrea Baccarelli, Qi Dai, Lifang Hou. Physical activity and epigenetic age among normative aging study participants. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A17.

Association between DNA Methylation in Whole Blood and Measures of Glucose Metabolism: KORA F4 Study.

Epigenetic regulation has been postulated to affect glucose metabolism, insulin sensitivity and the risk of type 2 diabetes. Therefore, we performed an epigenome-wide association study for measures of glucose metabolism in whole blood samples of the population-based Cooperative Health Research in the Region of Augsburg F4 study using the Illumina HumanMethylation 450 BeadChip. We identified a total of 31 CpG sites where methylation level was associated with measures of glucose metabolism after adjustment for age, sex, smoking, and estimated white blood cell proportions and correction for multiple testing using the Benjamini-Hochberg (B-H) method (four for fasting glucose, seven for fasting insulin, 25 for homeostasis model assessment-insulin resistance [HOMA-IR]; B-H-adjusted p-values between 9.2x10-5 and 0.047). In addition, DNA methylation at cg06500161 (annotated to ABCG1) was associated with all the aforementioned phenotypes and 2-hour glucose (B-H-adjusted p-values between 9.2x10-5 and 3.0x10-3). Methylation status of additional three CpG sites showed an association with fasting insulin only after additional adjustment for body mass index (BMI) (B-H-adjusted p-values = 0.047). Overall, effect strengths were reduced by around 30% after additional adjustment for BMI, suggesting that this variable has an influence on the investigated phenotypes. Furthermore, we found significant associations between methylation status of 21 of the aforementioned CpG sites and 2-hour insulin in a subset of samples with seven significant associations persisting after additional adjustment for BMI. In a subset of 533 participants, methylation of the CpG site cg06500161 (ABCG1) was inversely associated with ABCG1 gene expression (B-H-adjusted p-value = 1.5x10-9). Additionally, we observed an enrichment of the top 1,000 CpG sites for diabetes-related canonical pathways using Ingenuity Pathway Analysis. In conclusion, our study indicates that DNA methylation and diabetes-related traits are associated and that these associations are partially BMI-dependent. Furthermore, the interaction of ABCG1 with glucose metabolism is modulated by epigenetic processes.

Detecting glycogen in peripheral blood mononuclear cells with periodic acid schiff staining.

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Effects of dietary phytogenic product on the performance and immune response of pigs

Abstract The aim of the study was to determine the effects of supplementation of sows’ and weaners’ diet with Stresomix, preparation containing extracts from Magnifera indica, Withania somnifera, Phyllanthus emblica, and Ocimum sanctum, on pig performance and immunity under field condition. The hypothesis was that anti-inflammatory, antistress, and immunomodulatory properties of the herbs would enhance production parameters and immune response, according to the manufacturer's claim. The study was performed on 16 sows and 160 piglets. The following parameters were recorded: concentration and proportion of white blood cells and their populations, concentration of serum immunoglobulins, specific humoral postvaccinal response after vaccination against swine influenza and swine erysipelas, and main production parameters. No significant differences among treatment groups were found with regard to concentrations of leukocyte subpopulations and immunoglobulins, as well as all investigated production parameters (P&gt;0.05). In conclusion, the results of the study did not confirm that the investigated polyherbal product, administered at dose recommended by manufacturer, is able to significantly improve the performance and postvaccinal humoral response in clinically healthy pigs under field condition.

Abstract 3642: Profiles of DNA methylation in peripheral blood are associated with bladder cancer survival.

Abstract Bladder cancer is a common solid tumor, with greater than 71,000 cases diagnosed in the United States last year, and greater than 14,000 deaths attributable to this disease. In addition to this mortality, the morbidity of this disease can be great, as the non-invasive form of this disease, making up &amp;gt;75% of the incidence, recurs frequently, and therefore requires frequent invasive monitoring through cystoscopy at a great cost to the health care system. There are critical gaps in our understanding of the predictors of its recurrence and survival, and identifying molecular alterations associated with these outcomes is essential for improving patient morbidity and the care of patients with this disease. We have shown that profiles of DNA methylation in peripheral blood are associated with human bladder cancer and have recently demonstrated that much, but not all, of the variation in DNA methylation is attributable to disease-associated changes in the underlying proportion of immune cells in the peripheral blood sample. We also have devised a novel statistical strategy that utilizes DNA methylation profiles to quantify the proportion of blood cell subtypes in archival DNA. This is able to disentangle the variability in DNA methylation profiles attributable to blood cell population differences from epigenetic variation arising within a cell population. We sought to determine if variation in DNA methylation identified in peripheral blood, reflective, in part, of altered white blood cell proportions, is associated with bladder cancer recurrence and survival in a population-based study of incident bladder cancers in New Hampshire, USA. Illumina Infinium 27K DNA methylation arrays were performed on whole peripheral blood derived DNA samples from 223 cases of bladder cancer, having a median follow-up time of approximately 13 years. Array data was subjected to quality control procedures and normalization using the ComBAT methodology to account for plate/BeadChip effects. Fitting a series of Cox proportional hazards models, stratified by histology (carcinoma in-situ, low-grade non-invasive, high-grade non-invasive, and invasive) and controlled for patient age and gender revealed 6,873 high-confidence (q &amp;lt; 0.05) associations with bladder cancer specific death as the endpoint. In a principle components analysis, the first principal component, accounting for a striking 46% of the variability in methylation, was significantly associated with bladder cancer specific death. Ongoing analyses are examining how changes to underlying cell proportions mediate the relationship between DNA methylation and patient survival. These data suggest that peripheral blood DNA methylation profiles proximal to the time of diagnosis are strongly prospectively predictive of bladder cancer specific survival and suggest that these biomarkers may have important use as non-invasive prognostic tools worthy of continued study. Citation Format: Devin C. Koestler, Megan A. Murphy, Eugene A. Houseman, Margaret R. Karagas, Karl T. Kelsey, Carmen J. Marsit. Profiles of DNA methylation in peripheral blood are associated with bladder cancer survival. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3642. doi:10.1158/1538-7445.AM2013-3642

Urinary corticosterone responses to capture and toe-clipping in the cane toad ( Rhinella marina) indicate that toe-clipping is a stressor for amphibians

Toe-clipping, the removal of one or more toes, is a common method used to individually mark free-living animals. Whilst this method is widely used in studies of amphibians, the appropriateness of the method, and its potential detrimental effects have been the subject of debate. Here, we provide for the first time, evidence that toe-clipping is a stressor in a wild amphibian. We measured urinary corticosterone responses of male cane toads ( Rhinella marina) to capture and handling only, and to toe-clipping under field conditions. Urinary testosterone concentrations and white blood cell proportions were also measured. Urinary corticosterone metabolite concentrations increased 6 h after capture and handling only and remained high for 24 h; corticosterone returned to baseline levels after 48 h and remained low at 72 h post capture and handling. Corticosterone concentrations in toads subjected to toe-clipping increased at 6 h to significantly higher concentrations than after capture and handling only, then decreased more slowly than after capture and handling, and were still elevated (approximately double basal level) 72 h after toe-clipping. Testosterone did not change significantly after capture and handling only, whereas after toe-clipping testosterone decreased at 6 h and remained low at 72 h. There were weak short-term effects of toe-clipping compared with capture and handling only on white blood cell proportions. We have clearly shown that toe-clipping is a distinctly stronger stressor than capture and handling alone. This indicates that there is an ethical cost of toe-clipping, and this should be considered when planning studies of amphibians.

On the effects of the Fusarium toxin deoxynivalenol (DON) administered per os or intraperitoneal infusion to sows during days 63 to 70 of gestation

Six pregnant sows of 180.6 ± 5.6kg were fed either a Fusarium-contaminated (4.42mg DON and 48.3µg ZON per kg, DON per os, n = 3) or a control diet (0.15mg DON and 5µg ZON/kg) in the period of days 63 and 70 of gestation. On day 63 of gestation, sows fed the control diet were implanted with an intraperitoneal osmotic minipump (delivery rate of 10µL/h, for 7days) containing 50mg pure (98%) DON in 2ml 50% DMSO (DON ip, n = 3). Frequent plasma samples were taken to estimate the kinetics after oral and ip DON exposure. The intended continuous delivery of DON by the intraperitoneal minipump could not be shown, as there was a plasma peak (Cmax) of 4.2-6.4ng DON/mL either immediately (sow IP-2+3) or 2.5h (sow IP-1) after implantation of the pump followed by a one-exponential decline with a mean half-time (t1/2) of 1.75-4.0h and only negligible DON plasma concentrations after 12h. Therefore, the DON ip exposure has to be regarded as one single dose 1week before termination of experiment. The DON per os sows showed a mean basis level (after achieving a steady state) of DON plasma concentration of about 6-8ng/mL, as also indicated by the plasma DON concentration at the termination of the experiment. On day 70, caesarean section was carried out, the fetuses were killed immediately after birth, and samples of plasma, urine, and bile were taken to analyze the concentration of DON and its metabolite de-epoxy-DON. At necropsy there were no macroscopic lesions observed in any organ of either sows or piglets. Histopathological evaluation of sows liver and spleen revealed no alterations. The proliferation rate of peripheral blood mononuclear cells (PBMC) with or without stimulation was not affected by the kind of DON treatment. The exposure of pregnant sows at mid-gestation (days 63-70, period of organogenesis) to a Fusarium toxin-contaminated diet (4.42mg DON and 0.048mg ZON per kg) or pure DON via intraperitoneal osmotic minipump did not cause adverse effects on health, fertility, maintenance of pregnancy, and performance of sows and their fetuses. However, DON was detected in fetus plasma, indicating that this toxin can pass the placental barrier and may cause changes in the proportion of white blood cells (lower monocyte and neutrophil and higher lymphocyte proportion in DON per os fetuses).

Circulating white blood cells and platelets amplify oxidative stress in heart failure

Mitochondria of circulating white blood cells (WBC) and platelets sense oxidative stress during capillary passage and react by producing reactive oxygen species (ROS). Although evidence indicates that congestive heart failure (CHF) is associated with oxidative stress, the role of WBC and platelets as mediators in CHF has not been investigated. Patients with CHF (n = 15) and healthy volunteers (n = 9) were enrolled between 2006 and 2007 into this observational study. Arterial and venous blood samples from participants were incubated with probes to detect cytosolic and mitochondrial ROS. Fluorescence-activated cell sorting was used to measure the degree of fluorescence in WBC and platelets. Patients with CHF had a higher proportion of ROS-positive arterial WBC and platelets than did controls (67% +/- 47% versus 16% +/- 9%; P <0.005), as well as venous WBC and platelets (77% +/- 43% versus 38% +/- 13%; P <0.01). In the control group, the proportion of cytosolic ROS-positive arterial WBC and platelets was lower than that for ROS-positive venous WBC and platelets (16% +/- 9% versus 38% +/- 13%; P <0.005). CHF patients had a higher proportion of mitochondrial ROS-positive arterial and venous WBC and platelets than did controls. In CHF, the proportion of WBC and platelets that are ROS-positive is raised, possibly because cytosolic ROS-positive WBC and platelets are normally cleared in the lungs; this function is deficient in CHF while mitochondrial ROS production is increased. The raised numbers of circulating ROS-positive WBC and platelets amplify oxidative stress in CHF.

Endoglin Is Not Critical for Hematopoietic Stem Cell Engraftment and Reconstitution but Regulates Adult Erythroid Development

Endoglin is a transforming growth factor-beta (TGF-beta) accessory receptor recently identified as being highly expressed on long-term repopulating hematopoietic stem cells (HSC). However, little is known regarding its function in these cells. We have used two complementary approaches toward understanding endoglin's role in HSC biology: one that efficiently knocks down expression via lentiviral-driven short hairpin RNA and another that uses retroviral-mediated overexpression. Altering endoglin expression had functional consequences for hematopoietic progenitors in vitro such that endoglin-suppressed myeloid progenitors (colony-forming unit-granulocyte macrophage) displayed a higher degree of sensitivity to TGF-beta-mediated growth inhibition, whereas endoglin-overexpressing cells were partially resistant. However, transplantation of transduced bone marrow enriched in primitive hematopoietic stem and progenitor cells revealed that neither endoglin suppression nor endoglin overexpression affected the ability of stem cells to short-term or long-term repopulate recipient marrow. Furthermore, transplantation of cells altered in endoglin expression yielded normal white blood cell proportions and peripheral blood platelets. Interestingly, decreasing endoglin expression increased the clonogenic capacity of early blast-forming unit-erythroid progenitors, whereas overexpression compromised erythroid differentiation at the basophilic erythroblast phase, suggesting a pivotal role for endoglin at key stages of adult erythropoietic development.

Comparison of T Cell Subsets Between Somatic Cloned and Normal Cow

Somatic cloning technology is beneficial for genetically producing excellent animals. However, many developmental problems of somatically cloned animals have been described. Some of them may cause disorders of the immune system, resulting in the fluctuation of the proportion of white blood cells (WBC), different from that of normal animals in peripheral blood. In Holstein- cloned and normal cows, the fluctuation of granulocytes, monocytes, B cells and T cells, and further T cell subsets (CD4+, CD8+, gammadelta, CD8+gammadelta and WC1+gammadelta T cell) in peripheral blood were analyzed in early lactation stage (ELS) and mid to late lactation stage (MLS) by flow cytometry using specific monoclonal antibodies for cell surface markers. In both ELS and MLS, there were no significant differences in the proportions of granulocytes, monocytes, B cells and T cells between cloned and normal cows. In T cell subsets, gammadelta and WC1+gammadelta T cells in cloned cows were significantly less frequent than in normal cows in ELS. The decreased proportions of gammadelta and WC1+gammadelta T cells recovered to the level of normal cows in MLS. The population of granulocytes, monocytes, B cells and T cells, and T cell subsets except for gammadelta and WC1+gammadelta T cells in cloned cows fluctuated in a manner similar to those of normal cows during lactation. In ELS, the proportions of gammadelta and WC1+gammadelta T cells temporarily declined in cloned cows, suggesting that cloned cows may fall into an immunosuppressive state in ELS.