Proportion Of Leukocytes Research Articles

Itacitinib, a JAK1 Selective Inhibitor Preserves Graft-Versus-Leukemia (GVL), Enhances Survival and Is Highly Efficacious in a MHC-Mismatched Mouse Model of Acute GvHD

Introduction:Graft-versus-host disease (GvHD) is a severe complication arising in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Potent and selective modulation of JAK1/STAT-mediated signaling is an attractive therapeutic strategy for the management of acute GvHD and is currently being evaluated in clinical trials (GRAVITAS-301: NCT03139604; GRAVITAS-119: NCT03320642).Methods:Acute GvHD was induced in BALB/c mice using the established MHC-mismatched mouse model. BALB/c (H-2Kd) recipients were given an intravenous injection of a combination of splenocytes and T cell depleted bone marrow cells from allogeneic cell transfer from donor C57BL/6 (H-2Kb) mice. Animals were dosed orally with vehicle or the selective JAK1 inhibitor, itacitinib (60 mg/kg or 120 mg/kg twice daily). Engraftment was analyzed for the proportion of donor and host leukocytes (CD45+, H-2Kb, and H-2Kd). GvHD clinical scores were assessed by standard methods and inflammatory cytokine profiles in blood and colon quantified by multiplex analysis. Colon samples were sectioned and stained with the following immunohistochemical (IHC) markers: CD4, CD8, phosphoSTAT3 and CD3+phosphoSTAT3 (dual staining) for pharmacodynamic assessment of JAK/STAT pathway activity in colon and infiltrating T-cells. Effects of itacitinib on preservation of Graft-versus-Leukemia (GVL) were evaluated by injecting BALB/c mice with A20 lymphoma cells that are of H-2Kd phenotype along with combination of splenocytes and T cell depleted bone marrow from C57BL/6 (H-2Kb) mice.Results:Itacitinib administration was highly effective in both prophylactic (from day −3) and therapeutic (from day 14) dosing regimens in ameliorating body weight loss and improving GvHD scores. Itacitinib did not significantly impact donor engraftment as determined by CD45+/H-2Kb quantification by flow cytometry. Similar efficacy was observed with 60 mg/kg versus 120 mg/kg twice daily dosing regimens. Oral itacitinib administration achieved JAK1 IC50 coverage for 4 h and 12 h at 60 mg/kg twice daily and 120 mg/kg twice daily, respectively. Associated with GvHD progression, maximal upregulation of inflammatory cytokines were observed in peripheral blood on day 17 (IFN-γ, TNF-α, IL-6, IL-13) and in colon on day 28 (IFN-γ, TNF-α, IL-1β). Itacitinib (120 mg/kg twice daily) treatment significantly reduced the inflammatory cytokine milieu at these disease stages. No differences were observed in absolute number of CD4+ T cells and CD8+ T cells in blood and spleen with itacitinib treatment, but significant reductions were detected in CD4+ T cells and CD8+ T cells in the inflamed colon tissue along with significant JAK1/STAT3 inhibition as measured by reductions in normalized pSTAT3 in T cells and colonic epithelial cells. Itacitinib treatment did not negatively impact GVL responses, as evidence by T cell mediated reduction of tumor burden. Furthermore, itacitinib treatment enhanced the survival of the recipient BALB/c mice in comparison to the vehicle treated animals.Conclusions:Itacitinib, a selective JAK1 inhibitor ameliorated GvHD severity when administered prophylactically or therapeutically and had no detrimental effects on engraftment and preservation of GVL. Furthermore, itacitinib inhibited JAK1/STAT3 activation in diseased colon tissue and infiltrating T-cells, and reduced disease burden and improved survival by modulating levels of inflammatory cytokines important in the pathophysiology of acute GvHD. DisclosuresJuvekar:Incyte Corporation: Employment. Ruggeri:Incyte Corporation: Employment. Condon:Incyte Corporation: Employment. Borkowski:Biomodels LLC: Employment. Huber:Incyte Corporation: Employment. Smith:Incyte Corporation: Employment.

Therapeutic effects of different doses of methylprednisolone on smoke inhalation-induced acute lung injury in rats

To investigate the therapeutic effect of different doses of methylprednisolone (MP) in smoke inhalation-induced acute lung injury (SI-ALI). Adult male Sprague-Dawley (SD) rats were divided into control group (group A, n = 6), smoke inhalation group (group B, smoke inhalation 30 minutes, n = 30) and smoke + MP 40, 4, 0.4 mg/kg intervention group (groups C, D, E; intraperitoneal injection of MP at 1 hour before smoke inhalation, n = 30) according to random number table method. The survival status of rats in each group was observed at 24 hours, and murine smoke inhalation induced trauma score (MSITS) according to the symptoms and signs of rats at 3 hours after smoke inhalation were scored. The blood of abdominal aorta of rats was collected. Then the rats were sacrificed to harvest bronchoalveolar lavage fluid (BALF) and lung tissue. The levels of interleukin (IL-6, IL-17a) in plasma and BALF were detected by enzyme linked immunosorbent assay (ELISA); the total number of white blood cells and the proportion of leukocytes or macrophages in BALF were calculated; the histopathological changes of lung were observed and the lung injury score was given; the expression of myeloperoxidase (MPO) and high mobility group protein B1 (HMGB1) in lung tissue were detected by Western Blot. The 24-hour survival rate of group B rats was 33.67%. The survival rate of groups C, D and E (65.73%, 85.17%, 60.07%) were significantly higher than that of group B (all P < 0.05), and the survival rate of group D was significantly higher than that of groups C and E. Diffuse inflammatory cell infiltration, intra-alveolar hemorrhage and a large amount of edema fluid were seen in the lung tissue of group B; and the lung injury score was significantly higher than that of group A. Compared with group B, the lung injury in different doses of MP group were decreased to different degrees, while the lung injury scores in groups C and D were significantly decreased (3.31±1.37, 2.62±0.98 vs. 5.52±0.97, both P < 0.01); correlation analysis showed that MSITS score was significantly and positively correlated with lung injury score (r = 0.862, P < 0.001). The levels of plasma inflammatory factors and BALF protein, inflammatory cells and inflammatory factors, and the expression of MPO, HMGB1 in group B were significantly higher than those in group A. Compared with group B, the levels of inflammatory factors in plasma, and protein content, inflammatory cells and inflammatory factors in BALF in different doses of MP group were decreased to different degrees, with significant differences in groups C and D [plasma: IL-17a (pg/L): 49.28±27.12, 36.57±16.52 vs. 191.79±88.21; IL-6 (ng/L): 206.47±109.96, 197.52±113.86 vs. 669.00±299.60; BALF: protein content (mg/L): 892.0±164.5, 566.1±120.9 vs. 1 838.0±145.8; white blood cell count (×109/L): 5.40±1.67, 2.81±1.20 vs. 9.02±2.06; neutrophil ratio: 0.315±0.081, 0.273±0.080 vs. 0.590±0.096; IL-17a (ng/L): 22.63±8.62, 18.92±8.43 vs. 43.31±19.17; IL-6 (ng/L): 156.49±46.94, 123.66±64.91 vs. 253.43±80.03; all P < 0.01]; in addition, the expression of MPO and HMGB1 protein in lung tissues of MP groups with different doses were significantly decreased, the expression of MPO in group D was significantly lower than that in group E [MPO/β-actin (fold increase from group A): 2.14±0.97 vs. 4.35±0.87, P < 0.01], the expression of HMGB1 in groups C and D were significantly lower than that in group E [HMGB1/β-actin (fold increase from group A): 1.77±0.73, 1.23±0.67 vs. 3.65±1.08, both P < 0.05]. MP can significantly improve the survival rate of SI-ALI rats and reduce the acute pulmonary and systemic inflammatory response. The MP effect of 4 mg/kg was better than 40 mg/kg and 0.4 mg/kg.

Relationships among quarter milk leukocyte proportions and cow and quarter-level variables under different intramammary infection statuses.

The use of milk leukocyte differential (MLD) test has been proposed as a complement to somatic cell count (SCC) to assess the presence and the severity of intramammary infection. However, detailed information regarding the behavior of MLD under different physiological or pathological stages of the cow is nonexistent. The objective was to analyze the association between milk leukocyte proportions provided by a commercial automated MLD test and multiple cow and quarter-level variables. The study population consisted of 104 Holstein cows (32 primiparous and 72 multiparous) in one farm. Cows were categorized by days in milk as early (<50 DIM; n=29), middle (50–250 DIM; n=25), and late lactation (>250 DIM; n = 50). Milk from 416 quarters was collected and analyzed for lymphocytes (LYM), neutrophils (NEU), and macrophages (MAC) counts using an automated milk fluorescence microscopy system. Concurrently, a sterile composite milk sample was collected from each cow for pathogen identification through microbiological culture. Culture results were classified as no growth (NOG), gram-negative (GN), gram-positive (GP), or other (OTH). Milk leukocyte proportions varied depending on the level of total leukocyte counts (TLC; P < 0.001). Similarly, leukocyte ratios (NEU:LYM, NEU:MAC, and phagocyte:LYM) were different for multiple TLC categories (P < 0.05). There was no association between parity number and MLD; however, cows in early lactation had the greatest proportions of NEU and LYM. Leukocyte ratios varied depending on parity number and stage of lactation. Cows in the medium milk-yield category had the smallest proportions of NEU and LYM, and there was significant variation in leukocyte ratios, depending on the level of milk yield. In healthy quarters, MLD were not associated with quarter position; however, the NEU:MAC ratio was greater in rear quarters than in front quarters. In quarters with TLC >100,000, NEU% was greater in rear quarters than in front quarters (P = 0.03). For quarters with pathogen growth, TLC was greatest for GN followed by OTH and GP (P < 0.001). Milk LD depended on the isolated pathogen group, although the magnitudes of the differences were small. Although the changes in the proportions of leukocytes in milk were associated with categories of TLC, levels of milk yield, and mastitis-causing pathogen groups, the deviations were small in magnitude. Additional research is necessary to determine the potential applications for this methodology.

Usefulness of blood and cerebrospinal fluid laboratory testing to predict bacterial meningitis in the emergency department

IntroductionThe classic clinical presentation of bacterial meningitis (BM) is observed in less than half of the cases in adults, and symptoms are less specific in children, the elderly or immunocompromised, and other chronic patients. The usual signs and symptoms do not provide optimal sensitivity and specificity for distinguishing possible BM from viral meningitis (VM), which may lead to a delay in the appropriate antimicrobial therapy. Society therefore stands to benefit from the development of effective, objective, and rapid tools able to predict and identify patients with BM. These tools include laboratory tests for blood and cerebrospinal fluid (CSF). The aim of this review is to summarise recently published scientific evidence in order to clarify existing controversies and compare the usefulness and diagnostic ability of the different parameters used to predict BM. DevelopmentSystematic search of the main bibliographic databases and platforms to identify articles published between January 2000 and January 2016. We selected 59 articles that meet the objectives of this review. ConclusionsCSF lactate, proportion of polymorphonuclear leucocytes, and CSF glucose, as well as serum procalcitonin (PCT), are the independent factors most predictive of bacterial aetiology. The model that combines serum PCT and CSF lactate achieves the highest predictive power for BM, with a sensitivity and specificity exceeding 99%. We should consider BM when CSF lactate >33md/dL and/or PCT>0.25ng/mL.

An Epigenome-Wide Association Study of Obesity-Related Traits.

We conducted an epigenome-wide association study on obesity-related traits. We used data from 2 prospective, population-based cohort studies: the Rotterdam Study (RS) (2006-2013) and the Atherosclerosis Risk in Communities (ARIC) Study (1990-1992). We used the RS (n = 1,450) as the discovery panel and the ARIC Study (n = 2,097) as the replication panel. Linear mixed-effect models were used to assess the cross-sectional associations between genome-wide DNA methylation in leukocytes and body mass index (BMI) and waist circumference (WC), adjusting for sex, age, smoking, leukocyte proportions, array number, and position on array. The latter 2 variables were modeled as random effects. Fourteen 5'-C-phosphate-G-3' (CpG) sites were associated with BMI and 26 CpG sites with WC in the RS after Bonferroni correction (P < 1.07 × 10-7), of which 12 and 13 CpGs were replicated in the ARIC Study, respectively. The most significant novel CpGs were located on the Musashi RNA binding protein 2 gene (MSI2; cg21139312) and the leucyl-tRNA synthetase 2, mitochondrial gene (LARS2; cg18030453) and were associated with both BMI and WC. CpGs at BRDT, PSMD1, IFI44L, MAP1A, and MAP3K5 were associated with BMI. CpGs at LGALS3BP, MAP2K3, DHCR24, CPSF4L, and TMEM49 were associated with WC. We report novel associations between methylation at MSI2 and LARS2 and obesity-related traits. These results provide further insight into mechanisms underlying obesity-related traits, which can enable identification of new biomarkers in obesity-related chronic diseases.

Quantitation of the cellular content of saliva and buccal swab samples

Buccal swabs and saliva are the two most common oral sampling methods used for medical research. Often, these samples are used interchangeably, despite previous evidence that both contain buccal cells and blood leukocytes in different proportions. For some research, such as epigenetic studies, the cell types contributing to the analysis are highly relevant. We collected such samples from twelve children and twenty adults and, using Papanicolaou staining, measured the proportions of epithelial cells and leukocytes through microscopy. To our knowledge, no studies have compared cellular heterogeneity in buccal swab and saliva samples from adults and children. We confirmed that buccal swabs contained a higher proportion of epithelial cells than saliva and that children have a greater proportion of such cells in saliva compared to adults. At this level of resolution, buccal swabs and saliva contained similar epithelial cell subtypes. Gingivitis in children was associated with a higher proportion of leukocytes in saliva samples but not in buccal swabs. Compared to more detailed and costly methods such as flow cytometry or deconvolution methods used in epigenomic analysis, the procedure described here can serve as a simple and low-cost method to characterize buccal and saliva samples. Microscopy provides a low-cost tool to alert researchers to the presence of oral inflammation which may affect a subset of their samples. This knowledge might be highly relevant to their specific research questions, may assist with sample selection and thus might be crucial information despite the ability of data deconvolution methods to correct for cellular heterogeneity.

Determining the Significance of a Novel Disease Inducing Rodent Diet on Adenocarcinoma Growth In Vivo

Diet and diet‐induced morbidities (such as obesity) are leading risk factors for the development of various forms of cancer worldwide. However, the physiological mechanisms responsible for the interaction of diet and cancer growth are still unclear. Mechanistic studies in animals support the interaction of diet with cancer development and progression, however the animal diets used do not adequately model the true nutritional quality of many Americans. We have developed a novel disease inducing rodent diet referred to as the Americanized diet (AD) that embodies the complex nutritional profile of Americans while also accounting for the differences in human and rodent nutritional requirements. We set out to determine how consumption of the AD would influence the growth of the MC‐38 murine adenocarcinoma cells in vivo. All animal experiments were performed following animal protocols approved by the Liberty University IACUC and conform to the FASEB Statement of Principles for the use of animals in research and education. Weanling C57Bl/6 male mice were given ad libitum access to the AD (n=5) or standard rodent chow (n=5) and ddH2O. Food intake and body weights were calculated weekly and circulating total cholesterol (CHOL), HDL‐cholesterol (HDL), triglycerides, and glucose were quantified at baseline and again at the end of the study. After 6 weeks, mice were injected with 0.5×106 MC‐38 adenocarcinoma cells in the rear flank and injection site was monitored for the presence of a palpable tumor. Tumor volume was calculated using calipers and, after 3 weeks, tumor blood flow was estimated using contrast‐enhanced ultrasonography. Animals were then euthanized and tumor tissues collected for assessment of leukocyte content by flow cytometry and quantification of interleukin‐10 (IL‐10), major histocompatibility complex H2K, and vascular endothelial growth factor (VEGF) gene expression by RT‐PCR. Body weights and tumor volumes were analyzed using generalized linear models and the remaining parameters were analyzed using GLM multivariate analyses in SPSS. Mice assigned to ad libitum feeding of the AD consumed more calories and gained more weight (P=0.01) as compared to mice consuming standard chow. After 6 weeks of consuming the diet, mice fed the AD had greater HDL cholesterol (P=0.03); however this difference was lost after MC‐38 innoculation (P=0.2). MC‐38 growth was significantly reduced (P=0.004 and 0.07 at weeks 1 and 2, respectively) in mice consuming the AD as compared to mice consuming chow. Interestingly, no palpable tumor was observed in 2 of the 5 mice consuming the AD while all 5 mice consuming chow developed tumors. There was a greater relative tissue perfusion (P=0.02) in the tumor‐bearing mice fed the AD as compared to the mice fed chow. No difference was observed in the proportion of tumor leukocytes amongst the diets. Similarly, RT‐PCR revealed no difference in IL‐10, H2K or VEGF gene expression between treatment groups (P= 0.25, 0.98, 0.62 respectively). Based on this study, mice fed the AD had reduced growth of the MC‐38 murine adenocarcinoma cell line in vivo despite having greater body weight and caloric intake. While we were unable to detect a significant difference in tumor leukocyte content and/or the expression of IL10, H2K, and VEGF at the end of the study, it is possible these outcomes differed during earlier time points (weeks 1 and 2) and contributed to the reduced tumor growth observed in mice fed the AD.Support or Funding InformationThis work was supported by Liberty University, College of Osteopathic Medicine intramural grant #2016‐07.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

White reflection from cuttlefish skin leucophores

The highly diverse and changeable body patterns of cephalopods require the production of whiteness of varying degrees of brightness for their large repertoire of communication and camouflage behaviors. Leucophores are structural reflectors that produce whiteness in cephalopods; they are dermal aggregates of numerous leucocytes containing spherical leucosomes ranging in diameter from 200–2000 nm. In Sepia officinalis leucophores, leucocytes always occur in various combinations with iridocytes, cells containing plates that function as Bragg stacks to reflect light of particular wavelengths. Both spheres and plates contain the high-refractive-index protein reflectin. Four leucophore skin-patterning components were investigated morphologically and with spectrometry. In descending order of brightness they are: white fin spots, White zebra bands, White square, and White head bar. Different densities, thicknesses and proportions of leucocytes and iridocytes were correlated with the relative brightness measurements of the skin. That is, White fin spots and White zebra bands had leucocytes of the highest density, the greatest number of reflective cell layers, and the highest proportion of leucocytes to iridocytes. In contrast, the White square and White head bar had the lowest density of reflective cells, fewer cell layers and the lowest ratios of leucocytes to iridocytes. Leucophores are white in white light, yet reflect whatever colors are in the available light field: e.g. red in red light, green in green light, etc. Leucophores are physiologically passive, thus their ultrastructure alone is capable of diffusing all ambient wavelengths in all directions, regardless of the angle of incident light. However, the specific optical contributions of spherical leucosomes versus the associated plate-like iridosomes in producing whiteness versus brightness are yet to be determined. This study reveals complex morphological arrangements that produce white structural coloration for different brightnesses of skin by differentially combining spheres and plates.

Abstract MP61: A Multi-pollutant, Multi-ethnic, and Methylome-wide Association Study Highlights Epigenetic Effects of Exposure to Ambient Air Pollution Mixtures

Background: Ambient air pollution mixtures including fine particulates and gases have been associated with cardiovascular disease (CVD). Although mechanisms underlying the associations remain largely uncharacterized to date, examining epigenetic responses to air pollution mixtures may identify biological systems affected by exposure and provide insight into the pathophysiology and environmental epidemiology of CVD. Therefore, we undertook a multi-pollutant, methylome-wide study of associations between DNA methylation (DNAm) at 485,000 Cytosine-phosphate-Guanine (CpG) sites and pollutants regulated by the U.S. Environmental Protection Agency under the Clean Air Act. Methods: Data from the Atherosclerosis Risk in Communities Study and three sub-studies within the Women’s Health Initiative (WHI) included information on 8,567 participants, 83% of whom were women, 46% African American, and 9% Hispanic/Latino (mean age, 61.3 years). Daily mean, geocoded participant address-specific concentrations of ambient particulate matter with diameter ≤ 10μm (PM 10 ), ≤2.5μm (PM 2.5 ), and 2.5-10μm (PM 2.5-10 ); ozone (O 3 ); nitrogen dioxide (NO 2 ); nitrogen oxides (NO x ); carbon monoxide (CO); and sulfur dioxide (SO 2 ) were averaged over 2, 7, 28, and 365 days before measurement of leukocyte DNAm in whole blood. Methylome-wide, pollutant-DNAm associations were estimated separately at each averaging period in each stratum defined by race/ethnicity and study using multi-level, linear mixed models adjusting for socio-demographic, behavioral, and meteorological characteristics; estimated leukocyte proportions; and technical covariates. Estimates were combined across strata in pollutant- and period-specific, fixed-effects, inverse-variance weighted meta-analyses. Then multi-pollutant, period-specific effects were examined using the adaptive sum of powered scores (U) test (aSPU). CpG sites associated with multi-pollutant significance (aSPU P &lt; 1.0 x 10 -7 ) were characterized in silico to assess their putative function and biological plausibility. Results: Overall analyses identified a significant, 28-day mean, multi-pollutant association at cg15008743 near ZNF83 on chromosome 19 (p = 1x10 -7 ) and a highly-suggestive 28-day mean, multipollutant association at cg00489219 near ZNF621 on chromosome 3 (p = 5x10 -7 ). ZNF83 and ZNF621 encode zinc finger proteins with a potentially broad reach across biological systems given their transcription factor, metal, and DNA binding activities. ZNF621 also harbors rare genetic variants with large effects on blood pressure. Discussion: Methylome-wide associations of DNAm with EPA-regulated ambient air pollutants suggest biologically plausible pathways underpinning the increasingly well-documented ties between multi-pollutant mixtures and cardiovascular disease.

Abstract P105: Gaseous Air Pollutants and DNA Methlyation in a Methylome-wide Association Study of an Ethnically and Environmentally Diverse Population of U.S. Adults

Background: Epigenetic mechanisms underlying associations between coronary artery disease (CAD) and ambient exposures to carbon monoxide (CO), nitrogen oxides (NO 2 /NOx), ozone (O 3 ), and sulfur dioxide (SO 2 ) are incompletely understood. We therefore examined associations between these gaseous air pollutants and DNA methylation (DNAm)—a process that controls gene expression. Methods: We conducted a race/ethnicity- and study-stratified, methylome-wide association study of 12 Women’s Health Initiative and Atherosclerosis Risk in Communities subpopulations (N=8567, mean age 61.3 years, 83% female, 46% African American, 8% Hispanic/Latino American). In each subpopulation, we estimated daily mean ambient pollutant concentrations at geocoded participant addresses using national-scale, lognormal ordinary kriging. We averaged the concentrations over the 2, 28, and 365 days before phlebotomy, the source of leukocyte DNA used in estimating DNAm at 485,000 Cytosine-phosphate-Guanine (CpG) sites on the Illumina Infinium HumanMethylation450 BeadChip. We estimated pollutant-DNAm associations using multi-level, linear mixed-effects models adjusted for socio-demographic, behavioral, and meteorological factors; estimated leukocyte proportions; and technical covariates. Then we combined estimates from the 12 subpopulations in inverse variance-weighted meta-analyses and characterized CpG sites associated with significant associations and little evidence of heterogeneity among subpopulations ( p &lt;1.0x10 -7 ; p Cochran’s Q &gt;0.05). Results: Although we found no significant or suggestive CO-, O 3 -, or SO 2 -DNAm associations, we found a significant 28-day mean NO 2 -DNAm association at cg01885635 near ZNF621 on chr. 3 ( p =5.01x10 -8 ) and suggestive 365-day mean NO 2 -DNAm associations at cg12684684 near ARPC2 on chr. 2 ( p =9.3x10 -7 ), cg19693031 near TXNIP on chr. 1 ( p =7.3x10 -7 ), and cg01885635 near ZNF621 on chr. 3 ( p =6.7x10 -7 ). We also found 2- and 365-day mean NOx-DNAm associations at cg27032760 near KCTD16 on chr. 5 ( p =4.1x10 -7 ) and cg15008743 near ZNF83 on chr. 19 ( p =2.8x10 -7 ), respectively. ZNF621 and ZNF83 encode zinc finger proteins that possess transcription factor activity. ARPC2 encodes an actin-related protein complex subunit that has been associated with mean platelet volume, a known risk factor for myocardial infarction (MI) and post-MI mortality. TXNIP encodes a thioredoxin-binding protein that protects cells from oxidative stress, is highly glucose responsive, and has been associated with triglycerides, blood pressure, diabetes, and CAD. KCTD16 encodes a potassium channel tetramerization domain-containing protein. Conclusions: Pending replication, the preliminary findings suggest that biologically plausible epigenetic mechanisms may underlie associations between nitrogen oxide exposures and CAD in multi-racial/ethnic populations.

Abstract P101: Methylome-wide Association Study Provides Evidence of Particulate Matter Air Pollution-associated Dna Methylation at Cardiovascular Disease-related Genes

Background: DNA methylation (DNAm), a heritable but dynamic epigenetic modification that can influence gene expression without altering the genome, may underlie the associations between air pollution and cardiovascular disease risk. Therefore, our objective was to evaluate associations between DNAm and ambient concentrations of particulate matter (PM) ≤ 2.5 and ≤ 10 micrometers in diameter (PM 2.5; PM 10 ). Methods: We conducted a methylome-wide association study among twelve cohort- and race/ethnicity- stratified subpopulations from the Women’s Health Initiative and the Atherosclerosis Risk in Communities study (discovery n = 6,720; replication n = 1,936; mean age: 61.3 yrs; 83% female; 46% African American; 9% Hispanic/Latino American). We averaged geocoded address-specific estimates of daily and monthly mean PM concentrations over 2, 7, 28, and 365 days and 1 and 12 months before exams at which we measured leukocyte DNAm in whole blood. In each subpopulation, we estimated PM-DNAm associations at approximately 485,000 Cytosine-phosphate-Guanine (CpG) sites in multi-level, linear mixed-effects models adjusting for sociodemographic, behavioral, and meteorological characteristics; estimated leukocyte proportions; and technical covariates. We combined subpopulation-specific PM-DNAm associations in fixed-effects, inverse variance-weighted meta-analyses of the discovery, replication, and overall populations. Then we conducted in silico characterization of CpG sites at which PM-DNAm associations exceeded methylome-wide significance and were not heterogeneous ( P &lt; 1.0 x 10 -7 ; P Cochran’s Q &gt; 0.10) to assess their putative function and biological plausibility. Results: Discovery analyses identified significant PM 2.5 - and PM 10 -DNAm associations at four CpG sites, but none survived Bonferroni correction. Overall analyses identified significant associations at two CpG sites. On chromosome 20 near MATN4 , 28-day mean PM 10 was associated with increased DNAm at cg19004594 ( P all = 2.8 x 10 -8 ; P Cochran’s Q = 0.61). MATN4 is expressed in heart and lung tissues. It encodes Matrilin 4, a von Willebrand factor A domain-containing protein linked to cardiac remodeling. On chromosome 10 near ARPP21 , 1-month mean PM 10 was inversely associated with DNAm at cg24102420 ( P all = 4.8 x 10 -8 ; P Cochran’s Q = 0.51). ARPP21 is expressed in the brain/spinal cord and neutrophils. It encodes cAMP-regulated phosphoprotein 21, a regulator of calmodulin/calcium signaling. Conclusions: Findings from this methylome-wide association study suggest that ambient PM 10 concentrations affect DNA methylation at regions of the genome potentially related to cardiovascular disease among racially, ethnically and environmentally diverse populations of U.S. men and women. Further investigation is warranted to uncover epigenetic mechanisms of PM-associated cardiovascular traits.

Cytological endometritis in dairy cows: diagnostic threshold, risk factors, and impact on reproductive performance.

We determined the threshold proportion of polymorphonuclear leukocytes (PMNs) for a diagnosis of cytological endometritis (CEM), the risk factors for this condition, and its impact on reproductive performance in dairy cows. Uterine cytology was performed on 407 Holstein cows 4 weeks postpartum to determine the proportions of endometrial cells and PMNs. A receiver operator characteristics curve was used to determine the threshold above which the PMN proportion affected the likelihood of cows conceiving by 200 days postpartum. The optimal threshold was ≥ 14% PMN (sensitivity, 31.3%; specificity, 81.7%; p < 0.05). The farm identity, retained placenta (odds ratio [OR] = 1.87), and septicemic metritis (OR = 3.07) were risk factors for CEM (p < 0.05). Cows with CEM were less likely to resume cyclicity (OR = 0.58) and to conceive by 200 days postpartum (hazard ratio = 0.58). Cows with CEM tended (p < 0.1) to be less likely to become pregnant after their first insemination (OR = 0.65) and to require a greater number of inseminations per conception (2.3 vs. 2.2). In conclusion, a PMN threshold of 14% defined the presence of CEM at 4 weeks postpartum. The farm, retained placenta, and septicemic metritis were risk factors for CEM, which reduces subsequent reproductive performance.

Is it beneficial to use clinical scoring systems for acute appendicitis in adults?

Clinical scoring systems have been used to reduce negative appendectomy rate for several decades. However, the use of these systems has been questioned due to differences in their diagnostic accuracies. The aim of this prospective study was to develop a new clinical scoring system using a combination of all previously described variables for the diagnosis of acute appendicitis (AA). Consecutive patients who underwent emergency appendectomy for AA between December 2016 and April 2017 were prospectively included in the study. During admission, a prepared questionnaire including variables obtained from the previously used clinical scoring systems was administered. Histopathological analysis was regarded as the main outcome. Patients with no histopathological evidence of AA were defined as negative appendectomy. All variables were analyzed separately to assess their association with AA. A receiver operating characteristic curve with area under curve analysis was performed to obtain the cut-off values for numerical variables. There were 200 patients with a mean age of 30.8±12.8 years with a negative appendectomy rate of 5.5%. There was no significant association between the variables and the detection of histologically proven AA except increased white blood cell count >11.05/mm3 and proportion of the polymorphonuclear leukocytes >71.2% (p=0.003 and p=0.015, respectively). The present study shows that the development and/or use of scoring systems does not significantly improve the diagnostic accuracy of AA.

Endometritis subclínica y tasa de gestación en vacas lecheras en México

En el presente estudio se determinó la prevalencia de endometritis subclínica y su efecto en la tasa de gestación en vacas lecheras en manejo intensivo. Entre los días 35 y 45 posparto se seleccionaron 237 vacas sin patologías uterinas (vacas con útero involucionado y sin secreciones vaginales anormales); a todas ellas se les tomó una muestra citológica de endometrio mediante la técnica de Cytobrush. Los frotis se procesaron con la tinción de Diff-Quick. Se contaron un total de 200 células y se determinó la proporción de células polimorfo nucleares (PMN). Las vacas con ≥ 6 % de PMN se consideraron con endometritis subclínica. Cuarenta y tres por ciento (103/237) de las vacas presentó endometritis subclínica. Las vacas con puerperio patológico tuvieron mayor probabilidad de padecer endometritis subclínica que las vacas con puerperio normal (60 vs 36 %, respectivamente; razón de probabilidad= 2.69). Las vacas normales tuvieron mayor tasa de gestación en el primer servicio que la vacas con endometritis subclínica (31.4 vs 20.0 %; respectivamente; razón de probabilidad= 2.02) y la proporción de vacas gestantes en el día 120 posparto fue mayor en las vacas normales que en las vacas con endometritis subclínica (51.6 vs 37.9 %, respectivamente; razón de probabilidad= 1.98). Se concluye que la prevalencia de endometritis subclínica en vacas lecheras entre los días 35 y 45 posparto es de 43 %, lo cual disminuyó en un 11.4 % la tasa de gestación en el primer servicio y en un 13.7 % la tasa de gestación acumulada en el día 120 posparto.

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry.

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated ≥ 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated ≥ 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Physiological and proteomic responses to corticosteroid treatments in Eurasian perch, Perca fluviatilis: Investigation of immune-related parameters

The comparative effects of cortisol and 11-deoxycorticosterone (DOC), two major corticosteroids in fish, have yet received little attention in teleosts. We evaluated the proteomic and immune responses of Eurasian perch to chronic corticosteroid treatments. We implanted immature perch with cortisol (80mg/kg) or DOC (4mg/kg) and measured the proportions of blood leucocytes, immune indices in the plasma, spleen and liver (complement and lysozyme activity, total immunoglobulin and immune gene expression in the tissues) and differential proteome expression (corticosteroid versus control) in the liver and the spleen on days 2, 4 and 14 post-treatment. Implantation of cortisol decreased the ratio of blood leucocytes and depressed Ig levels in both organs while DOC modulated the proportion of leucocyte sub-populations (increase in lymphocytes and decrease in granulocytes). In contrast, the innate humoral immunity was not strongly influenced by any of corticosteroid implants. The only immune parameter that was significantly affected was lysozyme, after DOC treatment. A number of proteins were differentially regulated by these hormones and some were identified in the liver (21 for cortisol and 8 for DOC) and in the spleen (10 for cortisol and 10 for DOC). None of the proteins was directly linked to immunity, except the natural killer enhancing factor, which was repressed by cortisol in the spleen. Our results also confirm that the proteins involved in energetic and glucose metabolism are affected by corticosteroids. Furthermore, these corticosteroids differently regulate immune status in Eurasian perch and they primarily impact leucocytes, as opposed to innate immune function.

Maternal effector T cells within decidua: The adaptive immune response to pregnancy?

In human pregnancy the maternal immune system plays a critical role in the regulation of many aspects of human reproduction including implantation, placentation and defence against infection. Interest has been focussed on the role of uterine natural killer cells (uNK) in the maternal decidua whereas effector CD4+ and CD8+ T cells have received much less attention despite the observation that they represent a major proportion of decidual leucocytes in the latter phase of pregnancy. A range of recent studies have demonstrated that human decidual T cells are highly differentiated, express a range of cytokines and cytotoxic markers, and demonstrate a unique transcriptional profile characterized by high level expression of genes involved in interferon-signalling. Moreover, subpopulations of effector T cells demonstrate specificity for fetal tissue and are regulated through expression of inhibitory checkpoint proteins and T regulatory cells. Nevertheless, many questions remain to be answered, such as the potential role of maternal effector T cells in either supporting successful pregnancy or potentially clearing fetal cells that have entered the maternal circulation. In addition, there is an increasing interest in the role of maternal effector T cells in the pathogenesis of disorders such as chronic villitis miscarriage, stillbirth, fetal growth restriction and pre-eclampsia. Current debates in relation to these questions will be discussed within this review.

Regulatory effect of dietary intake of chromium propionate on the response of monocyte-derived macrophages from Holstein cows in mid lactation.

Chromium (Cr) has been reported to enhance immune function and improve insulin sensitivity and performance in beef and dairy cattle. However, its effect on bovine macrophage inflammatory and metabolic response is unknown. The objective of this study was to characterize the effect of dietary Cr on the inflammatory and metabolic response of polarized macrophages ex vivo. Twelve primiparous and 16 multiparous healthy Holstein cows in mid lactation (143 ± 37 d in milk) were enrolled in this study. Cows were fed a common total mixed ration once per day that was top-dressed with 200 g of ground corn containing 1 of 2 dietary treatments: control (CTL, no Cr supplementation) or Cr propionate (CrP, 8 mg of Cr/cow per day) for 35 d. At d 1, 17, and 35 of treatment, blood monocytes were isolated and cultured to obtain 3 monocyte-derived macrophage (MDM) phenotypes: M0 (non-polarized), M1 (pro-inflammatory; IFN-γ polarized) and M2 (anti-inflammatory; IL-4 polarized). The experiment was set in a randomized complete block design. Neither dry matter intake nor milk yield was affected by treatment. Plasma concentrations of metabolites and the metabolic and inflammatory response of MDM in spent media were not affected by treatment. Neither the whole blood cell population nor the specific proportion of leukocytes was affected by the main effect of treatment. However, we did observe a trend for fewer circulating neutrophils in cows fed CrP than in cows fed CTL for 35 d, which may be partly attributable to a greater influx of neutrophils into peripheral tissues, a reduced pro-inflammatory response during disease, or both; this warrants future study. Expression of IGFI was increased in MDM-M0, and expression of CXCL11 tended to increase in MDM-M2 from cows fed CrP compared with cows fed CTL. Expression of SLC2A3 also tended to increase in MDM-M2 from cows fed CrP compared with cows fed CTL at 17 d. Our results suggest that CrP has minimal effect on the inflammatory and metabolic response of MDM for Holstein dairy cows in mid lactation. Future studies are warranted to evaluate the differential regulation of Cr on the inflammatory and metabolic response of leukocytes from dairy cows at different stages of lactation and parity.

Aerobic vaginitis: no longer a stranger

Aerobic vaginitis (AV) is the name given in 2002 to a vaginal infectious entity which was not recognized as such before. It is characterized by abnormal (dysbiotic) vaginal microflora containing aerobic, enteric bacteria, variable levels of vaginal inflammation and deficient epithelial maturation. Although AV and bacterial vaginosis (BV) share some characteristics, such as a diminished number or absence of lactobacilli, increased discharge (fishy smelling in BV, while in severe forms of AV, a foul, rather rotten smell may be present) and increased pH (often more pronounced in AV), there are also striking differences between the two. There is no inflammation in women with BV, whereas the vagina of women with AV often appears red and edematous, and may even display small erosions or ulcerations. The color of the discharge in BV is usually whitish or gray and of a watery consistency, whereas in AV it is yellow to green and rather thick and mucoid. Women with BV do not have dyspareunia, while some women with severe AV do. Finally, the microscopic appearance differs in various aspects, such as the presence of leucocytes and parabasal or immature epithelial cells in AV and the absence of the granular aspect of the microflora, typical of BV. Despite all these differences, the distinction between AV and BV was not recognized in many former studies, leading to incomplete and imprecise diagnostic workouts and erroneous management of patients in both clinical and research settings.The prevalence of AV ranges between 7 and 12%, and is therefore less prevalent than BV. Although still largely undiagnosed, many researchers and clinicians increasingly take it into account as a cause of symptomatic vaginitis. AV can co-occur with other entities, such as BV and candidiasis. It can be associated with dyspareunia, sexually transmitted infections (such as human papilloma virus, human immunodeficiency virus, Trichomonas vaginalis and Chlamydia trachomatis), chorioamnionitis, fetal infection, preterm birth and cervical dysplasia. Many other possible pathological associations are currently under investigation.The diagnosis of AV is made using wet mount microscopy, ideally using phase contrast. An AV score is calculated, according to: lactobacillary grade, presence of inflammation, proportion of toxic leucocytes, characteristics of the microflora and presence of immature epithelial cells. To circumvent the hurdle of microscopic investigation, some groups have begun to develop nucleic-acid-based and enzymatic diagnostic tests, but the detailed information obtained with phase contrast microscopy is irreplaceable.The best treatment is not yet fully determined, but it must be tailored according to the microscopic findings and the patient's needs. There is a role for local estrogen therapy, corticosteroids, antimicrobials and probiotics. Further research will reveal more precise data on diagnosis, pathogenesis, management and prevention.